Patent Application
20200208125
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 2, 2018, is named PHOT_US_Sequences_031620_ST25.txt and is 314 Kbytes in size.
Disclosed embodiments of the present invention are in the field of improved performance of microalgae in the production of biological products such as but not limited to biofuels, biomass, pigments, starch, oils and the like through selection, mutagenesis or engineering to reduce expression or knockout the phototropin gene for example.
Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the unicellular green alga Chlamydomonas reinhardtii, phototropin (PHOT) plays a vital role in the progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, and chlorophyll binding proteins. The UV-A/blue light sensing phototropins mediate a variety of light responses and are responsible in higher plants for optimization of photosynthetic yields (Chen, Chory et al. 2004).
Phototropins are commonly composed of two domains, an amine terminal photosensory domain and a carboxy terminal serine/threonine protein kinase domain. The photosensory domain is a flavin mononucleotide binding domain, the LOV domain. Plants and green algae contain two of these domains in the phototropin regulatory sequence, LOV1 and LOV2 (Chen, Chory et al. 2004). LOV domain is a member of PAS domains and are about 110 amino acids. There is a conserved sequence within the LOV domain identified at amino acid position 238-245 of SEQ ID NO: 1 for example (Gly Arg Asn Cys Arg Phe Leu Gln Gly). (Salomon et al. 2000). A diagram of the phototropin protein is:
Phototropin knock-out mutants (PHOT K/O) have been made previously in plants (Suetsugu and Wada 2007, Moni, Lee et al. 2015) and algae (Zorin, Lu et al. 2009; Trippens, Greiner et al. 2012). However, all the PHOT K/O mutant prior art that has been located to date did not show improved productivity of the plant or alga.
In plants two phototropins have been reported, phot1 and phot2, these phototropins share sequence homology and have overlapping functions. These blue-light-sensitive receptors consist of two parts: a C-terminal serine-threonine kinase and two LOV domains that bind flavin mononucleotide as chromophores at the N-terminus. Recently, in the unicellular green alga, Chlamydomonas reinhardtii, a phototropin homolog was identified. It exhibits photochemical properties similar to those of higher plant phototropins and is also functional in Arabidopsis. Studies show that the basic mechanism of phototropin action is highly conserved, even though its apparent physiological functions are quite diverse.
Plants utilize several families of photoreceptors to better react to their environment, allowing them to fine tune pathways controlled by the photoreceptors—phototropin, phytochrome, and cryptochrome (Chen, Chory et al. 2004).
In higher plants phototropin mediates a variety of blue-light elicited physiological processes (Sullivan, Thomson et al. 2008). Phototropins are UV-A/blue light sensing photoreceptors that are known to optimize photosynthetic yields (Chen, Chory et al. 2004). The involvement of phototropin in photomovement in higher plants is well documented (Suetsugu and Wada 2007, Kagawa, Kimura et al. 2009). Studies involving Arabidopsis mutants lacking the phot1 and phot2 genes have revealed that in addition to regulating hypocotyl curvature of seedlings towards blue light, phototropins also regulate a diverse range of responses in flowering plants. These responses include chloroplast movements, nuclear positioning, stomatal opening, leaf expansion, leaf movements and leaf photomorphogenesis.
Phototropin knock-out mutants (PHOT K/O) have been made previously in plants (Suetsugu and Wada 2007, Moni, Lee et al. 2015). For instance in Physcomitrella patens (a moss) there are three PHOT genes and they have all been knocked out in different mutants (Suetsugu and Wada 2007). The focus of the P. patens study was the effect of PHOT K/O on phototropism (movement toward light) and the phenotypes they observed allowed them to determine which of the genes were necessary for phototropism (Suetsugu and Wada 2007).
PHOT expression was higher in darkness than in light, and phot1 Arabidopsis mutants was shown to increase the number of lateral roots produced (Moni, Lee et al. 2015). phot was also demonstrated to mediate phototropism, chloroplast relocation and leaf expansion (Matsuoka, Iwata et al. 2007). Using phot deficient Arabidopsis mutants, phototropin 2 was linked to palisade parenchyma cell development of leaves (Kozuka, Kong et al. 2011).
Another study looked at the role of phototropin under low photosynthetically active radiation (Takemiya, Inoue et al. 2005). They found that the wild-type and the PHOT1 mutant both showed increased but similar growth in low radiance blue light super imposed on red light. In white light there was no increase in biomass in both phot1 and phot2 mutants as well as in the double phot mutant.
A study by Folta and colleagues investigated the relationship between phot1 and phototropism and growth inhibition in Arabidopsis (Folta, Lieg et al. 2003). They found that the onset of phototropism and the phot1-mediated growth inhibition coincided and postulated that both were due to phot1 expression.
There is a substantial amount of patent literature around phototropin in higher plants. However, the focus has been on the commercial utility of the upstream, light regulated areas rather than on the phototropin gene itself. These light control domains that regulate PHOT expression—the light-oxygen-voltage-sensing (LOV) domains—have been carefully evaluated for potential commercial application in higher plants.
Shu & Tsien application (US20130330718) focused on using the LOV domain for control of proteins that generate singlet oxygen (SOGs). These fusion protein tags could be used for imaging under blue light for research purposes.
Other patents use light switchable regulatory sequences and contemplate the use of the phototropin LOV domain such as Yang and colleagues (EP2682469).
Hahn & Karginov (WO2011133493) focused on allosteric regulation of kinases using the light activated domains for control of expression in engineered fusion proteins (such as the LOV domains).
Hahn and colleagues (U.S. Pat. No. 8,859,232) demonstrated that the LOV domain of phototropin can be used as a light activated switch for the activation or inactivation of fusion proteins of interest. They contemplated using a LOV domain that could contain substantial portions of the phototropin molecule in addition to the LOV domain. They contemplated using the LOV domain isolated from algae and gave the specific example of Vaucheria frigida, a stramenopile or heterokont alga.
Kinoshita and colleagues (WO2014142334) demonstrated that overexpression of phototropin had no impact of stomatal opening in higher plants.
Bonger and colleagues (US20140249295) used the LOV domain as a fusion with another functional protein wherein the light switching ability of the LOV domain was used to control the stability and/or function of the fusion protein.
Folta and colleagues (WO2014085626) using mutants of phototropin 1 were able to show that the function of phot1 is mediation of the pathway in which green light reverses the effects of red and/or blue light on plant growth.
Schmidt & Boyden (US20130116165) describe a new group of fusion proteins with light regulatory regions derived from Avena sativa phototropin 1. These regulatory domains are used for altering channel function in membranes.
To date there is no disclosure of the use of PHOT knockout or knockdown (suppression) technology to improve or algae plant productivity.
Phototropin has already been well studied in several different algae including Chlamydomonas reinhardtii (Briggs and Olney 2001). However, there are indications that phototropins have diverged significantly or that the genes that function as phototropin are not very homologous to plant phototropin genes. For instance it was reported that in Thalassiosira pseudonana (a diatom) and Cyanidioschyzon merolae (unicellular red alga) no genes were found encoding the phototropins (Grossman 2005). However putative genes with photosensory LOV domains, aurechromes, have been reported for these and other photosynthetic stramenopiles (Table 1). Most aureochromes contain a single LOV domain and function as transcription factors that regulate cell division, chloroplast movement, pigment production, and phototropism. (Takahashi. J Plant Res (2016) 129:189-197)
In Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle (Huang and Beck 2003), control of the eye spot size and light sensitivity (Trippens, Greiner et al. 2012). Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. Phototropin has been localized to the flagella of Chlamydomonas reinhardtii (Huang, Kunkel et al. 2004). Phototropin is also known to be involved in expression of genes encoding chlorophyll and carotenoid biosynthesis and LHC apoproteins in Chlamydomonas reinhardtii Eberhard et al. 2006). The Chlamydomonas reinhardtii phototropin gene has been cloned and shown to function when expressed in Arabidopsis (Onodera, Kong et al. 2005).
Phototropin has been shown to control multiple steps in the sexual life cycle of Chlamydomonas reinhardtii (Huang and Beck 2003). PHOT knockdowns using RNAi were generated (Huang and Beck 2003). The entire focus of this study was on sexual mating and no mention of improved biomass, starch accumulation or photosynthesis rate was observed. It is also involved in the chemotaxis that is the initial phase of the sexual cycle of Chlamydomonas reinhardtii (Ermilova, Zalutskaya et al. 2004). However, no cell cycle implications of phototropin knockout or knockdowns have been published.
Detailed studies have carefully analyzed the function of the LOV domain in several algal species. An example is the Chlamydomonas reinhardtii mutant LOV2-C250S where careful studies of the light activation and regulation of this domain were carried out to better understand the mechanism of action (Sethi, Prasad et al. 2009).
Phototropin knock-out mutants (PHOT K/O) have been made previously in algae (Zorin, Lu et al. 2009 Trippens, Greiner et al. 2012). PHOT minus strains had larger eyespots than the parental strain (Trippens, Greiner et al. 2012). This study focused on the impact of PHOT on eyespot structure function. These authors used a knock-out mutant of PHOT to reduce expression of phototropin (Trippens, Greiner et al. 2012).
Novel phototropins have been described in the green alga Ostreococcus tauri and with a focus on their LOV domain structure/function (Veetil, Mittal et al. 2011).
Abad and colleagues (WO2013056212) provide the sequence for phototropin from a green alga, Auxenochiorella protothecoides, and indicate that the gene would be important for photosynthetic efficiency. However, they do not discuss the impact of deletion or inhibition of this gene on the alga.
Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the exemplary embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned are incorporated by reference in their entirety. In case of conflict, the present specification and definitions will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting for the practice of this invention.
Unless specifically referred to in the specification singular forms such as “a,” “an,” and “the,” include their plural forms. As an example, “an alga” includes its plural form “algae” and “a plant” includes the plural “plants.”
The term “algae” will refer to all organisms commonly referred to as algae including the prokaryotic cyanophyta (commonly called blue-green algae and cyanobacteria), prochlorophyta, glaucophyta, rhodophyta, heterokontophyta, haptophyte, cryptophyta, dinophyta, euglenophyta, chloroaracniophyta, chlorophyta, and those organisms of indeterminate nomenclature normally referred to as algae. A full description of these is found in the book “Algae An Introduction to Phycology” by Van Den Hoek, Mann & Jahns (1995), which is included by reference.
The term “expression” as used herein refers to transcription and/or translation of a nucleotide sequence within a host cell. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired polypeptide encoded by the selected sequence.
The term “overexpression” as used herein refers to excessive expression of a gene product (RNA or protein) in greater-than-normal amounts.
The term “homologous” refers to the relationship between two proteins that possess a “common evolutionary origin”, including proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species, as well as homologous proteins from different species.
As used herein, “identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions.
The term “sequence similarity” refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin (Reeck, de Haen et al. 1987). However, in common usage and in the current invention, the term “homologous”, when modified with an adverb such as “highly”, may refer to sequence similarity and may or may not relate to a common evolutionary origin.
In specific embodiments, two nucleic acid sequences are “substantially homologous” or “substantially similar” when at least about 75%, and more preferably at least 80%, and more preferably at least 85%, and more preferably at least about 90% or at least about 95% of the nucleotides (or any integer value in between) match over a defined length of the nucleic acid sequences, as determined by a sequence comparison algorithm such as BLAST, CLUSTAL, MUSCLE, etc. An example of such a sequence is an allelic or species variant of the specific phototropin gene of the present invention. Sequences that are substantially homologous may also be identified by hybridization, e.g., in a Southern hybridization experiment under stringency conditions as defined for that particular system. The homology may be as high as about 93-95%, 98%, or 99% (or any integer value in between). For example, the sequence to which homology is matched is a wild-type parental line and the length of the sequence is the full length of the sequence from wild-type parental line.
Similarly, in particular embodiments of the invention, two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 75% of the amino acid residues are identical wherein identical contemplates a conservative substitution at a nucleic acid position. In a preferred embodiment there is at least 80%, and more preferably at least 85%, and more preferably at least about 90% and more preferably at least about 90-95% of the amino acid residues are identical (or any integer value in between). Two sequences are functionally identical when greater than about 95% of the amino acid residues are similar. Preferably the similar or homologous polypeptide sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Version 7, Madison, Wis.) pileup program, or using any of the programs and algorithms described above. Conservative amino acid substitutions are among: acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; basic (positively charged) amino acids such as arginine, histidine, and lysine; neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; amino acids having aliphatic-hydroxyl side chains such as serine and threonine; amino acids having amide-containing side chains such as asparagine and glutamine; amino acids having aromatic side chains such as phenylalanine, tyrosine, and tryptophan; amino acids having basic side chains such as lysine, arginine, and histidine; amino acids having sulfur-containing side chains such as cysteine and methionine; naturally conservative amino acids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the homologs encoded by DNA useful in the transgenic plants or algae of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.
The term “knockout” or “gene knockout” refers herein to any organism and/or its corresponding genome where the gene of interest has been rendered unable to perform its function. This can be accomplished by both classical mutagenesis, natural mutation, specific or random inactivation, targeting in cis or trans, or any method wherein the normal expression of a protein is altered to reduce its effect. For example but not to limit the definition 1) one can use chemical mutagenesis to damage the gene and then select for organisms not expressing the gene, 2) one can target the gene and remove a portion or all of the gene by homologous recombination, 3) one can use RNAi methods to produce an inhibitor molecule for a particular protein and similar methods and 4) one can use genome editing tools (i.e. CRISPR-Cas) to specifically modify the gene.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature (Sambrook, Fritsch et al. 1989, Ausubel, Brent et al. 1997, Green and Sambrook 2012).
The term “transcriptome” refers to the set of RNA molecules present in a population of cells. It often reflects how an organism responds to particular situations and is looking at what genes are regulated under a particular condition. Examples of transcriptome analyses on algae are found in the following references (Hwang, Jung et al. 2008, Rismani-Yazdi, Haznedaroglu et al. 2011, Fu, Wang et al. 2014, Koid, Liu et al. 2014).
The term “biofuel” refers to any fuel made through the application of biological processes not on a geological timescale. Examples include but are not limited to conversion of algal biomass to biocrude through hydrothermal liquefaction, anaerobic digestion of spent algal biomass for conversion to methane, extraction of lipid from algal biomass to convert to biodiesel, and conversion of water to biohydrogen through biological processes.
The term “bioproduct” is any product produced from biological processes either in whole or in part.
The term biomass productivity or production as used herein refers to the rate of generation of biomass in an ecosystem. It is usually expressed in units of mass per unit surface (or volume) per unit time, for instance grams per square metre per day (g m−2 d−1). The mass unit may relate to biologically produced dry matter generated.
The term “sink molecules”, “sink compounds”, sink materials” refers to molecules used by an organism to store captured carbon. These can be but are not limited to sugars, starch, glycogen, lipids, fats, waxes, and similar biomolecules.
The publications discussed above are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosures by virtue of prior invention.
This and other unmet needs of the prior art are met by exemplary compositions and methods as described in more detail below.
One embodiment of the present invention provides for a method for increasing a biomass productivity of an algal strain wherein the expression or function of a Chlamydomonas reinhardtii phototropin gene, a gene substantially similar to the Chlamydomonas reinhardtii phototropin gene or a sequence substantially similar to SEQ ID NO 1-14, 51-66 and 69-128 is reduced or eliminated. In a preferred embodiment the gene substantially similar has greater than 75% homology, more preferably greater than 80%, or 85%, or 90% or 95% homology to the Chlamydomonas reinhardtii phototropin gene or the sequence identified in SEQ ID NO 1-14, 51-66 and 69-128.
For example, the biomass productivity of the algal strain is increased by greater than around 2-fold. The biomass production of storage product(s) in the algal strain is increased by greater than around 2-fold, for example the storage product(s) is selected from starch, lipid, pigments and other sink molecules and for example the productivity of biomass is increased by greater than around 2-fold. Further, the biomass productivity may be increased for bioproducts chosen from lipids, waxes, polysaccharides (e.g., starch, glycogen, mannans, glycans, cellulose, hemicellulose), pigments (e.g., xanthophyll). In a preferred embodiment the expression of the Chlamydomonas reinhardtii phototropin gene, the gene substantially similar to the Chlamydomonas reinhardtii phototropin gene or the sequence substantially similar to SEQ ID NO 1-14, 51-66 and 69-128 is reduced by example chemical mutagenesis and selection, genome editing, trans acting elements (e.g., RNAi), and/or an inducible basis through an inducible promoter.
Another embodiment of the present invention provides for an algal strain wherein relative to the wild-type parental line the expression of the phototropin gene or a substantially similar gene is reduced, the photosynthetic pigments making up the antenna complex are reduced, and/or the content of sink molecules is increased. In a preferred embodiment the phototropin gene or a substantially similar gene been rendered to be non-functional. In a preferred embodiment the non-functional gene has been substantially deleted or is rendered to be non-functional on an inducible basis through an inducible promoter. In a preferred embodiment the algal line having the phototropin gene deletion would generate sterile and stable diploid population of polyploid algae to avoid recombination of genetic material during sexual reproduction or in another embodiment would be used to generate stable transgene-stacking traits in polyploid algal strains. In a preferred embodiment the phototropin gene or a substantially similar gene is selected from SEQ ID NO 1-14, 51-66 and 69-128. In another preferred embodiment the gene or the gene substantially similar has greater than 75% homology, or greater than 80%, or 85%, or 90% or 95% homology to the Chlamydomonas reinhardtii phototropin gene or the sequence identified in SEQ ID NO 1-14, 51-66 and 69-128.
In another embodiment a method for increasing a biomass productivity of an algal strain wherein the expression or function of a Chlamydomonas reinhardtii NTR2 or NTRC gene, a gene substantially similar to a Arabidopsis NTR2 or NTRC gene or a sequence substantially similar to SEQ ID NO 35-50 and 67-68 is over expressed in the algal strain is provided. In a preferred embodiment the gene substantially similar has greater than 75% homology, or more than 80%, 85%, 90%, or 95% homology to the Arabidopsis NTR2 or NTRC gene or the sequence identified in SEQ ID NO 35-50 and 67-68.
For example, the biomass productivity of the algal strain is increased by greater than around 2-fold. The biomass production of storage product(s) in the algal strain is increased by greater than around 2-fold, for example the storage product(s) is selected from starch, lipid, pigments and other sink molecules and for example the productivity of biomass is increased by greater than around 2-fold. Further, the biomass productivity may be increased for bioproducts chosen from lipids, waxes, polysaccharides (e.g., starch, glycogen, mannans, glycans, cellulose, hemicellulose), pigments (e.g., xanthophyll).
In yet another embodiment a method for increasing a productivity of an algal strain wherein the expression or function of a Chlamydomonas reinhardtii KIN10 or KIN11 gene, a gene substantially similar to a Arabidopsis KIN10 or KIN11 gene or a sequence substantially similar to SEQ ID NO 15-34 is over expressed in the algal strain is provided. In a preferred embodiment the gene substantially similar has greater than 75% homology, or greater than 80%, 85%, 90%, or 95% homology to the Arabidopsis KIN10 or KIN11 gene or the sequence identified in SEQ ID NO 15-34. For example, the biomass productivity of the algal strain is increased by greater than around 2-fold. The biomass production of storage product(s) in the algal strain is increased by greater than around 2-fold, for example the storage product(s) is selected from starch, lipid, pigments and other sink molecules and for example the productivity of biomass is increased by greater than around 2-fold. Further, the biomass productivity may be increased for bioproducts chosen from lipids, waxes, polysaccharides (e.g., starch, glycogen, mannans, glycans, cellulose, hemicellulose), pigments (e.g., xanthophyll).
Exemplary embodiments of the compositions, systems, and methods disclosed herein wherein algae are treated so as to reduce or eliminate the expression of phototropin or a heterologous gene with the same function such that improved productivity is achieved.
In one aspect, embodiments of the present invention provide an organism and the method to use such organism where the phototropin gene is knocked out and the photosynthetic rate is improved and the biomass productivity improves.
In a further aspect, the mutant is produced from Chlamydomonas reinhardtii and the biomass productivity is doubled.
Another embodiment of the present invention provides an organism with reduced PHOT expression wherein the sexual cycle is arrested and the genetic stability of the algal cell culture line is improved.
In a further embodiment the organism is derived from Chlamydomonas reinhardtii and has reduced promiscuity resulting in a more stable genotype and phenotype.
In one aspect, embodiments of the present invention provide an organism with reduced phototropin gene expression and the method to use such organism which as improved non-photochemical quenching providing the ability for better response to high light levels.
In one aspect, embodiments of the present invention provide an organism with reduced phototropin expression and the method to use such organism that results in higher levels of sink molecules, such as but not limited to lipid and starch.
In a further embodiment the organism has enhanced cell division compared to wild-type.
In a further embodiment the organism is derived from Chlamydomonas reinhardtii.
In another embodiment of the method wherein the expression of the Chlamydomonas reinhardtii phototropin gene is reduced by genome editing (i.e. CRISPR/Cas).
In another embodiment of the method wherein the expression of the Chlamydomonas reinhardtii phototropin gene is reduced by trans acting elements (e.g., RNAi).
In a further embodiment the gene downstream of PHOT has substantial homology to the Arabidopsis KIN10 or KIN11 genes or a portion thereof (Snf1 related kinases, SNRK) and can be overexpressed to increase the productivity of an algal strain.
In yet a further embodiment the KIN10 and KIN11 genes or a portion thereof are chosen from genes substantially homologous to a nucleic acid sequence identified in SEQ ID NO 15 to 34 or a nucleic acid sequence encoding for an amino acid sequence identified in SEQ ID NO15 to 34.
In a further embodiment the gene downstream of phot has substantial homology to the Arabidopsis NTRC and NTR2 gene(s) or a portion thereof and can be overexpressed to increase the productivity of an algal strain.
In yet a further embodiment the NTRC and NTR2 genes or a portion thereof are chosen from genes substantially homologous to a nucleic acid sequence identified in SEQ ID NO 35 to 50 or a nucleic acid sequence encoding for an amino acid sequence selected in SEQ ID NO 35 to 50.
A better understanding of the exemplary embodiments of the invention will be had when reference is made to the accompanying drawings, and wherein:
While there have been numerous studies on algal phototropin (Huang and Beck 2003, Ermilova, Zalutskaya et al. 2004, Huang, Kunkel et al. 2004, Im, Eberhard et al. 2006, Sethi, Prasad et al. 2009, Veetil, Mittal et al. 2011, Trippens, Greiner et al. 2012) to date there has been no correlation of the reduction or knock-out of phototropin to higher levels of biomass production and increased production of sink molecules/products such as starch and lipid.
The transcriptome of a Chlamydomonas reinhardtii phototropin knock out (PHOT K/O) mutant and the wild-type parent were compared to analyze differences in gene expression in high light grown cultures (500 μmol photons m−2 s−1). An up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes was observed in the PHOT K/O mutants. Referring now to
Using a variety of methods exemplary embodiments of the invention are directed at improving the productivity of algal systems based on control of the phototropin gene and genes similar to phototropin in algal systems. This is particularly applicable to improving biomass productivity in algal mass culturing either for production of algal biofuels or bioproducts.
Productivity is a central issue in algae production and a doubling of the productivity could be very attractive to groups who hope to cross the threshold of commercial viability. However, one should note that widespread adoption of transgenic algae as a production system is not yet embraced. Several companies (for example Algenol, Ft. Meyers, Fla.) are using transgenic algae (cyanobacteria) in closed tube reactors outdoors and, presumably, have a track to (national) regulatory approval. Use of transgenic algae has been approved in Florida and approvals have recently been granted by the US EPA for GMO field trials for Sapphire Energy Company.
Production of bioproducts using this invention, owing to the observed doubling of productivity in biomass and sink molecules/compounds, could be pivotal in reaching commercial viability. The observed increase in starch production by this invention is especially important as it shows sink molecules/compounds are enhanced by the methods of this invention.
Alternative genome editing technologies such as CRISPR/Cas 9, Talen and Zinc finger nuclease approaches could also be used to inhibit expression of phototropin (Gaj, Gersbach et al. 2013, Sizova, Greiner et al. 2013).
It is possible to make PHOT knockouts using non-GMO approaches such as classical mutagenesis using chemical mutagens such as methylnitronitroso guanidine and ethyl methane sulfonate (Yan, Aruga et al. 2000).
To date, supporting data for this invention have been limited to the green alga, Chlamydomonas reinhardtii. Compared to wild-type C. reinhardtii, PHOT K/O mutants of the invention show:
Additionally, PHOT K/O mutants were unable to undergo sexual mating, which was attributed to an impact of the PHOT K/O on the cell cycle—effectively blocking meiosis while accelerating photosynthetic and cell division rates.
Chlamydomonas reinhardtii PHOT knockout lines were generated in different parental backgrounds. PHOT K/O line G5 was made in cw15 parental background and A4 mutant line was made in UV4 background (Zorin, Lu et al. 2009).
Chlorophyll (Chl) and carotenoids are the central pigments of the photosynthetic apparatus. These pigments are associated with light-harvesting complexes and reaction-center complexes in photosynthetic organisms. The light environment plays a major role in governing the pigment composition of pigment-protein complexes of the photosynthetic apparatus. Blue light is especially important in modulating the synthesis of Chl and carotenoids, as well as the biogenesis of the photosynthetic apparatus in microalgae and vascular plants. Consistent with phototropin regulation of pigment biosynthetic pathways C. reinhardtii PHOT K/O lines showed:
Carotenoid content: When grown under low light intensities PHOT K/O lines showed a 30-40% reduction in carotenoid content compared to parent wild. The changes in xanthophyll cycle pigments were analyzed since the xanthophyll cycle pigments play an important role as antioxidants and for non-photochemical quenching of excess energy captured by the light harvesting complex. Both PHOT K/O lines show higher accumulation of photoprotective pigments in high light compared to their respective WT parents. Referring now to
De-epoxidation rates: Consistent with the xanthophyll cycle pigment accumulation PHOT K/O lines show higher De-epoxidation in high light conditions as compared to their respective wild-type under high light (
In C. reinhardtii, the peripheral PSII antenna is able to migrate laterally between PSII and PSI, in a process known as state transitions, to balance the excitation energy distribution between the two photosystems and to regulate the ratio of linear and cyclic electron flows. Linear electron transfer produces ATP and NADPH, while cyclic electron transfer driven by PSI produces only ATP. Increasing the antenna size of the PSI complex facilitates cyclic electron transfer and has been shown to enhance ATP production and support the optimal growth of Chlamydomonas. To assess the impact of reduced pigment content on the ability to carry out state transitions, chlorophyll (Chl) fluorescence induction kinetics were measured in low-light grown parent wild-type (
Referring now to
The transcriptomic analysis of the PHOT K/O mutants compared to wild-type parental strains provided information on the different genes impacted by the elimination of phototropin expression (
Most importantly, phototropin knock out lines (open boxes), had twice the cell density (
Carbon fixation is the main pathway for storing energy and accumulating biomass in algae and plants. Many rate limiting genes were up-regulated in PHOT K/O lines (
Thioredoxins are small ubiquitous redox proteins, which are crucial components of the regulatory redox networks in all living cells. Thioredoxins are reduced by different reductases, depending on their subcellular localization. Among these reductases, NADPH-dependent thioredoxin reductases (NTR) genes are known to regulate multiple gene targets involved in photosynthesis, non-photochemical quenching (NPQ), Calvin-Benson cycle, starch biosynthesis, cold stress tolerance and thermotolerance.
KIN10 or KIN11 ((Snf1 related kinases, SNRK) are one of the very well-studied central regulators of energy and stress metabolism in plants. SNRK1 proteins play central roles in coordinating energy balance and nutrient metabolism in plants. A 10-fold increase was observed for the PHOT K/O mutants.
Cell cycle genes are up regulated in Chlamydomonas reinhardtii PHOT K/O mutants may enhance cell division in these lines contributing to the higher biomass in these lines (
Glycolysis is the first step in the breakdown of glucose to extract energy for cellular metabolism, which converts glucose to pyruvate and generates ATP (energy) and NADH (reducing power). Many important genes of this pathway show higher expression in PHOT K/O mutants.
We compared the chloroplast ultrastructure of the parental and PHOT K/O cells to determine whether there were changes in thylakoid membrane structure and starch accumulation. Starch represents the most widespread storage polysaccharide found in the plastids of both photosynthetic and non-photosynthetic cells of plants and algae. PHOT K/O lines exhibited higher accumulation of starch grains compared to their respective parent strains as well as up-regulation of starch synthesis genes (
Chlamydomonas reinhardtii PHOT K/O mutants have higher starch accumulation due to up-regulation of the following genes involved in starch biosynthesis is
A structural hallmark of thylakoid membranes in plants and microalgae is the stacking of the membranes associated with the localization of the PSII complex. The stromal membranes extending from the stacks are enriched in PSI and ATPase complexes. This arrangement of LHCII complexes provides functional flexibility, enabling their primary light harvesting function as well as ability to participate in multilevel regulatory mechanisms involving highly efficient energy dissipation through pigment interactions such as chlorophyll-xanthophyll interactions. These regulatory processes require a significant reorganization in the membrane, and a substantial degree of structural flexibility in thylakoid membranes to carry out short-term adaptations and long-term acclimations in response to change in light and environmental stimuli.
An electron micrograph illustration showing the thylakoid membrane structure in both parent strain and PHOT K/O line is drastically altered in PHOT K/O lines. These results are in concert with the phototropin involvement in regulation of LHC protein biosynthesis and pigment biosynthesis. When thylakoid membranes are tightly stacked, they are densely packed with proteins and inhibit efficient protein diffusion including diffusions of the electron transport carrier protein plastocyanin. This protein mobility is required for efficient photosynthetic electron transfer, as well as regulation and repair of photodamaged photosynthetic apparatus. In parent cells thylakoid membranes are very tightly stacked giving very little space for the movement of the molecules). In contrast, PHOT K/O lines have parallel grana stacks and wide luminal spacing
The following genes involved in lipid metabolism are up regulated in PHOT K/O mutants:
The methyl erythritol 4-phosphate (MEP) pathway is the source of isoprenoid precursors for the chloroplast. The precursors lead to the formation of various isoprenoids having diverse roles in different biological processes. Some isoprenoids have important commercial uses. Isoprene, which is made in surprising abundance by some trees, plays a significant role in atmospheric chemistry. Multiple genes involved in MEP/DOXP pathway were up regulated in PHOT K/O mutants (
Note that all data so far were generated in cell wall free mutants of Chlamydomonas reinhardtii. Metabolomic analyses in C. reinhardtii clarified the pathways and gene up-regulation in high light in C. reinhardtii PHOT K/O mutants of this invention:
The Chlamydomonas reinhardtii phototropin gene has already been sequenced and a provisional version is available publically (GenBank 5718965). Additional algal genes are available that have either been shown to be a phototropin, contain blue light receptors, have some homology to phototropin or are putative blue light receptors similar to phototropin (Table 1). Additional phototropin genes in two other production strains of microalgae are known.
Chlorella sp. Strain1412. Is a strain developed by the National Alliance of Biofuels and Bio-products (NAABB) consortium and is housed at UTEX Culture Collection Of Algae at the University of Texas at Austin (UTEX). The amino acid sequence is provided as SEQ ID NO. 1 and the nucleotide sequence as SEQ ID NO. 2. The phototropin B gene of Chlorella sorokiniana. Strain 1412 is provided as SEQ ID NO. 3 and nucleotide as SEQ ID NO. 4.
Chlorella sp. sorokiniana strain 1230. Is a UTEX strain. The amino acid sequence of phototropin A is provided as SEQ ID NO. 5 and the nucleotide sequence as SEQ ID NO. 6. The amino acid sequence of phototropin B is provided as SEQ ID NO. 7 and the nucleotide sequence as SEQ ID NO. 8.
Chlorella sp. sorokiniana strain 1228. The amino acid sequence of phototropin A is provided as SEQ ID NO. 9 and the nucleotide sequence as SEQ ID NO. 10. The amino acid sequence of phototropin B is provided as SEQ ID NO. 11 and the nucleotide sequence as SEQ ID NO. 12.
Picochlorum soloecismus (DOE101). The amino acid sequence is provided as SEQ. ID NO. 13 and the nucleotide sequence as SEQ. ID NO. 14.
Micromonas pusila CCMP1545
Volvox carteri f. nagariensis
Auxenochlorella protothecoides
Auxenochlorella protothecoides
Bathycoccus prasinos
Ostrecoccus tauri
Micromonas sp, RCC299
Cyanidioschyzon merolae 10D
Galdieria sulphuraria
Galdieria sulphuraria
Coccomyxa subellipsoidea C-169
Chlorella variabilis
Ostreococcus lucimarinus
Guillarida theta CCMP2712
Thalassiosira pseudonana
Thalassiosira pseudonana
Phaeodactylum tricornutum CCAP
Ectocarpus siliculosus
Nannochloropsis gaditana
Vaucheria frigida
Additional PHOT downstream signal transduction targets can be use as alternatives to the knockout or reduction in phot expression to generate the desirable phenotypes of this invention, including but not limited to improved photosynthetic efficiency, higher biomass productivity, increase yield of sink molecules/compounds, and improved genetic stability. An example of this could be the algal gene homologous to the Arabidopsis KIN10 and KIN11 kinases (Baena-Gonzalez, Rolland et al. 2007). Genes substantially homologous to the Chlorella genes in SEQ ID 15 to 27 and the Chlamydomonas genes in SEQ ID 28-34 would be applicable to this current invention.
Additional gene targets can be used as alternatives to the knockout or reduction in phot expression to generate the desirable phenotypes of this invention with desirable phenotypes having but not limited to improved photosynthetic efficiency, higher biomass productivity, increase yield of sink molecules. These genes could include the algal genes homologous to the Arabidopsis NADPH thioredoxin reductase C (NTRC) and NADPH thioredoxin reductase 2 genes (Toivola et al. 2013) Genes substantially homologous to the Chlorella genes in SEQ ID NO 35-40, 43-44 and 47 to 50 and the Chlamydomonas genes in SEQ ID 67-68 would be applicable to this current invention
Certain embodiments of the invention will be described in more detail through the following examples. The examples are intended solely to aid in more fully describing selected embodiments of the invention, and should not be considered to limit the scope of the invention in any way.
Chlamydomonas reinhardtii parental strains (cw15 and UV4) and the phototropin knockout (PHOT K/O) mutants (CW15 and A4) were grown at 25° C. in 250 mL Erlenmeyer flasks containing 100 mL of High-Salt (HS) or Tris-Acetate-Phosphate (TAP) media and shaken at 150 rpm (world wide web at chlamy.org/media.html). Cultures were typically inoculated from a log phase culture using 1 mL of cells. Flasks were illuminated using fluorescent light at the light intensities as indicated for each experiment.
Photoautotrophic growth of the parent strains CW15 and UV4) and the phototropin knock out mutants (G5 and A4) was measured in environmental photobioreactors (“ePBRs”) (obtained from Phenometrics, Inc.) in 500 mL of liquid HS media. All experiments were done in triplicates for each time point and each treatment. Light intensity was programmed for a 12 h sinusoidal light period with a peak mid-day intensity of 2,000 μmol photons m−2 s−1. Temperature was a constant 25° C., and the ePBRs were stirred with a magnetic stir bar at 200 rpm. Filtered air was bubbled constantly through the growing cultures. The optical density of the cultures was monitored on a daily basis at 750 nm using a Cary 300 Bio UV—Vis spectrophotometer (Agilent). After completion of growth measurements, the total contents of individual ePBRs were harvested by centrifugation at 11,000 rpm for 15 min. Cell pellets were frozen immediately in liquid N2 and later freeze-dried using a Microprocessor Controlled Lyophilizer (Flexi-Dry). After drying, pellets were weighed for total biomass.
For Chl fluorescence induction analysis, cell suspensions of the parental wild-type and transgenic Chlamydomonas strains were adjusted to a Chl concentration of ˜2.5 pg/mL. Quenching of Chl fluorescence was measured using the FL-3500 fluorometer (Photon System Instruments) (Kaftan, Meszaros et al. 1999). The cells were dark adapted for 10 min prior to the measurement. Chl fluorescence was induced using non-saturating continuous illumination and Chl fluorescence levels were measured every 1 μs using a weak pulse-modulated measuring flash. For the state transition experiments, low light grown cultures were dark adapted or pre-illuminated with 715 nm light for 10 min prior to the induction of Chl fluorescence. The actinic flash duration for this experiment was set to 50 μs and Chl fluorescence was measured every 1 μs.
CO2-supported rates of oxygen evolution were determined for low light (50 μmol photons m−2 s−1) HS grown log-phase cultures (0.4-0.6 OD750 nm) using a Clark-type oxygen electrode (Hansatech Instruments). Cells were re-suspended in 20 mM HEPES buffer (pH 7.4) and air-saturated rates of oxygen evolution were measured as a function of light intensity (650 nm) at 50, 150, 300, 450, 600, 750 and 850 μmol photons m−2 s−1. The same experiment was repeated in the presence of 10 mM NaHCO3. Light saturation curves were normalized on the basis of Chl as well as cell density (A750 nm). Chl was determined by method described by Arnon (Arnon 1949).
Chlamydomonas cultures were grown at low (50 μmol photons m31 2s−1) and high (saturating) light (500 μmol photons m31 2s−1) intensities for 5 days in HS media in shaker flasks. Cells were centrifuged at 3,000 rpm for 3 min and immediately frozen in liquid nitrogen and lyophilized. Carotenoids and chlorophylls were extracted with 100% acetone in the dark for 20 min. After incubation samples were centrifuged at 14,000 rpm for 2 min in a microfuge and the supernatant was transferred to a glass tube and dried under vacuum. The dried samples were re-suspended in 1 mL of acetonitrile:water:triethylamine (900:99:1, v/v/v) for HPLC analysis. Pigment separation and chromatographic analysis were performed on a Beckman HPLC equipped with a UV-Vis detector, using a C18 reverse phase column at a flow rate of 1.5 ml/min. Mobile phases were (A) acetonitrile/H20/triethylamine (900:99:1, v/v/v) and (B) ethyl acetate. Pigment detection was carried out at 445 nm with reference at 550 nm (Tian and DellaPenna 2001). Individual algal pigments were identified on the basis of their retention times and optical absorbance properties and quantified on the basis of their integrated absorbance peaks relative to known carotenoid standards. Carotenoid standards were purchased from DHI, Denmark. Pigments were standardized on the basis of dry weight of three replicates.
Cells were prepared for electron microscopy by immobilizing cells in 3% sodium alginate (w/v) and the alginate beads were then solidified by incubation in cold 30mM CaCl2 for 30min. We used alginate encapsulated algal cells to keep cells intact as well as to protect from direct and harmful effect of chemicals during fixation processes. These cells were fixed using 2% glutaraldehyde for 1.5-2 hours and after fixation, these cells were post fixed in buffered 2% osmium tetroxide for 1.5 hours. After dehydration these cells were embedded in Spurr's resin. Thin sections were stained with uranyl acetate and lead citrate. LEO 912 transmission electron microscope was used to view and collect images at 120 kv and a Proscan digital camera.
Total RNA was extracted from 100 mg of cells/sample, flash frozen in liquid nitrogen, grown at high light (500 μmol photons m31 2s−1) intensities for 5 days in HS media in shaker flasks) using the Direct-zol RNA-miniprep kit (ZYMO, P/N 2051) according to the manufacturer's instructions. Each total RNA sample was enriched for mRNA by hybridizing the poly(A) tail to oligo d(T)25 probes covalently coupled to magnetic beads, followed by elution (NEB, P/N S1419S). The enriched mRNA fractions were prepared for Illumina sequencing using the ScriptSeq V.2 RNA-seq Library Preparation Kit (Epicentre, P/N SSV21106) and sequenced on a Hi-Seq 2000 (2×150 bp), multiplexed at 6 samples per lane. The resultant sequence reads were trimmed for quality and mapped to the coding sequences present in version 9 of the Chlamydomonas reinhardtii genome annotation at web address phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Creinhardtii using bowtie2. The relative transcript abundance of each gene (mean of 3 biological samples) was determined using RSEM and differential expression values (UV4 vs A4) were calculated using EdgeR. All genes identified as differentially expressed were mapped to KEGG biochemical maps using the v.9 annotation assignments.
Phototropin genes were identified in three Chlorella species (herein designated as strain 1412, strain 1228 and Chlorella sorokiniana UTEX1230) and a Picochlorum soloecismus (DOE101) by conducting homologous BLASTp searches against the annotations of Chlorella species using Chlamydomonas reinhardtii phototropin genes/proteins (NP_851210) and Arabidopsis thaliana protein sequences (Accession #AED97002.1 and AEE78073) as query proteins. The Chlorella spp. and Picochlorum phototropin homologs were aligned to other phototropin amino acid sequences using CLUSTALW, then truncated based on conserved sequence alignments and phylogenetically analyzed using a Maximum-Likelihood algorithm. Each Chlorella strain contains two paralogous copies of photoropin and Picochlorum soloecismus. (DOE101) was found to contain 1 homolog of phototropin. These sequences are provided as SEQ ID Nos. 1-14. Additional phototropin sequences and functional homologs are provided in Table 1 and SEQ ID NO 51-66 and SEQ ID NO 69-128.
One method to reduce expression of algal PHOT gene(s) is to use RNAi technology driving the expression of double stranded, fold-back RNA elements to reduce the PHOT expression. A strong gene promoter such as psaD or other strong constitutive gene promoters could be used to drive expression of the RNAi construct similar to methods used previously in Chlamydmonas for modulation of light harvesting antennae complex (Perrine, Negi et al. 2012).
PHOT gene knockouts could be potentially generated by traditional mutagenesis approaches including chemical, UV, random insertional mutagenesis screened by TILLING (Comai, Young et al. 2004, Nieto, Piron et al. 2007), and by targeted knock outs using CRISPR/cas9 (Wang, Yang et al. 2013, Xiao, Wang et al. 2013, Dubrow 2014). Pooled PHOT-based PCR screening coupled with sequencing of PHOT PCR products could be used to screen for PHOT mutants.
Classical chemical mutagenesis is carried out using N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). This mutagen makes nucleotide changes in the DNA and these changes, depending on their position, can have effects that are either positive or negative in the use of the strain being treated. By careful observation of phenotypes produced, as well as implementation of selective pressure, one selects mutants with improved traits for the desired purpose. This method has been applied to algae previously (Yan, Aruga et al. 2000).
Identifying strains of algae that grow rapidly and produce high starch is used as a selection marker for PHOT K/O mutants. Because this approach does not involve adding foreign DNA (in fact is focused only on existing genetic potential of the strain being mutagenized), strains generated by chemical mutagenesis are not considered to be “genetically modified”, allowing deployment in the field without additional government regulation.
N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was chosen based on its proven use for modifying blue-green algae, as well as its ability to eliminate toxicity by degradation in dilute acid. First, the conditions required to result in approximately 99% lethality for Chlorella protothecoides are determined; this degree of lethality generated optimal mutation frequency in blue-green algae (Chapman and Meeks 1987). Two treatments, exposure to 0.25 mg/mL MNNG for 30 minutes and 0.025 mg/mL MNNG for 60 minutes, result in approximately 99% lethality for this strain (unpublished data). Both treatments are used to generate mutagenized populations of Chlorella using enrichment strategies.
Approximately 108 cells are mutagenized with four concentrations of MNNG and incubated for three different durations. After rinsing out the mutagen, approximately 104 cells are spread plated on nutrient plates, and the number of colonies scored after 12 days. Treatments with approximately 100 surviving colonies, representing 99% lethality, are chosen as optimal for generating mutations.
PHOT K/O mutants are expected to be more rapidly growing and to produce excess sink molecules/material. In C. protothecoides the sink is lipid which could be used as a screen for selection of cells representing high lipid cells. Numerous methods are in the literature for such selection such as Nile red (Pick and Rachutin-Zalogin 2012) and BODIPY 493/503 (Ohsaki, Shinohara et al. 2010). High lipid cells are selected by flow cytometry and then placed in flask for cell culture. Rapid growing high lipid cells will dominate the culture and should be PHOT K/O as determined in this invention.
Recently, it has been demonstrated that CRISPR/cas9 genome editing techniques can be used to knock out genes of interest in Chlamydomonas when the Cas9 gene is expressed constitutively. By incorporating multiple guide RNA elements to specifically recognize the PHOT gene high efficiencies of gene mutagenesis can occur during miss-repair of the double stranded break in the target gene catalyzed by Cas/9 by the endogenous repair enzymes. By targeting repair of a recognized restriction endonuclease site, inhibition of the digestion of the PHOT-specific PCR product by the diagnostic restriction endonuclease can be used as an effective screen for PHOT mutants. Similarly, DNA repair mistakes that occur following double stranded DNA breaks in the PHOT gene generated by TALEN complexes can be used to generate PHOT-specific mutants.
The following references and others cited herein, to the extent that they provide exemplary procedural and other details supplementary to those set forth herein, are specifically incorporated herein by reference and include US published patent applications and published patents: US 20130116165; US 20140249295; US 20130330718; U.S. Pat. No. 8,859,232 and other patent related documents EP2682469; WO 2011133493; WO 201408626; and WO 2013056212 and other publications listed:
This application is a continuation-in-part of U.S. patent application Ser. No. 15/831,178, entitled “Productivity and Bioproduct Formation in Phototropin Knock/Out Mutants in Microalgae”, filed Dec. 4, 2017, which is a continuation of International Patent Application No. PCT/162016/054466, entitled “Improved Productivity and Bioproduct Formation in Phototropin Knock/Out Mutants in Microalgae”, filed on Jul. 26, 2016, which claims priority to and benefit of U.S. Provisional Patent Application No. 62/171,176 entitled “Improved Productivity and Bioproduct Formation in Phototropin Knock/out Mutants in Microalgae” filed on Jun. 4, 2015, and the specification and claims thereof are incorporated herein by reference.
This invention was made with government support under grants Nos. Prime Contract No. DE-AC52-06NA25396 and NMC subcontract No. 277529. The U.S. government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62171176 | Jun 2015 | US |
| Number | Date | Country | |
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| Parent | PCT/IB2016/054466 | Jul 2016 | US |
| Child | 15831178 | US |
| Number | Date | Country | |
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| Parent | 15831178 | Dec 2017 | US |
| Child | 16820062 | US |
