Pichia pastoris light-operated transcriptional regulation system and application thereof

Abstract

The invention provides a novel pichia pastoris light-operated transcriptional regulation system and application thereof in protein production and metabolic engineering. The inducer of the novel pichia pastoris light-operated transcriptional regulation system does not depend on induction of chemical inducers such as methanol, the expression level and the expression time of recombinant protein are accurately regulated and controlled in an optogenetics mode, and the problems existing in a traditional methanol inducible promoter AOX1 are solved; a safe, flexible and efficient transcriptional regulation tool is provided for recombinant protein, and the method can be applied to the fields of protein production and metabolic engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein engineering or synthetic biology in biotechnology, in particular to a light-controlled transcriptional regulation system of Pichia pastoris and its application, more particularly to a novel light-controlled repressor protein, a light-controlled gene Pichia pastoris transcriptional expression system constructed by using the light-controlled repressor protein, and the application of the light-controlled gene Pichia pastoris expression system in protein production and metabolic engineering. BACKGROUND

[0002] A common way to regulate the expression of foreign genes is to use inducible promoters. Pichia pastoris is one of the most commonly used expression hosts for producing heterologous proteins, and is also a commonly used expression system in metabolic engineering practice, which can secrete high-titer foreign proteins. In Pichia pastoris, the commonly used inducible promoter for expressing foreign proteins is AOX1, which is strongly induced by methanol, and the expressed heterologous proteins can account for 30% of the total soluble proteins of the cell. Although the AOX1 promoter has high expression and strict regulation, it also has the following defects: the inducer of the AOX1 promoter, methanol, is toxic and flammable and explosive, which increases the difficulty of production and purification of food and drugs, and also poses a serious safety hazard during transportation, storage and use; when using the AOX1 promoter, pure oxygen needs to be introduced, and a large amount of heat is generated during methanol metabolism, which increases the cost of oxygen supply and cooling.

[0003] Vogl Thomas et al. (Vogl Thomas, Sturmberger Lukas, Fauland Pia C et al. Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors. [J]. Biotechnol Bioeng, 2018, 115: 1037-1050) studied the regulatory mechanism of AOX1 promoter, which was strongly induced by methanol. In the derepressed state (inhibitory carbon sources such as glucose and glycerol were depleted), three transcriptional activators Mxr1, Mit1 and Prm1 in Pichia pastoris can strongly activate the AOX1 promoter alone. This study achieved overexpression of a single transcriptional activator, converting Pichia pastoris into glucose / glycerol regulation, thereby not relying on methanol regulation. However, its yield is lower than that of methanol induction, and foreign proteins can only be expressed when glucose / glycerol is depleted, which cannot be transcribed in real time. Zhu Qiaoyun et al. (Zhu Qiaoyun, Liu Qi, Yao Chaoying et al. Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol. [J]. Nucleic Acids Res, 2022, 50: 10187-10199) synthesized Pichia pastoris transcription devices by fusing bacterial DNA binding proteins with yeast transcriptional activation domains (i.e. transcriptional activators removing DNA binding domains, only retaining activation domains) and connecting bacterial binding sequences with yeast core promoters. By fine-tuning the copy number of bacterial binding sequences, spacers and input promoters, a constitutive transcription device library (cTRDL) composed of 126 transcription devices was constructed, with expression intensity of 16-520% of AOX1 promoter; and an inducible TRDL (iTRDL) composed of 162 methanol-induced transcription devices was constructed, with expression intensity of 30-500% of AOX1 promoter. However, this expression system still relies on methanol regulation.Liu Qi et al. (Liu Qi, Song Lili, Peng Qiangqiang et al. A programmable high-expression yeast platform responsive to user-defined signals. [J]. Sci Adv, 2022, 8: eabl5166) also used the synthetic transcription signal amplification device (iTSAD) constructed by Zhu Qiaoyun et al. in Pichia pastoris, which has a bacterial-yeast trans-acting factor and a bacterial-yeast cis-acting element. On this basis, CRISPR activation and interference devices were designed to strictly regulate iTSAD. Although it can achieve methanol-independent regulation, and the yield is similar to traditional methanol induction, it still needs to add exogenous chemical inducers for induction expression. Wang Zhiqian et al. constructed a new type of optogenetic transcriptional regulation system in Pichia pastoris based on light-sensitive protein EL222. EL222 was fused with the activation domain of transcription activator VP16 to form a trans-acting factor, and nucleic acid sequence C120 was combined with a truncated constitutive promoter to form a cis-acting element. EL222 forms a homodimer under blue light, specifically binds to the C120 sequence, and activates the expression of the downstream promoter. However, this system expresses exogenous proteins under blue light, and the blue light has limited penetration ability in high-density fermentation of Pichia pastoris, limiting its practical application.

[0004] Therefore, as described above, although the AOX1 promoter has high expression and strict regulation, there are also many safety defects and costs of oxygen supply and cooling. Chemical induction regulation has good effects and high expression efficiency, but has many side effects and cytotoxicity. A few gene expression systems using light-sensitive proteins also need to add exogenous chemicals due to low expression of the target protein. Therefore, it is urgent to explore a new transcription regulation method for Pichia pastoris foreign protein expression. Light is an inducer that is easy to operate in time and space, generally has no toxicity to cells, and can introduce light-regulated proteins or light-sensitive proteins from distant evolutionary species into eukaryotic cells, and modify them into light-regulated transcription factors to facilitate light-regulated expression of target genes. However, there are few reports on this aspect at present. The currently known other light-sensitive proteins are mainly divided into three categories: one is the light receptor protein containing a light-oxygen-voltage (LOV) domain, such as a light-sensitive pigment; two are photolyase-like cryptochromes; and the third is a blue light using FAD (BLUF) discovered in recent years. The light-sensitive pigment is the most common light receptor protein containing the LOV domain, and the most studied ones are phototropin 1, white-collar-1 (WC-1), white-collar-2 (WC-2), PYP (photoactive yellow protein), Phy3, VIVID, etc. SUMMARY

[0005] In view of the problems of cell toxicity, high cost, and inconvenient regulation existing in the expression system, the present application develops a new type of transcription regulation device in Pichia pastoris that is not dependent on methanol induction, which is a high-efficiency light-regulated transcription expression system composed of two parts of recombinant light-sensitive transcription factor and recombinant cis-acting element, and has good gene expression regulation ability to solve the problems existing in the traditional methanol-induced AOX1 promoter, and provides a safe, flexible, and efficient transcription regulation tool for recombinant protein production.

[0006] In one aspect, the present application provides a new transcription regulation system of Pichia pastoris: the LacO nucleic acid sequence from the lactose operon and the cAOX1 promoter constitute a cis-promoter, and the LacI protein from the light control element LOV2 (i.e. OptoLacI) and the transcription activator are fused and expressed to constitute a trans-activator.

[0007] In certain embodiments, the recombinant light-sensitive transcription factor is a light- controlled repressor protein comprising a DNA-binding domain and a light-sensitive domain, the light-controlled repressor protein comprising a LacI protein and a LOV domain inserted between adjacent amino acids of Loop-1, Loop-2, or Loop-3 region of the LacI protein.

[0008] In certain embodiments, the LOV domain is a LOV2 domain; preferably, the LOV domain is selected from the LOV2 domain of the oat photosensory protein 1 gene (AsLOV2), the light-sensitive protein EL222 from Rhodobacter sphaeroides, the light-sensitive domain LOV2 from Arabidopsis thaliana, or a mutant thereof; preferably, the mutant is a circularly permuted mutant or a mutant that alters the speed of response; preferably, the LOV domain is AsLOV2, e.g., comprising the sequence set forth in SEQ ID NO: 1; preferably, the LOV domain is cpLOV27, e.g., comprising the sequence set forth in SEQ ID NO: 2.

[0009] In certain embodiments, the insertion position is selected from the following amino acid positions of the LacI protein: between positions 335 and 336, between positions 314 and 315, between positions 315 and 316, between positions 316 and 317, between positions 334 and 335, between positions 336 and 337, between positions 337 and 338, between positions 338 and 339, between positions 152 and 153; preferably, the insertion position is between positions 335 and 336 of the LacI protein.

[0010] In certain embodiments, the insertion position is selected from the following amino acid positions of the LacI protein: between positions 311 and 312, between positions 153 and 154; preferably, the insertion position is between positions 311 and 312 of the LacI protein.

[0011] In certain embodiments, the LacI protein is wild-type; preferably, the wild-type LacI protein comprises the sequence set forth in SEQ ID NO: 14.

[0012] In certain embodiments, the LacI protein is a modified LacI protein comprising an amino acid substitution compared to a wild-type LacI protein, the amino acid substitution being selected from (i) an amino acid substitution at position 220 to F, and / or, (ii) an amino acid substitution at position 84 to E, C, S, T, or I; preferably, the modified LacI protein comprises the sequence set forth in SEQ ID NO: 15 or 16, or a sequence comprising an amino acid substitution at position 84 to C, S, T, or I compared to SEQ ID NO: 15 or 16.

[0013] In certain embodiments, the N-terminus and / or C-terminus of the LOV domain is optionally linked to the LacI protein via a peptide linker.

[0014] In certain embodiments, the light-controllable repressor protein comprises a sequence as set forth in any one of SEQ ID NOs: 8, 17, 4-6, 7, 9-11, 12 or a sequence as set forth in SEQ ID NO: 3 or 13; preferably, the light-controllable repressor protein comprises a sequence as set forth in SEQ ID NO: 8 or 17; preferably, the light-controllable repressor protein comprises a sequence as set forth in SEQ ID NO: 3.

[0015] In certain embodiments, the second polypeptide is a transcription factor protein with the ability to independently recruit RNA polymerase; preferably, VP16, VP64, VPR, Mit1; preferably, the nucleotide sequence of the VP16 gene comprises SEQ ID NO: 18 or a degenerate sequence thereof.

[0016] In certain embodiments, the third polypeptide is a photo-sensitive degron (PSD) comprising a LOV domain and an ornithine decarboxylase (ODC).

[0017] In certain embodiments, the photo-sensitive degron (PSD) comprises a LOV domain, which is a LOV2 domain; preferably, the LOV domain is selected from the LOV2 domain of the oat photoreceptor 1 gene (AsLOV2), the light-sensitive protein EL222 from Rhodobacter sphaeroides, the light-sensitive domain LOV2 from Arabidopsis thaliana, or a mutant thereof; preferably, the photo-sensitive degron (PSD) comprises a sequence as set forth in SEQ ID NO: 21.

[0018] In one aspect, the present application provides a nucleic acid construct comprising a nucleotide sequence encoding the light-controllable gene expression system as described above.

[0019] In one aspect, the present application provides a vector comprising the nucleic acid construct as described above.

[0020] In certain embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a nucleotide sequence encoding a first polypeptide, a second polypeptide and / or a third polypeptide, and the second nucleic acid construct comprises a LacO operator, a promoter, a gene of interest, and a terminator; preferably, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes.

[0021] In some embodiments, the first polypeptide and the second polypeptide comprised in the first nucleic acid construct are connected by a peptide linker. Preferably, the peptide linker connecting the first polypeptide and the second polypeptide in the first nucleic acid construct is a GGGGS linker.

[0022] In some embodiments, the second polypeptide and the third polypeptide comprised in the first nucleic acid construct are connected by a peptide linker. Preferably, the peptide linker connecting the first polypeptide and the second polypeptide in the first nucleic acid construct is a GGGGS linker.

[0023] In some embodiments, the LacO operator comprised in the second nucleic acid construct is selected from LacOl, LacOid or a combination thereof.

[0024] In some embodiments, the LacO operator comprised in the second nucleic acid construct comprises at least one copy (e.g. 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, e.g. 3-5) of the LacOl operator sequence; preferably, the LacOl operator sequence comprises the sequence set forth in SEQ ID NO: 19.

[0025] In some embodiments, the promoter comprised in the second nucleic acid construct is a DNA sequence that is recognized, bound and transcribed by RNA polymerase in the presence of the second polypeptide of claim 1 or 10. Preferably, the promoter nucleic acid sequence comprised in the second nucleic acid construct is selected from AOXl minimal promoter, GAP minimal promoter, TEF minimal promoter, HIS3 minimal promoter, CYC minimal promoter. Preferably, the promoter nucleic acid sequence comprised in the second nucleic acid construct comprises the sequence set forth in SEQ ID NO: 20.

[0026] In some embodiments, the first nucleic acid construct further comprises a promoter and a terminator operably linked to the nucleotide sequence encoding the first polypeptide, the second polypeptide and / or the third polypeptide; preferably, the promoter is selected from TEF promoter, GAP promoter, ADH2 promoter, cP AOX1 Preferably, the terminator is selected from AOXl promoter, RPL15A promoter, CYC1 promoter.

[0027] In one aspect, the present application provides a host cell comprising the nucleic acid construct or the vector described above, preferably, the host cell is a eukaryotic cell; preferably, the host cell is Pichia pastoris.

[0028] In one aspect, the present application provides the light-controlled repressor protein, the nucleic acid construct, the vector, the host cell, the system, or the kit described above for use in regulating the expression of a gene of interest; preferably, the use comprises regulating the expression of a protein; preferably, the use comprises regulating a metabolic pathway and / or biosynthesis.

[0029] The present application constructs a Pichia pastoris light-controlled transcriptional regulation system that does not rely on chemical inducers such as methanol, and precisely regulates the expression level and expression time of recombinant proteins through optogenetics, which can be applied in the fields of protein production and metabolic engineering. The novel light-controlled gene expression system provided by the present application comprises a LacO nucleic acid sequence from a lactose operon and a cAOX1 promoter to form a cis promoter, and a LacI protein (i.e. OptoLacI) of a light-controlled element LOV2 and a transcriptional activator to form a trans activator. The system meets the requirements of single-component regulation of gene expression, realizes high temporal and spatial resolution of gene expression regulation, and has two systems of blue light and dark induction. The LacI and the operon sequence form a modular light-controlled system, which can be used immediately and has stronger system compatibility. The Pichia pastoris light-controlled transcriptional regulation system that does not rely on chemical inducers such as methanol precisely regulates the expression level and expression time of recombinant proteins through optogenetics, which can be applied in the fields of protein production and metabolic engineering.

[0030] The Pichia pastoris light-controlled gene expression system constructed based on the light-controlled repressor protein can regulate the expression intensity of the target gene by changing the frequency and intensity of blue light irradiation. The blue light-induced gene expression system of the present application has extremely strict induction advantages and little expression leakage. The dark-induced gene expression system of the present application has good application effect in protein expression and better induction effect than methanol induction. The dark-induced gene expression system of the present application has excellent application effect in metabolic regulation and has more strict regulation on metabolic pathways. Both the dark-induced gene expression system and the blue light-induced gene expression system of the present application have excellent spatial strictness. BRIEF DESCRIPTION OF DRAWINGS

[0031] The present application is further described in the following non-limiting drawings:

[0032] Figure 1 : Schematic diagram of the genome structure of the light-controlled Pichia pastoris with different LacO copy numbers.

[0033] Figure 2 : Performance test of the light-controlled Pichia pastoris strains with different LacO copy numbers.

[0034] Figure 3 : Schematic diagram of the genome structure of the light-controlled Pichia pastoris with optimized elements.

[0035] Figure 4 Optimization of the performance of the light-controlled Pichia pastoris strains.

[0036] Figure 5 Trends of the induction intensity and fold of the light-controlled Pichia pastoris strains over time.

[0037] Figure 6 Comparison of the expression intensity of the light-controlled transcriptional activation device and the methanol-inducible P AOX1 Comparison of the expression intensity. DETAILED DESCRIPTION

[0038] DEFINITIONS

[0039] Unless otherwise defined, scientific and technical terms used in this text have the meanings commonly understood by one of ordinary skill in the art. In order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0040] As used herein, the term "comprising" and variations thereof, are synonymous with the term "including" and variations thereof, and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of and "consisting of can be used in place of "comprising" and "including" to provide more specific embodiments, and are also disclosed.

[0041] As used herein, the sequence of the term "LacI protein" is well known to those skilled in the art and can be found in various public databases, for example, GenBank: CAF2498598.1. In this text, when referring to the amino acid position of the LacI protein, the description is made with reference to the sequence shown in SEQ ID NO: 14. For example, the expression "the amino acid residue at position 314 of the LacI protein" refers to the amino acid residue at position 314 of the sequence shown in SEQ ID NO: 14 and its corresponding position. The corresponding position refers to the position in the sequence to be compared that is equivalent to the particular amino acid position in SEQ ID NO: 14, when the sequence to be compared is optimally aligned (i.e., to obtain the highest percentage of identity) with SEQ ID NO: 14.

[0042] The terms "light-controlled", "light-sensitive" and "light-induced" proteins have the same meaning in this text and are used interchangeably, referring to proteins that are sensitive to light, which can be irradiated with light of the corresponding wavelength at different intensities or different frequencies, adjusting the conformation or configuration of the protein and thus affecting its activity, including activating, enhancing or repressing its activity.

[0043] "Host" refers to a eukaryote, including single-cell eukaryotes such as yeast, such as Pichia pastoris.

[0044] An exogenous protein can also be referred to as a "protein of interest" and refers to any useful protein, e.g., a useful eukaryotic protein that is desired to be expressed in a eukaryotic host cell, including naturally occurring or artificially modified or mutated useful proteins, particularly proteins that must be post-translationally modified (e.g., glycosylated, amidated, etc.) to be active, which modifications are obtained when the proteins are expressed in eukaryotic cells.

[0045] As used herein, the term "LOV domain" refers to a light-oxygen-voltage (LOV) domain, typically derived from a photoreceptor protein of a plant, alga, or bacterium. Preferably, the LOV domain is a LOV2 domain, preferably from Avena sativa, Rhodobacter sphaeroides, or Arabidopsis thaliana. In certain exemplary embodiments, the LOV domain has the amino acid sequence set forth in SEQ ID NO: 1 or 2 or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0046] As used herein, the term "circular permutation" refers to the linking of the original N- and C-termini of a protein by a linker and the introduction of new termini by cleavage of the protein's original peptide bond. The new protein thus formed is called a circularly permuted mutant.

[0047] As used herein, the term "gene" is used broadly to refer to a DNA nucleic acid associated with a biological function. As used herein, a "gene of interest" refers to any nucleic acid sequence that can be and / or will be of interest for controlling the level of its transcription.

[0048] As used herein, the term "transcription" refers to the synthesis of RNA from a DNA template; the term "translation" refers to the synthesis of a polypeptide from an mRNA template. Together, transcription and translation are referred to as "expression."

[0049] As used herein, the term "operably linked" refers to functional linkage between elements, wherein the elements are in a relationship that allows them to function in their intended manner. For example, for a promoter, the functional linkage between the promoter and a gene of interest is such that the promoter sequence is able to initiate transcription of the gene of interest.

[0050] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements of genetic material it carries in the host cell. Vectors typically contain one or more restriction enzyme recognition sites and / or sites for site-specific recombination into which foreign DNA fragments can be cut and ligated. Vectors can contain a variety of expression control elements, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, vectors can contain a replication origin.

[0051] As used herein, the term "expression cassette" refers to a combination of a gene to which control elements are operably linked for expression. Control elements can include, for example, promoter sequences, initiation codons, termination codons, terminators. Expression cassettes can include additional regulatory sequences and other sequences, such as enhancers, signal sequences, enhancers, introns, IRES sequences, etc.

[0052] As used herein, the term "promoter" refers to an expression control element that permits binding of RNA polymerase and initiation of transcription.

[0053] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, fungal cells.

[0054] In one aspect, the present application provides a light-controlled repressor protein comprising a LacI protein and a LOV domain inserted between adjacent amino acids of Loop-1, Loop-2 or Loop-3 region of the LacI protein.

[0055] In certain embodiments, the N- and / or C-terminus of the LOV domain is optionally linked to the LacI protein by a peptide linker. In certain embodiments, the N- and / or C-terminus of the LOV domain is linked to the LacI protein by a peptide linker, e.g., a peptide linker comprising one or several flexible amino acids.

[0056] LOV domain

[0057] Any LOV domain known in the art can be used in the present application. In certain embodiments, the LOV domain is a LOV2 domain. LOV2 domains of different species origin are all photore- sponsive domains, play the same role in different species; and are structurally highly similar in three- dimensional structure and the corresponding allosteric principle is the same. Therefore, LOV2 domains of different species origin are all suitable for use in the present application.

[0058] In certain embodiments, the LOV domain is selected from the LOV2 domain of the oat photoreceptor 1 gene (AsLOV2), the light-sensitive protein EL222 from Rhodobacter sphaeroides, the light-sensing domain LOV2 from Arabidopsis thaliana, or a mutant thereof.

[0059] In certain embodiments, the mutant is a circularly permuted mutant. In this context, "circularly permuted" has its art-recognized meaning, which refers to the original N- and C-termini of a protein being linked by a linker and introducing new termini by cleaving the original peptide bond of the protein.

[0060] In certain embodiments, the LOV domain is AsLOV2. In certain embodiments, the AsLOV2 comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0061] In certain embodiments, the LOV domain is a circularly permuted mutant of AsLOV2. In certain embodiments, the circularly permuted mutant of AsLOV2 is cpLOV27, which comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0062] In certain embodiments, the LOV domain is the light-sensitive protein EL222 from Rhodobacter sphaeroides.

[0063] In certain embodiments, the LOV domain is the light-sensing domain LOV2 from Arabidopsis thaliana.

[0064] LacI protein

[0065] In certain embodiments, the LacI protein is a wild-type LacI protein. In certain embodiments, the wild-type LacI protein comprises the sequence set forth in SEQ ID NO: 14.

[0066] In certain embodiments, the LacI protein is a modified LacI protein comprising an amino acid substitution selected from (i) an amino acid substitution at position 220 to F, and / or, (ii) an amino acid substitution at position 84 to E, C, S, T, or I, as compared to a wild-type LacI protein.

[0067] In certain embodiments, the modified LacI protein comprises the sequence set forth in SEQ ID NO: 15 or 16, or comprises an amino acid substitution at position 84 to C, S, T, or I as compared to SEQ ID NO: 15 or 16. In certain embodiments, the modified LacI protein comprises the sequence set forth in SEQ ID NO: 15 or 16.

[0068] Light-responsive repressor protein responsive to darkness

[0069] In certain embodiments, the light-controllable repressor protein of the present application has a dark-responsive activity. In this context, "dark-responsive activity" refers to the light-controllable repressor protein does not exert a repressive effect under dark conditions and allows the expression of a gene of interest. The light-controllable repressor protein preferably exerts a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) conditions and inhibits the expression of a gene of interest.

[0070] In certain embodiments, the insertion position in the dark-responsive light-controllable repressor protein is selected from the following amino acid positions of the LacI protein: between positions 335 and 336 (such as Loop-2 (Q335-T336)), between positions 314 and 315 (such as Loop-1 (K314-G315)), between positions 315 and 316 (such as Loop-1 (G315-N316)), between positions 316 and 317 (such as Loop-1 (N316-Q317)), between positions 334 and 335 (such as Loop-2 (T334-Q335)), between positions 336 and 337 (such as Loop-2 (T336-A337)), between positions 337 and 338 (such as Loop-2 (A337-S338)), between positions 338 and 339 (such as Loop-2 (S338-P339)), and between positions 152 and 153 (such as Loop-3 (D152-Q153)).

[0071] In certain embodiments, the insertion position in the dark-responsive light-controllable repressor protein is between positions 335 and 336 (such as Loop-2 (Q335-T336)) of the LacI protein.

[0072] In certain embodiments, the dark-responsive light-controllable repressor protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 8, 17, 4-6, 7, 9-11, 12. In certain embodiments, the dark-responsive light-controllable repressor protein comprises the amino acid sequence set forth in SEQ ID NO: 8 or 17.

[0073] Light-responsive repressor protein responsive to blue light

[0074] In certain embodiments, the light-controllable repressor protein of the present application has a blue light responsive activity. In this context, the "blue light responsive activity" refers to the light-controllable repressor protein does not exert a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) conditions and allows the expression of a gene of interest. The light-controllable repressor protein preferably exerts a repressive effect under dark conditions and inhibits the expression of a gene of interest.

[0075] In certain embodiments, the insertion position in the blue light responsive light-controllable repressor protein is selected from the following amino acid positions of the LacI protein: between positions 311 and 312 (such as Loop-1 (Q311-A312)), between positions 153 and 154 (such as Loop-3 (Q153-T154)).

[0076] In certain embodiments, the insertion position in the blue light responsive light-controllable repressor protein is between positions 311 and 312 (such as Loop-1 (Q311-A312)) of the LacI protein.

[0077] In certain embodiments, the blue light responsive light-controllable repressor protein comprises the amino acid sequence set forth in SEQ ID NO: 3 or 13. In certain embodiments, the blue light responsive light-controllable repressor protein comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0078] The light-controllable repressor protein of the present application can be produced by various methods known in the art, for example, by a genetic engineering method (recombinant technology), or by a chemical synthesis method (e.g., Fmoc solid phase method). The light-controllable repressor protein of the present application is not limited by the way it is produced.

[0079] Nucleic acid constructs and vectors

[0080] In another aspect, the present application provides a nucleic acid construct comprising a nucleotide sequence encoding the light-controllable repressor protein of the present application.

[0081] In another aspect, the present application provides a vector comprising the nucleic acid construct as described above.

[0082] In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is a plasmid.

[0083] In certain embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a nucleotide sequence encoding the light-controllable repressor protein of the present application, and the second nucleic acid construct comprises a LacO operator, a promoter, a gene of interest, and a terminator.

[0084] In certain embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a nucleotide sequence encoding the light-controllable repressor protein of the present application, and the second nucleic acid construct comprises a promoter, a LacO operator, and a cloning site (e.g., a multiple cloning site, MCS) for integration of a gene of interest.

[0085] In certain embodiments, the second nucleic acid construct comprises a LacO operator selected from the group consisting of LacOl, LacOid, or a combination thereof.

[0086] In certain embodiments, the second nucleic acid construct comprises a LacO operator comprising at least one copy (e.g., 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, e.g., 3-5) of a LacOl operator sequence. In certain embodiments, the second nucleic acid construct comprises a LacO operator comprising 3-5 copies of a LacOl operator sequence. In certain embodiments, the LacOl operator sequence comprises the sequence set forth in SEQ ID NO: 19.

[0087] In certain embodiments, the second nucleic acid construct comprises a LacO operator comprising LacOl and LacOid. In certain embodiments, the LacOid is located downstream of the LacOl. In certain embodiments, the LacO operator comprises LacOl and LacOid in the 5' to 3' direction.

[0088] In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controllable repressor protein of the present application. In certain embodiments, the promoter comprised by the first nucleic acid construct can be any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, i.e., as long as it uses the RNA polymerase of the host cell. In certain embodiments, the promoter is selected from the group consisting of a TEF promoter, a GAP promoter, an ADH2 promoter, a cP AOX1 promoter.

[0089] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes of the same vector.

[0090] Host cells

[0091] In another aspect, the present application provides a host cell comprising the nucleic acid construct or the vector as described above.

[0092] In certain embodiments, the host cell is a fungus. In certain embodiments, the host cell is Pichia pastoris.

[0093] System for regulating expression of a gene of interest

[0094] In another aspect, the present application provides a system for regulating expression of a gene of interest, comprising:

[0095] (1) a first nucleic acid construct comprising a nucleotide sequence encoding a light- controlled repressor protein of the present application;

[0096] (2) a second nucleic acid construct comprising a promoter, a LacO operator, and a second nucleic acid construct of a gene of interest.

[0097] In certain embodiments, the second nucleic acid construct comprises a LacO operator selected from the group consisting of LacOl, LacOid, or a combination thereof.

[0098] In certain embodiments, the second nucleic acid construct comprises a LacO operator comprising at least one copy (e.g., 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, e.g., 3-5) of a LacOl operator sequence. In certain embodiments, the second nucleic acid construct comprises a LacO operator comprising 3-5 copies of a LacOl operator sequence. In certain embodiments, the LacOl operator sequence comprises the sequence set forth in SEQ ID NO: 19.

[0099] In certain embodiments, the second nucleic acid construct comprises a LacO operator comprising LacOl and LacOid. In certain embodiments, the LacOid is located downstream of the LacOl. In certain embodiments, the LacO operator comprises LacOl and LacOid in a 5’ to 3’ orientation.

[0100] In certain embodiments, the second nucleic acid construct comprises a plurality of genes of interest arranged in tandem. In certain embodiments, additional promoters are optionally inserted between the plurality of genes of interest.

[0101] In certain embodiments, the light-controlled repressor protein is selected from the group consisting of a light-controlled repressor protein of the present application that responds to darkness. In such embodiments, the system for regulating expression of a gene of interest is a dark-induced gene expression system, the light-controlled repressor protein does not exert a repressive effect under dark conditions and allows expression of the gene of interest, and preferably exerts a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) conditions and inhibits expression of the gene of interest.

[0102] In certain embodiments, the light-controllable repressor protein is selected from the blue light responsive light-controllable repressor proteins of the present application. In such embodiments, the system for modulating expression of a gene of interest is a blue light inducible gene expression system, the light-controllable repressor protein does not exert a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) conditions and allows expression of the gene of interest, and preferably exerts a repressive effect under dark conditions and inhibits expression of the gene of interest.

[0103] Kit

[0104] In one aspect, the present application provides a kit comprising a host cell and an expression vector, wherein: the host cell has integrated into its genome an exogenous nucleotide sequence encoding a light-controllable repressor protein of the present application; and, the expression vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises an exogenous nucleotide sequence encoding the light-controllable repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator, and a cloning site (e.g., a multiple cloning site, MCS) for integration of a gene of interest.

[0105] In another aspect, the present application provides a kit comprising a host cell and an expression vector, wherein: the host cell has integrated into its genome an exogenous nucleotide sequence encoding a light-controllable repressor protein of the present application; and, the expression vector comprises the second nucleic acid construct.

[0106] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, preferably, the host cell is a Pichia pastoris.

[0107] In another aspect, the present application provides a kit comprising a host cell and an expression vector, wherein: the host cell has not integrated into its genome the exogenous nucleotide sequence encoding a light-controllable repressor protein; and, the expression vector comprises the first nucleic acid construct and a second nucleic acid construct.

[0108] In certain embodiments, the second nucleic acid construct of any of the above aspects, wherein the LacO operator is selected from LacOl, LacOid, or a combination thereof.

[0109] In certain embodiments, the second nucleic acid construct of any of the above aspects, wherein the LacO operator comprises at least one copy (e.g., 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, e.g., 3-5) of a LacOl operator sequence. In certain embodiments, the LacO operator comprises 3-5 copies of a LacOl operator sequence. In certain embodiments, the LacOl operator sequence comprises the sequence set forth in SEQ ID NO: 19.

[0110] In certain embodiments, the second nucleic acid construct of any of the above aspects, wherein the LacO operator further comprises LacOid. In certain embodiments, the LacOid is located downstream of LacOl. In certain embodiments, the LacO operator comprises LacOl and LacOid from 5' to 3' direction.

[0111] In certain embodiments, the kit of any of the above aspects is used for the purpose of regulating expression of a gene of interest.

[0112] In certain embodiments, the light-controlled repressor protein is selected from the group consisting of the light-controlled repressor proteins of the present application that are responsive to darkness. In such embodiments, the kit is used for dark-induced gene expression, i.e. does not exert a repressive effect and allows expression of the gene of interest under dark conditions, and preferably exerts a repressive effect and inhibits expression of the gene of interest under blue light (e.g. 430-495 nm, such as 450-480 nm) conditions.

[0113] In certain embodiments, the light-controlled repressor protein is selected from the group consisting of the light-controlled repressor proteins of the present application that are responsive to blue light. In such embodiments, the kit is used for blue light-induced gene expression, i.e. does not exert a repressive effect and allows expression of the gene of interest under blue light (e.g. 430-495 nm, such as 450-480 nm) conditions, and preferably exerts a repressive effect and inhibits expression of the gene of interest under dark conditions.

[0114] Methods and uses for regulating expression of a gene of interest

[0115] In another aspect, the present application provides the use of a light-controlled repressor protein, a nucleic acid construct, a vector, a host cell, a system, or a kit of the present application for the purpose of regulating expression of a gene of interest. The regulation of expression of a gene of interest is performed in vitro. The use is for non-therapeutic purposes.

[0116] The gene of interest involved in the present application preferably encodes a protein of interest. There is no limitation as to the protein of interest. The protein of interest can be a polypeptide that is not naturally occurring in the host cell, i.e. a heterologous protein, or can be native to the host cell, i.e. a homologous protein of the host cell. The protein of interest involved in the present application can be any peptide with biological activity, including but not limited to enzymes, regulatory proteins, receptors, hormones, cytokines, membrane proteins, transport proteins, antigens, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, antibodies or derivatives thereof, and the like. In certain embodiments, the protein of interest can be any protein suitable for therapeutic or prophylactic purposes in a mammal (e.g. a human).

[0117] Furthermore, it is known to those skilled in the art that microorganisms can produce some important compounds through anabolic metabolism, and thus by modulating the anabolic pathway in a microbial cell, the amount of the metabolic product can be altered (e.g., increased). Thus, the light-controllable repressor protein, nucleic acid construct, vector, host cell, system, or kit of the present application can also be used to modulate a metabolic pathway in a microorganism. In certain embodiments, the metabolic pathway can be endogenous or exogenous to Pichia pastoris. Thus, in certain embodiments, the gene of interest contemplated by the present application also includes a gene involved in the biosynthesis of a compound or an anabolic pathway in a microorganism, thereby allowing modulation of the biosynthesis of a compound or an anabolic pathway in a microorganism.

[0118] In another aspect, the present application provides a method for modulating the expression of a gene of interest, comprising:

[0119] (1) providing a system for modulating the expression of a gene of interest of the present application in a host cell;

[0120] (2) culturing the host cell under conditions that allow the expression of the gene of interest and inducing the expression of the gene of interest.

[0121] In certain embodiments, step (1) comprises: providing an expression vector comprising the first nucleic acid construct and the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding the light-controllable repressor protein of the present application integrated in its genome.

[0122] In certain embodiments, step (1) comprises: providing an expression vector comprising the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell comprises the exogenous nucleotide sequence encoding the light-controllable repressor protein integrated in its genome.

[0123] In certain embodiments, step (1) comprises: providing an expression vector comprising the first nucleic acid construct and the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell does not comprise the exogenous nucleotide sequence encoding the light-controllable repressor protein integrated in its genome.

[0124] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell is Pichia pastoris.

[0125] In certain embodiments, the step of introducing the expression vector into a host cell described in any of the above embodiments can be performed by any means known in the art, such as transfection, transformation, or transduction.

[0126] In certain embodiments, the second nucleic acid construct described in any of the above embodiments, wherein the LacO operator is selected from the group consisting of LacOl, LacOid, or a combination thereof.

[0127] In certain embodiments, the second nucleic acid construct described in any of the above embodiments, wherein the LacO operator comprises at least one copy (e.g., 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, e.g., 3-5) of a LacOl operator sequence. In certain embodiments, the LacO operator comprises 3-5 copies of a LacOl operator sequence. In certain embodiments, the LacOl operator sequence comprises the sequence set forth in SEQ ID NO: 19.

[0128] In certain embodiments, the second nucleic acid construct described in any of the above embodiments, wherein the LacO operator further comprises a LacOid. In certain embodiments, the LacOid is located downstream of the LacOl. In certain embodiments, the LacO operator comprises, from 5’ to 3’, a LacOl and a LacOid.

[0129] In certain embodiments, the light-controlled repressor protein is selected from the group consisting of the dark-responsive light-controlled repressor proteins of the present application; the inducing condition described in step (2) comprises culturing the host cell under dark condition to induce expression of the gene of interest. In certain embodiments, the method further comprises culturing the recombinant host cell under blue light (e.g., 430-495 nm, such as 450-480 nm) condition to suppress expression of the gene of interest.

[0130] In certain embodiments, the light-controlled repressor protein is selected from the group consisting of the blue light-responsive light-controlled repressor proteins of the present application; the inducing condition described in step (2) comprises culturing the host cell under blue light (e.g., 430-495 nm, such as 450-480 nm) condition to induce expression of the gene of interest. In certain embodiments, the method further comprises culturing the host cell under dark condition to suppress expression of the gene of interest.

[0131] In certain embodiments, the blue light described herein refers to light having a wavelength between 430-495 nm (e.g., 450-480 nm). The blue light described herein can be emitted by a light source, including LED light panels, light strips, light tubes, etc.

[0132] In certain embodiments, the expression vector comprising the first nucleic acid construct and the second nucleic acid construct can be obtained by the following steps: providing a vector comprising a first expression cassette comprising the first nucleic acid construct and a second expression cassette comprising a promoter, a LacO operator, and a cloning site (e.g., a multiple cloning site, MCS) for integration of a gene of interest, and integrating the gene of interest into the cloning site to provide a recombinant expression vector having the gene of interest integrated.

[0133] In certain embodiments, the expression vector comprising the second nucleic acid construct can be obtained by the following steps: providing a vector comprising a promoter, a LacO operator, and a cloning site (e.g., a multiple cloning site, MCS) for integration of a gene of interest, and integrating the gene of interest into the cloning site to provide a recombinant expression vector having the gene of interest integrated.

[0134] Examples

[0135] Those skilled in the art will appreciate that the examples describe the application in terms of preferred embodiments, and that the application is not intended to be limited to the preferred embodiments. The experimental methods in the examples are routine methods unless otherwise specified. The specific conditions in the examples are routine conditions or conditions recommended by the manufacturer unless otherwise specified. The reagents or instruments used are routine products available on the market unless otherwise specified.

[0136] Example 1: Construction of a light-controlled Pichia pastoris strain

[0137] A light-controlled Pichia pastoris strain is a strain in which gene expression is regulated using a light-controlled transcriptional activation device. The light-controlled transcriptional activation device of Pichia pastoris consists of two parts, an activator cassette and a response cassette, both of which are integrated into the Pichia pastoris genome by homologous recombination single crossover.

[0138] The activation expression cassette uses the pGAPZ(alpha)A vector. The OptoLacI and VP16 fusion expression gene is constructed into the vector between the GAP promoter and the AOX1 terminator by the Gibson assembly method (Table 1). 10 μL of Gibson assembly product is transformed into DH5α competent cells, ice bath for 30 min, 42°C heat shock for 90 s, then ice bath for 5 min, add 700 μL of low-salt LB medium to the competent cells, incubate at 37°C constant temperature shaker for 1 h, take an appropriate amount of bacterial liquid and spread on a solid low-salt LB plate containing bleomycin resistance, and incubate in a 37°C constant temperature incubator overnight. The next day, pick a single colony into low-salt LB medium containing bleomycin, incubate at 37°C constant temperature shaker for 8-9 h, extract the plasmid, and send to a sequencing company for sequencing. The plasmid after successful sequencing is named pCD56.

[0139] Table 1 Gibson reaction ingredient table (10 μL)

[0140] Reagents Volume (μl) 5x CII buffer 2 Linearized pZH44 fragment (0.03 pmol) X Linearized LOV2 fragment (0.06 pmol) Y Exnase II 1 ddH2O To 10

[0141] The response expression cassette uses the pPIC9K vector. The LacO nucleic acid sequence with different copy numbers is replaced with the complete AOX1p as a cis-acting element in series with the cAOX1p core region, and eGFP is used as a reporter gene. The synthetic cis-acting element and the eGFP gene are constructed in the multiple cloning site of the pPIC9K vector by the Gibson assembly method (deletion of the original AOX1 promoter of the vector). 10 μL of Gibson assembly product is transformed into DH5α competent cells, ice bath for 30 min, 42°C heat shock for 90 s, then ice bath for 5 min, add 700 μL of LB medium to the competent cells, incubate at 37°C constant temperature shaker for 1 h, take an appropriate amount of bacterial liquid and spread on a solid LB plate containing kanamycin resistance, and incubate in a 37°C constant temperature incubator overnight. The next day, pick a single colony into LB medium containing kanamycin resistance, incubate at 37°C constant temperature shaker for 8-9 h, extract the plasmid, and send to a sequencing company for sequencing. 0-5 copies of LacO are constructed in series with cAOX1p to form a synthetic cis-acting element, and the corresponding plasmids with successful sequencing are named pCD70, pCD61, pCD62, pCD63, pCD64, and pCD39 (Table 2).

[0142] Table 2 Plasmid name corresponding table with different LacO copy numbers

[0143] Plasmid name LacO copy number pCD70 0 pCD61 1 pCD62 2 pCD63 3 pCD64 4 pCD39 5

[0144] On the basis of successful plasmid construction, strain construction was further carried out. First, the activated expression cassette was integrated into the genome of Pichia pastoris GS115 strain. pCD56 was linearized by Avr II single enzyme digestion (Table 3), and the linearized fragment was recovered. 1 μg of the linearized fragment was electroporated into Pichia pastoris GS115 competent cells, 500 μL of 1 M sorbitol solution and 500 μL of YPD medium were added, and the mixture was incubated at 30°C on a constant temperature shaker for 1 h. An appropriate amount of bacterial solution was spread on a solid YPD plate containing bleomycin, and the plate was incubated at 30°C in a constant temperature incubator for 3 d. After 3 d, the colonies grown on the plate were verified by PCR and sequencing. The strain with successful integration was named CDy56.

[0145] Table 3 Single enzyme digestion reaction component table (200 μL)

[0146] Reagent Amount added endonuclease 4 μL 10x rCutSmart buffer 20 μL plasmid 15-20 μg ddH2O To 200 μL

[0147] Subsequently, pCD70, pCD61, pCD62, pCD63, pCD64, and pCD39 were linearized by Sal I single enzyme digestion (Table 3), and the linearized fragments were recovered. 1 μg of the linearized fragment was electroporated into Pichia pastoris CDy56 competent cells, 1 mL of 1 M sorbitol solution was added, and the mixture was incubated at 30°C on a constant temperature shaker for 2 h. An appropriate amount of bacterial solution was spread on a SC-His plate, and the plate was incubated at 30°C in a constant temperature incubator for 3 d. After 3 d, the colonies grown on the plate were verified by PCR and sequencing. The strains with successful integration were named CDy70a, CDy61a, CDy62a, CDy63a, CDy64a, and CDy39a, respectively (Table 4). The strains obtained by integrating the light-controlled transcriptional activation device into the genome were referred to as light-controlled strains. Figure 1

[0148] Table 4 Correspondence table of Pichia pastoris strains with different numbers of LacO copies

[0149] Pichia strain name LacO copy number CDy70a 0 CDy61a 1 CDy62a 2 CDy63a 3 CDy64a 4 CDy39a 5

[0150] Example 2: Performance test of light-controlled Pichia pastoris strain

[0151] The single colony strain grown on the plate was picked into 1 mL of YPD medium in a 24-well plate and incubated at 30°C on a constant temperature dark shaker for 20 h to the growth plateau. The bacterial solution was transferred into two 24-well plates at a 1% inoculation amount, and each condition was repeated in 3 holes. The two 24-well plates were placed in blue light (90 μmol / m2 / s) and darkness, respectively, and incubated for 24 h. Subsequently, the green fluorescence intensity of different strains at an excitation wavelength of 488 nm and an emission wavelength of 530 nm was tested by an enzyme marker instrument, the difference between the GFP fluorescence values under blue light and darkness was calculated, and the light response gene expression activity of the light-controlled Pichia pastoris strain was determined.​

[0152] The results show that the strains containing 1-5 copies of LacO have strong GFP fluorescence signals under dark conditions, while the strain CDy70a without LacO has almost no fluorescence, indicating that the constructed transcription activation device depends on the specific binding of OptoLacI and LacO to activate the downstream promoter. Figure 2 The strains containing 1-5 copies of LacO have lower fluorescence signals under blue light conditions than under dark conditions, indicating that under blue light conditions, OptoLacI and LacO are dissociated, and the transcription level is reduced. Figure 2 With the increase of 1-3 copies of LacO, the expression intensity of GFP under dark conditions shows an increasing trend. Figure 2

[0153] Example 3: Optimization of the induction fold of the light-controlled Pichia pastoris strain under dark conditions compared with blue light conditions

[0154] The initially constructed light-controlled transcription activation device has strong induction intensity, but the induction fold needs to be optimized. When the constitutive promoter of the activation expression cassette is the strong promoter P GAP , OptoLacI may not be able to completely dissociate from the genomic LacO under blue light conditions due to excessive expression, resulting in more leakage expression under blue light. Therefore, on the basis of CDy61a, P GAP was replaced with the weak promoters P ADH2 and cP AOX1 , respectively (expression intensity P GAP >P ADH2 >cP AOX1 ), and the obtained strains were named CDy98 and CDy123, respectively. Figure 3

[0155] ​​In addition, the stability of the protein under blue light conditions can be regulated by using a photo-sensitive degradation sequence (PSD). The PSD used in the present application consists of the LOV2 domain of the photosynthetic protein 1 of Arabidopsis thaliana and a synthetic degradation sequence cODC1 derived from the carboxy terminus of mouse ornithine decarboxylase (ODC). ODC consists of 37 amino acids without secondary structure and a cysteine-alanine motif, which has been shown to be essential for proteasome binding. In the PSD module, a 23 amino acid long synthetic variant of ODC is fused to the carboxy terminus of the LOV2 Jα helix. In the dark, the PSD is inactive due to helix folding upstream of the cysteine-alanine motif; in blue light, the PSD is activated due to unfolding of the Jα helix and is degraded by the proteasome. The PSD sequence was fused to the carboxy terminus of the transcriptional activator VP16 in CDy98 and CDy123, respectively, resulting in strains CDy121 and CDy122, respectively. Figure 3

[0156] CDy98, CDy121, CDy123 and CDy122 were tested according to the strain performance test method in Example 2. The results are shown in Table 2. Figure 4 As shown in Table 2, when the promoter strength of OptoLacI was reduced, the expression strength of the light-controlled strain under dark and blue light conditions was reduced, and the induction fold of dark compared to blue light was increased (CDy123 compared to CDy98, CDy122 compared to CDy121). When the promoter strength of OptoLacI was the same, the expression of OptoLacI_VP16 fused with PSD could further increase the induction fold of dark compared to blue light (CDy121 compared to CDy98, CDy122 compared to CDy123). In general, the strain CDy122 with the lowest expression strength of OptoLacI and fused expression of PSD had the highest dark / blue light induction fold Figure 4

[0157] Example 4: Test of the induction intensity and fold of the light-controlled Pichia pastoris strain over time

[0158] ​​The CDy122 monoclonal strain grown on a flat plate was inoculated into 1 mL YPD medium in a 24-well plate and cultured at 30°C in a constant-temperature dark shaker for 20 h to the growth plateau, and then the bacterial liquid was inoculated into 1 mL YPD medium in a 1.5 mL EP tube at a 1% inoculation amount, temporarily stored in a 4°C refrigerator, and transferred into two 24-well plates at 24 h, 21 h, 18 h, 15 h, 12 h, 9 h, 6 h and 3 h before detection, respectively. The two 24-well plates were cultured under blue light (90 μmol / m2 / s) and darkness for 24 h, respectively. In order to avoid the influence of the absorbance of the bacterial liquid on the detection results, the mean fluorescence intensity (MFI) of the strain was detected by flow cytometry. The bacterial liquid was diluted to OD 600nm = 0.3 with PBS, and the Pichia pastoris was dispersed into a single-cell suspension by ultrasonic treatment (total power of ultrasonic crusher 950 W, using power 4%, ultrasonic 10 s, stop 10 s, for a total of 30 s). The FITC fluorescence data were collected using a Fortessa X-20 analytical flow cytometer, and 100,000 cells were analyzed for each sample.

[0159] The results are shown in Figure 5 From 9 h to 24 h, the mean fluorescence intensity of the light-controlled Pichia pastoris strain CDy122 under dark conditions steadily increased, the mean fluorescence intensity under blue light conditions remained at a low level, and the dark / blue light induction multiple gradually increased. The possible reason for the abnormally high MFI of the cells at 3 h and 6 h was the residual GFP fluorescence of the seed liquid.

[0160] Example 5: Comparison of the expression intensity of the light-controlled transcription activation device and the methanol-inducible PAOX1

[0161] The methanol-inducible promoter P AOX1 has high expression efficiency and is the most commonly used promoter for expressing exogenous proteins in Pichia pastoris. Based on the core region cP AOX1 of P AOX1 , a new light-controlled transcription activation device in Pichia pastoris was constructed by using a light-controlled activation module to regulate the expression of cP AOX1 . In order to compare the expression intensity of the light-controlled transcription activation device and P AOX1 , the eGFP gene was inserted into the multiple cloning site of the pPIC3.5K plasmid vector by the Gibson assembly method to obtain the plasmid pCD53; pCD53 was single-enzyme cut with Sac I and the linearized fragment was recovered, and integrated into the genome of Pichia pastoris GS115 by homologous recombination single exchange to obtain the strain CDy53.

[0162] The CDy53 and CDy122 monoclonal strains grown on the plates were respectively picked into 1 mL YPD medium in 24-well plates, and cultured at 30°C in a constant temperature dark shaker for 20 h to the growth plateau. The bacterial liquid was respectively inoculated into 1 mL YPD medium in multiple 1.5 mL EP tubes at an inoculation amount of 1%, temporarily stored in a 4°C refrigerator, and respectively transferred into 24-well plates at 24 h, 21 h, 18 h, 15 h and 12 h before detection, and cultured at 30°C in a constant temperature shaker in the dark for 24 h. The MFI was analyzed by flow cytometry in the manner described in Example 4.

[0163] The results are shown in Table 1. Figure 6 As shown in Table 1, the fluorescence value of CDy53 began to decrease from 15 h, while the fluorescence value of CDy122 gradually increased, and exceeded that of CDy53 from 17 h, indicating that the expression intensity of the light-controlled transcriptional activation device of the present application was higher than that of the P AOX1 The present application has the advantages.

Claims

1. A light-controlled gene expression system, comprising: a) A recombinant photosensitive transcription factor encoding a gene, wherein the recombinant photosensitive transcription factor comprises a first polypeptide as a DNA-binding and photosensitive domain, namely a photorepressor protein, and a second polypeptide as a transcriptional activation domain, and / or includes a third polypeptide as an auxiliary photoregulatory component; b) A recombinant cis-acting element comprising a nucleic acid sequence recognized and bound by the first polypeptide, and a promoter nucleic acid sequence regulated by the second polypeptide.

2. The light-controlled gene expression system of claim 1, wherein the recombinant light-sensitive transcription factor is a light-controlled repressor protein comprising a DNA-binding domain and a light-sensitive domain, the light-controlled repressor protein comprising a LacI protein and an LOV domain inserted between adjacent amino acids in the Loop-1, Loop-2 or Loop-3 region of the LacI protein; Preferably, the LOV domain of the light-controlled repressor protein is the LOV2 domain; Preferably, the LOV domain is selected from the LOV2 domain (AsLOV2) of the oat phytochrome 1 gene, the EL222 phytosensitive protein from *Rhodopsinia serratifolia*, the LOV2 phytosensitive domain from *Arabidopsis thaliana*, or mutants thereof; Preferably, the mutant is a cyclic rearrangement mutant or a mutant with altered response rate; Preferably, the LOV domain is AsLOV2, for example, containing the sequence shown in SEQ ID NO: 1; Preferably, the LOV domain is cpLOV27, for example, containing the sequence shown in SEQ ID NO: 2; Preferably, the insertion position is selected from the following amino acid positions of the LacI protein: between positions 335 and 336, between positions 314 and 315, between positions 315 and 316, between positions 316 and 317, between positions 334 and 335, between positions 336 and 337, between positions 337 and 338, between positions 338 and 339, and between positions 152 and 153. Preferably, the insertion position is between positions 335 and 336 of the LacI protein.

3. The light-controlled gene expression system according to claim 1 or 2, wherein, The insertion position is selected from the following amino acid positions in the LacI protein: between positions 311 and 312, and between positions 153 and 154; preferably, the insertion position is between positions 311 and 312 in the LacI protein.

4. The light-controlled gene expression system according to any one of claims 1-3, wherein, The LacI protein is wild-type; Preferably, the wild-type LacI protein comprises the sequence shown in SEQ ID NO: 14; Preferably, the LacI protein is a modified LacI protein that, compared to the wild-type LacI protein, contains amino acid substitutions selected from the following: (i) the amino acid at position 220 is replaced with F, and / or, (ii) the amino acid at position 84 is replaced with E, C, S, T, or I; Preferably, the modified LacI protein comprises the sequence shown in SEQ ID NO: 15 or 16, or comprises a sequence in which the amino acid at position 84 is replaced with C, S, T, or I compared to SEQ ID NO: 15 or 16; Preferably, the N-terminus and / or C-terminus of the LOV domain are optionally linked to the LacI protein via a peptide linker.

5. The light-controlled gene expression system according to any one of claims 1-8, comprising the sequence shown in any one of SEQ ID NOs: 8, 17, 4-6, 7, 9-11, 12 or the sequence shown in SEQ ID NO: 3 or 13; Preferably, the light-controlled repressor protein comprises the sequence shown in SEQ ID NO: 8 or 17; Preferably, the light-controlled repressor protein comprises the sequence shown in SEQ ID NO: 3; Preferably, the second polypeptide is a transcription factor protein with the ability to independently recruit RNA polymerase; more preferably, it is VP16, VP64, VPR, or Mit1; more preferably, the nucleotide sequence of the VP16 gene contains the sequence shown in SEQ ID NO: 18 or a degenerate thereof. The third polypeptide is a photo-sensitive degron (PSD) containing an LOV domain and an ornithine decarboxylase (ODC). Preferably, the LOV domain included in the light-controlled gene expression system is the LOV2 domain; Preferably, the LOV domain is selected from the LOV2 domain (AsLOV2) of the oat phytochrome 1 gene, the EL222 phytosensitive protein from *Rhodopsinia serratifolia*, the LOV2 phytosensitive domain from *Arabidopsis thaliana*, or mutants thereof; Preferably, the photosensitive degradation sequence PSD comprises the sequence shown in SEQ ID NO:

21.

6. A nucleic acid construct comprising a nucleotide sequence encoding the light-controlled gene expression system of any one of claims 1-5.

7. A vector comprising the nucleic acid construct of claim 6; Preferably, it comprises a first nucleic acid construct and a second nucleic acid construct, wherein, The first nucleic acid construct contains nucleotide sequences encoding a first polypeptide, a second polypeptide, and / or a third polypeptide, and the second nucleic acid construct contains a LacO operator gene, a promoter, a target gene, and a terminator; Preferably, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes; More preferably, the first polypeptide and the second polypeptide contained in the first nucleic acid construct are linked by a peptide linker; Preferably, the peptide linker connecting the first polypeptide and the second polypeptide in the first nucleic acid construct is a GGGGS linker; Optionally, the second and third polypeptides contained in the first nucleic acid construct are linked by a peptide linker; Preferably, the peptide linker connecting the first polypeptide and the second polypeptide in the first nucleic acid construct is a GGGGS linker; More preferably, the LacO operator gene contained in the second nucleic acid construct is selected from LacO1, LacOid, or a combination thereof; More preferably, the LacO operator gene contained in the second nucleic acid construct contains at least one copy (e.g., 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, such as 3 to 5) of the LacO1 operator sequence; Preferably, the LacO1 manipulation sequence comprises the sequence shown in SEQ ID NO:

19.

8. The second nucleic acid construct of claim 7, wherein the promoter comprises a DNA sequence that is recognized, bound, and transcribed by RNA polymerase under the action of the second polypeptide of claim 1 or 7; Preferably, the promoter nucleic acid sequence contained in the second nucleic acid construct is selected from the AOX1 minimum promoter, GAP minimum promoter, TEF minimum promoter, HIS3 minimum promoter, and CYC minimum promoter; Preferably, the promoter nucleic acid sequence contained in the second nucleic acid construct comprises the sequence shown in SEQ ID NO:

20.

9. The carrier according to claim 7, wherein, The first nucleic acid construct further includes a promoter and a terminator operatively linked to the nucleotide sequences encoding the first polypeptide, the second polypeptide, and / or the third polypeptide; Preferably, the promoter is selected from TEF promoter, GAP promoter, ADH2 promoter, and cPAOX1 promoter; Preferably, the terminator is selected from the AOX1 promoter, the RPL15A promoter, and the CYC1 promoter.

10. A host cell comprising the nucleic acid construct of claim 9 or the vector of any one of claims 14-21; Preferably, the host cell is a eukaryotic cell; Preferably, the host cell is Pichia pastoris.

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