Method for producing starch using carbon dioxide, recombinant microorganism and method for constructing a recombinant microorganism

Abstract

The present application relates to a method for producing starch using carbon dioxide and a recombinant microorganism, a method for constructing a recombinant microorganism, and a reagent. In the method for producing starch using carbon dioxide, (1) energy and a carbon source are provided to a microbial cell based on carbon dioxide and extracellular non-photosynthetic energy, and (2) starch is produced in the microbial cell based on at least one of up-regulated glucose-1-phosphate adenylyltransferase and starch synthase in the microbial cell. Thus, by using non-photosynthetic energy such as electric energy or hydrogen energy, starch can be effectively produced inside the microbial cell by fixing carbon dioxide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, in particular, the present application relates to a method for preparing starch using carbon dioxide and a recombinant microorganism, and a method for constructing a recombinant microorganism. BACKGROUND

[0002] Starch is the most important nutrient in food, providing more than 80% of the calories in the world, of the nearly 2 billion tons of grain food produced in the world each year, 12-14 billion tons are starch. At present, agricultural planting is still the only way to produce starch. Global food production requires 38% of the land and 70% of fresh water resources, and about 200 million tons of chemical fertilizers and 3 million tons of pesticides are consumed each year. With the increase of global population, it is predicted that the global food demand will increase by more than 70% by 2050, and the current agricultural planting method is difficult to meet the growing demand for food. It is of great significance to develop a method that can replace agricultural planting to produce starch.

[0003] Excessive emission of carbon dioxide leads to global warming, ocean acidification, and irreversible damage to the environment. Since the industrial revolution, the concentration of carbon dioxide in the atmosphere has increased from 277 ppm to 400 ppm, which is equivalent to an increase of 1 trillion tons, and it is increasing at a rate of 3 ppm per year. Photosynthetic organisms such as plants convert and fix about 100 billion tons of carbon dioxide per year using light energy, so the earth's own carbon cycle system cannot digest so much carbon dioxide. Only by breaking through the natural photosynthetic limit and creating a more efficient artificial photosynthetic system, significantly improving the efficiency of biological carbon sequestration, can we achieve rapid biological conversion of carbon dioxide.

[0004] Therefore, on the one hand, how to scientifically use carbon dioxide to help the carbon neutralization strategy, on the other hand, how to realize the biological industrial synthesis of starch to realize "agricultural industrialization" are difficult problems to be solved.

[0005] Microalgae and cyanobacteria in photosynthetic organisms can directly utilize light energy to fix carbon dioxide for growth, and due to their fast growth rate, high energy utilization efficiency and other advantages, they become excellent chassis cells for developing cell carbon fixation. Currently, there are related articles and patents reporting the use of algae to produce starch (Improving carbohydrate and starch accumulation in Chlorella sp. AE10 by a novel two-stage process with cell dilution; JP07059557A, NEW MICROALGAE), but due to the growth characteristics of algae, a large cultivation area is still needed to obtain solar energy. There are also articles reporting the synthesis of starch in Saccharomyces cerevisiae (Recreating the synthesis of starch granules in yeast), Escherichia coli and other microorganisms, but the raw materials used are agricultural starch or hydrolyzed glucose, which does not meet the purpose of the present application.

[0006] Therefore, the current means for preparing starch still needs to be improved. SUMMARY

[0007] The present application aims to at least partially solve one of the technical problems in the related art. Therefore, the present application proposes a means for preparing starch by fixing carbon dioxide using non-light energy.

[0008] In a first aspect of the present application, the present application proposes a method for preparing starch using carbon dioxide. According to some embodiments of the present application, the method comprises: (1) providing energy and carbon source for microbial cells based on carbon dioxide and extracellular non-light energy; and (2) generating starch in the microbial cells based on at least one of up-regulated glucose-1-phosphate adenylyltransferase and starch synthase in the microbial cells. Thus, by utilizing non-light energy such as electrical energy or hydrogen energy, starch can be effectively prepared inside microbial cells by fixing carbon dioxide, thereby achieving scientific utilization of carbon dioxide to help carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0009] In a second aspect, the present application provides a recombinant microorganism for producing starch using carbon dioxide. According to embodiments of the present application, the recombinant microorganism has: (1) an enzyme system for obtaining energy and carbon source based on carbon dioxide and extracellular non-phototrophic energy; and (2) at least one of starch synthase and glucose-1-phosphate adenylyltransferase up-regulated compared to the wild type of the microorganism. Thus, the recombinant microorganism can effectively implement the method for producing starch as described in the first aspect, so that starch can be effectively produced inside the microbial cell by fixing carbon dioxide by using non-phototrophic energy such as electrical energy or hydrogen energy, thereby achieving scientific utilization of carbon dioxide to help carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0010] It will be appreciated by those skilled in the art that the features and advantages described for the method for producing starch in the first aspect also apply to the recombinant microorganism, which will not be described here again.

[0011] In a third aspect, the present application provides a method for constructing the microorganism of the second aspect, characterized in that the method comprises up-regulating at least one of starch synthase and glucose-1-phosphate adenylyltransferase in a starting microorganism. Thus, the recombinant microorganism described in the second aspect can be effectively produced, and thus the recombinant microorganism can effectively implement the method for producing starch as described in the first aspect, so that starch can be effectively produced inside the microbial cell by fixing carbon dioxide by using non-phototrophic energy such as electrical energy or hydrogen energy, thereby achieving scientific utilization of carbon dioxide to help carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0012] It will be appreciated by those skilled in the art that the features and advantages described for the method for producing starch and the recombinant microorganism in other aspects also apply to the method for constructing the recombinant microorganism, which will not be described here again.

[0013] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0014] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0015] Figure 1 The figure shows the disruption efficiency of GA gene using different SgRNA according to one embodiment of the present application;

[0016] Figure 2Figure 6 shows the disruption efficiency of GP gene using different SgRNAs according to one embodiment of the present application;

[0017] Figure 3 Figure 7 shows the starch production of recombinant strains containing different promoter combinations of GlgA and / or GlgC according to one embodiment of the present application;

[0018] Figure 4 Figure 8 shows the starch production effect after disruption of GA based on pHis- EcGlgA-EcGlgC recombinant strain according to one embodiment of the present application; and

[0019] Figure 5 Figure 9 shows the starch production effect after disruption of GP based on pHis- EcGlgA-EcGlgC and disruption of GA according to one embodiment of the present application. DETAILED DESCRIPTION

[0020] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and should not be understood as limiting the present application.

[0021] DEFINITIONS

[0022] As used herein, the terms or expressions used herein have the following meanings unless otherwise specified.

[0023] The term "recombinant microorganism" as used herein refers to a microorganism that has been genetically modified or engineered so that the sequence of an endogenous nucleic acid or the expression thereof is altered, or an exogenous nucleic acid is expressed (the exogenous nucleic acid can be contained in a vector or integrated into the genome of the microorganism). By "altered" is meant that the expression of a gene, or the level of an RNA molecule, or the level of an equivalent RNA molecule encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits, is up-regulated or down-regulated so that the expression, level, or activity is greater or less than that observed in the absence of the alteration.

[0024] The term "starting strain (or starting microorganism)" as used herein refers to a strain that can be genetically modified or engineered to obtain a recombinant microorganism. For example, the starting strain or microorganism can be a wild-type strain, or a strain that has been previously subjected to one or more genetic modifications or engineering and has undergone changes in nucleic acids compared to the wild-type strain.

[0025] The term "exogenous" as used herein refers to a substance that is not originally present in the starting strain or is a substance that is distinguished from the corresponding substance in the starting strain, for example a nucleic acid that is not present in the starting strain or a nucleic acid that has a mutation compared to the corresponding genetic sequence in the starting strain. According to embodiments of the present application, "exogenous nucleic acid" includes nucleic acids from the same species as the starting strain, as well as heterologous nucleic acids (i.e. nucleic acids from a different species than the starting strain).

[0026] The term "nucleic acid" as used herein is used interchangeably with the term "polynucleotide" and refers to an organic polymer composed of two or more monomers, including nucleotides, nucleosides or analogs thereof, and includes but is not limited to single- or double-stranded, sense or antisense deoxyribonucleic acid (DNA), sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term "nucleotide" refers to any one of a variety of chemical compounds composed of ribose or deoxyribose combined with a purine or pyrimidine base, and bonded to a phosphate group, and they are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound composed of a purine or pyrimidine base combined with deoxyribose or ribose, such as guanosine or adenosine, and it exists in nucleic acids.

[0027] It should be understood that the meaning of "nucleic acid" described herein includes "genes", and the nucleic acids described herein also include "vectors" or "plasmids".

[0028] The term "gene" as used herein refers to a polynucleotide that encodes a specific amino acid sequence, which includes all or part of one or more proteins or enzymes, and can include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine, for example, under what conditions the gene is expressed. The transcribed region of a gene can include untranslated regions (including introns, 5' untranslated regions (UTRs) and 3' UTRs) as well as coding sequences.

[0029] The term "up-regulation" as used herein refers to the activity of a specific functional protein being increased compared to the corresponding activity of the starting strain, which can be achieved by increasing the expression amount of the specific functional protein in the microbial cell (for example, by additionally introducing an exogenous nucleic acid encoding the functional protein, or by adjusting the regulatory elements such as promoters, etc. of the functional protein-encoding nucleic acid), or by engineering the amino acid sequence of the functional protein to improve its biological activity.

[0030] The term "down-regulation" as used herein refers to a decrease in the activity of a particular functional protein as compared to the corresponding activity of the starting strain, which can be achieved by decreasing the expression of the particular functional protein in the microbial cell (e.g. by knocking out the nucleic acid encoding the functional protein, but also by adapting the regulatory elements such as promoters etc. of the nucleic acid encoding the functional protein), but also by engineering the amino acid sequence of the functional protein such that its biological activity is decreased.

[0031] "Up-regulation" or "down-regulation" as referred to herein can be constitutive, e.g. due to stable, permanent transgenic expression, or due to stable mutation of the encoding nucleic acid or the corresponding endogenous gene, or due to regulation of the expression or behavior of the gene encoding the polypeptide or for expression of the polypeptide; can be transient, e.g. due to transient transformation or temporary addition of a modulator (e.g. an agonist or antagonist); or can be inducible, e.g. by transformation with a nucleic acid molecule under the control of an inducible promoter or an inducible construct and addition of an inducer.

[0032] The term "expression" as used herein refers to the transcription and / or translation of a genetic segment or gene. Typically, the resulting product is mRNA or a protein. However, expression products can also include functional RNA, such as antisense strand nucleic acids, tRNA, snRNA, rRNA, RNAi, siRNA, ribozymes, etc. Expression can be global, local or transient.

[0033] In the context of additional introduction of further exogenous nucleic acids, the exogenous nucleic acid can be integrated into the genome of the microorganism, thereby functioning, e.g. by inserting the exogenous nucleic acid (gene) into an integration vector, such that the integration vector undergoes homologous recombination with the genome of the microorganism, thereby integrating the exogenous nucleic acid (gene) into the genome of the microorganism. Alternatively, the exogenous nucleic acid can be extrachromosomal, e.g. inserted into an expression vector, which does not need to be integrated into the genome to be expressed, and which can be maintained during propagation of the microorganism, e.g. by a plasmid, without being lost, e.g. by providing a selection gene, e.g. a drug resistance gene, on the expression vector, such that the microorganism is always maintained with the corresponding expression vector during propagation in a culture medium supplemented with the corresponding drug, e.g. penicillin or kanamycin.

[0034] The term "knock-out" as used herein refers to a modification or engineering of a gene such that it no longer functions as originally.

[0035] The term "promoter" as used herein refers to a DNA sequence recognized by RNA polymerase, which binds and initiates transcription. It contains conserved sequences required for specific binding by RNA polymerase and initiation of transcription. Most promoters are located upstream (towards the 5' direction of the sense strand of the DNA) from the transcription initiation site of the structural gene and are usually not transcribed themselves. Promoters controlling the expression of genes are located in the regulatory region of the gene, upstream from the transcription initiation site. They are usually about 100 to 1000 base pairs long. Promoters used in genetic engineering of microorganisms are classified into three types according to their mode of action and function: constitutive promoters (which maintain constant activity in most or all tissues), specific promoters (which are tissue-specific or development-stage-specific), and inducible promoters (which are regulated by external chemical or physical signals).

[0036] The term "operably linked to a promoter" as used herein refers to a nucleic acid molecule linked to a promoter, which is capable of being controlled by the promoter.

[0037] The term "about" as used herein refers to a range of 5% above and below the index value. For example, a value of about 100 means that the value can be 100 plus or minus 5.

[0038] Method for preparing starch using carbon dioxide

[0039] In the first aspect of the present application, a method for preparing starch using carbon dioxide is provided. According to some embodiments of the present application, the method comprises: (1) providing energy and carbon source to a microbial cell based on carbon dioxide and extracellular non-photosynthetic energy; and (2) generating starch in the microbial cell based on at least one of up-regulated glucose-1-phosphate adenylyltransferase and starch synthase in the microbial cell. Thus, by using non-photosynthetic energy such as electrical energy or hydrogen energy, starch can be effectively prepared inside the microbial cell by fixing carbon dioxide, thereby achieving scientific utilization of carbon dioxide to help carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0040] The method for preparing starch is described in detail below according to embodiments of the present application.

[0041] It should be noted that the "extracellular non-light energy" used herein refers to an energy form other than light energy, such as thermal energy, electrical energy, chemical energy (especially hydrogen energy), potential energy, magnetic energy, etc., which exists outside the cell and can be utilized by the microbial cell. According to some embodiments of the present application, the non-light energy that can be used includes, but is not limited to, at least one of hydrogen energy and electrical energy. Thus, the limitation of natural photosynthesis can be effectively broken through, a more efficient artificial photosynthesis system can be created, the biological carbon fixation efficiency can be greatly improved, and rapid biological conversion of carbon dioxide can be achieved. According to embodiments of the present application, the provision of hydrogen energy and electrical energy is not particularly limited, for example, hydrogen energy is provided in the form of hydrogen gas, and electrical energy is provided in the form of an electric current.

[0042] It should be noted that the way of fixing carbon dioxide by using extracellular non-light energy such as hydrogen energy and electrical energy is not particularly limited, and the microbial cell can directly take in hydrogen energy, for example, hydrogen gas is introduced into the culture environment of the microbial cell, or the microbial cell can directly take in electrical energy, for example, an electric current is introduced into the culture environment of the microbial cell. In addition, at least one of the extracellular non-light energy such as hydrogen energy and electrical energy can be chemically reacted with carbon dioxide outside the cell, and the microbial cell can further take in the reaction product of the chemical reaction. According to some embodiments of the present application, step (1) can further include: causing the microbial cell to take in the carbon dioxide as a carbon source and absorb the extracellular non-light energy; and / or using the reducing ability of the extracellular non-light energy to obtain a low-carbon compound based on the carbon dioxide, and causing the microbial cell to take in the low-carbon compound as a carbon source.

[0043] It should be noted that the term "carbon source" as used herein refers to a substance containing carbon element and capable of being utilized by microorganism growth and reproduction. It is known to those skilled in the art that different microorganisms can utilize different carbon sources. The role of carbon source in microorganism growth and metabolism mainly includes providing carbon skeleton of cells, providing energy required for cell life activities, and providing carbon skeleton for product synthesis. Carbon source plays an important role in preparing microbial culture medium or cell culture medium, and provides material basis for normal growth and division of microorganisms or cells. Therefore, "carbon source" can refer to carbon dioxide or low-carbon compounds synthesized from carbon dioxide by using electric energy and / or hydrogen energy. According to some embodiments of the present application, the low-carbon compounds are at least one selected from carbon monoxide, formic acid, methanol, methane, ethanol, formaldehyde, acetaldehyde, acetic acid, propanol, propane, propyl aldehyde, acetone, hydroxyacetone, dihydroxyacetone and glycerol. Thus, microorganisms capable of using these low-carbon compounds as carbon source, especially as the only carbon source, can be used to synthesize starch. The efficiency of preparing starch can be further improved. It should be noted that the term "only carbon source" should be understood in a broad sense. Some other carbon-containing materials can be added during the culture process to activate the relevant activity of microorganisms, for example, appropriate carbon-containing components can be added to activate the inducible promoter, for example, a small amount of methanol can be added to activate the AOX1 promoter. Of course, those skilled in the art can understand that, in order to improve the growth rate of microorganisms and the like, other conventional nutrients such as nitrogen source can also be provided for microorganisms. Details are not described here.

[0044] In addition, according to some embodiments of the present application, the microorganism is at least one of modified yeast and bacteria, which is adapted to be capable of utilizing these carbon sources for metabolism. For example, Escherichia coli or yeast can be modified to fix carbon dioxide by using non-photonic energy such as electric energy or hydrogen energy. For example, for microorganisms incapable of utilizing electric energy such as Escherichia coli, the relevant enzyme system of other microorganisms capable of utilizing electric energy can be introduced into Escherichia coli for expression, so as to enable Escherichia coli to utilize the relevant carbon source or energy. Thus, according to embodiments of the present application, Escherichia coli can be transformed to generate all biomass carbon from carbon dioxide, and industrial yeast Pichia pastoris can be transformed from heterotroph to autotroph capable of growing on carbon dioxide, for example, Escherichia coli can be transformed to grow only on methanol to become synthetic methylotroph. Specifically, the enzyme system related to Calvin carbon fixation cycle can be introduced into Escherichia coli or yeast species such as Pichia pastoris, or the enzyme system related to XuMP pathway of Pichia pastoris can be introduced into Escherichia coli.

[0045] A person skilled in the art can synthesize low-carbon compounds containing 1-3 carbon atoms, such as at least one selected from carbon monoxide, formic acid, methanol, methane, ethanol, formaldehyde, acetaldehyde, acetic acid, propanol, propane, propyl aldehyde, acetone, hydroxyacetone, dihydroxyacetone, and glycerol, by using hydrogen or electric current to synthesize carbon dioxide based on any known method. For example, hydrogen has strong reducing power, and under appropriate reaction conditions, carbon dioxide can be reduced to obtain carbon monoxide or a carbon-hydrogen compound with controllable carbon-hydrogen ratio. According to an embodiment of the present application, hydrogen can be obtained by splitting water using solar energy, and as a non-limiting example, carbon dioxide can be hydrogenated to synthesize methanol with high selectivity and high stability by using carbon dioxide and hydrogen as catalysts with a double-metal solid solution oxide (ZnO-ZrO2).

[0046] For the convenience of understanding, some types of microorganisms that can be used in the present application are listed below. According to some embodiments of the present application, microorganisms suitable for absorbing electrical energy include, but are not limited to, at least one of Geobacter, Sporomusa, Methanogens, Acetobacterium, etc.; microorganisms suitable for taking in acetic acid as a carbon source include, but are not limited to, Yarrowia lipolytica, microorganisms suitable for absorbing hydrogen energy include, but are not limited to, at least one of hydrogen-oxidizing bacteria, Ralstonia, and Clostridium; microorganisms suitable for taking in carbon monoxide as a carbon source include, but are not limited to, Clostridium; microorganisms suitable for taking in formic acid as a carbon source include, but are not limited to, Ralstonia; microorganisms suitable for taking in methanol as a carbon source include, but are not limited to, at least one of Pichia pastoris, Candida boidinii, and Hansenula polymorpha; microorganisms suitable for taking in methane as a carbon source include, but are not limited to, Methylobacter and Methylococcus; or microorganisms suitable for taking in ethanol as a carbon source include, but are not limited to, Saccharomyces cerevisiae. Thus, the efficiency of preparing starch can be further improved.

[0047] Further, after the intake of carbon dioxide or low-carbon compounds obtained based on carbon dioxide as a carbon source, the microorganism can synthesize substances necessary for the growth of the microorganism, such as sugars, proteins, fibers, glucose, enzymes, etc. through metabolic pathways in the cells of the microorganism. The metabolic pathways mentioned herein can be either those possessed by the wild-type microorganism itself or those obtained by introducing and expressing exogenous genes into the cells of the microorganism through genetic modification. According to some embodiments of the present application, carbon dioxide can be converted into intermediates for subsequent synthesis of starch, such as 1-phospho-glucose, through multiple pathways. For example, six carbon fixation pathways have been found in nature, which are the Calvin cycle, the 3-hydroxypropionate double cycle, the Wood-Ljungdahl pathway, the reductive (reverse) TCA cycle, the dicarboxylic acid / 4-hydroxybutyric acid cycle, and the 3-hydroxypropionic acid / 4-hydroxybutyric acid cycle, and two artificial pathways CETCH and reductive glycine pathway to obtain two or three carbon intermediates.

[0048] According to some embodiments of the present application, the microorganism uses the carbon dioxide or the low-carbon compound as a main carbon source.

[0049] According to some embodiments of the present application, after the microorganism cells are provided with energy and carbon sources based on carbon dioxide and extracellular non-photosynthetic energy, the up-regulated glucose-1-phosphate adenylyltransferase GlgC and starch synthase GlgA can be used to catalyze 1-phospho-glucose (G-1-P) in the cells of the microorganism, and the final product starch can be obtained. The preparation of starch is realized by using the same microorganism cells, which improves the production efficiency and reduces the production cost.

[0050] The inventors of the present application have found that, in the case where starch synthase and glucose-1-phosphate adenylyltransferase do not exist in the wild-type microorganism or the starting strain, the recombinant microorganism can express the relevant enzymes by introducing exogenous genes. In the case where starch synthase and glucose-1-phosphate adenylyltransferase are already expressed in the starting strain or the wild-type microorganism, the expression efficiency can be further improved by additionally introducing the coding genes of exogenous starch synthase and glucose-1-phosphate adenylyltransferase or changing the promoters or other regulatory elements thereof. In other words, according to some embodiments of the present application, at least one of the starch synthase and the glucose-1-phosphate adenylyltransferase is up-regulated relative to the wild-type of the microorganism. Thus, the preparation efficiency of starch can be further improved relative to the starting strain. According to some embodiments of the present application, the starch synthase and the glucose-1-phosphate adenylyltransferase are respectively expressed by exogenous gene sequences. Thus, the preparation efficiency of starch can be further improved relative to the starting strain.

[0051] According to embodiments of the present application, the starch synthase used has the following amino acid sequence:

[0052] MQVLHVCSEMFPLLKTGGLADVIGALPAAQIADGVDARVLLPAFPDIRRGVTDAQVVSRRDTFAGHITLLFGHYNGVGIYLIDAPHLYDRPGSPYHDTNLFAYTDNVLRFALLGWVGAEMASGLDPFWRPDVVHAHDWHAGLAPAYLAARGRPAKSVFTVHNLAYQGMFYAHHMNDIQLPWSFFNIHGLEFNGQISFLKAGLYYADHITAVSPTYAREITEPQFAYGMEGLLQQRHREGRLSGVLNGVDEKIWSPETDLLLASRYTRDTLEDKAENKRQLQIAMGLKVDDKVPLFAVVSRLTSQKGLDLVLEALPGLLEQGGQLALLGAGDPVLQEGFLAAAAEYPGQVGVQIGYHEAFSHRIMGGADVILVPSRFEPCGLTQLYGLKYGTLPLVRRTGGLADTVSDCSLENLADGVASGFVFEDSNAWSLLRAIRRAFVLWSRPSLWRFVQRQAMAMDFSWQVAAKSYRELYYRLK (SEQ ID NO: 1)

[0053] According to the embodiments of the present application, the gene sequence of the starch synthase can be the following base sequence, and the expression efficiency thereof in yeast, especially Pichia, is obviously higher than that of other base sequences:

[0054]

[0055] According to the embodiment of the present application, the glucose-1-phosphate adenylyltransferase amino acid sequence is as follows:

[0056] MVSLEKNDHLMLARQLPLKSVALILAGGRGTRLKDLTNKRAKPAVHFGGKFRIIDFALSNCINSGIRRMGVITQYQSHTLVQHIQRGWSFFNEEMNEFVDLLPAQQRMKGENWYRGTADAVTQNLDIIRRYKAEYVVILAGDHIYKQDYSRMLIDHVEKGARCTVACMPVPIEEASAFGVMAVDENDKIIEFVEKPANPPSMPNDPSKSLASMGIYVFDADYLYELLEEDDRDENSSHDFGKDLIPKITEAGLAYAHPFPLSCVQSDPDAEPYWRDVGTLEAYWKANLDLASVVPELDMYDRNWPIRTYNESLPPAKFVQDRSGSHGMTLNSLVSGGCVISGSVVVQSVLFSRVRVNSFCNIDSAVLLPEVWVGRSCRLRRCVIDRACVIPEGMVIGENAEEDARRFYRSEEGIVLVTREMLRKLGHKQER (SEQ ID NO: 3) The glucose-1-phosphate adenylyltransferase gene sequence can be the following base sequence, and the expression efficiency thereof in yeast, especially Pichia, is obviously higher than that of other base sequences:

[0057]

[0058] According to some embodiments of the application, the starch synthase and the glucose-1-phosphate adenylyltransferase are each independently operably linked to a constitutive promoter or an inducible promoter. Thereby, the efficiency of starch production can be further improved and controllability of starch production can be achieved.

[0059] According to some embodiments of the application, the inducible promoter is adapted to increase the level of transcription upon induction of the carbon source. According to some embodiments of the application, both the starch synthase and the glucose-1-phosphate adenylyltransferase are controlled by an inducible promoter. Thereby, the efficiency of starch production can be further improved.

[0060] According to some embodiments of the application, the constitutive promoter comprises at least one selected from the group consisting of ZWF1, glucose-6-phosphate 1-dehydrogenase promoter, TPI1, triosephosphate isomerase promoter, GSH1, glutathione synthetase promoter, POR1, mitochondrial porin promoter, TKL1, transketolase promoter, PGD1, 6-phosphogluconate dehydrogenase promoter, PGM1, phosphoglycerate mutase promoter, PK, pyruvate kinase promoter and GAP, glyceraldehyde-3-phosphate dehydrogenase promoter.

[0061] According to some embodiments of the application, the inducible promoter comprises at least one selected from the group consisting of CAT1 : catalase, catalase promoter, TAL1 : transaldolase, transaldolase 1 promoter, TAL2: transaldolase, transaldolase 2 promoter, ALD4: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase 4 promoter, DAK1 : dihydroxyacetone kinase, dihydroxyacetone kinase promoter, FDH1 : formate dehydrogenase, formate dehydrogenase promoter, ALD: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase promoter, DAS1 : dihydroxyacetone synthase 1, dihydroxyacetone synthase promoter 1, DAS2: dihydroxyacetone synthase 2, dihydroxyacetone synthase promoter 2, AOX1 : alcohol oxidase 1, alcohol oxidase 1 promoter and AOX2: alcohol oxidase 2, alcohol oxidase 2 promoter. Preferably, a methanol inducible promoter is used, such as AOX1.

[0062] According to some embodiments of the present application, the metabolic action of at least one of glucan 1,4-alpha-glucosidase and glycogen phosphorylase on starch is blocked. Specifically, the metabolic action of at least one of glucan 1,4-alpha-glucosidase and glycogen phosphorylase on starch is blocked by mutating the gene encoding at least one of 1,4-alpha-glucosidase and glycogen phosphorylase. Thus, the accumulation of the generated starch can be further improved. According to embodiments of the present application, the specific gene can be mutated by gene editing, for example, insertion or deletion of 1-3 bases. The term "gene editing" used herein refers to the modification (including but not limited to insertion, deletion or replacement) of a specific target gene (also referred to as "target gene" or "target") in the genome of an organism. According to embodiments of the present application, under the guidance of SgRNA (recognizing the target gene), a nuclease Cas9 as a "molecular scissors" is used to generate a site-specific double-strand break (DSB) at a specific position in the genome, and further induce the organism to repair the DSB through non-homologous end joining (NHEJ) or homologous recombination (HR) (also referred to as "HDR" (Homology directed repair, homology directed repair)), thereby achieving a specific modification. In order to improve the efficiency of homologous recombination-mediated double-strand DNA repair (HDR), an additional single-stranded oligodeoxyribonucleotide (ssODN) based on the sequence of the target gene is added as a homologous recombination repair template.

[0063] According to some embodiments of the present application, the gene encoding the glucan 1,4-alpha-glucosidase is mutated by using SgRNA selected from the following:

[0064] 7: gagtcgataacgatctcctt;

[0065] 10: gttgttgatgtagccgtcta; or

[0066] 32: ggacgtgatcagggaacatg,

[0067] Preferably, 32: ggacgtgatcagggaacatg.

[0068] According to some embodiments of the present application, the gene encoding the glycogen phosphorylase is mutated by using SgRNA selected from the following:

[0069] 1-509: ggccacctccgactcaatca;

[0070] 6-509: gttaataagagcgttgtcca;

[0071] or 10-1018: gagaagtcaaactcggtggt.

[0072] Therefore, the efficiency of destroying glucan 1,4-alpha-glucosidase and glycogen phosphorylase can be improved, the accumulation amount of starch can be further improved, the preparation efficiency of starch can be further improved, and the preparation cost of starch can be reduced.

[0073] Those skilled in the art can understand that after the microbial cells synthesize starch, the microbial cells can be lysed to collect the starch, or the microbial cells can be genetically modified to secrete the starch outside the cells, or the microbial cells can be directly used as edible products. Preferably, according to some embodiments of the present application, further comprising: collecting the starch after lysing the microbial cells. Therefore, the purification of the starch can be realized, thereby facilitating the wide application of the obtained starch.

[0074] Recombinant microorganism

[0075] In the second aspect of the present application, a recombinant microorganism for preparing starch by using carbon dioxide is provided. According to embodiments of the present application, the recombinant microorganism has (1) an enzyme system for obtaining energy and carbon source based on carbon dioxide and extracellular non-photosynthetic energy; and (2) at least one of starch synthase and glucose-1-phosphate adenylyltransferase up-regulated compared with the wild type of the microorganism. Therefore, the recombinant microorganism can effectively implement the method for preparing starch provided in the first aspect, so that by using non-photosynthetic energy such as electrical energy or hydrogen energy, starch can be effectively prepared inside the microbial cells by fixing carbon dioxide, thereby achieving scientific utilization of carbon dioxide to help carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0076] According to embodiments of the present application, the extracellular non-photosynthetic energy includes at least one of hydrogen energy and electrical energy.

[0077] According to embodiments of the present application, the microbial cells are adapted to intake the carbon dioxide as a carbon source and absorb the extracellular non-photosynthetic energy; and / or

[0078] The microorganism is adapted to intake a low-carbon compound as a carbon source, which is obtained based on the carbon dioxide by using the reducing capacity of the extracellular non-photosynthetic energy.

[0079] According to embodiments of the present application, the recombinant microorganism has at least one of glucan 1,4-alpha-glucosidase and glycogen phosphorylase down-regulated compared with the wild type of the microorganism.

[0080] According to embodiments of the present application, the microorganism is at least one of a yeast and a bacterium engineered to utilize the carbon source.

[0081] According to embodiments of the present application, the low carbon compound contains 1-3 carbon atoms, and optionally, the low carbon compound is at least one selected from carbon monoxide, formic acid, methanol, methane, ethanol, formaldehyde, acetaldehyde, acetic acid, propanol, propane, propyl aldehyde, propion, hydroxyacetone, dihydroxyacetone, and glycerol.

[0082] For the convenience of understanding, some types of microorganisms that can be used in the present application are listed below. According to some embodiments of the present application, microorganisms suitable for absorbing electrical energy include, but are not limited to, at least one of Geobacter, Sporomusa, Methanogens, Acetobacterium, etc.; microorganisms suitable for taking acetic acid as a carbon source include, but are not limited to, Yarrowia lipolytica, microorganisms suitable for absorbing hydrogen energy include, but are not limited to, at least one of Hydrogenoxidizing bacteria, Ralstonia, and Clostridium; microorganisms suitable for taking carbon monoxide as a carbon source include, but are not limited to, Clostridium; microorganisms suitable for taking formic acid as a carbon source include, but are not limited to, Ralstonia; microorganisms suitable for taking methanol as a carbon source include, but are not limited to, at least one of Pichia pastoris, Candida boidinii, and Hansenula polymorpha; microorganisms suitable for taking methane as a carbon source include, but are not limited to, Methylobacter and Methylococcus; or microorganisms suitable for taking ethanol as a carbon source include, but are not limited to, Saccharomyces cerevisiae. Thus, the efficiency of preparing starch can be further improved.

[0083] According to embodiments of the present application, the microorganism is adapted to use the carbon dioxide or the low carbon compound as a main carbon source.

[0084] According to embodiments of the present application, the starch synthase and the glucose-1-phosphate adenylyltransferase are respectively expressed by an exogenous gene sequence.

[0085] According to embodiments of the present application, the starch synthase and the glucose-1-phosphate adenylyltransferase are respectively independently operably linked to a constitutive promoter or an inducible promoter.

[0086] According to embodiments of the present application, the inducible promoter is adapted to increase the transcription level under the induction of the carbon source.

[0087] According to embodiments of the application, the starch synthase and the glucose-1- phosphate adenylyltransferase are each controlled by an inducible promoter.

[0088] According to some embodiments of the application, the constitutive promoter comprises at least one selected from the group consisting of ZWF1, glucose-6-phosphate 1- dehydrogenase, glucose-6-phosphate dehydrogenase promoter, TPI1, triosephosphate isomerase, phosphotriose isomerase promoter, GSH1, glutathione synthetase, glutathione synthetase promoter, POR1, mitochondrial porin, mitochondrial porin promoter, TKL1, transketolase, transketolase promoter, PGD1, 6-phosphogluconate dehydrogenase, 6-phosphogluconate dehydrogenase promoter, PGM1, phosphoglycerate mutase, phosphoglycerate mutase promoter, PK, pyruvate kinase, pyruvate kinase promoter and GAP glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglyceraldehyde dehydrogenase promoter.

[0089] According to some embodiments of the application, the inducible promoter comprises at least one selected from the group consisting of CAT1 : catalase, catalase promoter, TAL1 : transaldolase, transaldolase 1 promoter, TAL2: transaldolase, transaldolase 2 promoter, ALD4: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase 4 promoter, DAK1 : dihydroxyacetone kinase, dihydroxyacetone kinase promoter, FDH1 : formate dehydrogenase, formate dehydrogenase promoter, ALD: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase promoter, DAS1 : dihydroxyacetone synthase 1, dihydroxyacetone synthase 1 promoter, DAS2: dihydroxyacetone synthase 2, dihydroxyacetone synthase 2 promoter, AOX1 : alcohol oxidase 1, alcohol oxidase 1 promoter, and AOX2: alcohol oxidase 2, alcohol oxidase 2 promoter. Preferably, a methanol inducible promoter, such as AOX1, is used. According to embodiments of the application, the at least one of the genes encoding 1,4-alpha-glucosidase and glycogen phosphorylase carries a mutation to block the metabolic action of the at least one of the glucan 1,4-alpha-glucosidase and glycogen phosphorylase on starch.

[0090] According to some embodiments of the application, the gene encoding the glucan 1,4-alpha-glucosidase is mutated by using an SgRNA selected from the group consisting of:

[0091] SgRNA 7: gagtcgataacgatctcctt (SEQ ID NO: 5),

[0092] SgRNA 10: gttgttgatgtagccgtcta (SEQ ID NO: 6),

[0093] SgRNA 32: ggacgtgatcagggaacatg (SEQ ID NO: 7),

[0094] Preferably, SgRNA 32: ggacgtgatcagggaacatg (SEQ ID NO: 7) is used.

[0095] According to some embodiments of the application, the gene encoding the glycogen phosphorylase is mutated by using an SgRNA selected from the group consisting of:

[0096] SgRNA 1-509: ggccacctccgactcaatca (SEQ ID NO: 8),

[0097] SgRNA 6-509: gttaataagagcgttgtcca (SEQ ID NO: 9),

[0098] SgRNA 10-1018: gagaagtcaaactcggtggt (SEQ ID NO: 10). Thus, the efficiency of destroying glucan 1,4-alpha-glucosidase and glycogen phosphorylase can be improved, the accumulation amount of starch can be further improved, the preparation efficiency of starch can be further improved, and the preparation cost of starch can be reduced.

[0099] It can be understood by those skilled in the art that the features and advantages described in the first aspect for the method for preparing starch also apply to the recombinant microorganism, which will not be repeated here.

[0100] Method for constructing a microorganism

[0101] In the third aspect of the present application, a method for constructing the microorganism of the second aspect is provided, characterized in that the following operation is performed on the starting microorganism: up-regulating at least one of starch synthase and glucose-1-phosphate adenylyltransferase. Thus, the recombinant microorganism described in the second aspect can be effectively prepared, and thus the recombinant microorganism can effectively implement the method for preparing starch proposed in the first aspect, so that starch can be effectively prepared in the microbial cell by fixing carbon dioxide by using non-photonic energy such as electrical energy or hydrogen energy, thereby achieving scientific utilization of carbon dioxide to help the carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0102] According to the embodiments of the present application, the above-mentioned method for constructing a recombinant microorganism can further have the following additional technical features:

[0103] According to the embodiments of the present application, the metabolic system of the starting microorganism is modified to have an enzyme system for obtaining energy and carbon source based on carbon dioxide and extracellular non-photonic energy.

[0104] According to the embodiments of the present application, the extracellular non-photonic energy includes at least one of hydrogen energy and electrical energy.

[0105] According to the embodiments of the present application, the metabolic system of the starting microorganism is modified so that:

[0106] The microbial cell is adapted to intake the carbon dioxide as a carbon source and absorb the extracellular non-photonic energy; and / or

[0107] The microorganism is adapted to take in low-carbon compounds as carbon sources, which are obtained based on the carbon dioxide using the extracellular non-photosynthetic reduction capacity.

[0108] According to embodiments of the present application, the starting microorganism is at least one of yeast and bacteria, to be adapted to utilize the carbon sources.

[0109] According to embodiments of the present application, the low-carbon compounds contain 1-3 carbon atoms, and optionally, the low-carbon compounds are at least one selected from carbon monoxide, formic acid, methanol, methane, ethanol, formaldehyde, acetaldehyde, acetic acid, propanol, propane, propyl aldehyde, acetone, hydroxyacetone, dihydroxyacetone, and glycerol.

[0110] For the convenience of understanding, some types of microorganisms that can be used in the present application are listed below. According to some embodiments of the present application, the microorganism adapted to absorb electrical energy includes but is not limited to at least one of Geobacter, Sporomusa, Methanogens, Acetobacterium, etc.; the microorganism adapted to take in acetic acid as a carbon source includes but is not limited to Yarrowia lipolytica, the microorganism adapted to absorb hydrogen energy includes but is not limited to at least one of hydrogen-oxidizing bacteria, Ralstonia, and Clostridium; the microorganism adapted to take in carbon monoxide as a carbon source includes but is not limited to Clostridium; the microorganism adapted to take in formic acid as a carbon source includes but is not limited to Ralstonia; the microorganism adapted to take in methanol as a carbon source includes but is not limited to at least one of Pichia pastoris, Candida boidinii, and Hansenula polymorpha; the microorganism adapted to take in methane as a carbon source includes but is not limited to Methylobacter and Methylococcus; or the microorganism adapted to take in ethanol as a carbon source includes but is not limited to Saccharomyces cerevisiae. Thus, the efficiency of preparing starch can be further improved.

[0111] According to embodiments of the present application, the microorganism is adapted to use the carbon dioxide or the low-carbon compounds as a main carbon source.

[0112] According to embodiments of the present application, the starch synthase and the glucose-1-phosphate adenylyltransferase are respectively at least one of up-regulated starch synthase and glucose-1-phosphate adenylyltransferase expressed by an exogenous gene sequence.

[0113] According to embodiments of the application, the starch synthase and the glucose-1-phosphate adenylyltransferase are each independently operably linked to a constitutive promoter or an inducible promoter.

[0114] According to embodiments of the application, the inducible promoter is adapted to increase the level of transcription upon induction by the carbon source.

[0115] According to embodiments of the application, the starch synthase and the glucose-1-phosphate adenylyltransferase are each independently operably linked to a constitutive promoter or an inducible promoter.

[0116] According to some embodiments of the application, the constitutive promoter comprises at least one selected from the group consisting of ZWF1, glucose-6-phosphate 1-dehydrogenase promoter, TPI1, triosephosphate isomerase promoter, GSH1, glutathione synthetase promoter, POR1, mitochondrial porin promoter, TKL1, transketolase promoter, PGD1, 6-phosphogluconate dehydrogenase promoter, PGM1, phosphoglycerate mutase promoter, PK, pyruvate kinase promoter and GAP glyceraldehyde-3-phosphate dehydrogenase promoter.

[0117] According to some embodiments of the application, the inducible promoter comprises at least one selected from the group consisting of CAT1 : catalase, catalase promoter, TAL1 : transaldolase, transaldolase 1 promoter, TAL2: transaldolase, transaldolase 2 promoter, ALD4: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase 4 promoter, DAK1 : dihydroxyacetone kinase, dihydroxyacetone kinase promoter, FDH1 : formate dehydrogenase, formate dehydrogenase promoter, ALD: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase promoter, DAS1 : dihydroxyacetone synthase 1, dihydroxyacetone synthase 1 promoter, DAS2: dihydroxyacetone synthase 2, dihydroxyacetone synthase 2 promoter, AOX1 : alcohol oxidase 1, alcohol oxidase 1 promoter, and AOX2: alcohol oxidase 2, alcohol oxidase 2 promoter. Preferably, a methanol inducible promoter is used, such as AOX1. According to embodiments of the application, the at least one of the genes encoding 1,4-alpha-glucosidase and glycogen phosphorylase carries a mutation to block the metabolic action of the at least one of the glucan 1,4-alpha-glucosidase and glycogen phosphorylase on starch.

[0118] According to some embodiments of the application, the gene encoding the glucan 1,4-alpha-glucosidase is mutated by using an SgRNA selected from the group consisting of:

[0119] SgRNA 7: gagtcgataacgatctcctt (SEQ ID NO: 5),

[0120] SgRNA 10: gttgttgatgtagccgtcta (SEQ ID NO: 6),

[0121] SgRNA 32: ggacgtgatcagggaacatg (SEQ ID NO: 7),

[0122] Preferably, SgRNA 32: ggacgtgatcagggaacatg (SEQ ID NO: 7) is used.

[0123] According to some embodiments of the application, the gene encoding the glycogen phosphorylase is mutated by using an SgRNA selected from the group consisting of:

[0124] SgRNA 1-509: ggccacctccgactcaatca (SEQ ID NO: 8),

[0125] SgRNA 6-509: gttaataagagcgttgtcca (SEQ ID NO: 9),

[0126] SgRNA 10-1018: gagaagtcaaactcggtggt (SEQ ID NO: 10). Thus, the efficiency of destroying glucan 1,4-alpha-glucosidase and glycogen phosphorylase can be improved, the accumulation amount of starch is further improved, the preparation efficiency of starch is further improved, and the preparation cost of starch is reduced.

[0127] It can be understood by those skilled in the art that the features and advantages described for the method for preparing starch and the recombinant microorganism in other aspects are also applicable to the method for constructing the recombinant microorganism, which will not be described here.

[0128] In addition, it can be understood by those skilled in the art that the present application also proposes a kit for constructing the microorganism of the second aspect, according to the embodiments of the present application, the kit comprises: an exogenous gene expression vector for enabling the starting microorganism to express at least one of starch synthase and glucose-1-phosphate adenylyltransferase; and optionally, a protein function blocking reagent for blocking at least one function of glucan 1,4-alpha-glucosidase and glycogen phosphorylase. Thus, by using the kit, the recombinant microorganism described above can be effectively constructed, and thus the recombinant microorganism can effectively implement the method for preparing starch proposed in the first aspect, so that starch can be effectively prepared in the microbial cell by fixing carbon dioxide by using non-photonic energy such as electric energy or hydrogen energy, thereby achieving scientific utilization of carbon dioxide to help the carbon neutralization strategy, and can be used for biological industrial synthesis of starch to achieve "agricultural industrialization".

[0129] According to the embodiments of the present application, further comprising: a carbon source metabolism modification vector for modifying the metabolic system of the starting microorganism so as to have an enzyme system for obtaining energy and carbon source based on carbon dioxide and extracellular non-photonic energy.

[0130] According to the embodiments of the present application, the starch synthase and the glucose-1-phosphate adenylyltransferase are respectively independently operably linked to a constitutive promoter or an inducible promoter.

[0131] According to the embodiments of the present application, the inducible promoter is suitable for increasing the transcription level under the induction of the carbon source.

[0132] According to embodiments of the application, the starch synthase and the glucose-1- phosphate adenylyltransferase are each controlled by an inducible promoter.

[0133] According to some embodiments of the application, the constitutive promoter comprises at least one selected from the group consisting of ZWF1, glucose-6-phosphate 1- dehydrogenase, TPI1, triosephosphate isomerase, GSH1, glutathione synthetase, POR1, mitochondrial porin, TKL1, transketolase, PGD1, 6-phosphogluconate dehydrogenase, PGM1, phosphoglycerate mutase, PK, pyruvate kinase, and GAP glyceraldehyde-3- phosphate dehydrogenase.

[0134] According to some embodiments of the application, the inducible promoter comprises at least one selected from the group consisting of CAT1: catalase, catalase promoter, TAL1: transaldolase, transaldolase 1 promoter, TAL2: transaldolase, transaldolase 2 promoter, ALD4: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase 4 promoter, DAK1: dihydroxyacetone kinase, dihydroxyacetone kinase promoter, FDH1: formate dehydrogenase, formate dehydrogenase promoter, ALD: mitochondrial aldehyde dehydrogenase, acetaldehyde dehydrogenase promoter, DAS1: dihydroxyacetone synthase 1, dihydroxyacetone synthase promoter 1, DAS2: dihydroxyacetone synthase 2, dihydroxyacetone synthase promoter 2, AOX1: alcohol oxidase 1, alcohol oxidase 1 promoter, and AOX2: alcohol oxidase 2, alcohol oxidase 2 promoter. Preferably, the methanol inducible promoter, such as AOX1, is used. According to embodiments of the application, the at least one of the genes encoding 1,4-alpha-glucosidase and glycogen phosphorylase carries a mutation to block the metabolic effect of the at least one of the glucan 1,4-alpha-glucosidase and glycogen phosphorylase on starch.

[0135] Thus, the efficiency of destroying the glucan 1,4-alpha-glucosidase and glycogen phosphorylase can be improved, the accumulation of starch can be further improved, the preparation efficiency of starch can be further improved, and the preparation cost of starch can be reduced.

[0136] According to embodiments of the application, the constitutive promoter comprises at least one selected from the group consisting of GAP,

[0137] The inducible promoter comprises at least one selected from the group consisting of methanol inducible promoters, preferably AOX1.

[0138] According to embodiments of the application, the protein function blocking agent is adapted to carry a mutation to the at least one of the genes encoding 1,4-alpha-glucosidase and glycogen phosphorylase to block the metabolic effect of the at least one of the glucan 1,4-alpha-glucosidase and glycogen phosphorylase on starch.

[0139] According to embodiments of the application, the protein function blocking agent comprises sgRNAs, so that the SgRNA selected from the group consisting of the following is used to mutate the gene encoding the glucan 1,4-alpha-glucosidase:

[0140] SgRNA 7: gagtcgataacgatctcctt (SEQ ID NO: 5),

[0141] SgRNA 10: gttgttgatgtagccgtcta (SEQ ID NO: 6),

[0142] SgRNA 32: ggacgtgatcagggaacatg (SEQ ID NO: 7),

[0143] Among them, preferably SgRNA 32: ggacgtgatcagggaacatg (SEQ ID NO: 7).

[0144] According to some embodiments of the present application, the protein function blocking reagent comprises the following sgRNA, thereby mutating the gene encoding the glycogen phosphorylase:

[0145] SgRNA 1-509: ggccacctccgactcaatca (SEQ ID NO: 8),

[0146] SgRNA 6-509: gttaataagagcgttgtcca (SEQ ID NO: 9),

[0147] SgRNA 10-1018: gagaagtcaaactcggtggt (SEQ ID NO: 10).

[0148] It can be understood by those skilled in the art that the features and advantages described in other aspects for the method for preparing starch and the recombinant microorganism and the construction method thereof are also applicable to the kit for constructing the recombinant microorganism, which will not be repeated here.

[0149] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following description, or can be learned by practice of the application.

[0150] Example 1: Preparation of starch by using Pichia pastoris

[0151] Synthesis of GlgA, GlgC genes

[0152] According to the information of the amino acid sequence of GlgA (amylosucrase, EC 2.4.1.21) from E. coli in GenBank SEQ ID NO: 1 (AAC76454.1) and the sequence information of GlgC (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) SEQ ID NO: 3 (AAC76455.1), the inventors screened two preferred gene sequences SEQ ID NO: 2 and 4 for P. pastoris.

[0153] Construction of GlgA and / or GlgC expression vector

[0154] The following five expression vectors were constructed using strong constitutive promoter pGap and strong inducible promoter pAOX1, respectively. The five expression vectors were constructed using the same backbone plasmid pHis.

[0155]

[0156] Disruption of GA gene (CRISPR-Cas9)

[0157] The GA (glucan 1,4-alpha-glucosidase, EC 3.2.1.3) gene on the genome of P. pastoris was disrupted using CRISPR-Cas9 technology. According to the gene sequence of GA, three different SgRNAs were screened: 7, 10, 32 (sequences are described above), which were synthesized by Figure 1 It can be seen from the figure that the disruption efficiency of GA varies among different SgRNAs, ranging from 64% to 92%, with 1 bp insertion / deletion as the main mutation.

[0158] Disruption of GP gene (CRISPR-Cas9)

[0159] On the basis of disrupting GA, the GP (glycogen phosphorylase, EC 2.4.1.1) gene on the genome of P. pastoris was further disrupted using CRISPR-Cas9 technology. According to the gene sequence of GP, three different SgRNAs were screened: 1-509, 6-509, 10-1018 (sequences are described above), which were synthesized by Figure 2 It can be seen from the figure that for GP, 100% disruption efficiency can be obtained, there is no SgRNA difference, and the mutation diversity is balanced.

[0160] Preparation of P. pastoris competent cells

[0161] Inoculate Pichia pastoris strain GS115 into 500 mL YPD medium, cultivate at 30°C, 200 r / min until OD600 = 1.3-1.5. Collect the bacteria at 4°C, 4500 r / min for 5 min, wash with 500 mL, 250 mL pre-cooled sterilized water and 20 mL pre-cooled 1 mol / L sorbitol respectively, each for one time. After each washing, collect the bacteria at 4°C, 4500 r / min for 5 min, finally suspend with 1 mL pre-cooled 1 mol / L sorbitol, divide into 80 μL / tube, use immediately, that is, obtain the electrocompetent cells.

[0162] Obtaining of recombinant Pichia pastoris

[0163] Linearize about 10 μg of each of the five correctly constructed expression vectors containing different promoter combinations GlgA and / or GlgC by BspEI enzyme digestion, recover the linear DNA by ethanol precipitation and dissolve in 10 μL sterile water. Mix the above linearized DNA with 80 μL GS115 electrocompetent cells, transfer into pre-cooled 0.2 cm electrotransformation cup. Use the electrotransformation instrument of American BIO-RAD company, perform electroshock according to the preset parameters of Pichia pastoris of the used electrotransformation instrument, immediately add 1 mL pre-cooled 1 mol / L sorbitol into the electroshock cup after the electroshock is completed, then transfer all the solution in the electroshock cup into a sterile centrifuge tube, directly take 200-300 μL bacterial solution to spread on MD plate (13.4 g / L YNB, 4×10 -4 g / L biotin, 20 g / L glucose, 15 g / L agarose); for the transformation of CRISPR-Cas9 related expression vectors, use 1 μg of DNA, other steps are the same as described above, after transformation, spread on YPD resistance plate containing G418 (500 μg / mL). Invert cultivate at 30°C for 2-4 days until colonies appear, after verification by PCR, the recombinant strain can be obtained.

[0164] Synthesis of methanol by carbon dioxide

[0165] By using bimetallic solid solution oxide (zinc oxide-zirconium dioxide, ZnO-ZrO2) as catalyst, carbon dioxide and hydrogen are used to synthesize methanol by high selectivity and high stability hydrogenation of carbon dioxide. The produced methanol can be determined by gas chromatography (Agilent 7890B). The chromatograph is equipped with thermal conductivity detector (TCD) and flame ionization detector (FID). The chromatographic column specification is 2 m×3.175 mm (Agilent).

[0166] Culture of recombinant Pichia pastoris

[0167] Each recombinant strain was inoculated into 25 mL of BSM liquid medium (5 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 4 mL / L trace element mother liquor (copper sulfate pentahydrate 6 g / L, potassium iodide 0.08 g / L, manganese sulfate 2.68 g / L, boric acid 0.02 g / L, sodium molybdate dihydrate 0.2 g / L, zinc sulfate heptahydrate 20 g / L, ferrous sulfate heptahydrate 65 g / L, cobalt chloride hexahydrate 0.916 g / L, sulfuric acid 5 mL / L, biotin 0.2 g / L), pH adjusted to 6.0) and cultured at 30°C, 200 r / min on a shaker for 48 h to obtain a seed solution. The seed solution was inoculated into fresh BSM, the initial OD600 was adjusted to 1, and the culture was incubated at 30°C, 200 r / min on a shaker, and methanol was added daily to a final concentration of 1% (V / V).

[0168] Determination of artificial starch yield

[0169] The bacterial cells cultured at 30°C for 2 d were adjusted to a concentration of 10 OD / mL, centrifuged, resuspended in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.5) and subjected to ultrasonic disruption. The disruption program was 5 s of ultrasonic treatment followed by 5 s of rest, and the total time was 10 min. 50 μL of the disrupted solution was boiled at 100°C for 5 min, then incubated with 30 U / mL of α-amylase and 30 U / mL of glucoamylase for a period of time until the starch was completely hydrolyzed to glucose, and then the glucose content was determined by a glucose determination kit (Beijing Puli Lei). The yield of starch was represented by the glucose content.

[0170] Figure 3 The starch yields of the recombinant strains containing different combinations of GlgA and / or GlgC promoters are shown. Figure 4 The starch production effect after GA disruption based on the pHis-EcGlgA-EcGlgC recombinant strain is shown. Figure 5 The starch production effect after GP disruption based on the pHis-EcGlgA-EcGlgC and GA disruption is shown. The results show that the highest starch yield of each artificial starch cell factory can reach 450 mg / L (60 mg / g DCW), and the highest yield is obtained by GS115 / pHis-EcGlgA-EcGlgC (abbreviated as A+C). On this basis, the key enzyme gene GP for starch utilization was further knocked out to obtain the recombinant strain GS115 / pHis-EcGlgA-EcGlgC-GP-1-509 (abbreviated as A+C-GP), which was simultaneously induced and fermented with the initial strain A+C, and GS115 was used as a control. The results show that the starch yield of A+C-GP can be increased by 150% compared with A+C, reaching 750 mg / L (100 mg / g DCW).

[0171] Example 2 Preparation of starch using hydrogen-oxidizing bacterium Cupriavidus necator

[0172] Construction of recombinant hydrogen-oxidizing bacterial strains

[0173] According to the sequence information of GlgA (amylosucrase, EC 2.4.1.21) from Escherichia coli (AAC76454.1) and GlgC (glucose-1 -phosphatase, EC 2.7.7.27) (AAC76455.1) in GenBank, two codon-optimized gene sequences for Cupriavidus necator were obtained as shown in SEQ ID NO: 11 and 12. The GlgA and GlgC genes were cloned into pBBR1 and pCM vectors, respectively, by the method of Simple Cloning (You C, Zhang X-Z, Zhang Y-HP. 2012. Simple cloning via direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 78(5): 1593-1595.) to obtain the corresponding recombinant plasmids pBBR1-GlgA and pCM-GlgC. The recombinant plasmids were then transformed into Escherichia coli S17, and finally the recombinant plasmids pBBR1-GlgA and pCM-GlgC were integrated into Cupriavidus necator by conjugation transfer.

[0174] Production of starch by recombinant strains using carbon dioxide

[0175] Hydrogen-oxidizing bacteria Cupriavidus necator can utilize carbon dioxide as the sole carbon source for growth. The recombinant strain was inoculated into 40 mL medium (9.0 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 1.0 g / L (NH4)2SO4, 80 mg / L MgSO4·7H2O, 1 mg / L CaSO4·2H2O, 0.56 mg / L NiSO4·7H2O, 0.4 mg / L Ferriccitrate, 200 mg / L NaHCO3, 50 mg / L FeSO4·7H2O, pH 6.5) and cultured in the medium with 1 mL / L trace elements (100 mg / L ZnSO4·7H2O, 30 mg / L MnCl2·4H2O, 300 mg / L H3BO3, 200 mg / L NiCl2·6H2O, 30 mg / L NaMoO4·2H2O), and the initial OD was controlled at about 0.2, the ventilation ratio of H2:O2:CO2 was 7:1:1, the ventilation flow rate was about 20 mL / min, and the culture was performed at 30 degrees. OD and starch production were determined every 24 h.

[0176] The cultured strain was adjusted to a concentration of 10 OD / mL, centrifuged, resuspended in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.5), and subjected to ultrasonic disruption with a disruption program of 5 s of ultrasonic treatment and 5 s of rest, for a total of 10 min. 50 μL of the disrupted liquid was boiled at 100 degrees for 5 min, then incubated with 30 U / mL of α-amylase and 30 U / mL of glucoamylase for a period of time until the starch was completely hydrolyzed into glucose, and then the glucose content was determined by a glucose determination kit (Beijing Puli Lei). The starch production was represented by the glucose content.

[0177] The results showed that the strain constructed in this example could effectively utilize carbon dioxide to produce starch.

[0178] Example 3 Preparation of starch by using Corynebacterium glutamicum

[0179] Construction of Corynebacterium glutamicum host cell

[0180] According to the sequence information of GlgA (Amylomaltase, EC 2.4.1.21) and GlgC (Glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) from E. coli in GenBank (AAC76454.1 and AAC76455.1), two codon-optimized genes were obtained for C. glutamicum, and a recombinant expression vector pEC-XK99E-GlgA-GlgC was obtained using pEC as a backbone vector, in which the expression of GlgA and GlgC both used the same promoter pTrc.

[0181] On this basis, the RpiB gene on the genome was disrupted by homologous recombination, blocking the pentose phosphate pathway of the bacterial cells themselves. Further, the MDH, PHI and PHI genes required for the ribulose monophosphate methanol utilization pathway were introduced, realizing the growth of methanol-dependent strains.

[0182] Preparation of C. glutamicum competence

[0183] (1) Activation: Take -80℃ frozen C. glutamicum (ATCC 13032) on LB + 0.5% glucose solid plate, and culture at 30℃ to grow single colonies.

[0184] (2) Seed culture: pick a single colony and inoculate into 10mL LB + 0.5% glucose liquid medium, 30℃, 200r / m culture for about 12h.

[0185] (3) Transfer into 50mL liquid LB medium containing 3% glycine and 0.1% Tween 80, so that the initial cell OD600 reaches 0.3.

[0186] (4) Culture at 30℃, 200r / m to OD600 of cells reaches 0.9. After the end of cell culture, the bacterial solution is first ice-bathed for 15min, and then the bacterial cells are collected at 4℃, 5000r / m.

[0187] (5) Discard the supernatant, and wash the bacterial cells with 30mL pre-cooled 10% glycerol for 4 times.

[0188] (6) Discard the supernatant, and finally resuspend the cells with 0.2mL pre-cooled 10% glycerol, and divide the cells into 1.5mL centrifuge tubes, 80μL per tube, and store in -80℃ refrigerator for use.

[0189] Obtaining of C. glutamicum recombinant strain

[0190] Take a tube of -80 degrees Celsius frozen electrocompetent cells, melt on ice. Add 5 μL of correctly constructed recombinant expression vector, gently tap the tube wall to mix it evenly with the competent cells. Transfer into a 1 mm pre-cooled electrotransformation cup, and perform electroporation at a voltage of 1.8 kV. After the end of the electric shock, immediately add 915 μL of BHI liquid medium (18.5 g / L brain heart infusion, 91 g / L sorbitol), place in a 46 degree water bath for 6 min, then transfer to a 30 degree recovery culture for 2 h, finally take an appropriate amount of bacterial liquid to spread on LBHIS (5 g / L peptone, 5 g / L sodium chloride, 2.5 g / L yeast extract, 18.5 g / L brain heart infusion, 91 g / L sorbitol) solid plate containing 25 μg / mL kanamycin, 30 degrees inverted culture, and wait for the colonies to grow. After PCR verification, the corresponding recombinant strain can be obtained.

[0191] Corynebacterium glutamicum produces starch using carbon dioxide

[0192] By using bimetallic solid solution oxide (zinc oxide-zirconium dioxide, ZnO-ZrO2) as catalyst, carbon dioxide and hydrogen gas are used to synthesize methanol by high selectivity and high stability hydrogenation of carbon dioxide. The produced methanol can be determined by gas chromatography (Agilent 7890B), which is equipped with a thermal conductivity detector (TCD) and a flame ionization detector (FID), and the chromatographic column specification is 2 m x 3.175 mm (Agilent).

[0193] Inoculate the recombinant strain in CGX11 (20 g / L ammonium sulfate, 5 g / L urea, 1 g / L potassium dihydrogen phosphate, 1.3 g / L dipotassium hydrogen phosphate trihydrate, 42 g / L MOPS, 0.01 g / L calcium chloride, 0.25 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate monohydrate, 0.001 g / L zinc sulfate heptahydrate, 0.2 mg / L copper sulfate, 0.02 mg / L nickel chloride hexahydrate, 0.2 mg / L biotin, 0.03 g / L protocatechuic acid, 0.1 mg / L vitamin B1, 5 g / L xylose, 5 g / L methanol, and adjust the pH to 7.0), and culture at 30 degrees and 200 r / m.

[0194] The cultured strain was adjusted to 10 OD / mL, centrifuged, resuspended in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.5) and subjected to ultrasonic disruption for 10 min with a 5 s on, 5 s off cycle. 50 μL of the disrupted solution was boiled at 100 °C for 5 min, then incubated with 30 U / mL α-amylase and 30 U / mL glucoamylase until the starch was completely hydrolyzed to glucose. The glucose content was determined by a glucose assay kit (Beijing Puli Lei). The starch yield was expressed as the glucose content, and the results are shown in the following table.

[0195] Table 1. The starch accumulation level of recombinant C. glutamicum expressing glucose-1-phosphate adenylyltransferase (EC: 2.7.7.27) and starch synthase (EC: 2.4.1.21)

[0196]

[0197] Note: the more "+"s, the more starch accumulated.

[0198] The results show that the recombinant C. glutamicum constructed in this example can effectively produce starch using methanol.

[0199] Example 4. Preparation of starch using E. coli

[0200] Construction of recombinant E. coli strain

[0201] According to the sequence information of E. coli-derived GlgA (starch synthase, EC 2.4.1.21) (AAC76454.1) and GlgC (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) (AAC76455.1) in GenBank, primers were designed and the GlgA and GlgC genes were cloned into pET21b vector by enzyme digestion and ligation to obtain the corresponding recombinant plasmids pET21b-GlgA and pET21b-GlgC. These two plasmids were transformed into BL21 (DE3) by chemical transformation method to obtain the corresponding recombinant strains.

[0202] Starch production by recombinant E. coli strains using different carbon sources

[0203] Single colonies were picked and cultured in LB (0.5% YE, 1% NaCl, 1% yeast extract) at 37 °C and 220 r / m overnight, then transferred into culture medium containing different carbon sources (the base medium was M9, and the carbon sources were glucose, glycerol and DHA, respectively, with LB as the control) at an inoculation amount of 1%. When OD600=0.8, 0.5 mM IPTG was added for induction fermentation at 16 °C.

[0204] Take the cultured strain, adjust the concentration to 10 OD / mL, centrifuge, resuspend in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.5) for ultrasonic disruption, and the disruption procedure is: ultrasonic for 5 s, pause for 5 s, and the total time is 10 min. Take 50 μL of the disrupted liquid, boil for 5 min at 100 degrees, then incubate with 30 U / mL of α-amylase and 30 U / mL of glucose amylase for a period of time until the starch is completely hydrolyzed into glucose, and then detect the glucose content by glucose assay kit (Beijing Puli Lei). The starch yield is represented by the glucose content, and the results are shown in the following table.

[0205] Table 2 Starch accumulation level of recombinant E. coli fermentation

[0206]

[0207] The results show that the E. coli constructed in this example can effectively utilize different carbon sources to produce starch.

[0208] Example 5 Preparation of starch by Yarrowia lipolytica

[0209] Construction of recombinant Yarrowia lipolytica

[0210] According to the amino acid sequences of 9 pairs of exogenous glucose-1-phosphate adenosine transferase (EC: 2.7.7.27) and starch synthase (EC: 2.4.1.21) (Table 1), the codon optimization of the gene sequence was carried out with Yarrowia lipolytica as the host, and the gene fragments were synthesized into an integrative expression plasmid, and the two genes were respectively between the promoters PrTef and the terminators Tlip2 and the promoters PrGPD and the terminators Tcyc, forming 9 double expression cassette plasmids. The above plasmid was double digested to obtain an integrative DNA fragment. The chemical transformation method was used to integrate the above integrative DNA fragment into the chromosome of Yarrowia lipolytica, and the recombinant Yarrowia lipolytica strain was obtained.

[0211] Table 3 Amino acid sequences of glucose-1-phosphate adenosine transferase (EC: 2.7.7.27) and starch synthase (EC: 2.4.1.21) used in the application (in order of SEQ ID NO: 13-30)

[0212]

[0213]

[0214]

[0215]

[0216] Recombinant strains utilizing acetic acid to produce starch

[0217] The recombinant Yarrowia lipolytica strain was inoculated into a mineral salt medium with acetic acid as the sole carbon source (2.73 g / L sodium acetate, 6.75 g / L (NH4)2SO4, 13 g / L KH2PO4, 0.45 g / L MgSO4·7H2O, 4 mL / L trace element stock solution (3.0 g / L FeSO4·7H2O, 4.5 g / L ZnSO4·7H2O, 4.5 g / L CaCl2·2H2O, 1 g / L MnCl2·4H2O, 300 mg / L CoCl2·6H2O, 300 mg / L CuSO4·5H2O, 400 mg / L Na2MoO4·2H2O, 1 g / L H3BO3, 100 mg / L KI, 19 g / L Na2EDTA·2H2O), 1 mL / L vitamin stock solution (50 mg / L D-Biotin, 1.0 g / L D-Pantothenic acid hemicalcium salt, 1.0 g / L Thiamin-HCl, 1.0 g / L Pyridoxin-HCl, 1.0 g / L Nicotinic acid, 0.2 g / L 4-aminobenzoic acid, 25 g / L m-Inositol)), and the cells were cultured at 30°C and 220 r / m until the OD600 was about 1.5. The cells were then collected by centrifugation at 13,000 r / m for 2 min and washed twice with distilled water.

[0218] The cultured strain was adjusted to a concentration of 10 OD / mL, centrifuged, and resuspended in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.5) for ultrasonic disruption. The disruption procedure was 5 s of ultrasonic treatment followed by 5 s of rest, and the total time was 10 min. 50 μL of the disrupted liquid was boiled at 100°C for 5 min, then incubated with 30 U / mL of α-amylase and 30 U / mL of glucoamylase for a period of time until the starch was completely hydrolyzed to glucose. The glucose content was then detected using a glucose assay kit (Beijing Puli Lei). The starch yield was represented by the glucose content, and the results are shown in Table 2.

[0219] Table 4. The starch accumulation level of recombinant Yarrowia lipolytica strains heterologously expressing glucose-1-phosphate adenylyltransferase (EC: 2.7.7.27) and starch synthase (EC: 2.4.1.21)

[0220]

[0221] Note: The more "+"s, the more starch accumulated.

[0222] Thus, the recombinant Yarrowia lipolytica constructed by the present embodiment can effectively produce starch from acetic acid, and by selecting the source or specific sequence of glucose-1-phosphate adenosine transferase and starch synthase, the efficiency of starch production can be improved.

[0223] Example 6 Preparation of starch by using methylobacterium

[0224] Construction of recombinant methylobacterium

[0225] According to the sequence information of GlgA (starch synthase, EC 2.4.1.21) (AAC76454.1) and GlgC (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) (AAC76455.1) of Escherichia coli in GenBank, primers were designed respectively, and Gibson assembly method was used to clone GlgA and GlgC genes into pCM vector, wherein GlgA and GlgC genes were expressed using a separate pMxa promoter, to obtain recombinant plasmid pCM-GlgA-GlgC. The plasmid was transformed into methylobacterium by chemical transformation method to obtain recombinant methylobacterium.

[0226] Starch production by recombinant strains using methanol

[0227] The above-mentioned recombinant methylobacterium was inoculated into a culture medium with methanol as the carbon source (32 g / L methanol, 1 g / L KNO3, 0.717 g / L KH2PO4, 0.272 g / L Na2HPO4, 1 g / L MgSO4, 0.2 g / L CaCl2, 1 mL / L trace element solution (0.5 g / L EDTA, 0.2 g / L FeSO4, 0.01 g / L ZnSO4, 0.003 g / L MnCL2, 0.02 g / L CoCl2, 0.1 g / L CuSO4, 0.003 g / L Na2MoO4)), and cultured at 30 degrees and 220 r / m.

[0228] The cultured strain was adjusted to a concentration of 10 OD / mL, centrifuged, resuspended in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.5) and subjected to ultrasonic disruption, with a disruption program of 5 s of ultrasonic treatment and 5 s of rest, for a total of 10 min. 50 μL of the disrupted liquid was boiled at 100 degrees for 5 min, then incubated with 30 U / mL of α-amylase and 30 U / mL of glucoamylase for a period of time until the starch was completely hydrolyzed to glucose, and then the glucose content was detected by a glucose assay kit (Beijing Puli Lei). The starch yield was represented by the glucose content, and the results are shown in the following table.

[0229] Table 5. Recombinant Methylobacterium Fermentation Accumulation of Starch with Expression of Glucose-1-Phosphate Adenylyltransferase (EC: 2.7.7.27) and Starch Synthase (EC: 2.4.1.21)

[0230]

[0231] Note: The more "+"s, the more starch accumulated.

[0232] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms are not necessarily directed to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0233] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method for preparing starch using carbon dioxide, characterized in that, include: (1) Based on carbon dioxide and extracellular non-photovoltaic energy, provide energy and carbon source for microbial cells, wherein the carbon source is a low-carbon compound containing 1 to 3 carbon atoms; and (2) Starch is generated in the microbial cell based on at least one of glucose-1-phosphate adenosine transferase and starch synthase upregulated in the microbial cell; The microorganism is yeast or Geobacterium. Geobacter Musculus Sporomusa Methanogens Methanogens Acetic acid-producing bacteria Acetobacterium Lactobacillus Ralstonia Clostridium Clostridium Hydroxyhydrogen bacteria, hookworm copper-loving bacteria Cupriavidus necator Methanogenic bacteria Methylobacter , Methylococcus At least one of them, to be suitable for utilizing the carbon source.

2. The method according to claim 1, characterized in that, The extracellular non-photovoltaic energy includes at least one of hydrogen energy and electrical energy.

3. The method according to any one of claims 1-2, characterized in that, In the microorganism, the metabolism of starch by at least one of glucan 1,4-α-glycosidase and glycogen phosphorylase is blocked.

4. The method according to any one of claims 1-3, characterized in that, Step (1) further includes: The microbial cells take in the carbon dioxide as a carbon source and absorb the extracellular non-light energy; and / or By utilizing the extracellular non-photochemical reducing power, low-carbon compounds are obtained based on the carbon dioxide, and the microorganisms ingest the low-carbon compounds as a carbon source.

5. The method according to any one of claims 1-4, characterized in that, The microorganism is at least one of modified yeasts and bacteria to utilize the carbon source.

6. The method according to any one of claims 1-5, characterized in that, The low-carbon compound is selected from at least one of carbon dioxide, carbon monoxide, formic acid, methanol, methane, ethanol, formaldehyde, acetaldehyde, acetic acid, propanol, propane, propanaldehyde, acetone, hydroxyacetone, dihydroxyacetone, and glycerol.

7. The method according to any one of claims 1-6, characterized in that, The microorganism is selected from at least one of the following: Pichia pastoris Pichia pastoris Candida Boydin Candida boidinii Hansenula yeast Hansenula polymorpha , Yarrowia lipolytica Yarrowia lipolytica and brewing yeast Saccharomyces cerevisiae .

8. The method according to any one of claims 1-7, characterized in that, At least one of the starch synthase and the glucose-1-phosphate adenosine transferase is integrated into the genome of the microbial cell in the form of an exogenous gene, or In the microbial cells, at least one of the starch synthase and the glucose-1-phosphate adenosine monophosphate transferase is contained in a free expression vector.

9. The method according to claim 8, characterized in that, The starch synthase and the glucose-1-phosphate adenosine acyltransferase are each independently operatively linked to a constitutive promoter or an inducible promoter.

10. The method according to claim 9, characterized in that, The inducible promoter is adapted to enhance transcription levels under the induction of the carbon source.

11. The method according to any one of claims 9-10, characterized in that, Both the starch synthase and the glucose-1-phosphate adenosine monophosphate transferase are controlled by an inducible promoter.

12. The method according to claim 9, characterized in that, The constitutive promoter includes at least one selected from ZWF1 glucose-6-phosphate dehydrogenase promoter, TPI1 triose phosphate isomerase promoter, GSH1 glutathione synthase promoter, POR1 mitochondrial porin promoter, TKL1 transketolaldase promoter, PGD1 6-phosphate gluconate dehydrogenase promoter, PGM1 phosphoglycerate mutase promoter, PK pyruvate kinase promoter, and GAP3 phosphoglyceraldehyde dehydrogenase promoter.

13. The method according to any one of claims 9-11, characterized in that, The inducible promoter includes at least one selected from CAT1 catalase promoter, TAL1 aldolase 1 promoter, TAL2 promoter, ALD4 acetaldehyde dehydrogenase 4 promoter, DAK1 dihydroxyacetone kinase promoter, FDH1 formate dehydrogenase promoter, ALD acetaldehyde dehydrogenase promoter, DAS1 dihydroxyacetone synthase promoter 1, DAS2 dihydroxyacetone synthase promoter 2, AOX1 alcohol oxidase 1 promoter, and AOX2 alcohol oxidase 2 promoter.

14. The method according to any one of claims 3-13, characterized in that, The blocking of starch metabolism by at least one of glucan 1,4-α-glycosidase and glycogen phosphorylase is achieved by mutating the gene encoding at least one of 1,4-α-glycosidase and glycogen phosphorylase.

15. The method according to any one of claims 1-14, characterized in that, The starch synthase has the amino acid sequence shown in SEQ ID NO: 1, 13-21, and the glucose-1-phosphate adenosine monophosphate transferase has the amino acid sequence shown in SEQ ID NO: 3, 22-30.

16. The method according to any one of claims 1-14, characterized in that, The starch synthase has the gene coding sequence shown in SEQ ID NO: 2, 11, and the glucose-1-phosphate adenosine acyltransferase has the gene coding sequence shown in SEQ ID NO: 4, 12.

17. The method according to any one of claims 14-16, characterized in that, The gene encoding the 1,4-α-glycosidase of the aforementioned glucan was mutated using SgRNA selected from the following: SgRNA7:gagtcgataacgatctcctt, SgRNA10:gttgttgatgtagccgtcta, SgRNA 32:ggacgtgatcagggaacatg.

18. The method according to any one of claims 14-16, characterized in that, The gene encoding the glycogen phosphorylase was mutated using an SgRNA selected from the following: SgRNA1-509:ggccacctccgactcaatca, SgRNA6-509:gttaataagagcgttgtcca, SgRNA10-1018: gagaagtcaaactcggtggt.

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