A vpr mutant protein capable of activating gene expression in saccharomyces cerevisiae

Abstract

The present application relates to a VPR mutant protein capable of activating gene expression of saccharomyces cerevisiae, which is obtained by randomly mutating the commonly used activation protein VPR in saccharomyces cerevisiae, using the designed fluorescent protein and resistance gene as a screening marker, has higher transcription activation efficiency, and expands the application range of CRISPR technology in the field of gene regulation of saccharomyces cerevisiae.

Description

Technical Field

[0001] This invention belongs to the field of directed evolutionary modification of proteins and transcriptional regulation of genes, and relates to a VPR mutant protein that can activate gene expression in Saccharomyces cerevisiae and its applications. Background Technology

[0002] Saccharomyces cerevisiae is a eukaryotic model organism in molecular biology and synthetic biology, thanks to its simple, economical, and rapid cultivation, and its ability to adapt well to large-scale aerobic and anaerobic cultivation. Because yeast can grow on completely defined culture media, researchers have been able to isolate many auxotrophic yeasts. These auxotrophic yeasts have various applications; for example, they can be used to analyze the metabolic pathways of yeast cells and modify them into product production platform strains, or to introduce exogenous metabolic pathways to produce a series of biological products using Saccharomyces cerevisiae. However, the amount of target products obtained solely through the growth and metabolism of Saccharomyces cerevisiae is generally extremely limited. Therefore, it is necessary to artificially regulate key genes in metabolic pathways to increase the yield of target products. More efforts are needed to achieve efficient, precise, and effective regulation of Saccharomyces cerevisiae genes.

[0003] Transcription factors are a series of protein molecules that can bind to or recognize specific nucleotide sequences upstream of genes and regulate gene transcription by modulating the recruitment of RNA polymerase. Their molecules include a DNA-binding domain and an effector domain that recruits the polymerase. Based on whether they upregulate or downregulate RNA polymerase recruitment, transcription factors can be divided into activators (or activating domains) and repressors (or repressor domains). Currently, the most mature application in metabolic regulation of Saccharomyces cerevisiae is a fusion-activated domain (VPR), which can increase the expression level of the target gene by up to about 5-fold in Saccharomyces cerevisiae. However, this is far from meeting the metabolic regulation requirements of Saccharomyces cerevisiae cell factories.

[0004] Currently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) gene editing system is an important synthetic biology tool for metabolic pathway reconstruction and module optimization, significantly improving the efficiency of traditional DNA editing. It is also used to regulate gene expression and construct genome-scale screening libraries. The Cas9 protein in the CRISPR system can be modified into a non-cleaving dCas9 protein. By fusing specific functional domains, dCas9 can achieve targeted inhibition or activation of target genes, regulating gene expression without altering the DNA sequence. However, due to limitations in the activation domain, the achievable activation intensity is generally low, which greatly restricts the application of CRISPR in gene regulation research and yield enhancement.

[0005] Therefore, the current problem is the need to research and develop a highly efficient activation domain (VPR) for gene activation in Saccharomyces cerevisiae. Summary of the Invention

[0006] The technical problem to be solved by this invention is the low activation efficiency of existing activation domains for target genes in Saccharomyces cerevisiae. This invention provides a VPR mutant protein that can significantly activate gene expression in Saccharomyces cerevisiae. It is obtained by randomly mutating the commonly used activating protein VPR in Saccharomyces cerevisiae and using designed fluorescent proteins and resistance genes as selection markers. It has high transcriptional activation efficiency and expands the application scope of CRISPR technology in the field of gene regulation in Saccharomyces cerevisiae.

[0007] Therefore, the first aspect of the present invention provides an amino acid mutant that affects the activity of the activation domain VPR, comprising one or more of the following mutations in the amino acid sequence of the wild-type VPR as shown in SEQ ID NO 1: mutation at position 35 (Asp and His), position 114 (Thr and Arg), position 191 (Ala and Val), position 203 (Ala and Val), position 215 (Ala and Thr), position 234 (Ala and Ser), position 302 (Leu and Met), and a protein translation termination mutation at position 370 (Leu).

[0008] A second aspect of the present invention provides a mutant protein of the activation domain VPR, the sequence of which comprises one or more mutations in the amino acid sequence of the wild-type VPR as shown in SEQ ID NO 1, from the N-terminus to the C-terminus: Asp and His at position 35, Thr and Arg at position 114, Ala and Val at position 191, Ala and Val at position 203, Ala and Thr at position 215, Ala and Ser at position 234, Leu and Met at position 302, and a protein translation termination mutation generated at position 370 (Leu).

[0009] In some embodiments of the present invention, the mutant is a VPR mutant protein with a sequence containing a mutation at position 191 (Ala and Val) of the amino acid sequence of wild-type VPR as shown in SEQ ID NO 1 from the N-terminus to the C-terminus, and a protein translation termination mutation at position 370 (Leu).

[0010] In some specific preferred embodiments of the present invention, the sequence of the VPR mutant protein No. 1 is shown in SEQ ID NO 2.

[0011] In some embodiments of the present invention, the mutant is a VPR mutant protein of position 2, the sequence of which contains mutations at positions 35 (Asp and His), 114 (Thr and Arg), 191 (Ala and Val), 203 (Ala and Val), 215 (Ala and Thr), 302 (Leu and Met), and a protein translation termination mutation at position 370 (Leu).

[0012] In some specific preferred embodiments of the present invention, the sequence of the VPR mutant protein No. 2 is shown in SEQ ID NO 3.

[0013] In some embodiments of the present invention, the mutant is a VPR mutant protein of position 3, the sequence of which contains mutations at positions 35 (Asp and His), 114 (Thr and Arg), 191 (Ala and Val), 203 (Ala and Val), 215 (Ala and Thr), 234 (Ala and Ser), 302 (Leu and Met), and a protein translation termination mutation at position 370 (Leu).

[0014] In some specific preferred embodiments of the present invention, the sequence of the VPR mutant protein number 3 is shown in SEQ ID NO 4.

[0015] According to the present invention, the mutant protein further includes a fusion protein obtained by attaching a tag to the N-terminus or C-terminus of the mutant protein described above; preferably, the mutant protein further includes a fusion protein obtained by attaching dCas9 to the N-terminus or C-terminus of VPR mutant protein 1, VPR mutant protein 2 or VPR mutant protein 3.

[0016] A third aspect of the present invention provides a nucleotide mutant affecting the activity of the exogenous activator protein VPR in Saccharomyces cerevisiae, comprising one or more of the following mutations located in the nucleotide sequence encoding the wild-type VPR: G and C at position 104, G and A at position 369, A and G at position 455, C and T at position 572, C and T at position 608, G and A at position 643, G and T at position 700, C and A at position 904, and T and A at position 1109.

[0017] The fourth aspect of the present invention provides a nucleotide molecule encoding the mutant protein described in the second aspect of the present invention, the sequence of which includes one or more of the following mutations located in the nucleotide sequence of ...

[0018] In some embodiments of the present invention, the nucleotide molecule is a nucleotide molecule encoding the VPR mutant protein 1, the sequence of which contains a mutation at position 572 C and T, and a mutation at position 1109 T and A, in the direction from the 5′ end to the 3′ end of the nucleotide sequence as shown in SEQ ID NO 1 encoding the wild-type VPR.

[0019] In some specific preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule of the VPR mutant protein is shown in SEQ ID NO 6.

[0020] In some embodiments of the present invention, the nucleotide molecule is a nucleotide molecule encoding the VPR mutant protein 2, the sequence of which contains mutations at positions 104 (G and C), 369 (G and A), 455 (A and G), 572 (C and T), 608 (C and T), 643 (G and A), 904 (C and A), and 1109 (T and A) in the nucleotide sequence of ...

[0021] In some specific preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule of the VPR mutant protein No. 2 is shown in SEQ ID NO 7.

[0022] In some embodiments of the present invention, the nucleotide molecule is a nucleotide molecule encoding the VPR mutant protein 3, the sequence of which contains mutations at positions 104 (G and C), 369 (G and A), 455 (A and G), 572 (C and T), 608 (C and T), 643 (G and A), 700 (G and T), 904 (C and A), and 1109 (T and A) in the nucleotide sequence of ...

[0023] In some specific preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule of the VPR mutant protein No. 3 is shown in SEQ ID NO 8.

[0024] According to the present invention, the nucleic acid molecule is a DNA molecule as follows:

[0025] (a1) DNA molecules whose coding regions include nucleotide sequences as shown in SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8;

[0026] (a2) DNA molecules with nucleotide sequences as shown in SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8;

[0027] (a3) has 75% or more identity with the nucleotide sequence described in (a1) or (a2) and is a DNA molecule encoding the protein described in the second aspect of the invention;

[0028] (a4) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence described in (a1) or (a2) and encodes the protein described in the second aspect.

[0029] The fifth aspect of the present invention provides an expression cassette, recombinant vector, or recombinant microorganism containing nucleotide molecules as described in the fourth aspect of the present invention.

[0030] The sixth aspect of the present invention provides the application of mutant proteins as described in the second aspect, or mutant proteins derived from nucleotide molecules as described in the fourth aspect of the present invention, or mutant proteins derived from expression cassettes, recombinant vectors, or recombinant microorganisms as described in the fifth aspect of the present invention, in activating gene expression in Saccharomyces cerevisiae.

[0031] In some embodiments of the present invention, the application includes strains or the target product whose yield is increased by activating the expression of endogenous or exogenous genes in wild or recombinant organisms.

[0032] This invention provides a VPR mutant protein capable of activating gene expression in *Saccharomyces cerevisiae*. It is obtained by randomly mutating VPR using error-prone PCR and selecting it using a designed fluorescent protein and resistance gene as markers. This novel activating protein is suitable for gene regulation in *Saccharomyces cerevisiae* and can efficiently activate endogenous or exogenous genes in the yeast. Experiments show that this VPR mutant protein can stably induce a 14.7-fold activation of the target gene in *Saccharomyces cerevisiae* using the CRISPRa system, which is 2.7 times the initial VPR activation intensity. Attached Figure Description

[0033] The present invention will now be described in further detail with reference to the accompanying drawings:

[0034] Figure 1 This diagram illustrates the activation of gene transcription by the dCas9-VPR fusion protein (taking the expression activation of the bleomycin resistance gene as an example). dCas9 (dead CRISPR-associated protein 9) represents CRISPR-associated protein 9 that has lost its catalytic activity; gRNA (guide RNA) represents single-stranded guide RNA; RNAPII (RNA polymerase II) represents RNA polymerase II; CYC1 promoter represents the promoter of the cytochrome c1 gene; ADH1 terminator represents the terminator of the alcohol dehydrogenase gene; BleoR, Ble, and z represent the bleomycin resistance gene, bleomycin resistance protein, and bleomycin, respectively. This diagram shows that the activation domain VPR is targeted to the preprogenitor region via dCas9 and gRNA, thereby recruiting polymerase and increasing the expression of the bleomycin resistance gene, thus causing the strain to exhibit stronger bleomycin resistance.

[0035] Figure 2 This is a drop test diagram of wild-type VPR protein and endogenous activator resistance; where OD... 600 The absorbance value of the *Saccharomyces cerevisiae* culture at 600 nm represents the concentration of the culture. SC-ura represents the basal culture medium for uracil-deficient yeast. SC-ura+zeocin represents SC-ura with the addition of a certain concentration of bleomycin, enabling the selection of bleomycin-resistant yeast strains. MSN2 and MSN2-6 are the endogenous activators of *Saccharomyces cerevisiae* and their mutants, respectively. This figure shows that the exogenous activation domain (VPR) is more efficient than the endogenous activator.

[0036] Figure 3 The drop point experiment diagram shows the VPR mutant obtained in the first round of screening. The strain with the code e13 is a Saccharomyces cerevisiae strain containing the mutant protein of the VPR mutant. After activation and expression of the mutant VPR, it has the highest bleomycin resistance in the same batch of screening. NC is the control strain without resistance.

[0037] Figure 4 The drop point experiment diagram shows the VPR mutant obtained in the second round of screening. The strain with the code e137 is a Saccharomyces cerevisiae strain containing the mutant protein of the VPR mutant. After activation by the mutant VPR, it has the highest bleomycin resistance in the same batch of screening. NC is the control strain without resistance.

[0038] Figure 5 The drop point experiment diagram shows the VPR mutant obtained in the second round of screening. The strain with the code e13711 is a Saccharomyces cerevisiae strain containing the mutant protein of the VPR mutant. After activation by the mutant VPR, it has the highest bleomycin resistance in the same batch of screening. NC is the control strain without resistance.

[0039] Figure 6 The image shows the results of real-time quantitative PCR characterization of the mCherry gene activation fold of the control strain without the VPR gene, the Saccharomyces cerevisiae strain containing wild-type VPR, the Saccharomyces cerevisiae strain containing mutant protein 1, the Saccharomyces cerevisiae strain containing mutant protein 2, and the Saccharomyces cerevisiae strain containing mutant protein 3. The transcription intensity of the target gene in the control strain without VPR was set to 1. After VPR activation, the gene expression level was 5.4 times that of NC; after activation with VPR-1 mutant protein, the gene expression level was 7.8 times that of NC; after activation with VPR-2 mutant protein, the gene expression level was 11.6 times that of NC; and after activation with VPR-3 mutant protein, the gene expression level was 14.7 times that of NC. Detailed Implementation

[0040] To make the present invention easier to understand, the present invention will be described in detail below with reference to the accompanying drawings and embodiments. These embodiments are for illustrative purposes only and are not limited to the application scope of the present invention. Specific experimental methods not mentioned in the following embodiments are generally carried out according to conventional experimental methods.

[0041] I. Terminology

[0042] The term "dNTPs Mix" as used in this invention refers to reagents that can be used in polymerase chain reaction (PCR), sequencing and cDNA synthesis reactions, wherein dNTP stands for "2′-deoxynucleotide-5′-triphosphate", and Mix includes four nucleotides (dATP, dCTP, dGTP, dTTP).

[0043] In this invention, the term "nucleotide mutant" refers to the smallest unit in the nucleotide sequence of a gene that can undergo mutation.

[0044] Similarly, the term "amino acid mutant" as used in this invention refers to the smallest unit in the amino acid sequence of a protein that can be mutated.

[0045] The ">" symbol used in this invention represents a base substitution when indicating a nucleotide mutation. For example, the substitution site is located at one or more of the following mutations in the nucleotide sequence of SEQ ID NO 4, which encodes the amino acid sequence shown in SEQ ID NO 1, from the 5′ end to the 3′ end: G and C at position 104, G and A at position 369, A and G at position 455, C and T at position 572, C and T at position 608, G and A at position 643, G and T at position 700, C and A at position 904, and T and A at position 1109.

[0046] In this invention, the label “c.” represents the nucleotide sequence of the protein-coding gene.

[0047] The label “p.” used in this invention represents the amino acid sequence of a protein when indicating a protein mutant.

[0048] In this invention, the terms "protein" and "protein protein" are used interchangeably.

[0049] The asterisk (*) used in this invention indicates the termination of the protein translation process.

[0050] II. Implementation Plan

[0051] Existing activators have low activation efficiency for Saccharomyces cerevisiae genes. To address this issue, the inventors conducted extensive research on the activator VPR and obtained a VPR mutant protein with high transcriptional activation efficiency through random mutation and designed screening markers.

[0052] Furthermore, based on the fusion activator VPR gene, the inventors constructed a mutant library using error-prone PCR and obtained VPR mutant proteins by designing and adding fluorescent proteins and resistance genes into Saccharomyces cerevisiae as screening markers.

[0053] Specifically, the inventors obtained the VPR mutant protein with high transcriptional activation efficiency through the following steps:

[0054] Based on the fusion activator VPR gene, a random mutant library was constructed using error-prone PCR. Mutants with high fluorescence intensity and high bleomycin resistance were screened by high-throughput flow cytometry and transferred to 96-well cell culture plates. Effective mutations were verified by drop-point assays, qPCR quantification of activation efficiency, and gene sequencing, thereby providing useful biological information.

[0055] For more detailed procedures, please refer to Examples 1-4 below, which describe in detail the process of obtaining mutant proteins of the VPR activation domain with higher activation efficiency. As can be seen, this invention, based on the fusion activator VPR gene, successfully constructed mutants with activation intensities of 7.8-fold, 11.6-fold, and 14.7-fold (2.7-fold of the initial VPR) by continuously constructing error-prone PCR mutant libraries, followed by high-throughput screening of 5000 highly fluorescent single clones using flow cytometry into 96-well cell culture plates containing bleomycin, and then verification through bleomycin drop experiments. These mutants can efficiently activate the expression of exogenous genes in *Saccharomyces cerevisiae*. A schematic diagram of activation using the CRISPRa system is shown below. Figure 1 As shown.

[0056] Based on the above, in a first aspect of the present invention, the inventors have discovered that the following amino acid mutants affect the activation efficiency of the fusion activator VPR, including one or more of the following mutations located in the wild-type VPR: the Asp and His mutation at position 35 (p.Asp35His), the Thr and Arg mutation at position 114 (p.Thr114Arg), the Ala and Val mutation at position 191 (p.Ala191Val), the Ala and Val mutation at position 203 (p.Ala203Val), the Ala and Thr mutation at position 215 (p.Ala215Thr), the Ala and Ser mutation at position 234 (p.Ala215Ser), the Leu and Met mutation at position 302 (p.Leu302Met), and the protein translation termination mutation at position 370 (p.Leu370*).

[0057] In this invention, the wild-type fusion activation domain VPR is composed of VP64 [composed of four repeating domains of the herpes simplex virus protein VP16 (GeneBank ID: AAA45864.1)], p65 (also known as RelA, one of the five members constituting the NF-κB transcription factor family, GenBank ID: AAB47194.2), Rta (a trans-activator derived from EB virus, GeneBank ID: ABB89247.1) and the nuclear localization sequence NLS, and the amino acid sequence shown in SEQ ID NO 1 is the amino acid sequence of the wild-type VPR.

[0058] The mutant protein capable of significantly activating the VPR provided by the second aspect of the present invention contains amino acid mutants as described in the first aspect of the present invention. This can be understood as follows: when its sequence contains one or more of the following mutations in the amino acid sequence of wild-type VPR as shown in SEQ ID NO 1 from the N-terminus to the C-terminus: Asp and His at position 35, Thr and Arg at position 114, Ala and Val at position 191, Ala and Val at position 203, Ala and Thr at position 215, Ala and Ser at position 234, Leu and Met at position 302, and a protein translation termination mutation at position 370 (which results in the mutant VPR protein having a shorter peptide chain length than the wild-type VPR), the activation efficiency of VPR will show a significant change.

[0059] Specifically, mutant proteins of the following activation domain VPR have significantly improved activation efficiency relative to wild-type VPR.

[0060] The mutant protein is VPR mutant protein No. 1, whose sequence contains a mutation at position 191 (Ala and Val) and position 370 (Leu and translation termination mutation) of the amino acid sequence shown in SEQ ID NO 1 of the wild-type VPR (this mutation results in the peptide chain length of VPR mutant protein No. 1 being shorter than that of wild-type VPR); the sequence of VPR mutant protein No. 1 is shown in SEQ ID NO 2.

[0061] The mutant protein is VPR mutant protein No. 2, whose sequence contains the following mutations in the amino acid sequence of the wild-type VPR as shown in SEQ ID NO 1: Asp and His at position 35 from the N-terminus to the C-terminus; Thr and Arg at position 114; Ala and Val at position 191; Ala and Val at position 203; Ala and Thr at position 215; Leu and Met at position 302; and a protein translation termination mutation at position 370 (Leu) (this mutation results in the peptide chain length of VPR mutant protein No. 2 being shorter than that of wild-type VPR); the sequence of VPR mutant protein No. 2 is shown in SEQ ID NO 3.

[0062] The mutant protein is VPR mutant protein No. 3, whose sequence contains the following mutations in the amino acid sequence of the wild-type VPR as shown in SEQ ID NO 1: Asp and His at position 35, Thr and Arg at position 114, Ala and Val at position 191, Ala and Val at position 203, Ala and Thr at position 215, Ala and Thr at position 234, Leu and Met at position 302, and a protein translation termination mutation at position 370 (which results in the peptide chain length of VPR mutant protein No. 3 being shorter than that of wild-type VPR); the sequence of VPR mutant protein No. 3 is shown in SEQ ID NO 4.

[0063] According to some embodiments of the present invention, the fusion protein formed by linking the VPR mutant protein to the dCas9 protein can be targeted to the upstream segment of the target gene by means of the CRISPR system, thereby achieving activation and regulation of the target gene.

[0064] Based on the above, in this invention, the mutant protein also includes a fusion protein obtained by linking dCas9 to the N-terminus or C-terminus of the VPR mutant protein of this invention during its preparation process; preferably, the mutant protein also includes a fusion protein obtained by linking dCas9 to the N-terminus or C-terminus of VPR mutant protein 1, VPR mutant protein 2 and VPR mutant protein 3.

[0065] In a third aspect of the invention, the inventors have discovered that the following nucleotide mutants affect the activation efficiency of the activation domain VPR, including one or more of the following mutations in the nucleotide sequence of the amino acid sequence as shown in SEQ ID NO 1 encoding the wild-type VPR: G and C at position 104 (c.104G>C), G and A at position 369 (c.369G>A), A and G at position 455 (c.455A>G), C and T at position 572 (c.572C>T), C and T at position 608 (c.608C>T), G and A at position 643 (c.643G>A), G and T at position 700 (c.700G>T), C and A at position 904 (c.904C>A), and T and A at position 1109 (c.1109T>A).

[0066] As mentioned above, the wild-type VPR is composed of VP64, p65, Rta and the nuclear localization sequence NLS; the amino acid sequence shown in SEQ ID NO 1 is an amino acid sequence composed of VP64, p65, Rta and the nuclear localization sequence NLS; the nucleotide sequence encoding the wild-type VPR as shown in SEQ ID NO 1 and the nucleotide sequence shown in SEQ ID NO 5 is a nucleotide sequence composed of wild-type VP64, p65, Rta and the nuclear localization sequence NLS genes.

[0067] The nucleotide molecule encoding the mutant protein of the second aspect of the present invention provided in the fourth aspect of the present invention contains nucleotide mutants as described in the third aspect of the present invention in its sequence. This can be understood as follows: when its sequence contains one or more of the following mutations located in the nucleotide sequence of the amino acid sequence shown in SEQ ID NO 1 encoding the wild-type VPR: mutations at positions 104 (G and C), 369 (G and A), 455 (A and G), 572 (C and T), 608 (C and T), 643 (G and A), 700 (G and T), 904 (C and A), and 1109 (T and A), the activation efficiency of the activation domain VPR will show a significant change.

[0068] Specifically, the mutant protein of the second aspect of the present invention encoded by the following nucleotide molecules has a significantly improved activation efficiency compared to the wild-type VPR encoded by the nucleotide sequence of the fusion gene of wild-type VP64, p65, Rta and nuclear localization sequence NLS.

[0069] The nucleotide molecule is a nucleotide molecule encoding the VPR mutant protein No. 1, and its sequence contains a mutation at position 572 C and T and position 1109 T and A in the nucleotide sequence of the amino acid sequence of the wild-type VPR as shown in SEQ ID NO 1, in the direction from the 5′ end to the 3′ end; the nucleotide sequence of the nucleotide molecule encoding the VPR mutant protein No. 1 is shown in SEQ ID NO 6.

[0070] The nucleotide molecule is a nucleotide molecule encoding the VPR mutant protein of position 2, and its sequence contains mutations at positions 104 (G and C), 369 (G and A), 455 (A and G), 572 (C and T), 608 (C and T), 643 (G and A), 904 (C and A), and 1109 (T and A) in the nucleotide sequence of position 5, from the 5′ end to the 3′ end of the amino acid sequence of wild-type VPR as shown in SEQ ID NO 1; the nucleotide sequence of the nucleotide molecule encoding the VPR mutant protein of position 2 is shown in SEQ ID NO 7.

[0071] The nucleotide molecule is a nucleotide molecule encoding the VPR mutant protein of position 3, and its sequence contains mutations at positions 104 (G and C), 369 (G and A), 455 (A and G), 572 (C and T), 608 (C and T), 643 (G and A), 700 (G and T), 904 (C and A), and 1109 (T and A) in the nucleotide sequence of position 5, from the 5′ end to the 3′ end of the amino acid sequence of wild-type VPR as shown in SEQ ID NO 1; the nucleotide sequence of the nucleotide molecule encoding the VPR mutant protein of position 3 is shown in SEQ ID NO 8.

[0072] According to the present invention, the nucleic acid molecule is a DNA molecule as follows:

[0073] (a1) A DNA molecule whose coding region includes the nucleotide sequences shown in SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8.

[0074] (a2) DNA molecules with nucleotide sequences as shown in SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8;

[0075] (a3) has 75% or more identity with the nucleotide sequence described in (a1) or (a2) and is a DNA molecule encoding the protein described in the second aspect of the invention;

[0076] (a4) hybridizes under stringent conditions with the nucleotide sequence described in (a1) or (a2) and encodes a DNA molecule that encodes the protein described in the second aspect of the invention.

[0077] In a fifth aspect of the invention, an expression cassette, recombinant vector, or recombinant microorganism for preparing the mutant protein of the activation domain VPR described in the second aspect of the invention is provided, comprising:

[0078] (1) An expression cassette containing the nucleotide molecules described in the fourth aspect of the present invention;

[0079] (2) A recombinant plasmid containing the nucleotide molecule described in the fourth aspect of the present invention;

[0080] (3) Recombinant cells containing the nucleotide molecules or the above-mentioned recombinant plasmids described in the fourth aspect of the present invention, i.e. genetically engineered bacteria containing the nucleotide molecules or the above-mentioned recombinant plasmids described in the fourth aspect of the present invention.

[0081] The nucleotide molecules described in the fourth aspect of this invention can be used to construct recombinant expression plasmids, which can be transferred into host cells to express mutant proteins and obtain mutant proteins with the activation domain VPR described in the second aspect of this invention.

[0082] The recombinant expression vector described in this invention is pCEN-L-Kan-GFP, and the host cell is a modified Saccharomyces cerevisiae strain CEN.PK113-5D.

[0083] The VPR mutant protein provided by this invention is composed of the wild-type VPR protein, which is formed by the fusion of VP64, p65, Rta, and the nuclear localization sequence NLS. After the coding gene (cDNA) of the mutant protein is introduced into a host cell, the resulting mutant protein is an intracellular protein and needs to be obtained through methods such as sonication to disrupt the cell.

[0084] This invention constructs a recombinant VPR engineered strain derived from *Saccharomyces cerevisiae*. By randomly mutagenesing VPR using error-prone PCR and employing designed fluorescent proteins and resistance genes as selection markers, a novel activating protein suitable for gene regulation in *Saccharomyces cerevisiae* was obtained. This mutant protein stably induces a 14.7-fold activation of the target gene in *Saccharomyces cerevisiae* using the CRISPRa system, with the mutant protein exhibiting 2.7 times the activation intensity of the original VPR. The VPR mutant activating protein provided by this invention can efficiently activate endogenous or exogenous genes in *Saccharomyces cerevisiae*.

[0085] The sixth aspect of the present invention provides the application of mutant proteins as described in the second aspect, or mutant proteins derived from nucleotide molecules as described in the fourth aspect of the present invention, or mutant proteins derived from expression cassettes, recombinant vectors, or recombinant microorganisms as described in the fifth aspect of the present invention, in activating gene expression in Saccharomyces cerevisiae.

[0086] In some embodiments of the present invention, the application includes strains or the target product whose yield is increased by activating the expression of endogenous or exogenous genes in wild or recombinant organisms.

[0087] The detection method and instruments in this invention:

[0088] (1) PCR amplification was performed using a T100 PCR instrument (Bio-Rad, USA).

[0089] (2) The PCR products were detected and separated using a MINI-SUB CELL GT POWER PAC Basic agarose gel electrophoresis system (Bio-Rad, USA).

[0090] (3) The PCR products were detected and verified using a Gel Doc XR+ gel imaging system (Bio-Rad, USA).

[0091] (4) The absorbance was measured using a GENESYS 50 UV-Vis spectrophotometer (Thermo Fisher Scientific).

[0092] (5) DNA and RNA concentrations were determined using a NanoDrop One ultra-micro spectrophotometer (Thermo Fisher Scientific).

[0093] (6) Fluorescent cells were sorted using an Influx flow cytometer (BD, USA).

[0094] (7) The activation efficiency of mutant VPR was quantitatively tested using a Quant Studio 3 real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific).

[0095] III. Examples

[0096] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are conventional laboratory methods using conventional experimental equipment. Unless otherwise specified, the experimental materials described below are all commercially available.

[0097] In the following examples, the experimental base strain was Saccharomyces cerevisiae CEN.PK113-5D (Genotype:MATaMAL2-8c SUC2 ura3-52.Resource:P). The dCas9-VPR fusion protein was modified from the University of Frankfurt, Germany. Specifically, a weakened version of the bleomycin resistance gene BleoR (Genebank ID: QCF28883.1) was inserted into the X-3 integration site of chromosome CEN.PK113-5D (Jessop-Fabre MM, et al. Biotechnology Journal, 2016, 11(8):1110-7.), and the red fluorescent protein gene mCherry (Genebank ID: UFQ89828.1) was inserted into the XII-2 integration site of chromosome CEN.PK113-5D (Jessop-Fabre MM, et al. Biotechnology Journal, 2016, 11(8):1110-7.). Both genes are started by the same weak promoter, which has the same gRNA recognition site of the CRIPSR activation system. Therefore, the dCas9-VPR fusion protein can target the CYC1 promoter of both genes, thereby activating both genes simultaneously. By using dual screening tags on the basic strains of recombinant experiments, VPR mutation and efficient screening can be carried out.

[0098] The culture medium and reagent formulations involved in the following examples are as follows:

[0099] LB liquid medium: 1% peptone, 0.5% yeast extract, 1% NaCl;

[0100] LB solid medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 1.5% agar;

[0101] YPD liquid medium: 2% peptone, 1% yeast extract, 2% glucose;

[0102] YPD solid medium: 2% peptone, 1% yeast extract, 2% glucose, 1.5% agar;

[0103] SC medium amino acid powder mixture (without histidine, leucine, and uracil): adenine 0.5g, alanine 2.0g, arginine 2.0g, asparagine 2.0g, aspartic acid 2.0g, cysteine ​​2.0g, glutamine 2.0g, glutamic acid 2.0g, glycine 2.0g, inositol 2.0g, isoleucine 2.0g, lysine 2.0g, cysteine ​​2.0g, para-aminobenzoic acid 0.2g, phenylalanine 2.0g, proline 2.0g, serine 2.0g, threonine 2.0g, tryptophan 2.0g, tyrosine 2.0g, valine 2.0g.

[0104] SC-Ura liquid medium (1L): yeast nitrogen base (ammonium sulfate and amino acid mixture without) 1.7g, ammonium sulfate 5g, amino acid mixture (histidine, leucine and uracil without) 1.655g, histidine 0.086g, leucine 0.173g, glucose 2%.

[0105] SC-Ura solid medium (1L): yeast nitrogen base (without ammonium sulfate and amino acid mixture) 1.7g, ammonium sulfate 5g, amino acid mixture (without histidine, leucine and uracil) 1.655g, histidine 0.086g, leucine 0.173g, glucose 2%, agar 1.5%.

[0106] Example 1: Construction of Error-Prone PCR Mutant Library

[0107] Using the Errorprone PCR Kit (GeneMorph II Random Mutagenesis Kit (Angilent Technologies)), an Errorprone PCR mutation library was constructed with a base mutation rate of 1-2‰ (2-4 bases / gene) and an amino acid mutation rate of 1-2 amino acids / gene.

[0108] The reaction system for error-prone PCR is shown in Table 1, with a total volume of 50 μL.

[0109] Table 1 Commonly Misunderstood PCR Reaction Systems

[0110]

[0111] The sequences of upstream primer F and downstream primer R are as follows:

[0112] Upstream primer F: 5′-GGAGCTCCCGCTGCTTTTAAATATTTTG-3′

[0113] Downstream primer R: 5′-GCCGGTAGAGGTGTGGTCAATAAG-3′

[0114] The amplification program for error-prone PCR is shown in Table 2:

[0115] Table 2. Amplification program for error-prone PCR

[0116]

[0117] Error-prone PCR products were separated and tested by agarose gel electrophoresis, followed by column purification to obtain pure errorprone DNA products. The recombinant vector pCEN-L-Kan was digested with the restriction endonuclease BsaI-HFv2 and then purified by column purification to obtain pure digested vector products.

[0118] Error-prone PCR and vector enzyme digestion purification products were co-electroplated into a *Saccharomyces cerevisiae* strain containing the red fluorescent protein gene *mCherry* (activation intensity indicator gene) and the bleomycin resistance weakening gene *BleoR13*. The resulting plasmid was assembled using the endogenous homologous recombination repair mechanism of *Saccharomyces cerevisiae*. After recovery by incubation at 30°C for 5 hours, 4 ml of the electroporated bacterial solution was evenly spread onto four SC-Ura selection plates and incubated at 30°C for 2-3 days to obtain a random mutant library. Example 2: Screening of high activation intensity mutants from error-prone PCR libraries.

[0119] Since the experimental Saccharomyces cerevisiae strain has integrated the red fluorescent protein gene mCherry and the bleomycin resistance weakening gene BleoR13 into its genome, it is possible to use the red fluorescence intensity and bleomycin resistance intensity to perform high-throughput screening of the activator VPR with high activation intensity.

[0120] 96-hole plate primary sieve:

[0121] (1) Use a cell spreader and 2ml of sterile water to scrape the colonies from the random mutant library, i.e., four SC-Ura screening plates, into a 50ml shaker tube. After centrifugation at 3000rpm for 3min, remove the supernatant, add 4ml of SC-Ura liquid culture medium, and incubate overnight at 30℃ and 200rpm on a shaker.

[0122] (2) Measurement of absorbance (OD) of overnight cultured bacterial culture 600 Transfer to a new 4ml SC-Ura liquid medium, initial OD 600 The concentration was 0.1. The cells were cultured at 30℃ and 200rpm in a shaker for 1.5 days, and then flow cytometry was performed for sorting. At the same time, strains containing the original VPR plasmid were cultured as red fluorescence intensity control and bleomycin resistance intensity control, and strains without green fluorescence were cultured as plasmid-free negative control.

[0123] (3) Prepare a 96-well cell culture plate for sorting in advance. Use sterile, unsolidified SC-Ura solid medium. When the medium cools to about 50°C, add a certain concentration (100 mg / L for the first round of screening) of bleomycin for sorting. You can set a concentration gradient to improve the sorting effect. Add 200 μL of solid medium to each well for sorting.

[0124] (4) When sorting cells by flow cytometry, first select the green fluorescent positive cell clusters, i.e. cells containing the mutant activation domain plasmid. Then, select the cells with the highest red fluorescence intensity of the strains containing the mutant library and sort them into 50 cells in a 96-well cell culture plate. The strains containing the initial VPR plasmid are sorted into one plate under the same conditions, with one single clone in each well, and placed in a 30°C incubator for culture.

[0125] (5) 3 to 5 days after sorting, observe the growth of the 96-well culture plate. If the control bacteria cannot grow, the experimental bacteria containing the mutant library can grow at a lower frequency on the 96-well plate. Select the single clones that can grow and transfer them to the SC-Ura solid culture plate for enrichment culture in a 30°C incubator.

[0126] (6) Culture the strain containing the mutant VPR plasmid on a plate for 3-5 days to obtain an effective VPR mutant strain.

[0127] Dropping point test for further screening:

[0128] (1) Pick single clones from the above enrichment culture plate and inoculate them into 4 ml SC-Ura medium. At the same time, inoculate strains without VPR plasmid and strains with only initial VPR plasmid as controls. Incubate overnight in a shaker at 30℃ and 200 rpm.

[0129] (2) The above-mentioned overnight cultured bacteria were transferred to a new culture medium, and the initial absorbance value OD was measured. 600 The concentration was 0.1, and the culture was carried out at 30°C and 200 rpm in a shaker until the OD value was reached. 600 The drop point experiment was conducted with values ​​ranging from 0.8 to 1.2 OD to preliminarily determine the VPR activation intensity.

[0130] (3) For the above-mentioned culture to OD 600 For bacterial suspensions with a concentration of 0.8 to 1.2 OD, first dilute the suspension with sterile water to a concentration of OD. 600 The initial concentration was 0.1, the volume was 1 mL, and then it was diluted sequentially to OD. 600 10 -2 10 -3 10 -4 After vortexing and mixing at several concentration gradients, 5 μL of each concentration gradient is dropped onto SC-Ura culture plates with bleomycin concentrations of 100 mg / L and 200 mg / L. For the second and third rounds of screening, the concentration of bleomycin in the dropping experiments needs to be gradually increased. The plates are then incubated at 30°C for 3 days to observe growth (see...). Figures 2-5 ).

[0131] (4) Based on the growth of the mutant VPR strain in the above drop test, a mutant VPR strain with high bleomycin resistance was obtained. The mutant VPR plasmid was extracted and sequenced to determine the mutation site. The strain was then transferred back to the initial experimental strain and a drop test was conducted again to confirm the mutation.

[0132] (5) Based on the growth of the second drop plate, identify the high-efficiency VPR mutant.

[0133] Example 3: Quantitative real-time PCR (qPCR) for quantifying the activation intensity of mutant VPR

[0134] (1) Three single clones of yeast strain containing high-efficiency VPR mutant were inoculated into three tubes of 4 mL SC-Ura medium. At the same time, yeast strains without VPR and yeast strains with initial VPR were inoculated as controls. The cultures were incubated overnight in a shaker at 30°C and 200 rpm.

[0135] (2) The above-mentioned overnight cultured strains were transferred to fresh 4 mL SC-Ura medium, and the initial OD 600 The concentration was 0.1, and the culture was carried out at 30°C and 200 rpm in a shaker until the OD value was reached. 600 The concentration should be 0.9 to 1.1. Quickly take 1 mL of bacterial culture into an ice-water mixture, centrifuge at 4000 rpm for 3 min at 4℃, remove the supernatant, freeze quickly in liquid nitrogen, and store at -80℃ for later use. The entire process of collecting bacterial cells must be rapid and at low temperature.

[0136] (3) Total RNA was extracted using the above-mentioned cryopreserved bacteria and Trizol reagent (Thermo Fisher Scientific). RNase contamination should be avoided during this process. After extraction, genomic DNA was digested using DNase I enzyme (New England Biolab), and heated in a 37°C water bath for 30 minutes. The RNA was then purified again using Trizol reagent to remove DNase I. The final RNA solution was heated in a 55°C metal bath for 15 minutes, and its concentration was measured. The RNA solution was used for the next reaction or stored at -80°C.

[0137] (4) Genomic DNA digestion reaction is shown in Table 3. Total volume: 100 μL.

[0138] Table 3 Genomic DNA digestion reaction system

[0139]

[0140] (5) Take 500 ng of RNA from the above RNA solution for reverse transcription. In this experiment, the All-in-One First-Strand cDNA Synthesis (Reverse Transcription) Kit from GeneCopoeia, USA, was used for RNA-Primer Mix preparation and reverse transcription.

[0141] (6) The preparation reaction of RNA-Primer Mix is ​​shown in Table 4:

[0142] Table 4. Reaction system for RNA-Primer Mix preparation

[0143]

[0144] (7) After lightly mixing and briefly centrifuging the RNA-Primer Mix system, heat it in a 65°C metal bath for 10 minutes, and then immediately store it on ice for later use.

[0145] (8) The reverse transcription reaction system is shown in Table 5:

[0146] Table 5 Reverse Transcription Reaction System

[0147]

[0148] (9) The reverse transcription procedure is shown in Table 6:

[0149] Table 6 Reverse Transcription Procedure

[0150]

[0151] (10) Use the above reverse transcription reaction products for qPCR. The qPCR reagent used in this experiment is PowerUpSYBRGreen Master Mix from Thermo Fisher Scientific. Each RNA sample was performed in 3 technical parallels. The activation intensity test gene was the mCherry gene, and the internal control was the ACT1 gene. The reaction system and reaction procedure are shown in Table 7 and Table 8.

[0152] (11) The qPCR reaction system is shown in Table 7, with a total volume of 12.5 μL.

[0153] Table 7 qPCR reaction system

[0154]

[0155] The upstream and downstream primer sequences for the mCherry gene cDNA are as follows:

[0156] Upstream primer: 5′-GAGGAGGATAACATGGCCATCA-3′

[0157] Downstream primer: 5′-GCCCTCCATGTGCACCTT-3′

[0158] The upstream and downstream primer sequences for the ACT1 gene cDNA are as follows:

[0159] Upstream primer: 5′-TCGTTCCAATTTACGCTGGTT-3′

[0160] Downstream primer: 5′-CGGCCAAATCGATTCTCAA-3′

[0161] The amplification program for qPCR is shown in Table 8:

[0162] Table 8. qPCR Amplification Program

[0163]

[0164] By analyzing qPCR experimental data, the activation intensity of the mutant VPR was quantified, and high-activation-intensity mutant VPRs were obtained (see...). Figure 6 ).

[0165] Example 4: Determine whether to perform continuous random mutation based on actual needs or the intensity of the mutation VPR.

[0166] If the VPR mutants obtained according to Examples 1-3 do not meet the actual needs, or the activation intensity of the mutated VPR is low, the VPR gene can be used as a template for the next round of random mutation and screening until a mutant VPR protein with ideal activation intensity is obtained.

[0167] This experiment used three rounds of error-prone PCR to construct mutant libraries, followed by initial screening using a 96-well plate sorter, secondary screening using a dropper test, and qPCR to quantify activation intensity. Three VPR mutant proteins [named VPR-1 (SEQ ID NO2), VPR-2 (SEQ ID NO3), and VPR-3 (SEQ ID NO4), respectively] with progressively increasing activation intensity were obtained. The nucleotide sequences and amino acid mutations of the vectors containing the mutant VPRs were extracted and determined, as shown in Table 9.

[0168] Table 9 Sequencing results of the mutants

[0169]

[0170] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A VPR mutant protein capable of activating gene expression in Saccharomyces cerevisiae, the sequence of which includes the following mutations in the amino acid sequence of wild-type VPR as shown in SEQ ID NO 1: Asp at position 35 is mutated to His, Thr at position 114 is mutated to Arg, Ala at position 191 is mutated to Val, Ala at position 203 is mutated to Val, Ala at position 215 is mutated to Thr, Ala at position 234 is mutated to Ser, Leu at position 302 is mutated to Met, and a protein translation termination mutation is generated at position 370 (Leu); the sequence of the VPR mutant protein is shown in SEQ ID NO 4.

2. The VPR mutant protein according to claim 1, characterized in that, The mutant protein also includes a fusion protein obtained by attaching a tag to the N-terminus or C-terminus of the above-mentioned VPR mutant protein.

3. The VPR mutant protein according to claim 2, characterized in that, The mutant protein is a fusion protein obtained by linking dCas9 to the N-terminus or C-terminus of the VPR mutant protein.

4. A nucleotide molecule encoding the mutant protein of any one of claims 1-3, wherein the sequence comprises the following mutations located in the nucleotide sequence encoding wild-type VPR as shown in SEQ ID NO 5: G to C at position 104, G to A at position 369, A to G at position 455, C to T at position 572, C to T at position 608, G to A at position 643, G to T at position 700, C to A at position 904, and T to A at position 1109; the nucleotide sequence of the nucleotide molecule encoding the VPR mutant protein is shown in SEQ ID NO 8.

5. The nucleotide molecule according to claim 4, characterized in that, The nucleic acid molecule is the following DNA molecule: (a1) A DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO 8; (a2) DNA molecules with nucleotide sequences as shown in SEQ ID NO 8.

6. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleotide molecule of claim 4 or 5.

7. The use of the mutant protein as described in any one of claims 1-3, or the mutant protein obtained from the nucleotide molecule as described in claim 4 or 5, or the expression cassette, recombinant vector, or recombinant microorganism as described in claim 6, in activating the expression of exogenous genes in Saccharomyces cerevisiae.

8. The application according to claim 7, characterized in that, The applications include strains or the target product whose yield is increased by activating the expression of exogenous genes in wild or recombinant Saccharomyces cerevisiae.

Images