KR20220136947A - Recombinant microalga with highly improved tolerances towards high CO2 and/or low pH conditions - Google Patents

Abstract

The present invention relates to a genetically modified microalgal strain Chlamydomonas reinhardtii PMA4ΔCter-V4 derived from Chlamydomonas reinhardtii, and uses thereof. A gene encoding a PMA protein is introduced into recombinant microalgae according to the present invention.

Description

Recombinant microalga with highly improved tolerances towards high CO2 and/or low pH conditions

The present invention relates to a novel recombinant microalgal strain with improved resistance to high-concentration CO ₂ and/or low pH (acidic) environments, and more particularly, Chlamydomonas reinhardtii-derived genetically modified microalgal strain Clara Midomonas reinhati PMA4ΔCter-V4 and -V10 and uses thereof.

Various environmental problems such as global warming and ocean acidification are occurring due to the constantly increasing anthropogenic CO ₂ since the industrial revolution. To solve this problem, numerous CO ₂ capture, utilization, and storage technologies (CO ₂ capture, utilization, and storage; CCUS) have been developed and proposed over the past few decades. Among the various CCUS technologies, CO ₂ utilization technology has recently received a lot of attention, which can produce a variety of useful substances through CO ₂ conversion, which directly contributes to CO ₂ reduction and other CO _{2 2} Because it has the advantage of overcoming the lack of technological economics, the biggest problem of CO2 capture and storage (CCS) technology, that is, the technology that simply captures CO ₂ generated by industrial activities and isolates it in huge storage. to be. CO ₂ conversion technology can be largely classified into chemical conversion and biological conversion technology depending on the platform used. Among them, biological conversion technology is attracting attention because it is more stable and more sustainable than chemical conversion method. The reason is that photosynthesis, a representative mechanism of biological CO ₂ conversion, can be driven by using sunlight, which can be supplied without external energy, without external energy input at room temperature and pressure, so it is superior in terms of high economic efficiency and life cycle. This is because an increase in the CO ₂ reduction effect can be expected.

Among the various species of organisms that are the basis of biological conversion technology, microalgae (photosynthetic microorganisms) realize biological CO ₂ conversion technology in the near future due to their high growth potential and thus high CO ₂ fixation ability compared to other biological CO ₂ conversion platform organisms. It is attracting a lot of attention as a species to make it happen. In addition, microalgae are recognized for their inherent ability to produce most useful substances needed by mankind, such as biofuels, materials, high value-added nutritional supplements, and pharmacological substances. In addition, the microalgae culture process is a wet biological process, and its utility is evaluated as promising because it can intensively respond to a large amount of CO ₂ emitted by directly connecting and driving an industrial CO ₂ emission source.

However, despite the aforementioned advantages, since microalgae are practically utilized, low environmental tolerance due to biological characteristics, especially low CO ₂ tolerance, is intended to be used in earnest as a pivotal biological platform for microalgae CO ₂ reduction industry. It is the biggest obstacle to trying. In other words, CO ₂ is an important material used as the sole carbon source in the process of photo-independent culture of microalgae, but paradoxically, when CO ₂ (about 5% or more) above a certain concentration is continuously supplied, excessive acidification inside the cell is induced. By having a significant adverse effect on cell activity, CO ₂ conversion ability is reduced and eventually leads to cell death. The problem is that, in the case of exhaust gases from general industrial processes, most of them contain more than 10% of high-concentration CO ₂ , except for sources that emit a small number of low CO ₂ concentrations, such as LNG combustion. In this case, as mentioned above, in many cases, the result is reduced cell activity and death, and in this case, the subject to convert CO ₂ is removed from the CO ₂ conversion system. For the stable conversion of CO ₂ and the successful operation of the production process for biologically useful substances based thereon, securing resistance to high concentration of CO ₂ of microalgae is one of the most important problems to be overcome.

Due to the above-described technical necessity, the cause of microalgae showing low resistance to high concentration of CO ₂ was analyzed through biological mechanism analysis related to intolerance of microalgae to CO ₂ , and as a result, proton pump (proton) on the plasma membrane pump), that is, abnormal low expression of plasma membrane H+ -ATPase (PMA) was determined to be the key cause of excessive acidification in microalgal cells when high concentration of CO ₂ is supplied. To overcome this, exogenous PMA gene As a result of introducing CO 2 , it was confirmed that the survival rate was remarkably increased not only in CO ₂ resistance but also in a general acidic environment (low pH), and the present invention was completed.

One object of the present invention is to provide a recombinant microalgae into which a gene encoding a PMA protein is introduced.

Another object of the present invention is to provide a recombinant microalgae with enhanced resistance to high concentration CO ₂ or acid with enhanced PMA protein activity.

Another object of the present invention is to provide a method for producing biomass or biofuel comprising the step of culturing the recombinant microalgae.

Another object of the present invention is to provide a method for enhancing the productivity of biomass or biofuel comprising the step of culturing the recombinant microalgae.

Another object of the present invention is a fragment of a polynucleotide in which a portion of the C-terminal sequence encoding a self-repression site has been removed from a polynucleotide encoding a PMA protein; and a polynucleotide encoding a fluorescent protein operably linked to the fragment by a linker, to provide an expression cassette for overexpression of PMA protein.

Another object of the present invention is to provide an expression vector comprising the expression cassette.

Another object of the present invention is to provide a transformant into which the expression vector is introduced.

Another object of the present invention is to provide a method for producing biomass or biofuel comprising the step of culturing a transformant.

Each description and embodiment disclosed herein is also applicable to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

In addition, throughout the specification of the present application, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components unless otherwise stated. do.

The present invention is a novel recombinant microalgal strain with improved resistance to high-concentration CO ₂ and/or low pH (acidic) environment, Chlamydomonas reinhardtii-derived genetically modified microalgal strain Chlamydomonas reinhardtii PMA4 ΔCter-V4, And it relates to a method of converting CO ₂ into biomass and biofuel by using the same.

As a method to solve the problems of the prior art, the present invention analyzed the cause of microalgae showing low resistance to high concentration of CO ₂ through the analysis of biological mechanisms related to intolerance to CO 2 of microalgae, and the Results Aberrant and low expression of the proton pump of the plasma membrane, that is, plasma membrane H+ -ATPase (PMA), is judged to be a key cause of excessive acidification in microalgal cells when high-concentration CO ₂ is supplied. As a result of introducing a foreign PMA gene to overcome this, it was completed by confirming that the survival rate was dramatically increased not only in CO ₂ resistance but also in a general acidic environment (low pH).

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In order to achieve the above object, in one aspect, the present invention may provide a recombinant microalgae into which a gene encoding a PMA protein is introduced.

In addition, in another aspect, it is possible to provide a recombinant microalgae with enhanced resistance to high concentration CO ₂ or acid with enhanced activity of the PMA protein.

As used herein, the term “microalgae” refers to phytoplankton present in water that performs photosynthesis. It is reported that over 500,000 species exist in the ocean or freshwater, have vitality in harsh environments such as hot springs and deep sea, and have high utility as biofuels and functional proteins.

The microalgae of the present invention may be recombinant microalgae modified to express a specific protein in the microalgae.

Specifically, the "recombinant microalgae" may be microalgae modified to express PMA protein, and specifically, high-concentration CO ₂ and/or resistance to acidity is enhanced compared to the parent strain in which the PMA protein is not expressed. can

As used herein, the term “PMA protein” is a plasma membrane H+-ATPase (PMA), also called a proton pump only for plasma. Plasma membrane proton transport ATP lyase is a type of hydrolase ^and electrogenic pump mainly found in the plasma membrane of fungi or plants. It causes active transport. Since the activity of plasma membrane proton transport ATP lyase forms electrochemical gradients across the cell membrane, secondary transport of other metabolites and ions occurs. Various metabolic processes are promoted, including transport and the response of plants to the environment.

The PMA protein may be a PMA4 protein derived from Nicotiana plumbaginifolia. The PMA4 protein can be expressed without a species barrier.

The PMA4 protein can be identified in NCBI Genbank, a known database. In addition, if it is a protein of other origin while having a corresponding activity, it may be included without limitation. Specifically, the protein may be a protein comprising the amino acid sequence of SEQ ID NO: 1, a protein consisting of the amino acid sequence of SEQ ID NO: 1, a protein consisting of the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 It may be used in combination with a protein having, but is not limited thereto.

The protein has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 1 and/or SEQ ID NO: 1 (identity) may be an amino acid sequence having. In addition, it is apparent that an accessory protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the protein.

In addition, as a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of a nucleotide sequence encoding the polypeptide, PMA activity Polypeptides having a can also be included without limitation.

The PMA4 protein may be encoded by the polynucleotide sequence of SEQ ID NO: 2.

The PMA4 protein may include a functional fragment having the same function but different in sequence from the protein.

As used herein, the term “functional fragment” refers to an amino acid sequence exhibiting efficacy corresponding to the protein, and a protein having an amino acid sequence in which a partial sequence of the amino acid sequence of the protein is deleted, modified, substituted or added is also described above. If it has an activity corresponding to that of a protein, it is obvious that it is included within the scope of the present application, and it can be referred to as a functional fragment for the purpose of the present application.

Specifically, the functional fragment may be an amino acid sequence in which a partial sequence is deleted from the PMA4 protein, and more specifically, a fragment in which a portion of the C-terminal sequence encoding the self-repression site of the PMA4 protein is removed. Or, more specifically, the 100 amino acid sequence that is the self-repression domain of the protein may be removed from the C-terminus of the PMA4 full-length protein.

The sequence in which 100 amino acid sequences, which are self-repression domains of the protein, are removed from the C-terminus of the PMA4 full-length protein may be the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 3 is the polynucleotide sequence of SEQ ID NO: 4 It may be coded, but is not limited thereto.

As used herein, the term “self-repression domain” refers to a domain capable of inhibiting expression of the PMA4 protein itself.

The functional fragment of the protein has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 3 and/or SEQ ID NO: 3 ) or an amino acid sequence having identity. In addition, it is apparent that an accessory protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the protein.

In addition, it is apparent that the present invention may also include a sequence in which the genetic sequence is codon-optimized based on the codon appearance frequency of the parent strain.

In the present invention, the term "polynucleotide" has a meaning to encompass DNA or RNA molecules, and nucleotides, which are basic structural units in polynucleotides, may include not only natural nucleotides, but also analogs in which sugar or base sites are modified ( Scheit, Nucleotide Analogs, John Wiley, New York (1980); see Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

The polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO: 1 or 3 may be included without limitation as long as it is a sequence capable of encoding a protein having the activity of the PMA4 protein or a functional fragment thereof.

In the present application, the term, "to be expressed / to be expressed" a protein refers to a state in which the activity of the target protein is enhanced compared to that before intrinsic or modified when the target protein is introduced into a microorganism or exists in a microorganism.

Specifically, "introduction of protein" means that the activity of a specific protein that the microorganism did not originally have or exhibited an improved activity compared to the intrinsic activity or activity before modification of the protein. For example, a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a fragment or vector containing a polynucleotide encoding a specific protein may be introduced into the microorganism to exhibit its activity. "Intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before the transformation when the trait of a microorganism is changed due to genetic variation caused by natural or artificial factors.

The method for introducing, enhancing, and inactivating the activity of the specific protein and/or gene may be performed using a suitable method known in the art according to the characteristics of the microorganism.

As used herein, the term “enhancement” of the activity of a protein means that the activity of the protein is introduced or increased compared to the intrinsic activity. The "introduction" of the activity means that the activity of a specific polypeptide that the microorganism did not originally have naturally or artificially appears.

As used herein, the term "increased" in the activity of a protein compared to the intrinsic activity means that the activity is improved compared to the intrinsic activity or the activity before modification of the protein of the microorganism. The "intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain or unmodified microorganism before the transformation when the trait of the microorganism is changed due to genetic mutation caused by natural or artificial factors. It can be used in combination with the activity before modification. The increase in activity may include both introducing an exogenous protein and enhancing the activity of an intrinsic protein. Increasing/enhancing the activity of the protein may be achieved by increasing/enhancing the expression of a gene.

Specifically, in the present application, the increase in activity is

1) increasing the number of copies of the polynucleotide encoding the protein;

2) modification of the expression control sequence to increase the expression of the polynucleotide,

3) modification of the polynucleotide sequence on the chromosome to enhance the activity of the protein;

4) introduction of a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized mutant polynucleotide of the polynucleotide, or

5) It may be performed by a method of deforming to be strengthened by a combination thereof, but is not limited thereto.

1) The increase in the copy number of the polynucleotide is not particularly limited thereto, but may be performed in a form operably linked to a vector or inserted into a chromosome in a host cell. Specifically, the polynucleotide encoding the protein of the present disclosure is operably linked to a vector capable of replicating and functioning independently of the host and introduced into a host cell, or inserting the polynucleotide into a chromosome in the host cell. The polynucleotide is operably linked to a capable vector and introduced into a host cell, thereby increasing the number of copies of the polynucleotide in the chromosome of the host cell.

Next, 2) modification of the expression control sequence so as to increase the expression of the polynucleotide, but is not particularly limited thereto, deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or these It can be carried out by inducing a mutation in the sequence with a combination of The expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, and the like.

A strong heterologous promoter may be linked to the upper portion of the polynucleotide expression unit instead of the original promoter, but is not limited thereto. In addition, 3) the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence or a combination thereof to further enhance the activity of the polynucleotide sequence. It can be carried out by inducing a mutation in the phase, or by replacing it with an improved polynucleotide sequence to have stronger activity.

In addition, 4) introduction of a foreign polynucleotide sequence, a foreign polynucleotide encoding a protein exhibiting the same/similar activity as the protein, or a codon-optimized mutant polynucleotide thereof may be introduced into a host cell. The foreign polynucleotide may be used without limitation in origin or sequence as long as it exhibits the same/similar activity as the protein. In addition, the introduced foreign polynucleotide may be introduced into the host cell by optimizing its codon so that the optimized transcription and translation are performed in the host cell. The introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a protein and increase its activity.

Finally, 5) the method of modifying to be enhanced by the combination of 1) to 4) above, increasing the copy number of the polynucleotide encoding the protein, modifying the expression control sequence to increase its expression, the polynucleotide on the chromosome Modification of the sequence and modification of a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized mutant polynucleotide thereof may be applied together to perform at least one method.

The term "recombinant microalgae with enhanced CO ₂ tolerance and/or acid tolerance" used in the present invention means that, unlike microalgae with conventional CO ₂ intolerance or weak tolerance, the activity of PMA4 protein is enhanced or expressed By being modified as possible, it may mean a recombinant microalgae with enhanced resistance to CO ₂ and/or acid.

In addition, the "recombinant microalgae with enhanced resistance to CO ₂ resistance and / or acid" includes both wild-type microalgae or microalgae in which genetic modification has occurred naturally or artificially, and external genes are inserted or of endogenous genes Microalgae in which a specific mechanism is weakened or strengthened due to a cause such as activity is enhanced or inactivated, and genetic modification or activity is enhanced to enhance the desired CO ₂ resistance and/or resistance to acid can

Specifically, the type of the microalgae is not particularly limited, but specifically Chlamydomonas may be a genus, and more specifically, may be Chlamydomonas reinhardtii.

The “Chlamydomonas reinhati” is a single-celled green alga with a diameter of about 10 micrometers that swims with two flagella, a cell wall made of hydroxyproline-rich glycoprotein, a large cup-shaped chloroplast, a large pyrenoid, and light have a focus on

Microalgae of the genus Chlamydomonas are widely distributed worldwide in soil and freshwater, among which Chlamydomonas reinhati is a well-studied biological model organism, partly because of its ease of culture and ability to manipulate genetics. There is this. Chlamydomonas reinhati can grow photoautotrophically in the presence of light, but can also grow in the dark if organic carbon is supplied. Commercially, Chlamydomonas reinhati is interested in the production of biopharmaceuticals and biofuels, as well as known as a useful research tool for making hydrogen.

The Chlamydomonas reinhatti may be used in combination with Chlamydomonas reinhard tea.

The present inventors deposited a new strain resistant to carbon dioxide and/or acid to the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology and were given an accession number KCTC14511BP.

Another aspect of the present application provides a fragment of a polynucleotide in which a portion of the sequence at the C-terminus encoding a self-repression site is removed from a polynucleotide encoding a PMA protein; and a polynucleotide encoding a fluorescent protein operably linked to the fragment by a linker, to provide an expression cassette for overexpression of PMA protein.

The terms “PMA protein” and “a fragment of a polynucleotide in which a portion of the C-terminal sequence encoding the self-suppression site has been removed from the polynucleotide encoding the PMA protein” are as described above.

As used herein, the term “expression cassette” may refer to a platform including a polynucleotide sequence encoding a polypeptide, a linker, and a polynucleotide sequence encoding a fluorescent protein so that the PMA4 protein or a functional fragment thereof is expressed.

In addition, the expression cassette may include a promoter and optionally a sequence controlling the expression of itself, such as an enhancer sequence or a terminator sequence.

As used herein, the term “linker” refers to linking a polynucleotide sequence encoding a PMA4 protein or a functional fragment thereof and a polynucleotide sequence encoding a fluorescent protein, and may be included without limitation as long as the expression cassette is operably linked. , specifically, may be GGSGGGSG, but is not limited thereto.

The term "fluorescence protein" refers to a protein that exhibits fluorescence in the visible region by forming a chromophore in its own polypeptide sequence by three amino acids. A group of proteins that share such unique fluorescence properties and are structurally similar can all be defined as fluorescent proteins, and specifically, GFP and mVenus, but are not limited thereto.

In another aspect, the present invention may provide an expression vector including the expression cassette and a transformant into which the expression vector is introduced.

As used herein, the term vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A plasmid, a type of vector, refers to a circular double-stranded DNA loop into which additional DNA fragments can be linked.

The vector of the present invention can direct the expression of a gene encoding a target protein operably linked to, such a vector is referred to as an expression vector. Generally, in the use of recombinant DNA technology, the expression vector is in the form of a plasmid.

As the expression vector, the type of expression vector that can be used may be determined depending on the host cell. When microalgae are used as the host, for example, pChlamy_4 vector may be used as the expression vector, and Hsp70A-Rbc S2 may be used as a promoter. available, but not limited thereto.

As a method of introducing DNA into microalgae, for example, the electroporation method (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929) -1933 (1978)), the lithium acetate method (J. Bacteriol., 153, 163-168 (1983)), etc. are available.

In addition, the expression vector includes a fragment for suppression of expression having various functions for suppressing, amplifying, or inducing the expression of a target gene, a marker for selection of a transformant, a gene resistant to antibiotics, and outside the cell body. It may further include a gene encoding a signal for the purpose of secretion, a custom fusion factor suitable for a difficult-to-express protein, and the like.

As used herein, the term "transformation" refers to introducing DNA into a host cell so that the DNA becomes replicable as an extrachromosomal factor or by chromosomal integration completion.

As the host cell used for the transformation according to the present invention, any host cell well known in the art may be used, but a host having high transduction efficiency and expression efficiency of the PMA4 encoding gene of the present invention may be used. for example, microalgal, bacterial, fungal, yeast, plant or animal (eg mammalian or insect) cells. Preferably, the host cell may be a microalgae.

The microalgae are the same as described above.

In another aspect, the present invention may provide a biomass or biofuel production method comprising the step of culturing the recombinant microalgae of the present invention.

In addition, in another aspect, it is possible to provide a method for enhancing the productivity of biomass or biofuel comprising the step of culturing the recombinant microalgae.

In addition, in another aspect, it is possible to provide a method for producing biomass or biofuel comprising the step of culturing the transformant of the present invention.

The term, “recombinant microalgae” is the same as described above.

As used herein, the term “culture” is used to allow the recombinant microalgae to grow, and the medium and culture conditions for culturing the transformant of the present invention can be appropriately selected and used according to the host cell. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of host cells. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, and methanol. Examples of the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquer, peptone, casein, bovine extract, soybean bag, and potato extract. Other nutrients, such as sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, vitamins, and antibiotics (tetracycline, neomacin, ampicillin, and kanamycin) may be included as needed. . During culturing, conditions such as temperature, pH of the medium, and incubation time can be appropriately adjusted to be suitable for cell growth and mass production of proteins.

More specifically, the culture in the present invention may be a photo-independent culture utilizing industrial exhaust gas containing a high concentration of CO ₂ .

The term, “photoindependent culture” may refer to independent culture under light conditions.

The high-concentration CO ₂ culture may be performed under a temperature condition of 23° C. while continuously supplying 20% CO 2 to the culture head space. In addition, the high-concentration CO ₂ culture may be performed under a light condition of 50 μmol photons m- ² s ^-1 .

According to the present invention, it is possible to generate microalgal biomass and biofuel (biodiesel) derived from the organism by using high-concentration CO ₂ contained in industrial exhaust gas as a single carbon source. Here, it is possible to generate microalgal biomass and biofuel (biodiesel) derived from the organism by utilizing the high-concentration CO ₂ contained in the industrial exhaust gas as a single carbon source. Biomass and biofuel production using the actual industrial exhaust gas is carried out in a photobioreactor, and industrial exhaust gas containing high concentration of CO ₂ (CO ₂ about 13%, nitrogen oxides about 20 ppm, sulfur oxides about 32 ppm) is produced. It can be carried out by injecting 0.1 vvm into the corresponding photobioreactor.

In addition, the production of biomass and biofuel using the actual industrial exhaust gas may be performed under outdoor conditions in which fluid light irradiation and variable temperature conditions appear.

In a specific embodiment of the present invention, after recognizing through literature analysis that the low tolerance of microalgae to CO ₂ is due to severe internal acidification of cells by CO ₂ as an acidic gas, acidic and high-concentration CO ₂ environment Expression level (transcriptome) analysis of genes related to CO ₂ tolerance of microalgae known through previous studies was performed in the lower parent strain Chlamydomonas reinhardtii CC125. Through this, it was observed that PMA, which is known to be the most important cause of the intolerance of CO ₂ in the cell, which governs pH homeostasis, is rather underexpressed in an acidic and high-concentration CO ₂ environment, that is, in an environment where internal acidification easily occurs. did.

Accordingly, in the present invention, the core cause of CO ₂ intolerance of microalgae was defined as low pH and high concentration CO ₂ conditions, that is, low PMA expression in an internal acidification promoting environment, and can be expressed without a species barrier by previous studies. The genetic sequence of the known PMA4 protein from Nicotiana plumbaginifolia was codon-optimized based on the codon appearance frequency of the parent strain.

In particular, when the C-terminal 100 amino acid sequence (300bp) encoding the autoinhibitory domain of the PMA4 protein is deleted (PMAΔCter), the protein's proton (proton; H+) ejection ability is improved. Thus, the relevant part was removed. In addition, for easy identification of proteins expressed in cells in the future, Venus, one of the fluorescent proteins, was converted to a C-terminal removed PMA4 protein through a glycine-rich linker sequence. (PMA4ΔCter-V). Thereafter, the gene was inserted through electroporation to improve the proton excretion ability of microalgal cells through successful expression in Chlamydomonas reinhardtii CC125 wild-type strain, thereby conferring high resistance to acidic and high-concentration CO ₂ supply environments. , The ability to maintain the internal pH of the cell was improved even under the conditions of acidification inside the cell, enabling smooth photosynthesis, and eventually, the biomass productivity and the CO ₂ fixation rate were significantly improved. Chlamydomonas reinhardtii-derived mutation micro Algal strains, Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 were identified.

In addition, according to a specific embodiment of the present invention, the microalgal strain Chlamydomonas reinhardtii PMA4ΔCter-V4 having high tolerance to acidic conditions and high concentration CO ₂ environment is superior to the wild-type strain Chlamydomonas reinhardtii CC125 from which it is derived. High concentration CO ₂ It has high growth (more than 3.2 times the maximum growth standard) and viability in a low pH environment (pH 5.5 under a mixed nutrient culture environment fed with acetate) under a (20% CO ₂ ) environment.

In addition, as a result of directly applying and culturing the strain directly to coal combustion exhaust gas containing not only high concentration of CO ₂ but also various biohazardous acid gases (nitrogen oxide and sulfur oxide), the photo-independent growth potential and 2.22 times higher than that of the parent strain and 2.22 It was confirmed that the CO2 conversion rate was improved more than twice.

In addition, it was confirmed that the productivity of representative biodiesel (Fatty acid methyl ester; fatty acid methyl ester) was also increased by 4.99 times or more among various useful substances that were biosynthesized in cells through photosynthesis while simultaneously producing microalgal biomass.

Through this, the mutant strain produced in the present invention is very suitable for use as a platform strain that can substantially produce carbon-neutral useful substances while contributing to industrial carbon emission reduction by converting industrial exhaust gas-derived CO ₂ .

In addition, in the case of the strain according to the present invention, since it is based on the Chlamydomonas reinhardtii strain, which is easy to genetically mutate but has been evaluated as having poor recyclability due to low high-concentration CO ₂ resistance, additional genetic By giving the ability to directly produce more diverse materials as well as biofuels from CO ₂ through mutation, it is expected to contribute to advancing a microalgae-based sustainable society.

The microalgal strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 having high tolerance to low pH (acidic conditions) and high concentration CO ₂ environment according to the present invention are superior to the wild-type strain Chlamydomonas reinhardtii CC125 from which they are derived. % CO ₂ ) It was confirmed that it showed growth potential (more than 3.2 times the maximum growth standard) and viability in a low pH environment (pH 5.5 under a mixed nutrient culture environment supplied with acetate). In addition, as a result of direct application and cultivation of the strain to coal combustion exhaust gas containing not only high-concentration CO ₂ but also various biohazardous acid gases (nitrogen oxide and sulfur oxide), it has a photo-independent growth potential of 2.28 times higher than that of the parent strain and 2.22 times higher than that of the parent strain. It was also demonstrated that it is possible to improve the CO ₂ conversion rate. In addition, it was experimentally verified that the productivity of biodiesel (fatty acid methyl ester; fatty acid methyl ester), a representative of various useful substances biosynthesized in cells through photosynthesis while producing microalgal biomass, also increased by 4.99 times or more. Through this, the mutant strain produced in the present invention is very suitable for use as a platform strain that can substantially produce carbon-neutral useful substances while contributing to industrial carbon emission reduction by converting industrial exhaust gas-derived CO ₂ . In addition, since this strain is based on the Chlamydomonas reinhardtii strain, which is easy to genetically mutate but has been evaluated to have poor utility due to low high-concentration CO ₂ resistance, this strain is used as the parent strain and then through additional genetic mutation. It is expected to contribute to advancing a microalgae-based sustainable society by giving the ability to directly produce more diverse materials as well as biofuels from CO ₂ .

1 is a model microalgae and the parent strain Chlamydomonas reinhardtii CC125 for the present invention is a microalgae growth graph derived from the process of establishing conditions for use in transcriptome analysis for identifying the cause of CO ₂ intolerance.
2 is a transcript analysis result of the parent strain Chlamydomonas reinhardtii in a high-concentration CO ₂ environment (CO ₂ 20%) and low pH (acidity, pH 5.0) causing internal acidification of microalgae.
3 shows the results of confirming the reproducibility of gene expression results at the transcript and translation levels by qRT-PCR and immunoblotting, respectively.
4 is a schematic diagram of a gene inserted into Chlamydomonas reinhardtii CC125 to improve resistance to high concentration of CO ₂ and acidic environment.
5 is a PCR image of wild-type Chlamydomonas reinhardtii CC125 and mutant Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 to confirm stable insertion of the PMA4ΔCter-V4 gene into a microalgal strain. In FIG. 4, A is a gene sequence encoding an actin protein, which was used as an endogenous control, and P is a PCR amplification result for a sequence of interest, that is, PMA4ΔCter-V4 and PMA4ΔCter-V10.
6 is a graph showing the relative expression levels of the PMA4ΔCter-V gene in the transcript levels of wild-type Chlamydomonas reinhardtii CC125 and mutant Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10.
7 is a Western blot image showing the relative expression levels of the PMA4ΔCter-V gene at the protein level of wild-type Chlamydomonas reinhardtii CC125 and mutant Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10.
8 is a solid medium culture result comparing the acid (pH 5.5, mixed nutrient culture medium) resistance of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10.
9 is a liquid medium culture result comparing the acid (pH 5.5, mixed nutrient culture medium) resistance of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10.
10 is a graph showing the growth evaluation results derived from culturing in a liquid autotrophic medium under 20% CO2 supply conditions to confirm the high-concentration CO ₂ tolerance of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10.
11 is a graph confirming that photoautotrophic biomass production (top view) and CO ₂ fixation rate (RCO ₂ , bottom view) are also improved under very high CO ₂ conditions (20% CO ₂ ).
12 is a graph showing the results of comparison of photosynthetic oxygen production rates under normal neutral atmospheric conditions and high-concentration CO2 environments of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10.
13 is a graph showing the monitoring results of the internal pH (pHi) of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 according to time exposed to a high-concentration CO ₂ environment.
14 is a graph showing PMA activity measured from plasma membrane samples isolated from wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10, respectively.
15 is a visualization of the proton-discharging ability of cells of wild-type Chlamydomonas reinhardtii CC125 and mutant Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 on a solid medium using bromocresol purple, a pH indicator.
16 is an image of outdoor culture in a 10 L-scale photobioreactor of wild-type Chlamydomonas reinhardtii CC125 and mutant strain Chlamydomonas reinhardtii PMA4ΔCter-V4 to which actual industrial exhaust gas is applied as a carbon source.
FIG. 17 shows the growth (biomass productivity), CO ₂ fixation rate (CO ₂ ) of the wild-type Chlamydomonas reinhardtii CC125 and the mutant Chlamydomonas reinhardtii PMA4ΔCter-V4 in a 10 L-scale photobioreactor, in which actual industrial exhaust gas is applied as a carbon source. fixation rate), calorific productivity, biodiesel productivity, and total lipid productivity (lipid productivity) is a graph comparing.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Experimental Example 1: Microalgal strain, medium formulation and culture conditions

C. reinhardtii WT (wild-type) strain CC-125 was obtained from the Chlamydomonas Resource Center at the University of Minnesota, and all engineered cell lines used in the present invention were constructed from this strain.

Cultivation of microalgae at laboratory scale is performed with modified Tris-acetate-phosphate (TAP; hereinafter MTAP) and Tris-phosphate (TP; hereinafter MTP) medium, and the buffer capacity of the medium is used to strictly maintain the acidity of the culture. MES, an additional buffer material for This is because the pH is very low due to ammonium uptake, so that CO ₂ tolerance cannot be accurately evaluated.

Neutral pH mixed nutrient MTAP medium is 10 mM MES, 17.4 mM acetic acid, 7 mM NH ₄ Cl, 0.83 mM MgSO ₄ ·7H ₂ O, 0.45 mM CaCl ₂ ·2H ₂ O, 1.65 mM K ₂ HPO ₄ , 1.05 mM KH ₂ PO ₄ , 0.134 mM Na ₂ EDTA·2H ₂ O, 0.136 mM ZnSO ₄ ·7H ₂ O, 0.184 mM H ₃ BO ₃ , 40 μM MnCl ₂ ·4H ₂ O, 32.9 μM FeSO ₄ ·7H ₂ O, 12.3 consisting of μM CoCl ₂ .6H ₂ O, 10 μM CuSO ₄ .5H ₂ O, and 4.44 μM (NH ₄ )6MoO ₃ , the pH was adjusted with 25 mM Tris base. For reference, all these materials were purchased from Sigma-Aldrich (USA).

Instead of acetic acid, 0.35‰ HCl was added, and an autotrophic MTP medium (pH 7.0) was prepared in the same manner as for other MTAP preparations.

Cells were incubated with shaking in an incubator at 23 °C at a rotation speed of 120 rpm under continuous light illumination (50 μE m-2 s-1). The headspace was provided with air and 20% CO ₂ enriched air according to the purpose of each experiment, and cell growth was monitored daily at 800 nm using a UV-1800 UV spectrophotometer (Shimadzu, Japan).

The specific growth rate (μ; d-1) of microalgae was evaluated according to Equation 1 below.

[Equation 1]

Here, OD _800,F and OD _800,I are the final and initial optical densities at 800 nm measured at two specific time points, t _F and t _I .

Experimental Example 2: RNA preparation and RNA-seq analysis

To identify high CO ₂ and low pH sensitive genes, RNA samples of C. reinhardtii grown under specific CO ₂ (20% CO ₂ ) conditions and pH (pH 5.0) conditions that inhibited the growth rate of microalgae by 50% (atmospheric pH) 7.0) (effective concentration that caused a decrease in growth rate, EC50) was isolated (FIG. 1). EC50 values of CO ₂ and pH prior to incubation were determined in advance.

After 1 day of cell growth, the exposure time is determined as the short period during which the stress stimulus induces stress-induced growth inhibition at the target level, before the cells enter the center of the temporal adaptation phase (between 3 and 4 days), the microalgae Cells were harvested by centrifugation (1373 x g, 5 min) and then immediately frozen using liquid nitrogen.

In addition, to investigate condition-dependent PMA expression patterns at the protein level, these same cell samples were used for immunoblotting analysis. Then, RNA extraction was performed using the PureHelixTM Total RNA Purification Kit (Nanohelix, Korea), and RNA-seq-based transcript analysis was performed using 6 biological replicates in each condition.

To confirm the quantity and quality of RNA samples, a NanoDropTM spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) were used. For library preparation, RNA samples exhibiting an RNA integrity number (RIN) of approximately 6.0 were selected using the TruSeq RNA Library Preparation Kit (Illumina, USA), and then RNA-seq was performed using the Illumina NovaSeq™ 6000 sequencing system. Afterwards, a 150 bp paired-end read was generated. In addition, the resulting data were mapped to the reference genome of C. reinhardtii (Chlamydomonas reinhardtii v5.6, JGI) using Bowtie2 software with basic parameters.

After calculating the RPKM (reads per kilobase per million mapped reads) unit of each gene using the Bioconductor R software package edgeR and identifying the DEGs, significant DEG gene sets were used to perform GO enrichment analysis using the R package topGO using the R package topGO. and statistical significance was determined using Fisher's exact test method (p < 0.05).

To assign GO terms, related genes were annotated into heterogeneous group clusters (COG) categories using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and individual genes associated with CO ₂ tolerance (Figure 2) were identified in Chlamydomonas reinhardtii v5. 6 were selected and analyzed based on gene annotations provided in the database. Then, their expression patterns were profiled to comprehensively evaluate the effect of high CO ₂ conditions on cells at the transcript level.

Experimental Example 3: Preparation of PMA overexpression expression vector and transformation using the same

In order to prepare a PMA overexpression expression vector, PMA4, a PMA derived from N. plumbaginifolia, was selected, and its coding sequence was obtained from the National Center for Biotechnology Information (NCBI, USA) database.

The coding region of the C-terminal autorepression domain in the original sequence (100 amino acids, 300 bp), which could reduce H+ efflux, was removed to confer improved H+ extrusion ability to the microalgal strain.

In addition, in order to confirm the sequence of PMA4ΔCter-V, a localization and final expression target of the exogenous protein, mVenus, a derivative of green fluorescent protein (GFP), was attached to PMA4ΔCter by a Gly-rich GGSGGGSG flexible linker.

Then, the chimeric protein coding sequence was codon-optimized using GeneArt™ GeneOptimizer™ software (Thermo Fisher Scientific, USA) taking into account the codon usage preference of the C. reinhardtii nucleus and synthesized using GeneArt. Then, the gene cassette was digested with EcoRI and BglII and cloned into a pChlamy_4 protein expression vector (Thermo Fisher Scientific, USA) containing a constitutive promoter (Hsp70A-Rbc S2) and a zeocin resistance gene (Sh Ble). The PMA4ACter-V-bearing plasmid was linearized with ScaI prior to transformation. All restriction enzymes and T4 DNA ligases used in the cloning step were purchased from Thermo Fisher Scientific (USA).

To construct the mVenus coding insert, the corresponding sequence (ie, PMA4ACter and PMA4ACter-V without linker sequence) was cloned into the same expression vector backbone and then transformed into microalgae by electroporation.

Seeds were kept fresh and grown in 200 mL of conventional mixed nutrient (TAP) medium until the cell concentration reached ~1.5 × 106 cells mL-1, followed by gentle centrifugation at 1373 × g for 5 min. It was collected and resuspended in 10 mL of MAX Efficiency™ Transformation Reagent for Algae (Thermo Fisher Scientific, USA), and washed twice.

After electropermeabilization, cells were incubated for 15 min and transferred to 10 mL of TAP medium supplemented with 40 mM sucrose. After incubation in the dark for 16 h, cells were collected and solid medium consisting of TAP medium, 1.5% (w/v) plant agar (Duchefa Biochemie, The Netherlands) and 5 μg mL-1 Zeocin (Thermo Fisher Scientific, USA). placed on Cells in the plate were grown for 7 days at 23 °C in low light conditions (50 µE m ^-2 s ^-1 ).

Experimental Example 3: Immunoblotting

Agrisera's H+ ATPase (1:1,000; AS07 260) and AtpB (1:10,000; AS05 085) were used as the primary antibody, and Agrisera's HRP-conjugated goat anti-rabbit IgG (condition-dependent PMA) was used as the secondary antibody. 1:100,000 for quantification of expression and 1:1500 for detection of exogenous PMA protein; AS09 602) were used. The dilution ratio of the secondary antibody was adjusted according to the manufacturer's protocol of the developing reagent, and the band intensity in the quantitative analysis of protein expression level was measured using ImageJ software.

Experimental Example 4: Primer

A full list of all primers used in this study, including names and sequences, is shown in Table 1 below.

[Table 1]

Experimental Example 5: Flow cytometry

Quantitative fluorescence of each cell line was estimated using a flow cytometer (BD Accuri™ C6 Plus flow cytometer; BD Biosciences, USA) with BD Accuri™ C6 Plus software (BD Biosciences, USA) and Flowjo software (BD Biosciences, USA). Excitation with a 488 nm laser and emission detected using a 533/30 nm bandpass filter (FITC-A).

Experimental Example 6: Confirmation of cell resistance

The pH values of the acidic media used for the low pH tolerance test were adjusted with HCl (both MTAP and MTP media). For the spot plating culture, after autoclaving, a liquid medium of various pH values (ie, pH 7.0 and pH 5.5) was used to solidify using plant agar (1.5% w/v). Four serial 10-fold dilutions were performed starting at 5 × 10 ⁵ cells mL ⁻¹ , and then 20 μL aliquots of each dilution were spotted onto pH-adjusted plates. In addition, further acidification induced by organic carbon sources (i.e., acetic acid) was performed only in an autotrophic medium (i.e., MTP with an initial pH value of 7.0) under extremely _high CO2 conditions ( ₂₀ % enriched air) for CO2 tolerance testing. The effect is isolated.

Also, to rule out adaptive effects, cells were abruptly transferred from ambient to high CO ₂ conditions. The optical density (OD800) of the cell suspension was converted to the biomass concentration (g L ⁻¹ ) using the relevant calibration function plotted for each strain.

A linear regression curve was derived based on the correlation between cell culture dry cell weight (DCW) and turbidity. DCW was measured by a filter-based gravimetric method using a Whatman GF/C glass microfiber filter (GE Healthcare, USA). Cell samples were obtained over the course of autotrophic culture (at atmospheric pH 7.0) of each cell line, which was performed separately to establish correlation. Aliquots of 100 μL and 5 mL from each culture were sampled to determine turbidity and DW, respectively. Then, the biomass productivity (P; g L ⁻¹ d ⁻¹ ) was calculated using Equation 2 below.

[Equation 2]

Here, X _F (g L ⁻¹ ) and X _I (g L ⁻¹ ) represent the final and initial DCW measured at two specific time points t _F and t _I , respectively.

Given the carbon balance under autotrophic conditions, the CO2 fixation rate (RCO2; g CO2 L ^-1 d ^-1 ) was evaluated using Equation 3 below.

[Equation 3]

where C, M _CO2 and M _C represent the carbon content (% w/w), the molecular weight of CO2 (44.01 g mol ^-1 ) and the atomic mass of the carbon (12.01 g mol ^-1 ), respectively.

In the laboratory-scale tolerance test, the carbon content of each cell line was assumed to be 50%, and to accurately estimate the actual carbon fixation capacity of algal cells in field culture, the carbon content pellets of microalgae were actually analyzed using a 5E-CHN2200 elemental analyzer (Changsha Kaiyuan Instruments). , China) was used.

Experimental Example 7: Outdoor (rooftop) cultivation by injection of coal-fired flue gas

For the cultivation of the produced mutant (PMA4ΔCter-V4) of the present invention, a microalgal CO2 conversion plant was constructed and operated under the permission of the Korean government (permission number: LML 19-912). Proof-of-concept outdoor cultivation was performed in a demonstration plant ( FIG. 16 ) using 10 L (working volume) bubble column PBR and an autotrophic medium optimized for outdoor microalgal cultivation. Prior to inoculation of microalgal cells, the culture system containing PBR and 10 L of industrial water was sterilized with high alkalinity (~pH 12, supplemented with 10 mM KOH), and the flue gas stream from a neighboring coal-fired boiler was removed. Thereafter, the water was continuously sprayed with an aeration rate of 0.1 vvm.

The exhaust gas consisted of 80.57% N ₂ , 13.18% CO ₂ , 7.46% O ₂ , 20.27 ppm NOX and 32.04 ppm SOX. The interaction of the acid component with the KOH of the acid component of the flue gas (eg CO ₂ , NOX and SOX) causes the gas injection to neutralize the aqueous phase (~pH 7.11), while at the same time automatically providing a bicarbonate buffer system suitable for large-scale operations. is formed Then, as a culture medium, 7 mM NH ₄ Cl, 0.83 mM MgSO ₄ .7H ₂ O, 0.45 mM CaCl ₂ .2H ₂ O, 1.65 mM K ₂ HPO ₄ , 1.05 mM KH ₂ PO ₄ , 0.134 mM Na ₂ EDTA.2H ₂ O, 0.136 mM ZnSO ₄ 7H ₂ O, 0.184 mM H ₃ BO ₃ , 40 μM MnCl ₂ 4H ₂ O, 32.9 μM FeSO ₄ 7H ₂ O, 12.3 μM CoCl ₂ 6H ₂ O, 10 μM CuSO ₄ Prepared by adding 5H ₂ O, and 4.44 μM (NH ₄ )6MoO ₃ . The culture was started by inoculating 50 mg L-1 of fresh, air-grown algal seeds based on the autotrophic culture medium and continued for 2 weeks (14 days). The pH of the algal culture was maintained at ˜6.56 throughout the run. The resulting biomass was obtained by a continuous flow centrifuge (21,000 × g) and then lyophilized (at a pressure of 1 kPa and a temperature of -55 °C) for further analysis.

Experimental Example 8: Analysis method (total lipid content, total FAME content and calorific value)

Using biomass harvested from field cultivation, each lipid productivity (mg lipid L ^-1 d ^-1 ), biodiesel productivity (mg FAME L ^-1 d ^-1 ) and calorific value (kJ L ^-1 d ^-1 ) strain rate was calculated using Equation 4 below.

[Equation 4]

where P and LHV are biomass productivity (mg biomass L1 d1) and low calorific value (also called net calorific value). P(mg biomass L ^-1 d ^-1 ), total lipid content (% w/w), total FAME content (% w/w) and LHV (kJ g ^-1 ) values to calculate various metrics was decided. P was calculated using Equation 2 above.

The total lipid fraction of the dried biomass was extracted according to the modified BlighDyer method to determine the total lipid content of the biomass and the content was determined using gravimetric analysis. Prepare FAME (commonly referred to as biodiesel fraction) from the remaining total lipid fraction for biodiesel content analysis and use an Agilent 7890 A Gas Chromatography (GC) System (Agilent Technology, USA) equipped with a DB-23 GC column (Agilent Technology, USA) and flame Analysis was performed using an ionization detector (FID). After adding 10 mg of pentadecanoic acid (C15:0) as an internal standard to the extracted lipid sample, it was converted to FAME through transesterification using 3% methanolic sulfuric acid. The resulting converted lipids were then quantified by GC. Supelco ^® 37 component FAME mix, FAME RM-3 and FAME were mixed and Mix RM-5 (all purchased from Sigma-Aldrich, USA) was adopted as the FAME reference standard.

A mixture of helium, hydrogen, and high-purity air was used as the carrier gas and fuel gas of the FID, respectively.

GC operating conditions were as follows: injection volume, 1 μL; split ratio, 1:50; inlet temperature, 250°C; detector temperature, 280 °C; and oven temperature, hold at 50° C. for 1 minute, increase to 175° C. at a rate of 25° C. min ⁻¹ , increase to 230° C. at a rate of 4° C. min ^-1 , hold at 230° C. for 5 minutes.

The calorific value (LHV) evaluation of biomass was performed using a 5E-C5500 automatic bomb calorimeter (Changsha Kaiyuan Instruments, China).

Experimental Example 9: Statistics and Reproducibility

All experiments are indicated in the corresponding figure. Results are presented as mean values and error bars showing the standard deviation of the mean (mean ± SD). If necessary, a two-sided Student's t-test was performed to check whether there was a statistically significant difference between the mean values of the two groups.

In RNA-seq data analysis, genes with |log2FC|> 1 and p < 0.05 were considered DEGs. Statistically significant differences were determined by a generalized linear model (GLM) likelihood ratio (LR) test. GO terms were determined based on a cutoff value of p < 0.05 using Fisher's exact test method. The pHi values of the constructed strains and WT control strains were compared with box and whisker plots to demonstrate median, maximum and minimum points, and interquartile ranges. Gel and blot images, including PCR and immunoblotting results, were obtained in single replicates. Comparisons between protein samples in quantitative analysis of protein expression levels were performed in the same gel. Confocal images were acquired from three biologically independent samples.

Example 1: Transcript analysis of the parent strain Chlamydomonas reinhardtii CC125

The transcriptome of the parent strain Chlamydomonas reinhardtii CC125 was analyzed according to Experimental Example 2, and the results are shown in FIGS. 1 and 2 .

1 is a model microalgae and the parent strain Chlamydomonas reinhardtii CC125 for the present invention, a microalgae growth graph derived from the process of establishing conditions for use in transcript analysis to identify the cause of CO ₂ intolerance, arrows indicate RNA-seq ( Biomass sampling time points for n = 3) are indicated.

As can be seen in FIG. 1 , each condition is a condition in which biomass growth potential for 1 day after exposure to the corresponding condition is reduced by about 50% due to causes such as internal acidification of cells compared to when cultured in a neutral atmospheric condition. and this became a condition for subsequent transcriptome analysis.

Next, Figure 2 is a transcript analysis result of the parent strain Chlamydomonas reinhardtii in a high-concentration CO 2 environment (CO ₂ 20%) and low pH (acidity, pH 5.0) that induces intracellular acidification of microalgae to be.

Microalgae show five major cellular responses in response to a high-concentration CO ₂ environment, which is the overexpression of the first proton pump, the underexpression of the second carbon concentrating mechanism, the change of the third cell membrane component, and the fourth Activation of the protein repair mechanism, activation of the fifth ATP synthesis mechanism, and the like.

As can be seen in FIG. 2 , unlike the expected behavior of known transcript expression, abnormally low expression of the PMA2 and PMA3 genes, which encode proton pumps, which are known to play the most major role in maintaining cell internal pH, are rather underexpressed under conditions that cause internal acidification. appeared to be confirmed.

In addition, these results can also be confirmed through FIG. 3 .

As can be seen in FIG. 3 , a decrease in the expression of PMA such as PMA2 (Cre10.g459200; slightly down-regulated) and PMA3 (Cre03.g164600; statistically significant down-regulated) was confirmed in the high CO ₂ condition, which indicates that PMA is This suggests that it plays an important role in maintaining life during exposure to various acidic conditions. The reproducibility of gene expression results at the transcript and translation levels under high CO conditions was confirmed by qRT-PCR and immunoblotting, respectively, implying that mRNA and protein expression match, and at the same time, down-regulation is direct to innate low tolerance. suggests that it is influencing

Accordingly, the main cause of intolerance of Chlamydomonas reinhardtii, a strain susceptible to high CO ₂ , was defined as abnormal low expression of the PMA gene.

Example 2: Construction of microalgal strains with high CO2 resistance

According to Experimental Example 3, an expression cassette capable of overexpressing PMA protein was prepared in the parent strain (wild type) Chlamydomonas reinhardtii CC125, and an expression vector including the same was prepared, and the expression vector was transformed into microalgae to have high CO ₂ resistance A microalgal strain having a was prepared.

Specifically, according to Example 1, the overexpression target for increasing CO ₂ tolerance of C. reinhardtii is typical PMA, that is, plasma membrane H+-ATPase isoform 4 of Nicotiana plumbaginifolia (hereinafter referred to as PMA4), which is This is because they are well characterized and successfully expressed in various bio platforms such as

If activated in the microalgal system through this overexpression of PMA4, it could be a versatile approach for various microalgal species suffering from stagnant H+ extrusion and consequent CO2 intolerance due to the host-independent nature of PMA4 expression.

To this end, in order to confer resistance to very large acids in microalgal cells, the self-repression domain present at the C-terminus was deleted from the gene sequence encoding the PMA4 protein, and then the truncated form of PMA4 was named PMA4Cter.

Then, in order to label a reporter to investigate the intracellular localization of the expressed acid-secretion nanomachine, a Venus fluorescent protein (mVenus) coding gene was ligated to the 3'-coding end of the PMA4Cter coding gene using a linker (GGSGGGSG). and named it PMA4Cter-V. In addition, a strong promoter Hsp70A-Rbc S2 was ligated.

Thereafter, the codon-optimized insertion cassette was transformed into microalgal cells by electroporation, followed by colony PCR using the screening primer set of Experimental Example 4 in a selective medium plate containing Zeocin. Two independent recombinant microalgae that maintained genetic and phenotypic stability over time were screened. These were named PMA4Cter-V4 and PMA4Cter-V10, respectively ( FIGS. 4 and 5 ).

4 is a schematic diagram of the gene insertion position revealed as a result of next generation sequencing for confirming the gene insertion position on the genomic DNA of Chlamydomonas reinhardtii PMA4ΔCter-V4 or PMA4ΔCter-V10 strain, PMA4ΔCter-V4 is It was confirmed that 23 bp was deleted at position 2714330 of chromosome 7 (Chromosome 7; Chr 7) and then inserted, and PMA4ΔCter-V10 was inserted after dropping 5461 bp at position 2162725 of chromosome 6. For reference, in the case of P, Z, L, and V, respectively, a promoter, a Zeocin antibiotic resistance gene, a linker sequence, and a Venus fluorescent protein.

As a result, as can be seen in FIGS. 4 and 5 , the strains transformed using the expression vector containing the expression cassette prepared in the present invention stably inserted the PMA4ΔCter-V4 and PMA4ΔCter-V10 genes into the microalgal strain, respectively. was able to confirm that In contrast, expression of PMA4ΔCter-V was not confirmed in the parent strain, wild-type (WT) strain.

Example 3: Evaluation of relative expression levels of PMA4ΔCter-V genes in Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4

The relative expression levels of the PMA4ΔCter-V gene of Chlamydomonas reinhardtii CC125 and Chlamydomonas reinhardtii PMA4ΔCter-V4 were confirmed at the gene and protein level, respectively, using qRT-PCR and Western blot.

As a result, as can be seen in FIGS. 6 and 7 , the PMA4ΔCter-V gene was not expressed at all in the wild type, but it was confirmed that the PMA4ΔCter-V gene was expressed relatively high in both of the two mutant strains prepared in the present invention. , in particular, it was confirmed that the expression was higher in PMA4ΔCter-V4.

Example 4: Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4 and PMA4ΔCter-V10 acid and high concentration CO2 tolerance evaluation

In order to confirm the characteristics of the transformed cells of the present invention, the productivity of the cell lines grown at low pH was first evaluated.

Specifically, the pH tolerance evaluation of algal cells was performed in two independent culture modes of mixed nutrient and autotrophic conditions, and acetic acid was used as an organic carbon source for autotrophic and mixed nutrient culture.

As a result, as can be seen in FIGS. 8 and 9 , the cell line transformed at a pH value of 5.5 in both culture using a solid medium and culture using a liquid medium showed a clear difference in growth performance compared to WT cells. In particular, it was found that, in contrast to the marked decrease in the growth rate of WT cells in the mixed nutrient culture of pH 5.5, the productivity of the transformed strain of the present invention was high, and the acid resistance of the recombinant microalgae was successfully increased.

In addition, as can be seen in Figure 10, the growth of the transformant strain of the present invention at low pH was confirmed to be similar to the growth at neutral pH. Through this, it was found that the PMA overexpression strategy as in the present invention did not have a negative effect on cells, and the exogenous PMA expression mutant in photoautotrophic acidic culture also showed relatively improved growth at the same acidity (pH 5.5), but this It was confirmed that there was no dramatic difference in growth compared with the results obtained from nutrient culture.

On the other hand, as can be seen in FIG. 11 , photoautotrophic biomass production (upper drawing) and CO ₂ fixation rate (RCO2, lower drawing) were improved under very high CO ₂ conditions (20% CO2). These two graphs represent the same time scale, and arrows indicate the time point (color-coded) at which the cell line reached its maximum concentration. Data from duplicate cell cultures are shown, with each dot shown in the bottom figure representing two biological replicates. According to the culture results, in the end, WT increased productivity 1.09 times under high CO ₂ conditions (up to 6 days) compared to ambient CO ₂ conditions (up to 17 days), whereas the maximum biomass production was reduced by 0.66 times.

In contrast, the maximum biomass production capacity in the recombinant microalgae of the present invention was significantly higher than that found in the WT strain under the same CO ₂ condition, and it can be seen that PMA4ΔCter-V4 and PMA4ΔCter-V10 are improved by 3.22 times and 3.03 times, respectively. There was (top of Figure 11). In addition, the cumulative production and ratio of the transgenic strains were also much higher than the ambient CO ₂ conditions (ie, autotrophic pH 7.0) (up to day 17) under high CO ₂ conditions (up to day 7). Specifically, cumulative biomass production was improved by 1.12-fold and 1.09-fold in PMA4ΔCter-V4 and PMA4ΔCter-V10, respectively, and productivity by 2.77-fold and 2.62-fold, respectively. This suggests that the PMA expression mutation can advantageously utilize the excess CO ₂ while attenuating the intracellular acidification effect.

This growth improvement also indicates that the amount of CO ₂ fixation obtained with each mutant during the incubation period was significantly improved compared to that found in WT under high CO ₂ providing conditions (bottom of FIG. 11 ). In terms of CO ₂ fixation rate, PMA4ΔCter-V4 and PMA4ΔCter-V10 increased RCO ₂ values by 3.23-fold and 2.99-fold, respectively, when compared to WT strains in the exponential growth phase (between days 2 and 6) where effective CO ₂ fixation occurs. showed

Taken together, this resistance improvement strategy suggests to increase the maximum biomass concentration of the mutant during a short incubation period in a high CO ₂ supply condition, and to improve the CO ₂ removal rate and efficiency.

Example 5: Characterization of Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 in a high-concentration CO2 environment

Characteristics of Chlamydomonas reinhardtii CC125 and Chlamydomonas reinhardtii PMA4ΔCter-V4 or -V10 in a high-concentration CO ₂ environment were evaluated, and the results are shown in FIGS. 12 and 13 .

First, Figure 12 is a graph of the results of comparing the photosynthetic oxygen production rate under normal neutral atmospheric conditions and high concentration CO ₂ environment of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10. WT grown with CO ₂ The strain had a lower total photosynthetic rate (i.e., O2 generation + dark respiration) than the WT strain cultured in ambient conditions (approximately 50.4% reduction), indicating that the microalgal photosynthetic apparatus is severely damaged by _high CO2 conditions. . In contrast, however, the PMA4ACter-V4 and PMA4ACter-V10 strains grown in _high CO2 conditions showed increased rates of photosynthetic O2 generation compared to those found during growth in atmospheric conditions (approximately 58.6% and 37.3% increases, respectively). ) (Fig. 12).

In addition, FIG. 13 is a graph showing the monitoring results for cell internal pH (pHi) according to time exposed to a high-concentration CO ₂ environment of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10. While the pHi decreased sharply (pH median 6.56) during 2 hours of exposure to CO ₂ , the recombinant microalgae of the present invention showed a better ability to strongly maintain a near-neutral pH in acidic conditions. Specifically, intermediate pH values of 6.65 and 6.78 were confirmed for PMA4ΔCter-V4 and PMA4ΔCter-V10, respectively ( FIG. 13 ).

This indicates that the high CO ₂ condition significantly affects the viability of microalgae by lowering the cytoplasmic pH, and has a positive response to CO ₂ derived intracellular acidification. _After all, given that photosynthetic CO2 fixation systems _and O2 evolution systems are highly sensitive to cytoplasmic and subcellular pH as well as ambient pH and CO2 conditions, _the difference in cellular ability to maintain pHi under _high CO2 conditions is not significant for WT. and it suggests that it is the root cause of the resistance gap between the transformed strain of the present invention.

Example 6: Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 evaluation of PMA activity and proton excretion ability of cells

Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 were evaluated for PMA activity and proton ejection ability of cells, and the results are shown in FIGS. 14 and 15 . For reference, this evaluation was evaluated by confirming the ATPase activity in vitro and in vivo.

14 is a graph showing PMA activity measured from plasma membrane samples isolated from wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10, respectively. Similar to the results described above, it was confirmed that PMA4ΔCter-V4 and PMA4ΔCter-V10 exhibited about 2.5-fold and 2.6-fold higher ATPase-mediated ATP hydrolysis performance than the WT strain, respectively. This demonstrates that the transformant strain of the present invention has a substantially higher H+.

15 is a visualization of the proton-discharging ability of cells of wild-type Chlamydomonas reinhardtii CC125 and mutant strains Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 on a solid medium using bromocresol purple, a pH indicator. The bright yellow color outside the circular cell population shows the region relatively acidified by protons ejected from the cells. As can be inferred from the color change around the cells due to the released H+ and concomitant extracellular acidification, it was visually confirmed that the H+ flux was markedly improved in the PMA-expressing mutant of the present invention.

Example 7: Evaluation of outdoor culture of Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10

Outdoor cultures of Chlamydomonas reinhardtii CC125, and Chlamydomonas reinhardtii PMA4ΔCter-V4 and -V10 were evaluated.

16 is an image of outdoor culture in a 10 L-scale photobioreactor of wild-type Chlamydomonas reinhardtii CC125 and mutant strain Chlamydomonas reinhardtii PMA4Δ Cter-V4 to which actual industrial exhaust gas is applied as a carbon source.

Figure 17 shows the growth (biomass productivity), CO ₂ fixation rate (CO 2 ) of the wild-type Chlamydomonas reinhardtii CC125 and the mutant strain Chlamydomonas reinhardtii PMA4Δ Cter-V4 outdoor cultured in a 10 L-scale photobioreactor using actual industrial exhaust gas as a carbon source. ₂ fixation rate), calorific productivity, biodiesel productivity, and total lipid productivity (lipid productivity) is a graph comparing.

After 2 weeks of cell culture, various performance indices of the strain were evaluated as a whole using various calculation formulas. As a result, as can be seen in FIGS. 16 and 17, PMA4ΔCter-V4 exhibits a significantly improved phototrophic growth rate (2.28 times higher) than the WT strain, and the high concentration CO ₂ contained in the harmful gas stream. could be observed Furthermore, the biomass productivity increase in the transgenic algal strain of the present invention is microalgal CO ₂ fixation rate (2.23 times), total lipid productivity (2.21 times), total fatty acid methyl ester (FAME) productivity (4.68 times) and calorific value (2.20) It was found that it leads to the improvement of other essential parameters, including Acceleration of CO ₂ sequestration, FAME accumulation and calorific value production is a biological platform for the production of various types of biofuels (i.e. biodiesel and solid fuels for direct combustion), while simultaneously using the recombinant microalgal strains of the present invention for industrial applications. It shows the practicality _of reducing CO2 emissions.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Korea Institute of Bioscience and Biotechnology KCTC14511BP 20210325

<110> Korea University Research and Business Foundation <120> Recombinant microalga with highly improved tolerances towards high CO2 and/or low pH conditions <130> JKP1641R1 <150> KR 10-2021-0042862 <151> 2021-04-01 <160 > 4 <170> KoPatentIn 3.0 <210> 1 <211> 952 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PMA4 <400> 1 Met Ala Lys Ala Ile Ser Leu Glu Glu Ile Lys Asn Glu Thr Val Asp 1 5 10 15 Leu Glu Lys Ile Pro Ile Glu Glu Val Phe Glu Gln Leu Lys Cys Thr 20 25 30 Arg Glu Gly Leu Ser Ala Asp Glu Gly Ala Ser Arg Leu Gln Ile Phe 35 40 45 Gly Pro Asn Lys Leu Glu Glu Lys Asn Glu Ser Lys Ile Leu Lys Phe 50 55 60 Leu Gly Phe Met Trp Asn Pro Leu Ser Trp Val Met Glu Ala Ala Ala 65 70 75 80 Val Met Ala Ile Ala Leu Ala Asn Gly Asp Gly Lys Pro Pro Asp Trp 85 90 95 Gln Asp Phe Ile Gly Ile Ile Cys Leu Leu Val Ile Asn Ser Thr Ile 100 105 110 Ser Phe Ile Glu Glu Asn Asn Ala Gly Asn Ala Ala Ala Ala Leu Met 115 120 125 Ala Gly Leu Ala Pro Lys Thr Lys Val Leu Arg Asp Gly Arg Trp Ser 130 135 140 Glu Gln Glu Ala Ala Ile Leu Val Pro Gly Asp Ile Ile Ser Val Lys 145 150 155 160 Leu Gly Asp Ile Ile Pro Ala Asp Ala Arg Leu Leu Glu Gly Asp Pro 165 170 175 Leu Lys Ile Asp Gln Ser Ala Leu Thr Gly Glu Ser Leu Pro Val Thr 180 185 190 Lys Asn Pro Gly Asp Glu Val Phe Ser Gly Ser Thr Cys Lys Gln Gly 195 200 205 Glu Leu Glu Ala Val Val Ile Ala Thr Gly Val His Thr Phe Phe Gly 210 215 220 Lys Ala Ala His Leu Val Asp Ser Thr Asn Asn Val Gly His Phe Gln 225 230 235 240 Lys Val Leu Thr Ala Ile Gly Asn Phe Cys Ile Cys Ser Ile Ala Ile 245 250 255 Gly Met Leu Val Glu Ile Ile Val Met Tyr Pro Ile Gln His Arg Lys 260 265 270 Tyr Arg Asp Gly Ile Asp Asn Leu Leu Val Leu Leu Ile Gly Gly Ile 275 280 285 Pro Ile Ala Met Pro Thr Val Leu Ser Val Thr Met Ala Ile Gly Ser 290 295 300 His Arg Leu Ser Gln Gln Gly Ala Ile Thr Lys Arg Met Thr Ala Ile 305 310 315 320 Glu Glu Met Ala Gly Met Asp Val Leu Cys Ser Asp Lys Thr Gly Thr 325 330 335 Leu Thr Leu Asn Lys Leu Ser Val Asp Arg Asn Leu Val Glu Val Phe 340 345 350 Ala Lys Gly Val Asp Lys Glu Tyr Val Leu Leu Leu Ala Ala Arg Ala 355 360 365 Ser Arg Val Glu Asn Gln Asp Ala Ile Asp Ala Cys Met Val Gly Met 370 375 380 Leu Ala Asp Pro Lys Glu Ala Arg Ala Gly Ile Arg Glu Val His Phe 385 390 395 400 Leu Pro Phe Asn Pro Val Asp Lys Arg Thr Ala Leu Thr Tyr Ile Asp 405 410 415 Asn Asn Asn Asn Trp His Arg Ala Ser Lys Gly Ala Pro Glu Gln Ile 420 425 430 Leu Asp Leu Cys Asn Ala Lys Glu Asp Val Arg Arg Lys Val His Ser 435 440 445 Met Met Asp Lys Tyr Ala Glu Arg Gly Leu Arg Ser Leu Ala Val Ala 450 455 460 Arg Arg Thr Val Pro Glu Lys Ser Lys Glu Ser Pro Gly Gly Arg Trp 465 470 475 480 Glu Phe Val Gly Leu Leu Pro Leu Phe Asp Pro Pro Arg His Asp Ser 485 490 495 Ala Glu Thr Ile Arg Arg Ala Leu Asn Leu Gly Val Asn Val Lys Met 500 505 510 Ile Thr Gly Asp Gln Leu Ala Ile Ala Lys Glu Thr Gly Arg Arg Leu 515 520 525 Gly Met Gly Thr Asn Met Tyr Pro Ser Ala Ser Ala Ser Leu Leu Gly Gln Asp 530 535 540 Lys Asp Ser Ala Ile Ala Ser Leu Pro Ile Glu Glu Leu Ile Glu Lys 545 550 555 560 Ala Asp Gly Phe Ala Gly Val Phe Pro Glu His Lys Tyr Glu Ile Val 565 570 575 Lys Lys Leu Gln Glu Arg Lys His Ile Val Gly Met Thr Gly Asp Gly 580 585 590 Val Asn Asp Ala Pro Ala Leu Lys Lys Ala Asp Ile Gly Ile Ala Val 595 600 605 Ala Asp Ala Thr Asp Ala Ala Arg Gly Ala Ser Asp Ile Val Leu Thr 610 615 620 Glu Pro Gly Leu Ser Val Ile Ile Ser Ala Val Leu Thr Ser Arg Ala 625 630 635 640 Ile Phe Gln Arg Met Lys Asn Tyr Thr Ile Tyr Ala Val Ser Ile Thr 645 650 655 Ile Arg Ile Val Phe Gly Phe Met Phe Ile Ala Leu Ile Trp Lys Tyr 660 665 670 Asp Phe Ser Ala Phe Met Val Leu Ile Ile Ala Ile Leu Asn Asp Gly 675 680 685 Thr Ile Met Thr Ile Ser Lys Asp Arg Val Lys Pro Ser Pro Met Pro 690 695 700 Asp Ser Trp Lys Leu Lys Glu Ile Phe Ala Thr Gly Val Val Leu Gly 705 710 715 720 Gly Tyr Gln Ala Leu Met Thr Val Val Phe Phe Trp Ala Met His Asp 725 730 735 Thr Asp Phe Phe Ser Asp Lys Phe Gly Val Lys Ser Leu Arg Asn Ser 740 745 750 Asp Glu Glu Met Met Ser Ala Leu Tyr Leu Gln Val Ser Ile Ile Ser 755 760 765 Gln Ala Leu Ile Phe Val Thr Arg Ser Arg Ser Trp Ser Phe Leu Glu 770 775 780 Arg Pro Gly Met Leu Leu Val Ile Ala Phe Met Ile Ala Gln Leu Val 785 790 795 800 Ala Thr Leu Ile Ala Val Tyr Ala Asn Trp Ala Phe Ala Arg Val Lys 805 810 815 Gly Cys Gly Trp Gly Trp Ala Gly Val Ile Trp Leu Tyr Ser Ile Ile 820 825 830 Phe Tyr Leu Pro Leu Asp Ile Met Lys Phe Ala Ile Arg Tyr Ile Leu 835 840 845 Ser Gly Lys Ala Trp Asn Asn Leu Leu Asp Asn Lys Thr Ala Phe Thr 850 855 860 Thr Lys Lys Asp Tyr Gly Lys Glu Glu Arg Glu Ala Gln Trp Ala Leu 865 870 875 875 Gln Arg Thr Leu His Gly Leu Gln Pro Pro Glu Ala Thr Asn Leu 885 890 895 Phe Asn Glu Lys Asn Ser Tyr Arg Glu Leu Ser Glu Ile Ala Glu Gln 900 905 910 Ala Lys Arg Arg Ala Glu Met Ala Arg Leu Arg Glu Leu His Thr Leu 915 920 925 Lys Gly His Val Glu Ser Val Val Lys Leu Lys Gly Leu Asp Ile Glu 930 935 940 Thr Ile Gln Gln His Tyr Thr Val 945 950 <210> 2 <211> 2856 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide sequence of PMA4 <400> 2 atggccaagg ccatcagcct ggaggagatc aagaacgaga ccgtggacct ggagaagatc 60 cccatcgagg aggtgttcga gcagctgaag tgcacccgcg agggcctgag cgctgacgag 120 ggcgctagcc gcctgcagat tttcggcccc aacaagctgg aggagaagaa cgagagcaag 180 atcctgaagt tcctgggctt catgtggaac ccgctgagct gggtcatgga ggctgctgcc 240 gtgatggcta tcgctctggc taacggcgac ggcaagccgc cggactggca ggacttcatc 300 ggcatcattt gcctgctggt catcaacagc accatcagct tcattgagga gaacaacgcc 360 ggcaacgccg ctgccgctct gatggctggc ctggctccca agaccaaggt gctgcgcgac 420 ggccggtgga gcgagcagga ggccgctatt ctggtccccg gcgacatcat tagcgtgaag 480 ctgggcgaca ttatccccgc tgacgctcgc ctgctggagg gcgacccgct gaagattgac 540 cagtccgctc tgaccggcga gtcgctgccc gtgacgaaga acccgggcga cgaggtgttc 600 agcggctcga cctgcaagca gggcgagctg gaggctgtgg tcattgctac cggcgtgcac 660 accttcttcg gcaaggctgc tcacctggtg gacagcacca acaacgtggg ccacttccag 720 aaggtgctga ccgcgattgg caacttctgc atctgctcga tcgccatcgg catgctggtc 780 gagatcatcg tgatgtaccc catccagcac cgcaagtacc gcgacggcat tgacaacctg 840 ctggtgctgc tgattggcgg catccccatt gctatgccca ccgtgctgag cgtgaccatg 900 gctattggca gccaccgcct gagccagcag ggcgctatta ccaagcgcat gaccgctatc 960 gaggagatgg ccggcatgga cgtgctgtgc agcgacaaga ccggcacgct gaccctgaac 1020 aagctgtccg tggaccgcaa cctggtcgag gtgttcgcta agggcgtcga caaggagtac 1080 gtcctgctgc tggctgctcg cgcttcccgc gtcgagaacc aggacgctat tgacgcttgc 1140 atggtcggca tgctggctga ccccaaggag gctcgcgctg gcattcgcga ggtccacttc 1200 ctgccgttca accccgtgga caagcgcacg gctctgacct acatcgacaa caacaacaac 1260 tggcaccgcg ccagcaaggg cgctcccgag cagattctgg acc tgtgcaa cgctaaggag 1320 gacgtccgcc gcaaggtgca cagcatgatg gacaagtacg ctgagcgcgg cctgcgcagc 1380 ctggctgtgg ctcgccgcac ggtgcccgag aagtccaagg agagccccgg cggccgctgg 1440 gagttcgtgg gcctgctgcc gctgttcgac ccgccgcgcc acgacagcgc tgagaccatt 1500 cgccgcgctc tgaacctggg cgtgaacgtg aagatgatca ccggcgacca gctggcgatt 1560 gctaaggaga ccggccggcg cctgggcatg ggcaccaaca tgtacccctc cgcttcgctg 1620 ctgggccagg acaaggacag cgctattgct agcctgccga tcgaggagct gattgagaag 1680 gctgacggct tcgctggcgt gttccccgag cacaagtacg agatcgtgaa gaagctgcag 1740 gagcgcaagc acatcgtcgg catgaccggc gacggcgtga acgacgctcc ggctctgaag 1800 aaggccgaca ttggcattgc tgtggccgac gctaccgacg ccgctcgcgg cgctagcgac 1860 attgtcctga ccgagccggg cctgtccgtg attattagcg ctgtgctgac ctcgcgcgcc 1920 atcttccagc gcatgaagaa ctacaccatc tacgccgtgt ccatcaccat ccgcatcgtg 1980 ttcggcttca tgttcattgc cctgatctgg aagtacgact tcagcgcctt catggtgctg 2040 atcattgcca tcctgaacga cggcaccatc atgaccatca gcaaggaccg cgtgaagccc 2100 tcgccgatgc cggacagctg gaagctgaag gagattttcg ccaccggcg t ggtgctgggc 2160 ggctaccagg ctctgatgac cgtggtgttc ttctgggcca tgcacgacac cgacttcttc 2220 agcgacaagt tcggcgtgaa gtccctgcgc aacagcgacg aggagatgat gagcgcgctg 2280 tacctgcagg tgtcgatcat ctcgcaggcg ctgattttcg tgacccgcag ccgcagctgg 2340 tccttcctgg agcgcccggg catgctgctg gtcattgcct tcatgattgc ccagctggtg 2400 gcgaccctga tcgccgtgta cgctaactgg gctttcgctc gcgtgaaggg ctgcggctgg 2460 ggctgggctg gcgtgatctg gctgtactcc atcatcttct acctgccgct ggacatcatg 2520 aagttcgcca tccgctacat cctgagcggc aaggcttgga acaacctgct ggacaacaag 2580 accgccttca ccaccaagaa ggactacggc aaggaggagc gcgaggctca gtgggctctg 2640 gctcagcgca ccctgcacgg cctgcagccg ccggaggcta ccaacctgtt caacgagaag 2700 aacagctacc gcgagctgag cgagatcgcc gagcaggcta agcgccgcgc tgagatggct 2760 cggctgcgcg agctgcacac cctgaagggc cacgtcgaga gcgtggtcaa gctgaagggc 2820 ctggacatcg agaccatcca gcagcactac accgtg 2856 <210> 3 <211> 852 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PMA4 Cter <400> 3 Met Ala Lys Ala Ile Ser Leu Glu Glu Ile Lys Asn G lu Thr Val Asp 1 5 10 15 Leu Glu Lys Ile Pro Ile Glu Glu Val Phe Glu Gln Leu Lys Cys Thr 20 25 30 Arg Glu Gly Leu Ser Ala Asp Glu Gly Ala Ser Arg Leu Gln Ile Phe 35 40 45 Gly Pro Asn Lys Leu Glu Glu Lys Asn Glu Ser Lys Ile Leu Lys Phe 50 55 60 Leu Gly Phe Met Trp Asn Pro Leu Ser Trp Val Met Glu Ala Ala Ala 65 70 75 80 Val Met Ala Ile Ala Leu Ala Asn Gly Asp Gly Lys Pro Pro Asp Trp 85 90 95 Gln Asp Phe Ile Gly Ile Ile Cys Leu Leu Val Ile Asn Ser Thr Ile 100 105 110 Ser Phe Ile Glu Glu Asn Asn Ala Gly Asn Ala Ala Ala Ala Leu Met 115 120 125 Ala Gly Leu Ala Pro Lys Thr Lys Val Leu Arg Asp Gly Arg Trp Ser 130 135 140 Glu Gln Glu Ala Ala Ile Leu Val Pro Gly Asp Ile Ile Ser Val Lys 145 150 155 160 Leu Gly Asp Ile Ile Pro Ala Asp Ala Arg Leu Leu Glu Gly Asp Pro 165 170 175 Leu Lys Ile Asp Gln Ser Ala Leu Thr Gly Glu Ser Leu Pro Val Thr 180 185 190 Lys Asn Pro Gly Asp Glu Val Phe Ser Gly Ser Thr Cys Lys Gln Gly 195 200 205 Glu Leu Glu Ala Val Val Ile Ala Thr Gly Val His Thr Phe Phe Gly 210 215 220 Lys Ala Ala His Leu Val Asp Ser Thr Asn Asn Val Gly His Phe Gln 225 230 235 240 Lys Val Leu Thr Ala Ile Gly Asn Phe Cys Ile Cys Ser Ile Ala Ile 245 250 255 Gly Met Leu Val Glu Ile Ile Val Met Tyr Pro Ile Gln His Arg Lys 260 265 270 Tyr Arg Asp Gly Ile Asp Asn Leu Leu Val Leu Leu Ile Gly Gly Ile 275 280 285 Pro Ile Ala Met Pro Thr Val Leu Ser Val Thr Met Ala Ile Gly Ser 290 295 300 His Arg Leu Ser Gln Gln Gly Ala Ile Thr Lys Arg Met Thr Ala Ile 305 310 315 320 Glu Glu Met Ala Gly Met Asp Val Leu Cys Ser Asp Lys Thr Gly Thr 325 330 335 Leu Thr Leu Asn Lys Leu Ser Val Asp Arg Asn Leu Val Glu Val Phe 340 345 350 Ala Lys Gly Val Asp Lys Glu Tyr Val Leu Leu Leu Ala Ala Arg Ala 355 360 365 Ser Arg Val Glu Asn Gln Asp Ala Ile Asp Ala Cys Met Val Gly Met 370 375 380 Leu Ala Asp Pro Lys Glu Ala Arg Ala Gly Ile Arg Glu Val His Phe 385 390 395 400 Leu Pro Phe Asn Pro Val Asp Lys Arg Thr Ala Leu Thr Tyr Ile Asp 405 410 415 Asn Asn Asn Asn Trp His Arg Ala Ser Lys Gly Ala Pro Glu Gln Ile 420 425 430 Leu Asp Leu Cys Asn Ala Lys Glu Asp Val Arg Arg Lys Val His Ser 435 440 445 Met Met Asp Lys Tyr Ala Glu Arg Gly Leu Arg Ser Leu Ala Val Ala 450 455 460 Arg Arg Thr Val Pro Glu Lys Ser Lys Glu Ser Pro Gly Gly Arg Trp 465 470 475 480 Glu Phe Val Gly Leu Leu Pro Leu Phe Asp Pro Pro Arg His Asp Ser 485 490 495 Ala Glu Thr Ile Arg Arg Ala Leu Asn Leu Gly Val Asn Val Lys Met 500 505 510 Ile Thr Gly Asp Gln Leu Ala Ile Ala Lys Glu Thr Gly Arg Arg Leu 515 520 525 Gly Met Gly Thr Asn Met Tyr Pro Ser Ala Ser Ala Ser Leu Leu Gly Gln Asp 530 535 540 Lys Asp Ser Ala Ile Ala Ser Leu Pro Ile Glu Glu Leu Ile Glu Lys 545 550 555 560 Ala Asp Gly Phe Ala Gly Val Phe Pro Glu His Lys Tyr Glu Ile Val 565 570 575 Lys Lys Leu Gln Glu Arg Lys His Ile Val Gly Met Thr Gly Asp Gly 580 585 590 Val Asn Asp Ala Pro Ala Leu Lys Lys Ala Asp Ile Gly Ile Ala Val 595 600 605 Ala Asp Ala Thr Asp Ala Ala Arg Gly Ala Ser Asp Ile Val Leu Thr 610 615 620 Glu Pro Gly Leu Ser Val Ile Ile Ser Ala Val Leu Thr Ser Arg Ala 625 630 635 640 Ile Phe Gln Arg Met Lys Asn Tyr Thr Ile Tyr Ala Val Ser Ile Thr 645 650 655 Ile Arg Ile Val Phe Gly Phe Met Phe Ile Ala Leu Ile Trp Lys Tyr 660 665 670 Asp Phe Ser Ala Phe Met Val Leu Ile Ile Ala Ile Leu Asn Asp Gly 675 680 685 Thr Ile Met Thr Ile Ser Lys Asp Arg Val Lys Pro Ser Pro Met Pro 690 695 700 Asp Ser Trp Lys Leu Lys Glu Ile Phe Ala Thr Gly Val Val Leu Gly 705 710 715 720 Gly Tyr Gln Ala Leu Met Thr Val Val Phe Phe Trp Ala Met His Asp 725 730 735 Thr Asp Phe Phe Ser Asp Lys Phe Gly Val Lys Ser Leu Arg Asn Ser 740 745 750 Asp Glu Glu Met Met Ser Ala Leu Tyr Leu Gln Val Ser Ile Ile Ser 755 760 765 Gln Ala Leu Ile Phe Val Thr Arg Ser Arg Ser Trp Ser Phe Leu Glu 770 775 780 Arg Pro Gly Met Leu Leu Val Ile Ala Phe Met Ile Ala Gln Leu Val 785 790 795 800 Ala Thr Leu Ile Ala Val Tyr Ala Asn Trp Ala Phe Ala Arg Val Lys 805 810 815 Gly Cys Gly Trp Gly Trp Ala Gly Val Ile Trp Leu Tyr Ser Ile Ile 820 825 830 Phe Tyr Leu Pro Leu Asp Ile Met Lys Phe Ala Ile Arg Tyr Ile Leu 835 840 845 Ser Gly Lys Ala 850 <210> 4 <211> 2556 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide sequence of PMA4 Cter <400> 4 atggccaagg ccatcagcct ggaggagatc aagaacgaga ccgtggacct ggagaagatc 60 cccatcgagg aggtgttcga gcagctgaag tgcacccgcg agggcctgag cgctgacgag 120 ggcgctagcc gcctgcagat tttcggcccc aacaagctgg aggagaagaa cgagagcaag 180 atcctgaagt tcctgggctt catgtggaac ccgctgagct gggtcatgga ggctgctgcc 240 gtgatggcta tcgctctggc taacggcgac ggcaagccgc cggactggca ggacttcatc 300 ggcatcattt gcctgctggt catcaacagc accatcagct tcattgagga gaacaacgcc 360 ggcaacgccg ctgccgctct gatggctggc ctggctccca agaccaaggt gctgcgcgac 420 ggccggtgga gcgagcagga ggccgctatt ctggtccccg gcgacatcat tagcgtgaag 480 ctgggcgaca ttatccccgc tgacgctcgc ctgctggagg gcgacccgct gaagattgac 540 cagtccgctc tgaccggcga gtcgctgccc gtgacgaaga acccgggcga cgaggtgttc 600 agcggctcga cctgcaagca gggcgagctg gaggctgtgg tcattgctac cggcgtgcac 660 accttcttcg gcaaggctgc tcacctggtg gacagcacca acaacgtggg ccacttccag 720 aaggtgctga ccgcgattgg caacttctgc atctgctcga tcgccatcgg catgctggtc 780 gagatcatcg tgatgtaccc catccagcac cgcaagtacc gcgacggcat tgacaacctg 840 ctggtgctgc tgattggcgg catccccatt gctatgccca ccgtgctgag cgtgaccatg 900 gctattggca gccaccgcc t gagccagcag ggcgctatta ccaagcgcat gaccgctatc 960 gaggagatgg ccggcatgga cgtgctgtgc agcgacaaga ccggcacgct gaccctgaac 1020 aagctgtccg tggaccgcaa cctggtcgag gtgttcgcta agggcgtcga caaggagtac 1080 gtcctgctgc tggctgctcg cgcttcccgc gtcgagaacc aggacgctat tgacgcttgc 1140 atggtcggca tgctggctga ccccaaggag gctcgcgctg gcattcgcga ggtccacttc 1200 ctgccgttca accccgtgga caagcgcacg gctctgacct acatcgacaa caacaacaac 1260 tggcaccgcg ccagcaaggg cgctcccgag cagattctgg acctgtgcaa cgctaaggag 1320 gacgtccgcc gcaaggtgca cagcatgatg gacaagtacg ctgagcgcgg cctgcgcagc 1380 ctggctgtgg ctcgccgcac ggtgcccgag aagtccaagg agagccccgg cggccgctgg 1440 gagttcgtgg gcctgctgcc gctgttcgac ccgccgcgcc acgacagcgc tgagaccatt 1500 cgccgcgctc tgaacctggg cgtgaacgtg aagatgatca ccggcgacca gctggcgatt 1560 gctaaggaga ccggccggcg cctgggcatg ggcaccaaca tgtacccctc cgcttcgctg 1620 ctgggccagg acaaggacag cgctattgct agcctgccga tcgaggagct gattgagaag 1680 gctgacggct tcgctggcgt gttccccgag cacaagtacg agatcgtgaa gaagctgcag 1740 gagcgcaagc acatcgtcgg catga ccggc gacggcgtga acgacgctcc ggctctgaag 1800 aaggccgaca ttggcattgc tgtggccgac gctaccgacg ccgctcgcgg cgctagcgac 1860 attgtcctga ccgagccggg cctgtccgtg attattagcg ctgtgctgac ctcgcgcgcc 1920 atcttccagc gcatgaagaa ctacaccatc tacgccgtgt ccatcaccat ccgcatcgtg 1980 ttcggcttca tgttcattgc cctgatctgg aagtacgact tcagcgcctt catggtgctg 2040 atcattgcca tcctgaacga cggcaccatc atgaccatca gcaaggaccg cgtgaagccc 2100 tcgccgatgc cggacagctg gaagctgaag gagattttcg ccaccggcgt ggtgctgggc 2160 ggctaccagg ctctgatgac cgtggtgttc ttctgggcca tgcacgacac cgacttcttc 2220 agcgacaagt tcggcgtgaa gtccctgcgc aacagcgacg aggagatgat gagcgcgctg 2280 tacctgcagg tgtcgatcat ctcgcaggcg ctgattttcg tgacccgcag ccgcagctgg 2340 tccttcctgg agcgcccggg catgctgctg gtcattgcct tcatgattgc ccagctggtg 2400 gcgaccctga tcgccgtgta cgctaactgg gctttcgctc gcgtgaaggg ctgcggctgg 2460 ggctgggctg gcgtgatctg gctgtactcc atcatcttct acctgccgct ggacatcatg 2520aagttcgcca tccgctacat cctgagcggc aaggct 2556

Claims (31)

Recombinant microalgae into which the gene encoding the PMA protein has been introduced. According to claim 1,
The PMA protein is a functional fragment in which a portion of the C-terminal sequence encoding the self-repression site of the PMA protein is removed, recombinant microalgae. 3. The method of claim 2,
The part of the sequence is 100 amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 1, recombinant microalgae. 3. The method of claim 2,
The functional fragment is a polynucleotide comprising SEQ ID NO: 4, recombinant microalgae. According to claim 1,
The microalgae are of the genus Chlamydomonas, recombinant microalgae. 6. The method of claim 5,
Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), the recombinant microalgae. According to claim 1,
The recombinant microalgae will be deposited with accession number KCTC14511BP, recombinant microalgae. A method for producing biomass or biofuel comprising the step of culturing the microalgae of any one of claims 1 to 7. 9. The method of claim 8,
The biofuel is biodiesel, the production method. A method of enhancing biomass or biofuel productivity comprising the step of culturing the microalgae of any one of claims 1 to 7. Recombinant microalgae with enhanced resistance to high concentrations of CO ₂ or acid with enhanced PMA protein activity. 12. The method of claim 11,
The microalgae, the PMA protein is the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof, recombinant microalgae. 13. The method of claim 12,
The functional fragment is that the self-replicating domain is removed from the C-terminus of the amino acid sequence of SEQ ID NO: 1, recombinant microalgae. 13. The method of claim 12,
The functional fragment consists of the amino acid sequence of SEQ ID NO: 3, recombinant microalgae. 12. The method of claim 11,
The recombinant microalgae has enhanced resistance to high concentration CO ₂ or acid compared to the parent strain in which the activity of the PMA protein is not enhanced, recombinant microalgae. 12. The method of claim 11,
The microalgae are of the genus Chlamydomonas, recombinant microalgae. 17. The method of claim 16,
Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), the recombinant microalgae. 12. The method of claim 11,
The recombinant microalgae will be deposited with accession number KCTC14511BP, recombinant microalgae. A method for producing biomass or biofuel comprising the step of culturing the microalgae of any one of claims 11 to 18. a fragment of a polynucleotide in which a portion of the C-terminal sequence encoding the self-repression site has been removed from the polynucleotide encoding the PMA protein; and
An expression cassette for overexpressing PMA protein comprising a polynucleotide encoding a fluorescent protein operably linked to the fragment by a linker. 21. The method of claim 20,
The expression cassette, wherein the fragment of the polynucleotide encoding the PMA protein comprises SEQ ID NO: 4. 21. The method of claim 20,
The linker is GGSGGGSG, the expression cassette. 21. The method of claim 20,
The fluorescent protein is VENUS, the expression cassette. 24. An expression vector comprising the expression cassette of any one of claims 20-23. A transformant into which the expression vector of claim 24 is introduced into a host cell. 26. The method of claim 25,
The host cell is a microalgae, the transformant. 27. The method of claim 26,
The microalgae are of the genus Chlamydomonas, transformants. 28. The method of claim 27,
Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), the transformant. 26. The method of claim 25,
The transformant was deposited under the accession number KCTC14511BP, the transformant. 30. A method for producing biomass or biofuel comprising the step of culturing the transformant of any one of claims 25 to 29. 31. The method of claim 30,
The culture is a photo-independent culture, the production method.

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