Recombinant microorganism for fixing carbon dioxide and use thereof

Abstract

Provided is a polypeptide combination, which comprises a Rubisco polypeptide from a species of the family Chlamydomonadaceae, and a fusion polypeptide comprising an EPYC1 polypeptide of a species of the family Chlamydomonadaceae and a mineralizing peptide, wherein the mineralizing peptide can convert a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide. Further provided are a polynucleotide or a vector encoding the polypeptide combination, a microorganism containing the polypeptide combination, the polynucleotide or the vector, and a method of using the microorganism.

Description

Recombinant microorganisms for fixing carbon dioxide and their applications Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a recombinant microorganism expressing a eukaryotic carbon-fixing protein. Background Art

[0002] In recent years, microbial cell factories have been developed and utilized as a green and sustainable method for producing biofuels and chemicals from renewable feedstocks using autotrophic and heterotrophic microorganisms. Autotrophic microorganisms can utilize carbon dioxide as the sole carbon source for growth and chemical synthesis, but their production efficiency is relatively low. Heterotrophic microorganisms, on the other hand, synthesize products from a variety of carbon sources. However, due to carbon losses during their central metabolic processes, carbon yield is one of the most important parameters for evaluating their manufacturing performance.

[0003] Carbon dioxide fixation in heterotrophic microorganisms is a key research area in synthetic biology, aiming to convert carbon dioxide into useful organic products, thereby addressing carbon emissions and sustainable energy issues. Furthermore, introducing exogenous carbon fixation pathways to create mixed carbon sources of carbon dioxide and sugars for chemical production is a viable approach to increase carbon yield.

[0004] Common microbial factories, such as Escherichia coli, are widely used in biomanufacturing. Since the natural metabolic pathways of microbial factories usually do not include efficient CO2 fixation pathways, existing solutions are to genetically modify microorganisms to introduce exogenous CO2 fixation pathways to improve carbon utilization. Therefore, improving CO2 fixation efficiency is crucial for achieving efficient microbial synthesis and sustainable production. Among them, the most common exogenous CO2 fixation pathway is the Calvin-Benson-Bassham cycle (CBB), whose core carbon fixation enzyme is ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), which converts CO2 into organic matter and even achieves autotrophy through the catalytic reactions of multiple enzymes (Antonovsky et al., Cell, 2016; Gleizer et al., Cell, 2019). By introducing key enzyme genes for the CBB cycle into Escherichia coli, researchers have successfully achieved a certain degree of carbon dioxide fixation, but its efficiency is low. This is because Rubisco has a lower affinity for CO2 than O2 under natural conditions, resulting in a preference for photorespiration with O2 under current atmospheric conditions, leading to low carbon dioxide fixation efficiency. Therefore, improving the efficiency of the existing pathway has become an important research direction.

[0005] The eukaryotic algae Chlamydomonas has evolved a carbon-concentrating mechanism based on protein-based liquid-liquid phase separation, which increases carbon fixation efficiency by promoting carboxylation reactions by increasing the CO2 concentration around Rubisco. However, current research on liquid-liquid phase separation has focused solely on elucidating the molecular mechanisms, and it remains uncertain whether this mechanism can be successfully expressed and applied in microorganisms, such as bacteria, to enhance heterologous carbon fixation efficiency in recombinant microorganisms.

[0006] Artificial photosynthesis harnesses sunlight to create high-value chemicals from abundant resources, but achieving high activity and selectivity in the production of multi-carbon products remains challenging. Consequently, the recent emergence of semi-artificial photosynthetic systems, leveraging the efficient light absorption properties of semiconductors combined with living biocatalysts to convert carbon dioxide into high-value-added products powered by solar energy, offers a promising solution to addressing environmental and energy challenges.

[0007] Semi-artificial photosynthetic systems primarily include cell membrane hybrid systems and intracellular hybrid systems. The most typical existing cell membrane hybrid system is CdS nanoparticles deposited on cell biofilms for acetic acid production (Sakimoto et al., Science, 2016). This system transfers photoexcited electrons from an extracellular semiconductor into the cell to generate reducing equivalents involved in carbon fixation, resulting in slow mass transfer and energy consumption across the cell membrane at the extracellular bio-inorganic interface. To address this limitation, intracellular biohybrid systems have been developed (Zhang et al., Nature Nanotechnology, 2018; Wu et al., Joule, 2022). However, in these intracellular hybrid systems, light absorbers (such as AuNCs and C3N4 QDs) are synthesized and then taken up by bacteria through endocytosis, resulting in their random distribution in the cytoplasm. Despite improvements in energy conversion efficiency, interference and disruption of cellular physiological metabolism by nanoparticles remain unavoidable.

[0008] Therefore, there is still a need for improved recombinant microorganisms that contain light absorbers and carbon fixation enzymes concentrated in specific areas within the cell to improve the efficiency of light conversion and carbon fixation while reducing the impact on the physiological metabolism of the microbial cells. Summary of the Invention

[0009] To meet these needs, the present invention generates semiconductor nanoparticles through in situ mineralization within membraneless organelles within cells, co-localizing the nanoparticles with carbon-fixing enzymes within these membraneless organelles. This not only improves the efficiency of photo-electro-chemical energy conversion, but also reduces the material's impact on cellular physiological metabolism.

[0010] In a first aspect, the present invention provides a combination of polypeptides comprising a Rubisco polypeptide from a species of the Chlamydomonas family, and a fusion polypeptide comprising an EPYC1 polypeptide from a species of the Chlamydomonas family and a mineralizing peptide capable of converting a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide.

[0011] In some embodiments, the EPYC1 polypeptide and the mineralization peptide are connected by a peptide linker. In some embodiments, the peptide linker is a flexible linker, such as a GS linker.

[0012] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the same species. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

[0013] In some embodiments, the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, and the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

[0014] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0015] In some embodiments, the mineralizing peptide is a Car9 peptide. In some embodiments, the Car9 peptide comprises the amino acid sequence of SEQ ID NO:4.

[0016] In some embodiments, the fusion polypeptide further comprises an sfGFP polypeptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0017] In a second aspect, the present invention provides an isolated polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding a combination of polypeptides of the present invention.

[0018] The present invention also provides a vector or a combination of vectors, preferably an expression vector or a combination of expression vectors, comprising the polynucleotide or a combination of polynucleotides of the present invention.

[0019] In a third aspect, the present invention provides a recombinant microorganism, eg, a bacterium or yeast, comprising a combination of polypeptides, a polynucleotide or a combination of polynucleotides, or a vector or a combination of vectors of the present invention.

[0020] In some embodiments, the microorganism further comprises a molecular chaperone that helps polypeptide folding and / or a polynucleotide encoding the molecular chaperone. In some embodiments, the molecular chaperone comprises a GroELS system and rbcX.

[0021] In some embodiments, the microorganism further comprises a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to produce ribulose-1,5-bisphosphate, or a polynucleotide encoding the Prk polypeptide.

[0022] In a fourth aspect, the present invention provides a method for fixing carbon dioxide, comprising the steps of:

[0023] i) culturing the microorganism of the present invention in the presence of a metal-containing compound; and

[0024] ii) contacting the microorganism with carbon dioxide, preferably under light.

[0025] In some embodiments, the metal-containing compound is a titanium-containing compound, such as TiBALDH.

[0026] In a fifth aspect, the present invention provides a method for producing a chemical of interest, comprising the steps of:

[0027] i) introducing a polynucleotide or a combination of polynucleotides, or a vector or a combination of vectors of the present invention into a microorganism, such as a bacterium or yeast, to obtain a modified microorganism; and

[0028] ii) cultivating, preferably in the presence of a metal-containing compound and / or under conditions of light, the modified microorganism to produce the chemical of interest.

[0029] In some embodiments, step i) further comprises introducing into the microorganism an additional polynucleotide selected from a) a polynucleotide encoding a molecular chaperone that helps polypeptide folding, such as the GroELS system and rbcX, b) a polynucleotide encoding an sfGFP polypeptide, c) a polynucleotide encoding a Prk polypeptide, and combinations thereof.

[0030] In some embodiments, the metal-containing compound is a titanium-containing compound, such as TiBALDH.

[0031] In some embodiments, the method further comprises introducing into the microorganism, before, simultaneously with, or after step i), a polynucleotide encoding a polypeptide involved in the biosynthesis of the chemical of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1 and 2 show maps of expression vectors pET22b-LS and pBAD33-prk-G-rbcX, respectively.

[0033] Figure 3 shows the carbon sequestration mechanism based on the CBB pathway.

[0034] Figure 4 shows the growth curve of recombinant Escherichia coli (Prk+Rubisco) co-transformed with expression vectors pET22b-LS and pBAD33-prk-G-rbcX. The blank control represents untransformed Escherichia coli, and Prk represents recombinant Escherichia coli transformed only with pBAD33-prk-G-rbcX.

[0035] Figures 5 and 6 show maps of the expression vectors pET22b-LSE and pET22b-LSEV, respectively.

[0036] Figure 7 shows confocal microscopy (lower row, left panel), structured light microscopy (lower row, middle panel), and transmission electron microscopy (lower row, right panel) images of recombinant E. coli co-transformed with pET22b-LSE or pET22b-LSEV and pBAD-prk-G-rbcX. The upper two panels are enlarged fragments of the structured light microscopy images.

[0037] Figure 8 shows the activity of Rubisco in recombinant E. coli (Rubisco + EPYC1) co-transformed with pET22b-LSE and pBAD-prk-G-rbcX. The control represents untransformed E. coli, and Rubisco represents recombinant E. coli co-transformed with pET22b-LS and pBAD33-prk-G-rbcX.

[0038] Figure 9 shows the rate of CO2 consumption in recombinant E. coli (Rubisco + EPYC1) co-transformed with pET22b-LSE and pBAD-prk-G-rbcX. The control represents untransformed E. coli, and Rubisco represents recombinant E. coli co-transformed with pET22b-LS and pBAD33-prk-G-rbcX.

[0039] FIG10 shows a map of the expression vector pET22b-LSE-sfGFP-Car9.

[0040] FIG11 shows the effect of adding different concentrations of TiBALDH to the culture medium on the growth of recombinant E. coli.

[0041] FIG12 shows transmission electron microscopy images of mineralized recombinant E. coli (upper panel) and elemental analysis of particles within the cells (lower panel).

[0042] Figure 13 shows high-angle annular dark field scanning transmission electron microscopy (HAADF-TEM) analysis, elemental analysis, high-angle annular dark field transmission electron microscopy (HRT-TEM) analysis, selected electron diffraction (SEAD), and crystal lattice spacing measurements of mineralized recombinant E. coli ultrathin sections. The first image from the left in the top row shows the HAADF-TEM image, and images 2-5 show the elemental analysis results. The first image from the left in the bottom row shows the HRT-TEM image, the second image shows a magnified image of the compartment indicated by the arrow in the first image, the third image shows the SEAD image, and the fourth image shows the crystal lattice spacing measurement.

[0043] Figure 14 shows the photoconversion effect of mineralized recombinant E. coli. Control 1 is unmineralized E. coli, and Control 2 is titanium dioxide semiconductor powder extracted from mineralized bacteria. The small panels in Figure 14 are local highlights of the curves for each sample.

[0044] FIG15 shows the effect of light on the redox state within mineralized recombinant E. coli cells.

[0045] FIG16 shows the effect of light on the mineralized recombinant E. coli cells containing 13 The blank control was untransformed E. coli.

[0046] FIG17 shows the biosynthetic pathway of 2,3-butanediol.

[0047] Figure 18 shows the yield of 2,3-butanediol produced by photosynthetic carbon-fixing recombinant E. coli. Reference: Yield reported in prior art. Control: Recombinant E. coli co-transformed with the plasmids pET22b-RABC and pBAD-prk-G-rbcX. Experimental: Recombinant E. coli co-transformed with the plasmids pET22b-LSE-sfGFP-Car9-RABC and pBAD-prk-G-rbcX.

[0048] Figure 19 shows the carbon yield of 2,3-butanediol produced by photosynthetic carbon-fixing recombinant E. coli. Reference: Carbon yield reported in prior art. Control group: Recombinant E. coli co-transformed with the plasmids pET22b-RABC and pBAD-prk-G-rbcX. Experimental group: Recombinant E. coli co-transformed with the plasmids pET22b-LSE-sfGFP-Car9-RABC and pBAD-prk-G-rbcX.

[0049] FIG20 shows maps of plasmids pET22b-LSE-sfGFP-Car9-RABC and pET22b-RABC.

[0050] Figure 21 shows a map of the expression vector 301-LSE-prk.

[0051] Figure 22 shows a map of the expression vector 302-G-rbcX.

[0052] Figure 23 shows the laser confocal microscopy imaging of the recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) of 301-LSE-prk and 302-G-rbcX co-transformation.Left width is a representative visual field imaging, and right width is an amplified image showing the carbon-fixing module (aggregate) with carbon concentration mechanism.

[0053] Detailed Description of the Invention

[0054] 1. Definition

[0055] Unless otherwise indicated, all terms used herein have the same meanings as understood by one skilled in the art, and the practice of the present invention will employ conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of one skilled in the art.

[0056] The term "organelle" is generally considered to refer to microstructures or organs with specific morphology and function that are dispersed within the cytoplasm. They constitute the basic structure of the cell and enable it to carry out normal physiological functions. Organelles in cells include membrane-containing organelles such as mitochondria, endoplasmic reticulum, chloroplasts, and Golgi apparatus, as well as membrane-less organelles.

[0057] As used herein, the term "membraneless organelle" refers to a compartment within a cell that lacks a membrane structure and where molecules aggregate through interactions. Examples include the nucleolus and promyelocytic leukemia protein bodies in the nucleus, and the cytoplasmic processing bodies and germ granules in the cytoplasm. The Chlamydomonas phase separation protein (EPYC1) interacts with Rubisco, accumulating in specific regions of the cell to form membraneless organelles that function in carbon fixation.

[0058] As used herein, the term "peptide" means a chain comprising at least two amino acids linked by peptide bonds. The term "polypeptide" is interchangeable with "protein" and means a chain comprising ten or more amino acid residues. All peptide and polypeptide formulas or sequences are written from left to right, showing the direction from amino terminus to carboxyl terminus.

[0059] The term "mineralizing peptide" refers to a peptide that can convert metal-containing compounds in solution (including cytoplasm) into mineral particles. Examples of mineralizing peptides include, but are not limited to, Car9 peptide and R5 peptide (which can convert titanium-containing compounds into TiO2). "Metal-containing compound" refers to a compound containing a metal element.

[0060] The term "amino acid" includes naturally occurring amino acids and non-natural amino acids in proteins. Conventional nomenclature (single-letter and three-letter) for naturally occurring amino acids in proteins is used, as described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0061] As used herein, the term "fusion polypeptide" is a recombinant product comprising two or more peptide fragments that are not present in a single native polypeptide. The fragments can be fused directly or via a linker, such as a flexible linker (e.g., a GS linker). Generally, a fusion polypeptide can be produced by expressing a polynucleotide comprising a nucleotide sequence encoding two or more peptide fragments and a linker (if present) in the desired order.

[0062] As used herein, the term "polynucleotide" generally refers to a nucleic acid molecule (e.g., 100 nucleotides and up to 30k nucleotides in length) and a sequence that is complementary (antisense) or identical (sense) to the sequence of a messenger RNA (mRNA) or miRNA fragment or molecule. The term can also refer to a transcribed or non-transcribed DNA or RNA molecule.

[0063] As used herein, the term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide that is isolated from a naturally occurring gene or modified to contain a non-naturally occurring nucleic acid segment. When a polynucleotide construct contains the control sequences required for expression of a coding sequence of the present invention, the polynucleotide construct comprises an "expression cassette."

[0064] As used herein, the term "exogenous polynucleotide" refers to a nucleotide sequence that is not derived from the host in which it is located. It may be identical to the host's DNA or be heterologous. An example is a sequence of interest that is inserted into a vector. Such exogenous DNA sequences can be derived from a variety of sources, including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also encompass DNA sequences encoding antisense oligonucleotides.

[0065] As used herein, the term "expression cassette" refers to a fragment comprising a polynucleotide encoding a polypeptide operably linked to additional nucleotide sequences that provide for the expression of the polynucleotide, eg, control sequences.

[0066] As used herein, the term "encoding" means that the polynucleotide directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with an ATG start codon or other start codon (e.g., GTG, TTG) and ends with a stop codon (e.g., TAA, TAG, TGA). The coding sequence can be a DNA, cDNA, or recombinant nucleotide sequence.

[0067] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0068] "Control sequences" include all elements necessary or beneficial for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, an enhancer, a signal peptide sequence, and a transcription terminator. A control sequence includes, at a minimum, a promoter and a signal for terminating transcription and translation.

[0069] For example, the control sequence can be a suitable promoter sequence, a nucleotide sequence that is recognized by the host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter sequence contains a transcriptional control sequence that mediates polypeptide expression. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, such as the lac operon of Escherichia coli. Promoters also include mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0070] As used herein, the term "operably linked" refers herein to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence such that the control sequence directs the expression of the polypeptide coding sequence.

[0071] Various manipulations can be performed on the polynucleotide encoding the polypeptide of interest to improve expression of the polypeptide. Before insertion into a vector, it may be desirable or necessary to manipulate the polynucleotide according to the expression vector or host (e.g., codon optimization). The techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0072] As used herein, the term "recombinant" refers to a nucleic acid, vector, polypeptide or protein that is produced using DNA recombination (cloning) methods and is distinct from a native or wild-type nucleic acid, vector, polypeptide or protein.

[0073] As used herein, the term "hybridize" refers to nucleotide sequences that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other and typically remain hybridized to each other under given stringent hybridization and washing conditions.

[0074] For the present invention, in order to determine the percentage identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, a gap can be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Then, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are the same length.

[0075] Those skilled in the art will appreciate that various computer programs can be used to determine the identity between two sequences.

[0076] "Percent identity" or "percent sequence identity" refers to a comparison between the amino acids of two polypeptides or between the nucleotides of two polynucleotides, and when optimally aligned, the two polypeptides or polynucleotides have approximately the specified percentage of identical amino acids. For example, "95% identity" refers to a comparison between the amino acids of two polypeptides or between the nucleotides of two polynucleotides, and when optimally aligned, 95% of the amino acids in the two polypeptides or 95% of the nucleotides in the two polynucleotides are identical.

[0077] Those skilled in the art are aware of various hybridization conditions, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, Sambrook et al., 1989, supra.

[0078] Of course, the polynucleotides of the present invention do not include polynucleotides that hybridize only to a poly A sequence (such as the 3' end poly (A) of mRNA) or a complementary extension of poly T (or U) residues.

[0079] As used herein, the term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients for vectors. The term includes progeny of the original cell that has been transduced. Thus, as used herein, "host cell" generally refers to a cell that has been transduced with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be identical to the original parent in morphology or in genome or total DNA complement due to natural, accidental, or deliberate mutations.

[0080] 2. Combination of peptides

[0081] The present invention provides a polypeptide combination comprising a Rubisco polypeptide from a Chlamydomonas species, and a fusion polypeptide comprising an EPYC1 polypeptide from a Chlamydomonas species and a mineralizing peptide capable of converting metal-containing compounds into compounds capable of converting light energy into chemical energy, such as metal oxides.

[0082] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas, such as Chlamydomonas reinhardtii.

[0083] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the same species. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

[0084] In some embodiments, the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide.

[0085] In some embodiments, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0086] In some embodiments, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 2, or a biologically active fragment thereof.

[0087] In some embodiments, the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3, or a biologically active fragment thereof.

[0088] It is known in the art that EPYC1 and Rubisco polypeptides from different Chlamydomonas species can also interact with each other. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0089] In some embodiments, the mineralizing peptide is a Car9 peptide. In some embodiments, the Car9 peptide comprises the amino acid sequence of SEQ ID NO:4.

[0090] In some embodiments, the mineralizing peptide is an R5 peptide. In some embodiments, the R5 peptide comprises the amino acid sequence of SEQ ID NO:15.

[0091] In some embodiments, the fusion polypeptide further comprises an sfGFP polypeptide. In some embodiments, the sfGFP polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 9, or a biologically active fragment thereof, and the sfGFP is capable of promoting the mineralization of the Car9 peptide.

[0092] In some embodiments, the fusion polypeptide comprises a peptide linker connecting the polypeptides. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, for example (G m S) n , wherein m and n are each independently 1, 2, 3 or 4.

[0093] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, an sfGFP polypeptide, a peptide linker, and a Car9 peptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0094] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, a sfGFP polypeptide, a peptide linker, and an R5 peptide.

[0095] 3. Polynucleotides and vectors expressing polypeptide combinations

[0096] The present invention provides polynucleotides encoding the fusion polypeptides of the present invention.

[0097] In some embodiments, the polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 16, or a degenerate variant thereof.

[0098] In some embodiments, the polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 17, or a degenerate variant thereof.

[0099] In some embodiments, the polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO: 18, or a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 18, or a degenerate variant thereof.

[0100] In some embodiments, the polynucleotide of the present invention comprises the nucleotide sequence of SEQ ID NO: 20, or a degenerate variant thereof.

[0101] The polypeptide combination of the present invention can be encoded by a single polynucleotide or a combination of polynucleotides.

[0102] The polynucleotides or combinations of polynucleotides of the present invention can be amplified using cDNA, mRNA or genomic DNA as templates and suitable oligonucleotide primers according to standard PCR amplification techniques. The polynucleotides amplified as above can be cloned into a suitable vector and characterized by DNA sequence analysis.

[0103] A polynucleotide or combination of polynucleotides of the invention can be prepared by standard synthetic techniques, for example, by using an automated DNA synthesizer.

[0104] The present invention also relates to complementary strands of the polynucleotides described herein.A nucleic acid molecule that is complementary to another nucleotide sequence is a nucleic acid molecule that is sufficiently complementary to the nucleotide sequence to hybridize with the other nucleotide sequence to form a stable duplex.

[0105] Also provided are polynucleotide constructs and vectors (eg, expression vectors) comprising a polynucleotide or combination of polynucleotides of the present invention for expression of the polypeptide combinations of the present invention.

[0106] In some embodiments, the polynucleotide of the present invention is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.

[0107] In some embodiments, the expression vector comprises a Lac operator.

[0108] In some embodiments, the combination of polypeptides can be expressed in a single expression cassette (transcribed into a single mRNA). Techniques for translating multiple polypeptides from a single mRNA are well known in the art. For example, an internal ribosome entry site (IRES), a ribosome binding site (RBS), and / or a nucleotide sequence encoding a 2A peptide can be added between the coding sequences of the individual polypeptides.

[0109] In some embodiments, the combination of polypeptides may be expressed in multiple expression cassettes (transcribed into multiple mRNAs).

[0110] The polynucleotides or combinations of polynucleotides encoding the polypeptides of the present invention may be subjected to various manipulations to allow expression of the polypeptides. It may be desirable or necessary to manipulate the polynucleotides prior to insertion into a vector, depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0111] In order to identify and select host cells containing the expression vectors of the present invention, the vectors of the present invention preferably contain one or more selectable markers that allow for easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product confers antimicrobial or viral resistance, heavy metal resistance, complements an auxotrophic deficiency, etc. For example, a bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

[0112] The vectors of the present invention can be integrated into the host cell genome or can replicate independently in the cell independent of the genome. Elements required for integration into the host cell genome or for independent autonomous replication are known in the art (see, for example, Sambrook et al., 1989, supra). In a second aspect, the present invention provides an isolated polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding a combination of polypeptides of the present invention.

[0113] The present invention also provides a vector or a combination of vectors, preferably an expression vector or a combination of expression vectors, comprising the polynucleotide or a combination of polynucleotides of the present invention.

[0114] 4. Recombinant Host Cells

[0115] The present invention provides a recombinant host cell, which is capable of fixing carbon dioxide and, preferably, forming membraneless organelles, and more preferably, achieving in situ generation of semiconductor materials within the cell.

[0116] In some embodiments, the recombinant host cell comprises a Rubisco polypeptide and / or a polynucleotide or vector encoding a Rubisco polypeptide, preferably an expression vector. In some embodiments, the recombinant host cell further comprises a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide or a polynucleotide encoding the Prk polypeptide.

[0117] In some embodiments, the recombinant host cell comprises a Rubisco polypeptide from a Chlamydomonas species, and a fusion polypeptide comprising an EPYC1 polypeptide from a Chlamydomonas species and a mineralization peptide capable of converting a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide.

[0118] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas, such as Chlamydomonas reinhardtii.

[0119] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the same species. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

[0120] In some embodiments, the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide.

[0121] In some embodiments, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0122] In some embodiments, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 2, or a biologically active fragment thereof.

[0123] In some embodiments, the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3, or a biologically active fragment thereof.

[0124] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0125] In some embodiments, the mineralizing peptide is a Car9 peptide. In some embodiments, the Car9 peptide comprises the amino acid sequence of SEQ ID NO:4.

[0126] In some embodiments, the mineralizing peptide is an R5 peptide. In some embodiments, the R5 peptide comprises the amino acid sequence of SEQ ID NO:15.

[0127] In some embodiments, the fusion polypeptide further comprises an sfGFP polypeptide. In some embodiments, the sfGFP polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 8, or a biologically active fragment thereof, and the sfGFP is capable of promoting the mineralization of the Car9 peptide.

[0128] In some embodiments, the fusion polypeptide comprises a peptide linker connecting the polypeptides. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, for example (G m S) n , wherein m and n are each independently 1, 2, 3 or 4.

[0129] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, an sfGFP polypeptide, a peptide linker, and a Car9 peptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0130] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, a sfGFP polypeptide, a peptide linker, and an R5 peptide.

[0131] In some embodiments, the recombinant host cell further comprises a molecular chaperone that assists in polypeptide folding and / or a polynucleotide encoding the same. In some embodiments, the molecular chaperone comprises the GroELS system and rbcX. The GroELS system generally comprises a GroEL subunit and a GroES subunit. In some embodiments, the GroEL subunit comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:6, or a biologically active fragment thereof. In some embodiments, the GroES subunit comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:7, or a biologically active fragment thereof. In some embodiments, the rbcX comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:8, or a biologically active fragment thereof.

[0132] In some embodiments, the microorganism further comprises a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to produce ribulose-1,5-bisphosphate, or a polynucleotide encoding the Prk polypeptide. In some embodiments, the Prk polypeptide comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 10, or a biologically active fragment thereof.

[0133] The example of host cell of the present invention includes but is not limited to bacterial cell, yeast cell and vegetable cell (such as the vegetable cell of multicellular plant).In some embodiments, described host cell is bacterial cell, for example aerobic bacteria.The example of described bacterial cell is to include but is not limited to the bacterium from Escherichia (for example intestinal bacteria), Bacillus (for example subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), wax shape bacillus (Bacillus cereus) etc.) or Corynebacterium (for example Corynebacterium glutamicum).

[0134] 5. Methods for fixing carbon dioxide and producing chemicals

[0135] The inventors unexpectedly discovered that introducing the carbon-concentrating mechanism of Chlamydomonas into microorganisms, such as bacteria (e.g., Escherichia coli), can form membraneless organelles, enabling efficient carbon dioxide fixation in E. coli. The inventors also discovered that metal oxide nanoparticles capable of converting light energy into chemical energy can be formed within the membraneless organelles of these microorganisms, providing energy for carbon dioxide fixation, thus achieving light-driven carbon dioxide fixation.

[0136] The present invention provides a method for fixing carbon dioxide, which comprises the following steps:

[0137] i) culturing a recombinant host cell comprising a Rubisco polypeptide and / or a polynucleotide or vector encoding a Rubisco polypeptide, preferably an expression vector; and

[0138] ii) contacting the microorganism with carbon dioxide.

[0139] In some embodiments, the recombinant host cell further comprises a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide or a polynucleotide encoding the Prk polypeptide.

[0140] The present invention provides a method for fixing carbon dioxide, which comprises the following steps:

[0141] i) culturing a recombinant host cell comprising a Rubisco polypeptide from a species in the family Chlamydomonas and a fusion polypeptide comprising an EPYC1 polypeptide from a species in the family Chlamydomonas and a mineralizing peptide capable of converting the metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide, in the presence of a metal-containing compound; and

[0142] ii) contacting the microorganism with carbon dioxide, preferably under light.

[0143] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas, such as Chlamydomonas reinhardtii.

[0144] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the same species. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

[0145] In some embodiments, the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide.

[0146] In some embodiments, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0147] In some embodiments, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 2, or a biologically active fragment thereof.

[0148] In some embodiments, the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3, or a biologically active fragment thereof.

[0149] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0150] In some embodiments, the mineralizing peptide is a Car9 peptide, and the metal-containing compound is a titanium-containing compound, such as TiBALDH. In some embodiments, the Car9 peptide comprises the amino acid sequence of SEQ ID NO:4.

[0151] In some embodiments, the mineralizing peptide is an R5 peptide. In some embodiments, the R5 peptide comprises the amino acid sequence of SEQ ID NO:15.

[0152] In some embodiments, the fusion polypeptide further comprises an sfGFP polypeptide. In some embodiments, the sfGFP polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 9, or a biologically active fragment thereof, and the sfGFP is capable of promoting the mineralization of the Car9 peptide.

[0153] In some embodiments, the fusion polypeptide comprises a peptide linker connecting the polypeptides. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, for example (G m S) n , wherein m and n are each independently 1, 2, 3 or 4.

[0154] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, an sfGFP polypeptide, a peptide linker, and a Car9 peptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0155] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, a sfGFP polypeptide, a peptide linker, and an R5 peptide.

[0156] In some embodiments, the recombinant host cell further comprises a molecular chaperone that assists in polypeptide folding and / or a polynucleotide encoding the same. In some embodiments, the molecular chaperone comprises the GroELS system and rbcX. The GroELS system generally comprises a GroEL subunit and a GroES subunit. In some embodiments, the GroEL subunit comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:6, or a biologically active fragment thereof. In some embodiments, the GroES subunit comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:7, or a biologically active fragment thereof. In some embodiments, the rbcX comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:8, or a biologically active fragment thereof.

[0157] In some embodiments, the microorganism further comprises a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to produce ribulose-1,5-bisphosphate, or a polynucleotide encoding the Prk polypeptide. In some embodiments, the Prk polypeptide comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 10, or a biologically active fragment thereof.

[0158] The present invention provides a method for fixing carbon dioxide, which comprises the following steps:

[0159] i) introducing a polynucleotide or a combination of polynucleotides, or a vector or a combination of vectors into a host cell to obtain a modified microorganism;

[0160] ii) culturing, preferably in the presence of a metal-containing compound and / or under light, the modified microorganism; and

[0161] iii) contacting the cultured microorganisms with carbon dioxide,

[0162] The polynucleotide or combination of polynucleotides, or vector or combination of vectors encodes a combination of polypeptides, comprising a Rubisco polypeptide from a species of Chlamydomonas, and an EPYC1 polypeptide from a species of Chlamydomonas, or a fusion polypeptide comprising an EPYC1 polypeptide from a species of Chlamydomonas and a mineralizing peptide, wherein the mineralizing peptide is capable of converting a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide.

[0163] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas, such as Chlamydomonas reinhardtii.

[0164] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the same species. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

[0165] In some embodiments, the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide.

[0166] In some embodiments, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0167] In some embodiments, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 2, or a biologically active fragment thereof.

[0168] In some embodiments, the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3, or a biologically active fragment thereof.

[0169] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0170] In some embodiments, the mineralizing peptide is a Car9 peptide, and the metal-containing compound is a titanium-containing compound, such as TiBALDH. In some embodiments, the Car9 peptide comprises the amino acid sequence of SEQ ID NO:4.

[0171] In some embodiments, the mineralizing peptide is an R5 peptide. In some embodiments, the R5 peptide comprises the amino acid sequence of SEQ ID NO:15.

[0172] In some embodiments, the fusion polypeptide further comprises an sfGFP polypeptide. In some embodiments, the sfGFP polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 9, or a biologically active fragment thereof, and the sfGFP is capable of promoting the mineralization of the Car9 peptide.

[0173] In some embodiments, the fusion polypeptide comprises a peptide linker connecting the polypeptides. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, for example (G m S) n , wherein m and n are each independently 1, 2, 3 or 4.

[0174] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, an sfGFP polypeptide, a peptide linker, and a Car9 peptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0175] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, a sfGFP polypeptide, a peptide linker, and an R5 peptide.

[0176] In some embodiments, step i) further comprises introducing an additional polynucleotide into the microorganism, the additional polynucleotide being selected from the group consisting of a) a polynucleotide encoding a molecular chaperone that assists polypeptide folding, such as the GroELS system and rbcX, b) a polynucleotide encoding an sfGFP polypeptide, c) a polynucleotide encoding a Prk polypeptide, and combinations thereof. The GroELS system generally comprises a GroEL subunit and a GroES subunit. In some embodiments, the GroEL subunit comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:6, or a biologically active fragment thereof. In some embodiments, the GroES subunit comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:7, or a biologically active fragment thereof. In some embodiments, the rbcX polypeptide comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 8, or a biologically active fragment thereof. In some embodiments, the Prk polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 9, or a biologically active fragment thereof.

[0177] The present invention provides a method for producing a chemical of interest, comprising the steps of:

[0178] i) introducing a polynucleotide or a combination of polynucleotides, or a vector or a combination of vectors into a host cell to obtain a modified microorganism; and

[0179] ii) cultivating, preferably in the presence of a metal-containing compound and / or under conditions of light, said modified microorganism to produce said chemical of interest,

[0180] The polynucleotide or combination of polynucleotides, or vector or combination of vectors encodes a combination of polypeptides, comprising a Rubisco polypeptide from a species of Chlamydomonas, and an EPYC1 polypeptide from a species of Chlamydomonas, or a fusion polypeptide comprising an EPYC1 polypeptide from a species of Chlamydomonas and a mineralizing peptide, wherein the mineralizing peptide is capable of converting a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide.

[0181] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas, such as Chlamydomonas reinhardtii.

[0182] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the same species. In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

[0183] In some embodiments, the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide.

[0184] In some embodiments, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0185] In some embodiments, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 2, or a biologically active fragment thereof.

[0186] In some embodiments, the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3, or a biologically active fragment thereof.

[0187] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0188] In some embodiments, the mineralizing peptide is a Car9 peptide, and the metal-containing compound is a titanium-containing compound, such as TiBALDH. In some embodiments, the Car9 peptide comprises the amino acid sequence of SEQ ID NO:4.

[0189] In some embodiments, the mineralizing peptide is an R5 peptide. In some embodiments, the R5 peptide comprises the amino acid sequence of SEQ ID NO:15.

[0190] In some embodiments, the fusion polypeptide further comprises an sfGFP polypeptide. In some embodiments, the sfGFP polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 9, or a biologically active fragment thereof, and the sfGFP is capable of promoting the mineralization of the Car9 peptide.

[0191] In some embodiments, the fusion polypeptide comprises a peptide linker connecting the polypeptides. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, for example (G m S) n , wherein m and n are each independently 1, 2, 3 or 4.

[0192] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, an sfGFP polypeptide, a peptide linker, and a Car9 peptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0193] In some embodiments, the fusion polypeptide comprises (preferably from N-terminus to C-terminus) an EPYC1 polypeptide, a sfGFP polypeptide, a peptide linker, and an R5 peptide.

[0194] In some embodiments, step i) further comprises introducing an additional polynucleotide into the microorganism, the additional polynucleotide being selected from the group consisting of a) a polynucleotide encoding a molecular chaperone that assists polypeptide folding, such as the GroELS system and rbcX, b) a polynucleotide encoding an sfGFP polypeptide, c) a polynucleotide encoding a Prk polypeptide, and combinations thereof. The GroELS system generally comprises a GroEL subunit and a GroES subunit. In some embodiments, the GroEL subunit comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:6, or a biologically active fragment thereof. In some embodiments, the GroES subunit comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:7, or a biologically active fragment thereof. In some embodiments, the rbcX polypeptide comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 8, or a biologically active fragment thereof. In some embodiments, the Prk polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 9, or a biologically active fragment thereof.

[0195] In some embodiments, the method further comprises introducing into the microorganism, before, simultaneously with, or after step i), a polynucleotide encoding a polypeptide involved in the biosynthesis of the chemical of interest.

[0196] In some embodiments, the chemical of interest is 2,3-butanediol. In some embodiments, the method further comprises, before, simultaneously with, or after step i), introducing into the microorganism genes encoding α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC), or butanediol dehydrogenase (BDH).

[0197] In some embodiments, the ALS comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 12, or a biologically active fragment thereof.

[0198] In some embodiments, the ALDC comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 13, or a biologically active fragment thereof.

[0199] In some embodiments, the BDH comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 14, or a biologically active fragment thereof.

[0200] In some embodiments, the host cell is a bacterial cell, such as a plant cell of a multicellular plant. In some embodiments, the host cell is a bacterial cell, such as an aerobic bacterium. The example of the bacterial cell is an antibacterial cell including but not limited to a bacterium from Escherichia (such as Escherichia coli), Bacillus (such as subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus etc.) or Corynebacterium (such as Corynebacterium glutamicum).

[0201] Implementation Plan

[0202] Embodiment 1. A recombinant host cell comprising a Rubisco polypeptide from a species of the Chlamydomonas family and an EPYC1 polypeptide from a species of the Chlamydomonas family.

[0203] Embodiment 2: The recombinant host cell of embodiment 1, further comprising a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide or a polynucleotide encoding the Prk polypeptide.

[0204] Embodiment 3. The recombinant host cell of embodiment 1 or 2, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from species of the genus Chlamydomonas.

[0205] Embodiment 4. The recombinant host cell of any one of embodiments 1-3, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from the same species, such as Chlamydomonas reinhardtii.

[0206] Embodiment 5. The recombinant host cell of any one of embodiments 1-4, wherein the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide, for example, wherein the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, or a biologically active fragment thereof, for example, wherein the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 2, or a biologically active fragment thereof.

[0207] Embodiment 6. The recombinant host cell of any one of embodiments 1-5, wherein the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 3, or a biologically active fragment thereof.

[0208] Embodiment 7. The recombinant host cell of any one of embodiments 1-3, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

[0209] Embodiment 8. The recombinant host cell of any one of embodiments 1-7, wherein the recombinant host cell further comprises a molecular chaperone that assists polypeptide folding and / or a polynucleotide encoding the molecular chaperone.

[0210] Embodiment 9. The recombinant host cell of embodiment 8, wherein the molecular chaperone comprises the GroELS system and rbcX.

[0211] Embodiment 10. The recombinant host cell of embodiment 9, wherein the GroELS system generally comprises a GroEL subunit and a GroES subunit, for example, wherein the GroEL subunit comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 6, or a biologically active fragment thereof, and the GroES subunit comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 7, or a biologically active fragment thereof.

[0212] Embodiment 11. The recombinant host cell of embodiment 9, wherein the rbcX comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 8, or a biologically active fragment thereof.

[0213] Embodiment 12, the recombinant host cell of any one of embodiments 1-11, wherein the microorganism further comprises a phosphoribosylpyrophosphate aminotransferase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to produce ribulose-1,5-bisphosphate, or a polynucleotide encoding the Prk polypeptide, for example, wherein the Prk polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 10, or a biologically active fragment thereof.

[0214] Embodiment 13. The recombinant host cell of any one of embodiments 1-12, which is selected from bacterial cells, yeast cells and plant cells (such as plant cells of multicellular plants).

[0215] Embodiment 14. The recombinant host cell of any one of embodiments 1-13, wherein the host cell is a bacterial cell, such as an aerobic bacterium.

[0216] Embodiment 15, the recombinant host cell of embodiment 14, wherein the bacterial cell is a bacterium from the genus Escherichia (e.g., Escherichia coli), the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, etc.), or the genus Corynebacterium (e.g., Corynebacterium glutamicum).

[0217] Embodiment 16: A method for fixing carbon dioxide, comprising the following steps:

[0218] i) culturing the recombinant host cell of any one of embodiments 1-15; and

[0219] ii) contacting the microorganism with carbon dioxide.

[0220] Beneficial effects of the present invention

[0221] This invention demonstrates that the carbon-concentrating mechanism found in Chlamydomonas can function in bacteria such as Escherichia coli and Corynebacterium glutamicum, significantly improving the efficiency of carbon dioxide fixation compared to microorganisms that do not perform carbon concentration. Furthermore, by forming titanium dioxide nanoparticles within membraneless organelles that can convert light energy into chemical energy, this invention utilizes light energy to promote carbon dioxide fixation, significantly avoiding the interference and disruption of the nanoparticles on cellular physiological metabolism. This carbon dioxide fixation also significantly increases chemical production and carbon yield. Example

[0222] The following examples are provided for illustration only and are not intended to limit the present application in any way.

[0223] Example 1: Construction and expression of the eukaryotic CBB carbon fixation pathway from Chlamydomonas in recombinant Escherichia coli

[0224] The purpose of this example is to verify whether the CBB carbon fixation pathway derived from eukaryotic Chlamydomonas can be functionally expressed in Escherichia coli.

[0225] 1.1 Functional Expression of Rubisco and Prk from Chlamydomonas in Escherichia coli

[0226] Based on commercially available vectors, expression vectors for functional expression of Rubisco and Prk in Escherichia coli were constructed.

[0227] Specifically, the expression vector pET22b-LS (Figure 1, the nucleotide sequences encoding the polypeptides are connected by ribosome binding sites) for expressing Rubisco large and small subunits (chrrbcL, SEQ ID NO: 1, chrbcS, SEQ ID NO: 2) was constructed based on the high copy vector pET22b; the expression vector pBAD33-prk-G-rbcX (Figure 2, the nucleotide sequences encoding the polypeptides are connected by ribosome binding sites) for expressing Prk (SEQ ID NO: 10), GroEL (SEQ ID NO: 6), GroES (SEQ ID NO: 7) and rbcX (SEQ ID NO: 8) was constructed based on the medium copy vector pBAD. DNA encoding the above sequences was synthesized (Shanghai Sangon Biotech Co., Ltd.) and assembled by Gibson assembly (using Gibson Master Mix NEB) was cloned into the vector backbone.

[0228] The two constructed vectors were co-transformed into competent Escherichia coli BL21 (DE3) (purchased from Quanshijin) using the heat shock method. The transformed E. coli were screened using LB solid medium supplemented with ampicillin (100 mg / L) and chloramphenicol (50 mg / L), and positive clones were confirmed by sequencing (Sangon Biotechnology (Shanghai) Co., Ltd.).

[0229] Cultures were shaken at 37°C (220 rpm) and induced with 0.2% L-arabinose and 1 mM IPTG. OD600 values ​​were measured at 4, 8, 12, 16, 20, and 24 hours, and growth curves of the recombinant E. coli were plotted. Untransformed E. coli and E. coli transformed only with pBAD33-prk-G-rbcX served as controls. Ribulose-1,5-bisphosphate (RuBP), the product of the Prk enzyme-catalyzed reaction, arrests the growth of E. coli. Rubisco uses RuBP as a substrate to react with CO2, achieving carbon fixation (see Figure 3). Consequently, the bacterial growth curves show that when only the prk enzyme is expressed, bacterial growth is arrested. However, when both Rubisco and Prk are expressed, bacterial growth resumes and grows faster than untransformed bacteria (Figure 4), demonstrating the functional expression of both Rubisco and Prk in E. coli and the successful construction of a carbon fixation module.

[0230] 1.2 Construction and characterization of recombinant Escherichia coli expressing the carbon fixation module and phase separation protein

[0231] In order to co-express the phase separation protein EPYC1 (SEQ ID NO: 3) with Rubisco and thus construct membraneless organelles in recombinant Escherichia coli, the nucleotide sequence encoding EPYC1 or EPYC1-venus fusion protein (SEQ ID NO: 11) was inserted into the pET22b-LS vector constructed in Example 1.1 (synthesized by Sangon Biotech (Shanghai) Co., Ltd.) by Gibson assembly to obtain the vectors pET22b-LSE and pET22b-LSEV ( Figures 5 and 6 ).

[0232] According to the method of Example 1.1, pET22b-LSE or pET22b-LSEV and pBAD-prk-G-rbcX were co-transformed into competent BL21 (DE3).

[0233] The transformed bacteria were inoculated into LB liquid culture medium supplemented with ampicillin (100 mg / L) and chloramphenicol (50 mg / L) and cultured at 37°C in a shaker (220 rpm) until OD600 was between 0.6 and 0.8. Then, inducers 20% L-arabinose (final concentration 0.2%) and 1 M IPTG (final concentration 1 mM) were added and cultured at 37°C in a shaker (220 rpm) overnight (12-16 hours).

[0234] Recombinant E. coli cells co-transformed with pET22b-LSEV and pBAD-prk-G-rbcX were observed using confocal microscopy, structured light illumination microscopy, and transmission electron microscopy. Specifically, 100 μL of overnight bacterial culture was centrifuged (3000-5000 rpm, 5 minutes); the bacterial cells were collected and washed twice with ultrapure water, then resuspended in 500 μL of ultrapure water; 10 μL of the bacterial suspension was dropped onto a glass slide and observed using a confocal microscope (Nikon A1R, 60X oil objective) and a structured light microscope (Nikon N-SIM S) at a wavelength of 488 nm; and 10 μL of the bacterial suspension was dropped onto a copper grid, dried with an infrared lamp, and observed using a TEM (FEI Tecnai F30).

[0235] The results showed that obvious fluorescent clustered compartment structures were formed in the recombinant Escherichia coli cells (Figure 7), indicating that protein liquid-liquid phase separation occurred in the bacterial cells, indicating that the carbon concentration mechanism in Chlamydomonas was successfully introduced into Escherichia coli, forming a carbon fixation module with a carbon concentration mechanism.

[0236] 1.3. Quantitative characterization of carbon fixation efficiency of recombinant Escherichia coli.

[0237] In order to characterize the carbon fixation ability of recombinant Escherichia coli, the effect of phase separation protein on the enzymatic activity of Rubisco was first determined.

[0238] Specifically, 1 mL of overnight bacterial culture (recombinant E. coli co-transformed with pET22b-LSEV and pBAD-prk-G-rbcX) was centrifuged (3000-5000 rpm, 5 minutes); the bacterial cells were collected, washed twice with ultrapure water, and resuspended in 1 mL of 100 mM Tris; the suspension was sonicated for 30 minutes to disrupt the bacterial cells, then centrifuged and the supernatant (containing Rubisco) was added to the following reaction system:

[0239] The reaction mixture (containing 100 mM Tris (pH 8.0), 20 mM MgCl2, 10 mM KCl, 25 mM NaHCO3, 3.5 mM ATP, 5 mM creatine phosphate, 2 mM DTT, 5 U / mL 3-phosphoglycerate kinase, 5 U / mL creatine phosphokinase, 5 U / mL glyceraldehyde 3-phosphate dehydrogenase, and an appropriate amount of supernatant containing Rubisco) was activated at 30°C for 10 min. The reaction was then initiated by the addition of 0.5 mM RuBP and 0.25 mM NADH and immediately monitored in a microplate reader (Wallac / Spectromax) at 340 nm (30°C) for 5 minutes before recording the change in absorbance.

[0240] The CO2 consumption rate was determined as follows: 0.5 mL of overnight cultured bacteria (recombinant E. coli co-transformed with pET22b-LSEV and pBAD-prk-G-rbcX) was inoculated into 50 mL of 5% L-arabinose, 20 μM IPTG, 20 mM 13 C-labeled NaHCO3, 1 μg / mL VB1, and 1 g / L peptone were cultured in M9 medium at 37°C in a shaker (220 rpm) for 12 hours. 2 mL of the bacterial solution before and after culture (0 hour and 12 hours) were collected by centrifugation, and the bacterial pellet was dried and weighed. The supernatant was subjected to NMR detection of HCO3 - and CO2 concentrations. The concentrations were calculated based on the corresponding peak area and the standard curve of NaHCO3 with gradient concentrations (0.5mM, 1mM, 5mM, 10mM, 20mM). The consumption rate was calculated using the formula:

[0241] (0 hours HCO3 - and CO2 concentration -12 hours HCO3 - and CO2 concentration) / 12h*dry weight

[0242] The dry weight is the difference between the dry weight of bacteria in 2 mL of bacterial solution after 12 hours of culture and the dry weight of bacteria in 2 mL of bacterial solution at 0 hours.

[0243] The results showed that the presence of the phase separation protein EPYC1 significantly increased the activity of Rubisco (Figure 8) and the CO2 consumption rate (Figure 9), indicating that the carbon concentration mechanism improved the carbon fixation efficiency of recombinant Escherichia coli.

[0244] Example 2: Construction and characterization of mineralized recombinant Escherichia coli containing a semiconductor-enzyme complex

[0245] The purpose of this embodiment is to construct a recombinant Escherichia coli that can form membraneless organelles, realize the in situ generation of semiconductor materials within the cell and fix carbon dioxide.

[0246] 2.1. Preparation of biomineralized recombinant Escherichia coli

[0247] Based on the high-copy vector pET22b, the expression vector pET22b-LSE-sfGFP-Car9 was constructed to express the Rubisco large and small subunits (SEQ ID NOs: 1 and 2) and the EPYC1-sfGFP-Car9 fusion protein (SEQ ID NO: 5) ( Figure 10 , the nucleotide sequences encoding each polypeptide are connected by a ribosome binding site).

[0248] According to the method described in Example 1, the vectors pET22b-LSE-sfGFP-Car9 and pBAD-prk-G-rbcX were co-transformed into Escherichia coli competent BL21 (DE3), and the transformed Escherichia coli were screened.

[0249] A mineralizing peptide (Car9) converts TiBALDH into non-metallic (N, C)-doped titanium dioxide (TiO2) within the cell, forming a semiconductor-enzyme complex. Upon mineralization, recombinant E. coli cells form membraneless organelles composed of protein aggregates containing Rubisco and the semiconductor-doped TiO2.

[0250] Specifically, the screened recombinant Escherichia coli was inoculated into LB liquid culture medium and cultured in a shaking incubator at 37°C until the OD was between 0.6 and 0.8. Then, 20% L-arabinose (final concentration 0.2%), 1M IPTG (final concentration 1mM) and different concentrations (0, 0.1mM, 0.2mM, 0.4mM, 0.8mM) of TiBALDH (Sigma) were added as inducers. The culture was shaken at 37°C for 12-16 hours, and the OD600 value of the culture was measured.

[0251] As shown in FIG11 , the recombinant E. coli without the addition of TiBALDH showed the highest cell density (OD600 value), and the cell density decreased as the TiBALDH concentration increased.

[0252] 2.2 Characterization of Biomineralized Recombinant E. coli

[0253] First, a transmission electron microscope (TEM, FEI Tecnai F30) was used to observe the mineralized recombinant E. coli prepared in Example 2.1. Specifically, the recombinant E. coli culture prepared in Example 2.1 was centrifuged at 3000-5000 rpm for 5 minutes, the cells were collected and washed twice with ultrapure water, resuspended with ultrapure water to an OD600 of 0.1, and then diluted 10 times with ultrapure water. 10 microliters of the suspension was dropped onto a copper grid, dried with an infrared lamp, and observed with a TEM. Aggregates were observed at both ends of the cells, and the aggregates contained nanoparticles with a size of 50-100 nanometers; elemental analysis (X-ray energy dispersive spectroscopy (TEM-EDS mapping) showed that the particles contained titanium (Ti) and oxygen (O) (Figure 12).

[0254] Furthermore, the bacteria were embedded and then ultrathinly sectioned. Specifically, the recombinant Escherichia coli culture prepared in Example 2.1 was centrifuged at 3000-5000 rpm for 5 minutes, the bacterial cells were collected and washed twice with ultrapure water. The washed bacterial cells were fixed with 2.5% glutaraldehyde for 15 minutes, washed three times with 0.1M PBS (pH 7.4), fixed with pre-cooled 1% osmium tetroxide for 2 hours at room temperature, and then washed three times with 0.1M PBS (pH 7.4). The fixed bacterial cells were dehydrated with gradient concentrations of ethanol (50%, 70%, 80%, 85%, 90%, 95%, 100%), and then dehydrated twice with 100% ethanol for 10 minutes each. The samples were then infiltrated with penetrants i) acetone: epoxy resin (2:1), ii) acetone: epoxy resin (1:1), and iii) epoxy resin for 12 hours each. The infiltrated sample was placed in a small capsule, and the embedding agent epoxy resin was added and cured at 60°C for 48 h. Then, the sample was cut into ultrathin sections with a thickness of 60 to 100 nm in a microtome.

[0255] The slice was measured by TEM scanning transmission electron microscopy high-angle annular dark field imaging (HAADF-TEM), and obvious compartment formation was observed. Elemental analysis (as described above), transmission electron microscopy high-angle annular dark field imaging (HRT-TEM), selected electron diffraction (SAED) and crystal lattice spacing measurement all indicated that the compartment contained generated titanium dioxide (Figure 13).

[0256] Example 3: Characterization of the photosynthetic carbon fixation function of biomineralized recombinant Escherichia coli

[0257] The purpose of this example is to characterize the photosynthetic carbon fixation function of the biomineralized recombinant Escherichia coli prepared in Example 2.1.

[0258] 3.1 Photoconversion Effect of Biomineralized Recombinant Escherichia coli

[0259] The bacterial suspension (40 μL, OD600 = 2) was evenly spread on one side of carbon paper (1 cm × 1 cm). After the suspension was dried, 20 μL of Nafion (0.05% absolute ethanol) was added to the carbon paper, and then the carbon paper was clamped with an electrode clamp. The working electrode (carbon paper with bacteria), the reference electrode (Ag / AgCl reference electrode) and the counter electrode (platinum sheet) were placed in a quartz electrolytic cell (volume 50 mL). The electrolytic cell was injected with an electrolyte solution (PBS, 40 mL), purged with nitrogen for 10 minutes, and then sealed. The photoreaction was carried out under an LED light source (white light, or monochromatic light of 450 nm or 620 nm with a bandpass filter, Perfect Light Source, China) with a light intensity of 20 k lux. The following photoelectrochemical analyses were performed on an electrochemical workstation (CHI760E, Shanghai Chenhua): (1) linear sweep voltammetry (LSV) with a voltage range of 0 to −1 V and 0 to 1 V, and a scan rate of 0.01 V s -1 ; (2) Ampere-time method (It), recording the current values ​​at potentials of -0.6 V and 0.6 V (relative to Ag / AgCl).

[0260] The photoconversion effect was measured by measuring the change in the photocurrent of the bacteria before and after illumination. The results showed that the complete system had an obvious high-response photocurrent curve (Figure 14).

[0261] 3.2 Reducing power in biomineralized recombinant Escherichia coli cells

[0262] The reducing power (NADPH and NADH) within the mineralized recombinant E. coli cells was measured using a Beyotime kit (WST-8 assay). The results showed that the NAD(P)H / NAD(P)- ratio showed a strong response to light, indicating photochemical energy conversion (Figure 15).

[0263] 3.3 Carbon Fixation in Biomineralized Recombinant Escherichia coli Cells

[0264] pass 13 The substrate NaHCO3 was labeled with C and the product glycerate 3-phosphate (3PGA) was detected by LC-MS.

[0265] Specifically, the mineralized recombinant E. coli cells were inoculated into a medium containing 0.2% L-arabinose, 1 mM IPTG, 20 mM 13Cells were cultured in M9 medium (Shanghai Sangon Biotech Co., Ltd.) containing C-labeled NaHCO₃ and 1 μg / mL vitamin B1 to an OD₀⁶ of 0.4–0.6. The cell suspension (20 ml) was collected, washed in 10 mL of cold (−20°C) 60% methanol (v / v), and collected by centrifugation to terminate cellular metabolism. The cells were resuspended in 160 μl of cold (−10°C) 60% methanol (v / v), and 200 μl of 0.3 M KOH was added. The mixture was frozen in liquid nitrogen for 5 minutes and stored at −80°C for at least 2 hours. The sample was thawed on ice and neutralized with 4 μL of acetic acid. After centrifugation (12,000 g, −10°C) for 10 minutes, the supernatant was transferred to a clean centrifuge tube for LC-MS / MS analysis.

[0266] An Agilent 6460 Series LC-MS / MS system equipped with an HPLC system and a triple quadrupole mass spectrometer was used. An Agilent XDC18 column (5 μM, 150 mm × 4.6 mm) was used for MS detection in negative ion and selected multiple reaction monitoring (MRM) modes. 3PGA standards were purchased from Sigma-Aldrich. Methanol was purchased from Fisher Scientific. The mobile phases were Solution A (water) and Solution B (methanol) (see Table 1). The flow rate was 0.6 mL min-1. The injection volume was 50 μL, and the column temperature was 40°C. The MS settings were as follows: gas temperature, 350°C; gas flow rate, 8 L min-1; nebulizer, 38 psi; sheath gas temperature, 350°C; sheath gas flow rate, 9 L min-1; capillary, -3500 V; nozzle voltage, 500 V. The dwell time was set to 200 ms. Metabolites were quantified using their standard curves.

[0267] Table 1. LC-MS / MS mobile phase conditions

[0268] As shown in Figure 16, the recombinant E. coli cultured under light contained 13 The 3PGA ratio of C was significantly higher than that of the recombinant E. coli cultured in the dark, indicating that light promoted carbon fixation in the recombinant E. coli, that is, effective photosynthetic carbon fixation was achieved.

[0269] Example 4: Production of 2,3-butanediol using photosynthetic carbon-fixing recombinant Escherichia coli

[0270] The purpose of this example is to verify the ability of the photosynthetic carbon-fixing recombinant Escherichia coli of the present invention to produce chemicals by producing 2,3-butanediol.

[0271] 4.1. Construction of a synthetic pathway for 2,3-butanediol

[0272] The recombinant Escherichia coli of the present invention can convert CO2 into 3PGA, which is further converted into pyruvate (pyr). As shown in Figure 17, pyruvate is a precursor for the synthesis of 2,3-butanediol. It is also necessary to express α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC), and butanediol dehydrogenase (BDH) in recombinant Escherichia coli for construction. The polynucleotides encoding ALS (SEQ ID NO: 12), ALDC (SEQ ID NO: 13), and BDH (SEQ ID NO: 14) were amplified by PCR (donated from the State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University) and ligated to the pET22b vector (pET22b-LSE-sfGFP-Car9) pre-attached with a carbon fixation module by the Gibson method to obtain the vectors pET22b-LSE-sfGFP-Car9-RABC and pET22b-RABC (Figure 20).

[0273] According to the method of Example 1, pET22b-LSE-sfGFP-Car9-RABC or pET22b-RABC and pBAD-prk-G-rbcX were co-transformed into Escherichia coli competent BL21 (DE3) and screened.

[0274] The selected recombinant E. coli were mineralized as described in Example 2.1 (final concentration of TiBALDH was 0.1 mM).

[0275] 4.2. Production of 2,3-Butanediol under Light

[0276] The mineralized recombinant E. coli culture prepared in Example 4.1 was centrifuged for 5 minutes (rotation speed 3000-5000rpm) and the cells were collected and washed twice with M9 liquid culture medium. The cleaned cells were collected and resuspended in M9 liquid culture medium supplemented with ampicillin (100mg / L) and chloramphenicol (50mg / L) and 50g / L glucose (cell density was OD 1-1.5), and then placed in a light incubator for light fermentation (light 2000lux, 30°C, 150-200rpm). After fermentation for 24 hours, 2mL of fermentation broth was centrifuged at 13000rpm for 5 minutes, the supernatant was retained, 1mL was used for butanediol extraction, and 1mL was used for residual glucose determination.

[0277] 1 mL of ethyl acetate was added to 1 mL of supernatant and sonicated for 30 minutes. After thorough mixing, the mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was aspirated for GC analysis (Agilent, GC8890, HP-5, nitrogen carrier gas). The injector and detector temperatures were set to 280°C. The column temperature was maintained at 40°C for 3 minutes and then increased to 240°C at 20°C / min. The injection volume was 1 μL. Product concentrations were calculated using the calibration curve.

[0278] Use the Biyuntian Glucose Detection Kit to measure residual glucose in the culture medium (O-toluidine method). Refer to the kit instructions for specific procedures. Dilute the original supernatant appropriately based on the amount of residual glucose. Calculate the carbon yield based on the carbon balance based on the butanediol production and residual glucose.

[0279] The results showed that compared with the reported yield (Xu et al., Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2,3-butanediol Metab Eng. 2014, 23: 22-33) and the control strain, the recombinant Escherichia coli of the present invention obtained the highest yield (51 g / L, Figure 18) and carbon yield (1.34 mol / mol, Figure 19) under light conditions, which was more than 2.5 times that of the control.

[0280] Example 5. Construction and characterization of recombinant Corynebacterium glutamicum expressing carbon fixation module and phase separation protein

[0281] The purpose of this example is to verify the formation of membraneless organelles (carbon concentrating mechanism) for carbon fixation in Corynebacterium glutamicum.

[0282] Based on the high-copy vector pec-xc99e (see Li et al., 2021, Heterologous production of α-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method, Bioresource Technology, 341: 125782), an expression vector 301-LSE-prk ( FIG21 , nucleotide sequences encoding each polypeptide are connected by a ribosome binding site) was constructed for expressing Rubisco large and small subunits (chrrbcL, SEQ ID NO: 1, chrbcS, SEQ ID NO: 2), Prk (SEQ ID NO: 10), and EPYC1-venus fusion protein (SEQ ID NO: 11) was constructed. Based on the medium-copy vector pz9 (see Li et al., 2021), an expression vector 301-LSE-prk ( FIG21 , nucleotide sequences encoding each polypeptide are connected by a ribosome binding site) was constructed for expressing GroEL (SEQ ID NO: 6), GroES (SEQ ID NO: 7), and rbcX (SEQ ID NO: 8) was constructed. NO:8) was synthesized using the expression vector 302-G-rbcX (Figure 22, the nucleotide sequences encoding the polypeptides were linked by ribosome binding sites). DNA encoding the above sequences was synthesized (Shanghai Sangon Biotech Co., Ltd.) and assembled using Gibson assembly (using Gibson Master Mix NEB) was cloned into the vector backbone. 2 μL of the assembled product was transformed into 50 μL of competent Escherichia coli DH5a cells using the heat shock method. The transformed cells were plated on LB plates containing 7.5 mg / L chloramphenicol and 25 mg / L kanamycin and cultured overnight. Positive clones were then picked and verified by sequencing (Shanghai Sangon Biotech Co., Ltd.).

[0283] With above-mentioned two plasmids (each 10 μ L) by electroporation method (Bio-rad XCell electroporator, electroporation parameter is 2500V, 5.0ms) successively transform into the competent cell of 100 μ L Corynebacterium glutamicum strain ATCC14067.Recover 3 hours in 900 μ L BHIS substratum, be coated on the BHISG (S is sorbitol, G is glucose) flat board containing 7.5mg / L chloramphenicol and 25mg / L kanamycin and cultivate 36 hours.Pick positive clone, and sequence verification (Sanggong Biotechnology (Shanghai) Co., Ltd.).

[0284] A single clone was picked from the plate cultured for 36 hours and inoculated into BMI medium supplemented with kanamycin (7.5 mg / L) and chloramphenicol (25 mg / L). The culture was cultured until the OD600 was between 0.6 and 0.8. The inducer IPTG (final concentration 0.1 mM) was added and cultured at 30°C for 24 hours.

[0285] 100 μL of bacterial culture was centrifuged (3000-5000 rpm, 3-5 minutes); the bacterial cells were collected and washed twice with ultrapure water, and then resuspended with 300-600 μL of ultrapure water; 10 μL of bacterial suspension was dropped onto a glass slide and observed with a confocal microscope (Nikon A1R, 60X oil objective) at a wavelength of 488 nm.

[0286] The results showed that obvious fluorescent clustered compartment structures were formed in the recombinant Corynebacterium glutamicum cells (Figure 23), indicating that protein liquid-liquid phase separation occurred in the bacterial cells, indicating that the carbon concentration mechanism in Chlamydomonas was successfully introduced into Corynebacterium glutamicum, forming a carbon fixation module with a carbon concentration mechanism.

[0287] sequence

Claims

1. A combination of polypeptides, which comprises a Rubisco polypeptide from a species of the family Chlamydomonadaceae, and a fusion polypeptide comprising an EPYC1 polypeptide and a mineralization peptide from a species of the family Chlamydomonadaceae, wherein the mineralization peptide is capable of converting a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide.

2. The combination of polypeptides according to claim 1, wherein the EPYC1 polypeptide and the mineralization peptide are linked by a peptide linker.

3. The combination of polypeptides according to claim 2, wherein the peptide linker is a flexible linker, such as a GS linker.

4. The combination of polypeptides according to any one of claims 1-3, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from a species of the genus Chlamydomonas.

5. The combination of polypeptides according to any one of claims 1-4, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from the same species.

6. The combination of polypeptides according to any one of claims 1-5, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from the species Chlamydomonas reinhardtii.

7. The combination of polypeptides according to any one of claims 1-6, wherein the Rubisco polypeptide comprises a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide, the Rubisco large subunit polypeptide comprises the amino acid sequence of SEQ ID NO:1, the Rubisco small subunit polypeptide comprises the amino acid sequence of SEQ ID NO:2, and the EPYC1 polypeptide comprises the amino acid sequence of SEQ ID NO:

3.

8. The combination of polypeptides according to any one of claims 1-4, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

9. The combination of polypeptides according to any one of claims 1-8, wherein the mineralization peptide is a Car9 peptide.

10. The combination of polypeptides according to claim 9, wherein the Car9 peptide comprises the amino acid sequence of SEQ ID NO:

4.

11. The combination of polypeptides according to any one of claims 1-10, wherein the fusion polypeptide further comprises an sfGFP polypeptide.

12. The combination of polypeptides according to any one of claims 1-10, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

5.

13. An isolated polynucleotide or combination of polynucleotides, which comprises a nucleotide sequence or combination of nucleotide sequences encoding the combination of polypeptides according to any one of claims 1-12.

14. A vector or combination of vectors, preferably an expression vector or combination of expression vectors, which comprises the polynucleotide or combination of polynucleotides according to claim 13.

15. A recombinant microorganism, such as a bacterium or yeast, which comprises the combination of polypeptides according to any one of claims 1-12, the polynucleotide or combination of polynucleotides according to claim 13, or the vector or combination of vectors according to claim 14.

16. The microorganism according to claim 15, which further comprises a molecular chaperone that helps polypeptide folding and / or a polynucleotide encoding the molecular chaperone.

17. The microorganism according to claim 16, wherein the molecular chaperone comprises a GroELS system and rbcX.

18. The microorganism according to any one of claims 15-17, further comprising a phosphoribosyl pyrophosphate aminotransferase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose 5-phosphate to produce ribulose 1,5-bisphosphate, or a polynucleotide encoding said Prk polypeptide.

19. A method for fixing carbon dioxide, comprising the following steps: i) culturing the microorganism according to any one of claims 15-18 in the presence of a metal compound; and ii) contacting said microorganism with carbon dioxide, preferably under illumination.

20. The method according to claim 19, wherein the metal compound is a titanium-containing compound, such as TiBALDH.

21. A method for producing a chemical of interest, comprising the following steps: i) introducing the polynucleotide according to claim 13 or a combination of polynucleotides, or the vector according to claim 14 or a combination of vectors, into a microorganism, such as a bacterium or yeast, to obtain a modified microorganism; and ii) culturing the modified microorganism, preferably in the presence of a metal compound and / or under illumination, to produce the chemical of interest.

22. The method according to claim 21, wherein step i) further comprises introducing an additional polynucleotide into said microorganism, the additional polynucleotide being selected from a) a polynucleotide encoding a molecular chaperone that aids polypeptide folding, such as the GroELS system and rbcX, b) a polynucleotide encoding an sfGFP polypeptide, c) a polynucleotide encoding a Prk polypeptide, and combinations thereof.

23. The method according to claim 21 or 22, wherein the metal compound is a titanium-containing compound, such as TiBALDH.

24. The method according to any one of claims 21-23, further comprising introducing, before, simultaneously with, or after step i), a polynucleotide encoding a polypeptide involved in the biosynthesis of the chemical of interest into said microorganism.

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