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 microorganism for fixing carbon dioxide and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering. In particular, the present application relates to recombinant microorganisms expressing eukaryotic carbon-fixing proteins. BACKGROUND

[0002] In recent years, the development and utilization of microbial cell factories as a green and sustainable method to produce biofuels and chemicals by autotrophic and heterotrophic microorganisms using renewable feedstocks. Autotrophic microorganisms can utilize carbon dioxide as the sole carbon source for growth and chemical synthesis, but the production efficiency is relatively low. While heterotrophic microorganisms synthesize products using various carbon sources as raw materials, the central metabolic process causes carbon loss, so carbon yield is one of the most important parameters to evaluate their manufacturing performance.

[0003] Carbon dioxide fixation in heterotrophic microorganisms is an important research direction in the field of synthetic biology, aiming to convert carbon dioxide into useful organic products, thereby solving problems such as carbon emissions and sustainable energy. At the same time, by introducing an exogenous carbon-fixing pathway to produce chemicals using carbon dioxide and sugar as mixed carbon sources is also a feasible path to improve carbon yield.

[0004] Common microbial factories, such as Escherichia coli, are widely used in biological manufacturing. Since the natural metabolic pathways of microbial factories usually do not include an efficient CO2 fixation pathway, existing solutions are to genetically modify microorganisms to introduce an exogenous CO2 fixation pathway to improve carbon utilization. Therefore, improving the efficiency of CO2 fixation 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-fixing enzyme is Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), which converts CO2 into organic matter through a series of enzyme-catalyzed reactions and even achieves autotrophy (Antonovsky et al., Cell, 2016; Gleizer et al., Cell, 2019). By introducing key enzyme genes of the CBB cycle in E. coli, researchers have successfully achieved a certain degree of carbon dioxide fixation, but the efficiency is not high, because under natural conditions, the affinity of Rubisco for CO2 is lower than that for O2, leading to a greater tendency to react with O2 for photorespiration under current atmospheric conditions, thereby resulting in low efficiency of fixed carbon dioxide. Therefore, improving the efficiency of existing pathways has become an important direction of current research.

[0005] Eukaryotic algae evolved a carbon concentration mechanism based on protein liquid-liquid phase separation to promote the carboxylation reaction by increasing the CO2 concentration around Rubisco, thereby improving carbon fixation efficiency. However, current research on liquid-liquid phase separation has only focused on the analysis of molecular mechanism, and it is still not possible to predict whether this mechanism can be successfully expressed and applied in microorganisms such as bacteria to improve the efficiency of heterologous carbon fixation in recombinant microorganisms.

[0006] Artificial photosynthesis uses sunlight to create high-value chemicals from abundant resources, but it is difficult to achieve high activity and high selectivity production of multi-carbon products. Therefore, in recent years, a semi-artificial photosynthesis system has emerged that uses the high-efficiency light absorption characteristics of semiconductors combined with living biological catalysts to convert carbon dioxide into high-value products powered by solar energy, providing a promising solution to environmental and energy challenges.

[0007] The semi-artificial photosynthesis system mainly includes cell membrane hybrid system and intracellular hybrid system. The most typical cell membrane hybrid system is the deposition of CdS nanoparticles on the cell membrane for acetic acid production (Sakimoto et al., Science, 2016). This system transfers photo-excited electrons from extracellular semiconductors to intracellular to produce reducing equivalents involved in the carbon fixation process, which leads to slow mass transport and energy consumption through the cell membrane across the extracellular biological-inorganic interface. To address this limitation, intracellular biological hybrid systems have been developed (Zhang et al., Nature nano, 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. Although the energy conversion efficiency has improved, the interference and destruction of nanoparticles to cell physiological metabolism are still inevitable.

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

[0009] To meet the above needs, the present application generates semiconductor nanoparticles in situ by mineralization in membraneless organelles within the cell, and co-localizes the nanoparticles with carbon fixation enzymes in the membraneless organelles. This not only improves the light-electricity-chemical energy conversion efficiency, but also reduces the impact of the material on cell physiological metabolism.

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

[0011] In some embodiments, the EPYC1 polypeptide and the mineralization peptide are linked 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 a species of 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 comprising the amino acid sequence of SEQ ID NO: 1 and a Rubisco small subunit polypeptide comprising 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 mineralization 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 a 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 application provides an isolated polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding the combination of polypeptides of the present application.

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

[0019] In a third aspect, the present application provides a recombinant microorganism, e.g., a bacterium or a yeast, comprising a combination of polypeptides of the present application, a combination of polynucleotides or polynucleotides, or a combination of vectors or vectors of the present application.

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

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

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

[0023] i) culturing a microorganism of the present application 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 application provides a method of producing a chemical of interest, comprising the steps of:

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

[0028] ii) culturing, preferably under the presence of a metal-containing compound and / or 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 the group consisting of a) a polynucleotide encoding a chaperone that helps polypeptide folding, such as a GroELS system and rbcX, b) a polynucleotide encoding a 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 to the microorganism, prior to, simultaneously with, or after step i), a polynucleotide encoding a polypeptide involved in the biosynthesis of the chemical of interest. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

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

[0038] Figure 9 shows the rate of CO2consumption in recombinant E. coli (Rubisco+EPYC1) co-transformed with pET22b-LSE and pBAD-prk-G-rbcX. Control represents untransformed E. coli, Rubisco represents recombinant E. coli co-transformed with pET22b-LS and pBAD33-prk-G-rbcX.

[0039] Figure 10 shows the map of expression vector pET22b-LSE-sfGFP-Car9.

[0040] Figure 11 shows the effect of adding different concentrations of TiBALDH to the medium on the growth of recombinant E. coli.

[0041] Figure 12 shows the transmission electron microscopy imaging of the mineralized recombinant E. coli (top panel) and elemental analysis of the granules in the cell (bottom panel).

[0042] Figure 13 shows the scanning transmission electron microscopy high-angle annular dark-field image (HAADF-TEM) measurement, elemental analysis, transmission electron microscopy high-angle annular dark-field image (HRT-TEM) measurement, selected electron diffraction (SEAD) and crystal lattice spacing measurement of the mineralized recombinant E. coli ultrathin section. The top row, left to right, shows the image of the HAADF-TEM measurement, and the bottom row, left to right, shows the image of the HRT-TEM measurement.

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

[0044] Figure 15 shows the effect of light on the redox state in the mineralized recombinant E. coli cells.

[0045] Figure 16 shows the effect of light on the percentage of 3PGA containing C in the mineralized recombinant E. coli cells, with the blank control being the untransformed E. coli. 13 C. The blank control is the untransformed E. coli.

[0046] Figure 17 shows the biosynthetic pathway of 2,3-butanediol.

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

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

[0049] Figure 20 shows the map of plasmid pET22b-LSE-sfGFP-Car9-RABC and pET22b-RABC.

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

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

[0052] Figure 23 shows the laser confocal microscopy imaging of recombinant Corynebacterium glutamicum co-transformed with 301-LSE-prk and 302-G-rbcX. The left panel is the representative field imaging, and the right panel is the magnified image showing the carbon-fixing module (aggregates) with carbon concentration mechanism.

[0053] DETAILED DESCRIPTION

[0054] 1. DEFINITIONS

[0055] Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art in the field of microbiology and recombinant DNA technology, and the practice of the present application will employ, unless otherwise indicated, conventional techniques of microbiology and recombinant DNA technology, which are within the skill of those in the art.

[0056] The term "organelle" is generally considered as a microstructure or microorgan with certain morphology and function scattered in the cytoplasm. They constitute the basic structure of the cell, enabling the cell to achieve normal physiological functions. The organelles in the cell include mitochondria, endoplasmic reticulum, chloroplast, Golgi body and other organelles containing membrane structures, and also include membraneless organelles.

[0057] As used herein, the term "membraneless organelle" refers to a compartment in the cell without membrane structure, in which molecules are aggregated by interaction. For example, nucleolus in the nucleus and promyelocytic leukemia protein body, cytoplasmic processing body and germ granule in the cytoplasm. The phase separation protein (EPYC1) of Chlamydomonas interacts with Rubisco to aggregate in a specific area in the cell to form a membraneless organelle with carbon fixation function.

[0058] As used herein, the term "peptide" means a chain comprising at least two amino acids connected by a peptide bond. The term "polypeptide" can be used interchangeably with "protein" and means a chain comprising ten or more amino acid residues. The chemical formula or sequence of all peptides and polypeptides herein is written in the order from left to right, showing the direction from the amino terminus to the carboxyl terminus.

[0059] The term "mineralizing peptide" refers to a peptide that is capable of converting a metal-containing compound in solution (including cytoplasm) into a mineral particle. Examples of mineralizing peptides include, but are not limited to, Car9 peptide and R5 peptide, which are capable of converting titanium-containing compounds into Ti02. A "metal-containing compound" refers to a compound that contains a metal element.

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

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

[0062] As used herein, the term "polynucleotide" refers generally to a nucleic acid molecule (e.g., 100 nucleotides in length and up to 30 k nucleotides) and a sequence that is complementary (antisense) or identical (sense) to a 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- or double- stranded polynucleotide that is isolated from a naturally occurring gene, or modified to contain non-naturally occurring nucleic acid fragments. A polynucleotide construct comprises an "expression cassette" when it contains control sequences required for expression of a coding sequence of the present application.

[0064] As used herein, the term "exogenous polynucleotide" refers to a nucleotide sequence that is not derived from the host in which it is present. It can be identical to or heterologous to the host's DNA. An example is a sequence of interest 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 that encode antisense oligonucleotides.

[0065] As used herein, the term "expression cassette" refers to a polynucleotide segment comprising a polypeptide-encoding polynucleotide operably linked to additional nucleotide sequences provided for expression of the polynucleotide, e.g., control sequences.

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

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

[0068] "Control sequences" include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the application. Each control sequence can 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, polyadenylation sequence, propeptide sequence, promoter, enhancer, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and a polyadenylation signal.

[0069] For example, a control sequence can be a suitable promoter sequence recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the application. A promoter sequence contains transcription control sequences that mediate the transcription of a polypeptide. The promoter can be any nucleotide sequence, which shows transcriptional activity in the host cell of choice including promoter

[0070] As used herein, the term "operably linked" means in this context that a control sequence is placed in the proper position in relation to the coding sequence of a polynucleotide sequence, whereby the control sequence directs expression of the polypeptide coding sequence.

[0071] Various manipulations of the polynucleotide encoding a polypeptide of interest can be performed so as to improve expression of the polypeptide. Manipulations of the polynucleotide, e.g., codon optimization, are desirable or necessary prior to its insertion into a vector, depending on the expression vector or host. Techniques used to modify polynucleotide sequences with recombinant DNA methods are well known in the art.

[0072] As used herein, the term "recombinant" refers to nucleic acids, vectors, polypeptides, or proteins that are produced using DNA recombination (cloning) methods and are not identical to naturally occurring or wild type nucleic acids, vectors, polypeptides, or proteins.

[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%, more preferably at least 98% homologous to each other, typically remain hybridized to each other under given stringent hybridization and washing conditions.

[0074] For purposes of the present application, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = # of identical positions / total # of positions (i.e., overlapping positions) x 100). Preferably, the two sequences are the same length.

[0075] Those of skill in the art know that various computer programs are available to determine identity between two sequences.

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

[0077] Those of skill in the art know various hybridization conditions, e.g., stringent hybridization conditions and highly stringent hybridization conditions. See, e.g., Sambrook et al., 1989, supra.

[0078] Of course, the polynucleotides of the present application do not include polynucleotides that hybridize only to poly A sequences (e.g., the 3' end poly(A) of mRNA) or 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, which can or have been used as recipients for vectors. The term includes the progeny of the original cell which 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 parental cell can not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0080] 2. A combination of polypeptides

[0081] The present invention provides a combination of polypeptides comprising a Rubisco polypeptide from a Chlamydomonadaceae species, and a fusion polypeptide comprising an EPYC1 polypeptide from a Chlamydomonadaceae species and a mineralization peptide capable of converting a metal-containing compound to a compound capable of converting light energy to chemical energy, such as a metal oxide.

[0082] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from a species of Chlamydomonas, such as C. 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 C. 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 mineralization 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 mineralization 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 a 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, the sfGFP being capable of facilitating mineralization of the Car9 peptide.

[0092] In some embodiments, the fusion polypeptide comprises a peptide linker connecting each polypeptide. 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, a 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 combinations of polypeptides

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

[0097] In some embodiments, the polynucleotide of the application 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 application 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 application 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 application comprises the nucleotide sequence of SEQ ID NO: 20, or a degenerate variant thereof.

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

[0102] The polynucleotide or combination of polynucleotides of the application can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The polynucleotides amplified by PCR as above can be cloned into suitable vectors and characterized by DNA sequence analysis.

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

[0104] The application also relates to the complementary strand of the polynucleotides described herein. A nucleic acid molecule which is complementary to another nucleotide sequence is a nucleic acid molecule which is sufficiently complementary to the other nucleotide sequence so as to be able to form a stable duplex with the other nucleotide sequence.

[0105] For the expression of the polypeptide combination of the application, polynucleotide constructs and vectors (e.g. expression vectors) comprising the polynucleotide or combination of polynucleotides of the application are also provided.

[0106] In some embodiments, the polynucleotide of the application 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 operon.

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

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

[0110] Various manipulations of the polynucleotide or combination of polynucleotides encoding the polypeptides of the application can be performed to allow for expression of the polypeptides. Manipulations of the polynucleotide prior to its insertion into a vector can be desirable or necessary for the purpose of the expression of the polynucleotide. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0111] To identify and select host cells containing the expression vectors of the application, the vector of the application preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or infected cells. A selectable marker is a gene that encodes a product that (1) confers resistance to a drug, (2) confers resistance to a virus, (3) confers resistance to heavy metals, (4) confers auxotrophy, or (5) confers some other trait that allows for easy identification of cells that contain the expression vector. For example, a bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers resistance to an antibiotic such as ampicillin, kanamycin, chloramphenicol, or tetracycline.

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

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

[0114] 4. Recombinant host cells

[0115] The present invention provides a recombinant host cell that is capable of sequestering carbon dioxide and, preferably, forming a membraneless organelle, and more preferably, effecting in situ production of a semiconductor material within the cell.

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

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

[0118] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from a species of 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 mineralization 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 mineralization 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 a 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, the sfGFP being capable of facilitating mineralization of the Car9 peptide.

[0128] In some embodiments, the fusion polypeptide comprises a peptide linker connecting each polypeptide. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, e.g., (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, a 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 chaperone that aids in polypeptide folding and / or a polynucleotide encoding the chaperone. In some embodiments, the chaperone comprises a 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 phosphoribosyl pyrophosphate amidotransferase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to ribulose-5-phosphate, 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] Examples of host cells of the present application include, but are not limited to, bacterial cells, yeast cells, and plant cells (e.g., plant cells of multicellular plants). In some embodiments, the host cell is a bacterial cell, e.g., an aerobic bacterium. Examples of the bacterial cell include, but are not limited to, bacteria from the genus Escherichia (e.g., E. coli), Bacillus (e.g., B. subtilis, B. licheniformis, B. amyloliquefaciens, B. cereus, etc.), or Corynebacterium (e.g., C. glutamicum).

[0134] 5. Method for sequestering carbon dioxide and producing a chemical

[0135] The inventors have surprisingly found that introducing into a microorganism, such as a bacterium (e.g., E. coli), the carbon concentrating mechanism from Chlamydomonas can form a membraneless organelle that enables efficient carbon dioxide fixation in E. coli. The inventors have also found that metal oxide nanoparticles capable of converting light energy into chemical energy can be formed in the membraneless organelle in the microorganism, providing the energy for carbon dioxide fixation, enabling light-driven carbon dioxide fixation.

[0136] The present invention provides a method of fixing carbon dioxide, comprising the steps of:

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

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

[0139] In some embodiments, said recombinant host cell further comprises a phosphoribosyl pyrophosphate amidotransferase (Prk) polypeptide or a polynucleotide encoding said Prk polypeptide.

[0140] The present invention provides a method of fixing carbon dioxide, comprising the steps of:

[0141] i) culturing a recombinant host cell, said recombinant host cell comprising a Rubisco polypeptide from a Chlamydomonas sp., and a fusion polypeptide comprising an EPYC1 polypeptide from a Chlamydomonas sp. 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, in the presence of said metal-containing compound; and

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

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

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

[0145] In some embodiments, said 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 mineralization 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 mineralization 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 a 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, the sfGFP being capable of facilitating mineralization of a Car9 peptide.

[0153] In some embodiments, the fusion polypeptide comprises a peptide linker connecting each polypeptide. 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, a 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 chaperone that helps fold the polypeptide and / or a polynucleotide encoding the chaperone. In some embodiments, the chaperone comprises a 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 ribulose phosphate kinase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to 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 disclosure provides a method of sequestering carbon dioxide, comprising the steps of:

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

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

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

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

[0163] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from a species of the genus Chlamydomonas, such as C. 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 C. 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 a 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, that is capable of facilitating mineralization of a Car9 peptide.

[0173] In some embodiments, the fusion polypeptide comprises a peptide linker linking each polypeptide. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, e.g., (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, a 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 to the microorganism an additional polynucleotide selected from the group consisting of a) a polynucleotide encoding a chaperone such as the GroELS system and rbcX that helps polypeptide folding, b) a polynucleotide encoding a sfGFP polypeptide, c) a polynucleotide encoding a Prk polypeptide, and combinations thereof. The GroELS system typically 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. 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 application provides a method of producing a chemical of interest, comprising the steps of:

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

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

[0180] wherein the polynucleotide or the combination of polynucleotides, or the vector or the combination of vectors, encodes a combination of polypeptides comprising a Rubisco polypeptide from a Chlamydomonadaceae species, and an EPYC1 polypeptide from a Chlamydomonadaceae species or a fusion polypeptide comprising an EPYC1 polypeptide from a Chlamydomonadaceae 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.

[0181] In some embodiments, the Rubisco polypeptide and the EPYC1 polypeptide are from a species of Chlamydomonas, e.g., C. 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 C. 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, e.g., 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, which sfGFP is capable of facilitating mineralization of the Car9 peptide.

[0191] In some embodiments, the fusion polypeptide comprises a peptide linker connecting each polypeptide. In some embodiments, the peptide linker is a flexible linker, such as a GS linker, e.g., (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, an sfGFP polypeptide, a peptide linker, and a R5 peptide.

[0194] In some embodiments, step i) further comprises introducing to the microorganism an additional polynucleotide selected from the group consisting of a) a polynucleotide encoding a chaperone such as the GroELS system and rbcX that helps polypeptide folding, b) a polynucleotide encoding a sfGFP polypeptide, c) a polynucleotide encoding a Prk polypeptide, and combinations thereof. The GroELS system typically 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. 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 to the microorganism, prior to, 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 introducing to the microorganism, prior to, simultaneously with, or after step i), a polynucleotide encoding an alpha-acetolactate synthase (ALS), an alpha-acetolactate decarboxylase (ALDC), a 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] Examples of the host cell include, but are not limited to, bacterial cells, yeast cells, and plant cells (e.g., plant cells of multicellular plants). In some embodiments, the host cell is a bacterial cell, e.g., an aerobic bacterium. Examples of the bacterial cell include, but are not limited to, bacteria from the genus Escherichia (e.g., E. coli), the genus Bacillus (e.g., B. subtilis, B. licheniformis, B. amyloliquefaciens, B. cereus, etc.), or the genus Corynebacterium (e.g., C. glutamicum).

[0201] Embodiments

[0202] Embodiment 1. A recombinant host cell comprising a Rubisco polypeptide from a Chlamydomonadaceae species, and an EPYC1 polypeptide from a Chlamydomonadaceae species.

[0203] Embodiment 2. The recombinant host cell of Embodiment 1, further comprising a phosphoribosyl pyrophosphate amidotransferase (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 a 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, e.g., C. 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, e.g., wherein 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, e.g., wherein 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.

[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 chaperone that aids in polypeptide folding and / or a polynucleotide encoding the chaperone.

[0210] Embodiment 9. The recombinant host cell of embodiment 8, wherein the chaperone comprises a 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, e.g., 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 ribulose phosphate kinase (Prk) polypeptide that catalyzes the phosphorylation of D-ribulose-5-phosphate to ribulose-1,5-bisphosphate or a polynucleotide encoding the Prk polypeptide, e.g., 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 the group consisting of a bacterial cell, a yeast cell, and a plant cell (e.g., a plant cell of a multicellular plant).

[0215] Embodiment 14. The recombinant host cell of any one of Embodiments 1-13, wherein the host cell is a bacterial cell, e.g., 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., E. coli), Bacillus (e.g., B. subtilis, B. licheniformis, B. amyloliquefaciens, B. cereus, etc.), or Corynebacterium (e.g., C. glutamicum).

[0217] Embodiment 16. A method of fixing carbon dioxide, comprising the steps of:

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

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

[0220] Advantages of the Invention

[0221] The present application proves that the carbon concentration mechanism in Chlamydomonas can work in bacteria such as Escherichia coli and Corynebacterium glutamicum, and significantly improves the efficiency of fixing carbon dioxide compared with microorganisms without carbon concentration. The present application also promotes the fixation of carbon dioxide by using light energy through the formation of titanium dioxide nanoparticles capable of converting light energy into chemical energy in a membrane-free organelle, and greatly avoids the interference and damage of nanoparticles to cell physiological metabolism. By fixing carbon dioxide, the yield of chemicals and carbon yield are also significantly improved. EMBODIMENTS

[0222] The following examples provided are only for illustration, not for any limitation of the present application.

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

[0224] The purpose of this example is to verify whether the eukaryotic CBB carbon fixation pathway from Chlamydomonas can achieve functional expression 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, based on the high-copy vector pET22b, the expression vector pET22b-LS (Figure 1, the nucleotide sequences encoding each polypeptide are connected by a ribosome binding site) for expressing Rubisco large and small subunits (chrbcL, SEQ ID NO: 1, chrbcS, SEQ ID NO: 2) was constructed; based on the medium-copy vector pBAD, the expression vector pBAD33-prk-G-rbcX (Figure 2, the nucleotide sequences encoding each polypeptide are connected by a ribosome binding site) 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. The DNA encoding the above sequences was synthesized (Shanghai Generay Biotech Co., Ltd.), and cloned into the vector backbone by Gibson assembly (Gibson Assembly® Master Mix NEB) was used).

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

[0229] 37℃ shaking incubation (220 rpm) was performed with 0.2% L-arabinose and 1 mM IPTG for induction, and OD600 of the culture was measured at 4, 8, 12, 16, 20 and 24 hours, and the growth curve of the recombinant E. coli was plotted, with untransformed and only pBAD33-prk-G-rbcX transformed E. coli as controls. The product of the reaction catalyzed by Prk enzyme, 1,5-diphosphoribulose (RuBP), causes the growth arrest of E. coli, and Rubisco reacts with CO2 with RuBP as substrate to achieve the carbon fixation process (see Figure 3). Therefore, the growth curve of the bacteria shows that when only prk enzyme is expressed, the bacteria stop growing, and when both Rubisco and Prk are expressed, the bacteria resume growing and the growth rate is faster than that of the untransformed bacteria (Figure 4), indicating that the functional expression of Rubisco and Prk in E. coli is achieved, and the carbon fixation module is successfully constructed.

[0230] 1.2, Construction and characterization of recombinant E. coli expressing carbon fixation module and phase separation protein

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

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

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

[0234] Recombinant E. coli cells co-transformed with pET22b-LSEV and pBAD-prk-G-rbcX were observed by confocal microscope, structured illumination microscope and transmission electron microscope. Specifically, 100 μΐ of overnight cultured bacteria were centrifuged (3000-5000 rpm, 5 min); the bacterial cells were collected and washed with ultrapure water for 2 times, then resuspended with 500 μΐ of ultrapure water; 10 μΐ of bacterial suspension was dropped onto a glass slide, and observed by confocal microscope (Nikon A1R, 60X oil lens) and structured light microscope (Nikon N-SIM S) at 488 nm wavelength; and 10 μΐ of bacterial suspension was suspended onto a copper grid, dried by infrared lamp and observed by TEM (FEI Tecnai F30).

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

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

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

[0238] Specifically, 1 mL of overnight cultured bacteria (recombinant E. coli co-transformed with pET22b-LSEV and pBAD-prk-G-rbcX) were centrifuged (3000-5000 rpm, 5 min); the bacterial cells were collected, washed with ultrapure water for 2 times, and resuspended with 1 mL of 100 mM Tris; the suspension was ultrasonicated for 30 min to break the bacterial cells, and then centrifuged to obtain the supernatant (containing Rubisco) which was added into the following prepared 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 phosphocreatine, 2 mM DTT, 5 U / mL 3-phosphoglycerate kinase, 5 U / mL creatine phosphokinase, 5 U / mL glyceraldehyde 3-phosphate dehydrogenase, and appropriate amount of supernatant containing Rubisco) was activated at 30°C for 10 min. Then, the reaction was initiated by adding 0.5 mM RuBP and 0.25 mM NADH, and the change in light absorption was recorded immediately in an enzyme marker (Wallac / Spectromax) at 340 nm (30°C) after monitoring for 5 minutes.

[0240] The CO2consumption rate was determined as follows. 0.5 mL of bacteria (recombinant E. coli co-transformed with pET22b-LSEV and pBAD-prk-G-rbcX) from overnight culture was inoculated into 50 mL of M9 medium containing 0.2% L-arabinose, 20 mM IPTG, 20 mM NaHCO3, 1 pg / mL VB1 and 1 g / L peptone, and incubated at 37°C with shaking (220 rpm) for 12 h. 2 mL of bacterial solution was taken before and after incubation (0 h and 12 h), and the supernatant was collected by centrifugation. The bacterial pellet was dried and weighed. The supernatant was subjected to NMR detection of HCO3 13 and CO2concentrations. The concentrations were calculated according to the standard curve of NaHCO3with corresponding peak area and gradient concentration (0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM). The formula for calculating the consumption rate is - (0 h HCO3

[0241] and CO2concentrations - 12 h HCO3 - and CO2concentrations) / 12 h * dry weight - (0 h HCO3

[0242] wherein the dry weight is the difference between the dry weight of bacteria in 2 mL of bacterial solution after 12 h incubation and the dry weight of bacteria in 2 mL of bacterial solution at 0 h.

[0243] The results show that the presence of phase separation protein EPYC1 significantly improves the activity of Rubisco (Figure 8) and the CO2consumption rate (Figure 9), indicating that the carbon concentrator mechanism improves the carbon fixation efficiency of recombinant E. coli.

[0244] Example 2, Construction and characterization of recombinant E. coli containing mineralized semiconductor-enzyme complex

[0245] The purpose of this example is to construct recombinant E. coli that can form membraneless organelles, achieve in-situ generation of semiconductor materials in cells, and fix carbon dioxide.

[0246] 2.1, Preparation of biomineralized recombinant E. coli

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

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

[0249] The TiBALDH was converted into non-metal element (N, C) doped titanium dioxide (TiO2) by the mineralized peptide (Car9) in the cell to form a semiconductor-enzyme complex. After the recombinant E. coli was mineralized, a membraneless organelle formed by protein aggregation was formed in the cell, which contained Rubisco and semiconductor-doped TiO2.

[0250] Specifically, the screened recombinant E. coli was inoculated into LB liquid medium and cultured at 37°C on a shaker 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, and the culture was cultured at 37°C on a shaker for 12-16 hours, and the OD600 value of the culture was measured.

[0251] As shown in Figure 11, the recombinant E. coli without the addition of TiBALDH showed the highest cell density (OD600 value), and the cell density decreased with the increase of the concentration of TiBALDH.

[0252] 2.2, Characterization of biomineralized recombinant E. coli

[0253] First, the mineralized recombinant E. coli prepared in Example 2.1 was observed using a transmission electron microscope (TEM, FEI Tecnai F30). Specifically, the recombinant E. coli culture prepared in Example 2.1 was centrifuged at 3000-5000 rpm for 5 minutes, the bacterial bodies were collected and washed twice with ultrapure water, resuspended to an OD600 of 0.1 with ultrapure water, then diluted 10-fold with ultrapure water, 10 microliters of the suspension was suspended on a copper grid, dried with an infrared lamp and observed with a TEM. Aggregates were observed at both ends of the cell, which contained nanoparticles with a size of 50-100 nanometers; elemental analysis (Energy Dispersive Spectrometer, TEM-EDS mapping) showed that the particles contained titanium element (Ti) and oxygen element (O) (Figure 12).

[0254] Further, the bacterial cells were embedded and ultra-thin sections were prepared. Specifically, the recombinant E. coli culture prepared in Example 2.1 was centrifuged at 3000-5000 rpm for 5 minutes, and the bacterial cells were collected and washed twice with ultrapure water. The washed bacterial cells were fixed with 2.5% glutaraldehyde for 15 min, 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 time. Subsequently, the samples were infiltrated with i) acetone:epoxy resin (2:1), ii) acetone:epoxy resin (1:1), and iii) epoxy resin for 12 hours each. The infiltrated samples were placed in a small capsule, and an embedding agent, epoxy resin, was added, and cured at 60°C for 48 hours. Then, the samples were cut into ultra-thin sections with a thickness of 60 to 100 nm in a microtome.

[0255] The sections were subjected to scanning transmission electron microscopy high-angle annular dark-field image (HAADF-TEM) measurement, and obvious compartment formation was observed. Elemental analysis (as described above), transmission electron microscopy high-angle annular dark-field image (HRT-TEM), selected area electron diffraction (SAED), and crystal lattice spacing measurement all showed that the compartments contained generated titanium dioxide (Figure 13).

[0256] Example 3, Characterization of the photosynthetic carbon fixation function of the biomineralized recombinant E. coli

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

[0258] 3.1, Photoconversion effect of the biomineralized recombinant E. coli

[0259] A bacterial suspension (40 μL, OD600 = 2) was evenly spread on one side of a 1 cm × 1 cm carbon paper. After drying the suspension, 20 μL of Nafion (0.05% absolute ethanol) was added to the carbon paper, and then the carbon paper was clamped with electrode clips. 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 (50 mL volume). The electrolyte solution (PBS, 40 mL) was injected into the electrolytic cell, purged with nitrogen for 10 minutes, and then sealed. The photoreaction was carried out under an LED light source (white light, or monochromatic light at 450 nm or 620 nm with a bandpass filter, Perfect Light Source, China) at an 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 -1V and 0 to 1V, and a scan rate of 0.01V s. -1 (2) Ampere time method (It), record the current values ​​at potentials of -0.6V and 0.6V (relative to Ag / AgCl).

[0260] The photoconversion effect was measured by the change in photocurrent of the bacterial cells before and after light exposure. The results showed that the intact system had a significant high-response photocurrent curve (Figure 14).

[0261] 3.2 Reducing power within recombinant biomineralized E. coli cells

[0262] The reducing power (NADPH and NADH) in mineralized recombinant E. coli cells was determined using Beyotime's kit (WST-8 method). The results showed that the NAD(P)H / NAD(P)- ratio had a very significant response to light, indicating a photochemical energy conversion (Figure 15).

[0263] 3.3 Carbon fixation of recombinant Escherichia coli cells after biomineralization

[0264] pass 13 The product, 3-phosphoric glycerate (3PGA), was detected by LC-MS using C-labeled substrate NaHCO3.

[0265] Specifically, mineralized recombinant E. coli cells were inoculated with a solution containing 0.2% L-arabinose, 1 mM IPTG, and 20 mM... 13C-labeled NaHC03and 1 pg / mL vitamin B1in M9 medium (Shanghai Genechem Co., Ltd.) to OD 600 = 0.4-0.6, collect cell suspension (20 ml), wash in 10 mL cold (-20 °C) methanol aqueous solution (60%, v / v) and centrifugal collection to terminate cell metabolism as soon as possible, resuspended with 160 μΐ cold (-10 °C) methanol aqueous solution (60%, v / v) after adding 200 μΐ 0.3 M KOH, the mixture was frozen in liquid nitrogen for 5 minutes, and stored at -80 °C for more than 2 hours. The sample was thawed on ice, and 4 μΐ^of acetic acid was added for neutralization. After centrifugation (12,000 g, -10 °C) for 10 minutes, the supernatant was transferred to a clean centrifuge tube for LC-MS / MS detection.

[0266] An Agilent 6460 series LC-MS / MS system equipped with an HPLC system and a triple quadrupole mass spectrometer, an Agilent XDB 18 column (5 uM, 150 mm x 4.6 mm) was used for MS detection in negative ion and selected multiple reaction monitoring (MRM) mode. The 3PGA standard was purchased from Sigma-Aldrich. Methanol was purchased from Fisher Scientific. The mobile phase was solution A (water) and solution B (methanol) (see Table 1). The flow rate was 0.6 mL min-1. The injection volume was 50 μΐ, and the column temperature was 40 °C. The settings of MS were as follows: gas temperature, 350 °C; gas flow, 8 L min-1; nebulizer, 38 psi; sheath gas temperature, 350 °C; sheath gas flow, 9 L min-1; capillary, -3500 V; nozzle voltage, 500 V. The dwell time was set to 200 ms. Metabolites were quantified by their standard curves.

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

[0268] As shown in Figure 16, the proportion of 3PGA containing C in the recombinant Escherichia coli cultured under light was significantly higher than that in the recombinant Escherichia coli cultured in the dark, indicating that the carbon fixation of the recombinant Escherichia coli was promoted under light, i.e. effective photosynthetic carbon fixation was achieved. 13 C of the recombinant Escherichia coli cultured under light, indicating that the carbon fixation of the recombinant Escherichia coli was promoted under light, i.e. effective photosynthetic carbon fixation was achieved.

[0269] Example 4, production of 2,3-butanediol by photosynthetic carbon fixation of recombinant Escherichia coli

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

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

[0272] The recombinant E. coli of the present application 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, and it is also necessary to express a-acetolactate synthase (ALS), a-acetolactate decarboxylase (ALDC), and butanediol dehydrogenase (BDH) in the recombinant E. coli to construct. The polynucleotides encoding ALS (SEQ ID NO: 12), ALDC (SEQ ID NO: 13), and BDH (SEQ ID NO: 14) were amplified by PCR (kindly provided by the State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University) and ligated to the pET22b vector (pET22b-LSE-sfGFP-Car9) which has been pre-connected with the carbon fixation module by the Gibson method, to obtain vectors pET22b-LSE-sfGFP-Car9-RABC and pET22b-RABC (Figure 20).

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

[0274] The mineralized recombinant E. coli culture prepared in Example 4.1 was centrifuged for 5 minutes (at 3000-5000 rpm) and the bacterial cells were collected, washed twice with M9 liquid medium. The washed bacterial cells were collected and resuspended with M9 liquid medium supplemented with ampicillin (100 mg / L) and chloramphenicol (50 mg / L) and 50 g / L glucose (cell density OD at 1-1.5), and then placed in a light incubator for light fermentation (light 2000 lux, 30°C, 150-200 rpm). After 24 hours of fermentation, 2 mL of fermentation broth was centrifuged at 13000 rpm for 5 minutes, the supernatant was retained, 1 mL was used for butanediol extraction, and 1 mL was used for residual glucose determination.

[0275] 1 mL of ethyl acetate was added to 1 mL of the supernatant and ultrasonicated for 30 minutes, after thorough mixing, centrifuged at 12000 rpm for 10 minutes, and the upper solution was taken for GC determination (Agilent, GC8890, HP-5, nitrogen was used as the carrier gas). The temperature of the injector and detector was set to 280°C. The column temperature of the chromatographic column was maintained at 40°C for 3 minutes, and then increased to 240°C at a rate of 20°C / min. The injection volume was 1 μL. The product concentration was calculated using a calibration curve. 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 (at 3000-5000 rpm) and the bacterial cells were collected, washed twice with M9 liquid medium. The washed bacterial cells were collected and resuspended with M9 liquid medium supplemented with ampicillin (100 mg / L) and chloramphenicol (50 mg / L) and 50 g / L glucose (cell density OD at 1-1.5), and then placed in a light incubator for light fermentation (light 2000 lux, 30°C, 150-200 rpm). After 24 hours of fermentation, 2 mL of fermentation broth was centrifuged at 13000 rpm for 5 minutes, the supernatant was retained, 1 mL was used for butanediol extraction, and 1 mL was used for residual glucose determination.

[0277] 1 mL of ethyl acetate was added to 1 mL of the supernatant and ultrasonicated for 30 minutes, after thorough mixing, centrifuged at 12000 rpm for 10 minutes, and the upper solution was taken for GC determination (Agilent, GC8890, HP-5, nitrogen was used as the carrier gas). The temperature of the injector and detector was set to 280°C. The column temperature of the chromatographic column was maintained at 40°C for 3 minutes, and then increased to 240°C at a rate of 20°C / min. The injection volume was 1 μL. The product concentration was calculated using a calibration curve.

[0278] Residual glucose in the culture medium was detected with the Biotecnos glucose detection kit (O-toluidine method), following the instructions of the kit. The original supernatant was diluted as appropriate according to the amount of residual glucose. Carbon yield was calculated according to carbon balance from butanediol production and residual glucose.

[0279] The results show that the recombinant E. coli of the application obtained the highest yield (51 g / L, Figure 18) and carbon yield (1.34 mol / mol, Figure 19) under light conditions, more than 2.5 times that of the control, compared to 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.

[0280] Example 5. Construction and characterization of recombinant C. glutamicum expressing carbon-fixing modules and phase separation proteins

[0281] The purpose of this example is to verify the formation of carbon-fixing membraneless organelles (carbon concentration mechanism) in C. glutamicum.

[0282] An expression vector 301-LSE-prk (Figure 21, nucleotide sequences encoding each polypeptide are connected with ribosome binding sites between them) for expressing Rubisco large subunit (chrbcL, 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 a high-copy vector pec-xc99e (see Li et al., 2021, Heterologous production of a-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method, Bioresource Technology, 341: 125782); an expression vector 302-G-rbcX (Figure 22, nucleotide sequences encoding each polypeptide are connected with ribosome binding sites between them) for expressing GroEL (SEQ ID NO: 6), GroES (SEQ ID NO: 7) and rbcX (SEQ ID NO: 8) was constructed based on a medium-copy vector pz9 (see Li et al., 2021, supra). DNA encoding the above sequences was synthesized (Shanghai Generay Biotech Co., Ltd.), cloned into the vector backbone by Gibson assembly (Gibson Assembly® Master Mix NEB) and transformed into 50 μL of competent cells of Escherichia coli strain DH5a by heat shock method. The transformed cells were spread on LB plates containing 7.5 mg / L chloramphenicol and 25 mg / L kanamycin for overnight culture, and positive clones were picked and verified by sequencing (Shanghai Generay Biotech Co., Ltd.).

[0283] The two plasmids (10 μL each) were transformed into 100 μL of competent cells of Corynebacterium glutamicum strain ATCC14067 by electroporation method (Bio-rad XCell Electroporator, electroporation parameters: 2500 V, 5.0 ms). The transformed cells were recovered in 900 μL of BHIS medium for 3 hours, and then spread on BHISG (S is sorbitol, G is glucose) plates containing 7.5 mg / L chloramphenicol and 25 mg / L kanamycin for 36 hours of culture. Positive clones were picked and verified by sequencing (Shanghai Generay Biotech Co., Ltd.).

[0284] ​Single colonies were picked from the above-mentioned plates cultured for 36 hours and inoculated in BMI medium supplemented with kanamycin (7.5 mg / L) and chloramphenicol (25 mg / L) and cultured to OD600 between 0.6-0.8 before adding inducer IPTG (final concentration 0.1 mM) and cultured at 30°C for 24 hours.

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

[0286] The results showed that obvious fluorescent aggregation compartmental structures were formed in the recombinant C. 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 C. glutamicum, forming a carbon fixation module with carbon concentration mechanism.

[0287] Sequence

Claims

1. A combination of polypeptides comprising a Rubisco polypeptide from a Chlamydomonadaceae species, and a fusion polypeptide comprising an EPYC1 polypeptide from a Chlamydomonadaceae species 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.

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

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

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

6. The combination of polypeptides of 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 of any one of claims 1-6, wherein the Rubisco polypeptide comprises a Rubisco large subunit polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and a Rubisco small subunit polypeptide comprising 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 of any one of claims 1-4, wherein the Rubisco polypeptide and the EPYC1 polypeptide are from different species.

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

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

4.

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

12. The combination of polypeptides of 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 comprising a nucleotide sequence or combination of nucleotide sequences encoding the combination of polypeptides of any one of claims 1-12.

14. A vector or combination of vectors, preferably an expression vector or combination of expression vectors, comprising the polynucleotide or combination of polynucleotides of claim 13.

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

16. The microorganism of claim 15, further comprising a chaperone that aids in folding of the polypeptides and / or a polynucleotide encoding the chaperone.

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

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

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

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

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

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

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

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

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