Engineered yeast as well as construction method and application thereof

Abstract

The invention relates to a yeast engineering bacterium as well as a construction method and application thereof.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a lysine-regulated yeast engineering bacterium and a construction method thereof. Background Art

[0002] Yeast is a unicellular fungus that is widely distributed throughout nature. It is a typical heterotrophic facultative anaerobic microorganism that can survive under aerobic and anaerobic conditions and is a natural fermentation agent. There are many known yeast species, among which Saccharomyces cerevisiae is the most mature yeast. Saccharomyces cerevisiae is a unicellular eukaryotic organism with spherical or oval cells and a diameter of 5-10μm. It is considered to be the most promising large-scale production strain and the first eukaryotic organism to complete genome sequencing. Saccharomyces cerevisiae has the advantages of clear genetic background, rapid growth, high stress resistance, and no phage contamination. It also contains a variety of proteins, amino acids, vitamins, biologically active substances and other rich nutrients. As a host system, it expresses a variety of exogenous gene products and is widely used in various industrial production and research purposes. At the same time, Saccharomyces cerevisiae is also a eukaryotic model organism for modern molecular and cell biology research.

[0003] Gene editing and transcriptional regulation play a very important role in the basic research and industrial application of Saccharomyces cerevisiae. Among them, exploring the highest possible expression of the target gene is often a necessary means to improve production yield or titer in industrial production. At present, many methods for gene regulation of Saccharomyces cerevisiae have been reported in the literature, such as the use of copper ions or temperature to induce gene expression. However, these methods are still some distance away from industrial production because they may cause gene instability or are difficult to implement in large-scale production. In addition, many gene regulation methods have the problem of leaky expression, which often has an adverse effect on cell growth when expressing certain cytotoxic proteins (reference: doi: 10.1093 / nar / 28.24.e108). In addition, high gene expression often causes cell stress, which in turn affects strain stability. Therefore, how to maximize the metabolic yield of the strain while maintaining strain stability is an urgent problem to be solved. An effective way to solve this problem is to artificially regulate the timing of gene expression, distinguish the growth phase (growth phase) and the production phase (production phase) of the strain, so that the bacterial community is mainly used for growth and accumulation of bacterial cells in the early stage of fermentation, and mass production of target products in the middle and late stages of fermentation.

[0004] At present, the most commonly used induction expression system in yeast is the yeast galactose induction system (GAL induction system). The regulation of the GAL system is quite strict. It is inhibited in the presence of glucose and activated in the presence of galactose. In the absence of galactose, the transcription repressor GAL80p inhibits the expression of the GAL gene by binding to the transcription activator GAL4. In order to avoid excessive production costs caused by the additional addition of galactose, some studies have knocked out the GAL80 transcription factor so that the induction of the GAL promoter does not require galactose, but is inhibited by high glucose and expressed in low glucose (in the presence of glucose, the transcription of the GAL4 gene is inhibited through the Snf1 signaling pathway, thereby inhibiting the activation of pGAL, and the bacteria mainly grow at this time; when the glucose is consumed, the pGAL promoter begins to express, and the target product can be mass-produced). This strategy solves the problem of the high cost of the system in industrial applications to a certain extent. However, glucose is a commonly used carbon source in fed-batch fermentation, and the instability of the process will cause large fluctuations in the expression of genes controlled by the GAL promoter, which is not conducive to the stability between batches in the production process.

[0005] Therefore, constructing a new yeast engineered strain does not require the use of expensive galactose for activation, thereby reducing production costs; at the same time, the growth and production stages of the isolated strains can be artificially regulated to maximize the yield of the target gene product, which is of great significance in the industrial production of yeast. SUMMARY OF THE INVENTION

[0007] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a lysine-regulated yeast engineering bacterium and its construction method and application. The yeast engineering bacterium disclosed in the present invention does not need to use expensive galactose to activate the galactose regulation system, thereby reducing production costs; lysine is used to distinguish the growth stage and production stage of the yeast engineering bacterium, so that the bacterial community is mainly used for growth in the early stage of fermentation, and a large amount of target products are accumulated in the middle and late stages of fermentation, thereby maximizing the yield of the target gene product.

[0008] In order to achieve the above objectives, the present disclosure adopts the following specific scheme:

[0009] In one aspect, the present disclosure provides a yeast engineered bacterium, wherein the yeast engineered bacterium comprises the following modifications:

[0010] (1) a modified endogenous GAL80 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL80 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL80 gene;

[0011] (2) a modified endogenous GAL4 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL4 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL4 gene;

[0012] (3) Introducing the GAL4 gene into the yeast engineering bacteria, wherein the GAL4 gene is operably linked to a lysine-regulated promoter, wherein the lysine-regulated promoter is derived from the upstream regulatory sequence of the LYS9 gene (pLYS9 promoter) and inhibits the expression of the GAL4 gene in the presence of lysine.

[0013] According to certain embodiments of the present disclosure, the pLYS9 promoter comprises the nucleotide sequence shown in SEQ ID NO:9.

[0014] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a sequence fragment or a combination of fragments of the pLYS9 promoter.

[0015] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a fragment of at least 300 bp from the 3′ end of the sequence of the pLYS9 promoter;

[0016] Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:11.

[0017] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter;

[0018] Preferably, the lysine-regulated promoter further comprises the sequence shown in SEQ ID NO:26.

[0019] According to certain embodiments of the present disclosure, a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter is placed upstream of a fragment of at least 300 bp at the 3′ end of the sequence of the pLYS promoter;

[0020] Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:19.

[0021] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a sequence as shown in any one of SEQ ID NOs: 27-30, or a combination thereof.

[0022] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises:

[0023] The combination of SEQ ID NOs: 28, 29 and 30;

[0024] The combination of SEQ ID NOs: 27, 29 and 30;

[0025] A combination of SEQ ID NOs: 27, 28 and 30; or

[0026] The combination of SEQ ID NOs: 27, 28 and 29.

[0027] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a sequence as shown in any one of SEQ ID NOs: 9-11 and SEQ ID NOs: 16-24;

[0028] Preferably, the lysine-regulated promoter comprises a sequence as shown in SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.

[0029] According to certain embodiments of the present disclosure, the modified endogenous GAL80 gene and / or its regulatory sequence is selected from a knocked-out endogenous GAL80 gene and / or its regulatory sequence, and a mutation-inactivated endogenous GAL80 gene and / or its regulatory sequence;

[0030] and / or

[0031] The modified endogenous GAL4 gene and / or its regulatory sequence is selected from a knocked-out endogenous GAL4 gene and / or its regulatory sequence, and a mutated inactivated endogenous GAL4 gene and / or its regulatory sequence.

[0032] According to certain embodiments of the present disclosure, the modified endogenous GAL80 gene and / or its regulatory sequence comprises a knocked-out endogenous GAL80 gene and / or its regulatory sequence, and / or

[0033] The modified endogenous GAL4 gene and / or its regulatory sequence includes the promoter of the endogenous GAL4 gene being replaced by a terminator.

[0034] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the genome of the yeast engineered bacteria;

[0035] Preferably, the GAL4 gene is integrated into the downstream non-coding region of the HPR1, ECM1 3, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene in the genome of the yeast engineered bacteria.

[0036] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the region 0-2000 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene of the yeast genome.

[0037] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the region 0-1000 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene in the genome of the yeast engineered bacteria.

[0038] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the region 0-500 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene in the genome of the yeast engineered bacteria.

[0039] According to certain embodiments of the present disclosure, the yeast engineering bacteria further comprises a yeast galactose inducible system (GAL inducible system), wherein the GAL inducible system comprises a GAL4-induced activated promoter, and a gene of interest operably linked to the GAL4-induced activated promoter.

[0040] According to certain embodiments of the present disclosure, the GAL4-induced activated promoter is selected from the group consisting of a pGAL 1 promoter, a pGAL2 promoter, a pGAL3 promoter, a pGAL4 promoter, a pGAL7 promoter, a pGAL 10 promoter, and a pGAL80 promoter.

[0041] According to certain embodiments of the present disclosure, the yeast is selected from Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis; preferably, the yeast is Saccharomyces cerevisiae.

[0042] In another aspect, the present disclosure provides use of engineered yeast in producing a target product encoded by the gene of interest.

[0043] In another aspect, the present disclosure provides a method for producing a target product, the method comprising:

[0044] 1) pre-culturing the yeast engineering bacteria of the present disclosure in a medium supplemented with lysine under conditions suitable for the growth of the yeast engineering bacteria;

[0045] 2) culturing the yeast engineered bacteria under conditions suitable for expressing a target product from the gene of interest; and

[0046] 3) Recovering the target product from the yeast engineered bacteria cells and / or culture medium.

[0047] According to certain embodiments of the present disclosure, the amount of lysine added to the culture medium in step 1) is about 0.2-1 g / L;

[0048] Preferably, the amount of lysine added is about 0.3-0.5 g / L.

[0049] In another aspect, the present disclosure provides a method for constructing the yeast engineering bacteria, the method comprising:

[0050] (1) modifying the endogenous GAL80 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL80 gene, and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL80 gene;

[0051] (2) modifying the endogenous GAL4 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL4 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL4 gene;

[0052] (3) Introducing the GAL4 gene and the lysine-regulated promoter into the yeast engineering bacteria, wherein the GAL4 gene is operably linked to the lysine-regulated promoter.

[0053] According to certain embodiments of the present disclosure, the modification or introduction in steps (1), (2) and / or (3) is achieved using the CRISPR / Cas9 gene editing system.

[0054] According to certain embodiments of the present disclosure, the Cas9 protein coding sequence of the CRISPR / Cas9 gene editing system is integrated into the genome of the yeast engineered bacteria.

[0055] On the other hand, the present disclosure provides a lysine-regulated promoter, which is derived from the upstream regulatory sequence of the LYS9 gene (pLYS9 promoter), wherein the lysine-regulated promoter comprises a sequence fragment or a combination of fragments of the pLYS9 promoter, wherein the pLYS9 promoter comprises the nucleotide sequence shown in SEQ ID NO: 9.

[0056] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a fragment of at least 300 bp from the 3′ end of the sequence of the pLYS9 promoter;

[0057] Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:11.

[0058] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter;

[0059] Preferably, the lysine-regulated promoter further comprises the sequence shown in SEQ ID NO:26.

[0060] According to certain embodiments of the present disclosure, a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter is placed upstream of a fragment of at least 300 bp at the 3′ end of the sequence of the pLYS promoter;

[0061] Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:19.

[0062] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a sequence as shown in any one of SEQ ID NOs: 27-30, or a combination thereof.

[0063] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises:

[0064] The combination of SEQ ID NOs: 28, 29 and 30;

[0065] The combination of SEQ ID NOs: 27, 29 and 30;

[0066] A combination of SEQ ID NOs: 27, 28 and 30; or

[0067] The combination of SEQ ID NOs: 27, 28 and 29.

[0068] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a sequence as shown in any one of SEQ ID NOs: 9-11 and SEQ ID NOs: 16-24;

[0069] Preferably, the lysine-regulated promoter comprises a sequence as shown in SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 : Flowchart of the technology for gene editing in Saccharomyces cerevisiae.

[0071] Figure 2 :Schematic diagram of the integration structure of CRISPR / Cas9 gene.

[0072] Figure 3 : Schematic diagram of the design of pLYS9 promoters of different lengths.

[0073] Figure 4: The effect of lysine-regulated pLYS9 promoters of different lengths expressing the transcription activator GAL driving GFP expression at 8 hours.

[0074] Figure 5 : The effect of lysine-regulated pLYS9 promoters of different lengths expressing the transcription activator GAL driving GFP expression at 24 hours.

[0075] Figure 6 : The effect of lysine-regulated pLYS9 promoters of different lengths expressing the transcription activator GAL driving GFP expression at 32 hours.

[0076] Figure 7 : The effect of lysine-regulated pLYS9 promoters of different lengths expressing the transcription activator GAL driving GFP expression at 48 hours.

[0077] Figure 8 : Schematic diagram of pLYS9 promoter design with different deletion fragments.

[0078] Fig. 9 : The effect of lysine regulation on the expression of transcription activator GAL driven GFP expression by pLYS9 promoter with different deletion fragments at 48 hours.

[0079] Fig.10 : Leakage expression results of pLYS9 promoter with different deletion fragments under the regulation of 1g / L lysine at 48 hours.

[0080] Fig.11 :Effects of different concentrations of lysine (0-5g / L) on the expression regulation of GAL4 induced by the 300bp pLYS9 promoter.

[0081] Fig.12 :Effects of different concentrations of lysine (0-0.5g / L) on the expression regulation of GAL4 induced by the 300bp pLYS9 promoter.

[0082] Fig.13 : Schematic representation of lysine regulation of gene expression of interest during fermentation and distinguishing between growth and production phases.

[0083] Fig.14 : Effect of pLYS9 promoter with different lengths and sequence deletions on inducing GAL4 expression under the regulation of 0.5 g / L lysine.

[0084] Fig.15 : Leakage expression results of pLYS9 promoter with different lengths and sequence deletions at 48 hours under the regulation of 0.5g / L lysine. DETAILED DESCRIPTION OF THE INVENTION

[0086] All publications and patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference into the present disclosure and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with the cited publication.

[0087] Each of the embodiments described and illustrated herein has separate components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any described method can be executed in the order of events described or in any other order that is logically possible.

[0088] The experimental methods for the specific conditions not specified in the following examples of the present disclosure are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Various commonly used chemical and biological reagents used in the examples are all commercially available products. Unless otherwise specified, the present disclosure is not limited to specific materials, reagents, tools, etc.

[0089]

[00136] Embodiments of the present disclosure will employ, unless otherwise indicated, chemical, biological, and other techniques within the skill of the art.

[0090] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as those generally understood by those skilled in the art of the present disclosure. The terms used in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. It is also possible in the present disclosure that the steps can be performed in different orders where it is logically feasible.

[0091] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0092] Where a numerical range is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly indicates otherwise), between the upper and lower limits of the range and any other intervening values ​​stated or within the stated range are encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. When a stated range includes one or both limits, ranges excluding either or both of these included limits are also encompassed within the disclosure.

[0093] The terms "comprising," "containing," "including," and "having," and any variations thereof, of the present disclosure are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps inherent to the process, method, product, or device. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0094] The term "plurality" used in this disclosure refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0095] With respect to the recitation of numerical ranges herein, each intermediate number having the same precision therebetween is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0096] The term "MM medium" used in the present disclosure refers to a minimal medium (MM) that can only meet the growth requirements of wild-type strains of microorganisms. The minimal culture media of different microorganisms are different. The minimal culture medium used in the embodiments of the present disclosure is a minimal culture medium suitable for the cultivation of brewer's yeast.

[0097] The term "galactose regulatory mechanism" used in the present disclosure refers to the GAL regulatory mechanism, which specifically refers to the transcriptional activation initiated by the promoters pGAL 1 promoter, pGAL2 promoter, pGAL3 promoter, pGAL4 promoter, pGAL7 promoter, pGAL 10 promoter and pGAL80 promoter in the galactose regulatory system of yeast, which is regulated by the positive activation of the GAL4 transcription activator, and the activity of the GAL4 transcription factor is inhibited by the GAL80 negative regulatory protein, and the activity of the GAL80 protein is regulated by galactose in the culture medium. When galactose exists in the cell growth environment, galactose can competitively bind to the GAL80 protein, release the GAL4 transcription factor, and GAL4 further activates the transcription of the promoters pGAL1 promoter, pGAL2 promoter, pGAL3 promoter, pGAL4 promoter, pGAL7 promoter, pGAL10 promoter and pGAL80 promoter in the system. Therefore, in the GAL regulatory system, galactose is an activator of the GAL promoter.

[0098] The term "galactose inducible (GAL) system" used in the present disclosure includes GAL promoters (such as pGAL1, pGAL2, pGAL3, pGAL4, pGAL7, pGAL10 and pGAL80) and their regulatory networks (for a detailed description of the GAL promoter, see Deng, J., Wu, Y, Zheng, Z. et al. A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae. Microb Cell Fact 20, 202 (2021). https: / / doi.org / 10.1186 / s12934-021- 01691-3, which is incorporated herein by reference in its entirety). Deng et al. divided the genes involved in the GAL induction system into two categories, one of which is related to galactose metabolism, including GAL1, GAL2, GAL7 and GAL10, and the other is responsible for the regulation of the former, including GAL4, GAL80 and GAL3. In this system, GAL4p is a transcriptional activator and GAL80p is an inhibitor of GAL4p. The GAL promoter needs to be activated by the transcriptional activator GAL4 to initiate transcription, and the activity of the transcriptional activator GAL4 is inhibited by the GAL80 negative regulatory protein. Generally speaking, GAL4p is docked on the GAL promoter. In the absence of galactose, GAL80p binds to GAL4p and inhibits the activation of the GAL promoter. When galactose is added, GAL3p is a transcriptional regulator that forms a complex with GAL80p to relieve the inhibition of GAL4p by GAL80p and activate the activity of the GAL promoter. However, this activation may be hindered by glucose inhibition. The activity of GAL80 protein is regulated by galactose in the culture medium. When galactose is present in the cell growth environment, galactose can competitively bind to GAL80 protein, releasing GAL4 transcription factor, which further activates the transcription of GAL promoters (such as pGAL1, pGAL2, pGAL3, pGAL4, pGAL7, pGAL10 and pGAL80) in the system. Therefore, in the GAL regulatory system, galactose is an activator of the GAL promoter. However, since galactose is expensive, the galactose-induced method is not suitable for industrial application due to its high cost.

[0099] The term "engineered yeast" used in the present disclosure refers to yeast modified by genetic engineering methods.

[0100] The term "Kluyveromyces lactis" as used in the present disclosure refers to Kluyveromyces lactis, which also has a GAL regulatory mechanism (see Pannala, VR, Ahammed Sherief, KY, Bhartiya, S. et al. Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae. SVst Synth Biol 5, 69-85 (2011). https: / / doi.org / 10.1007 / s11693-011-9082-7, which is incorporated herein by reference in its entirety). Saccharomyces cerevisiae GAL4 can activate the lactose-galactose regulatory mechanism of Kluyveromyces lactis (see Riley, MI, Hopper, JE, Johnston, SA, & Dickson, RC (1987). GAL4 of Saccharomyces cerevisiae Activates the Lactose-Galactose Regulon of Kluyveromyces lactis and Creates a New Phenotype: Glucose Repression of the Regulon. Molecular and Cellular Biology, 7(2), 780-786. https: / / doi.org / 10.1128 / mcb.7.2.780-786.1987, which is incorporated herein by reference in its entirety).

[0101] The term "Pichia pastoris" used in the present disclosure refers to Pichia pastoris. The Saccharomyces cerevisiae GAL4 sequence is very similar to the Pichia pastoris PpATT1 sequence, and the PpATT1 gene can regulate the thermotolerance of Pichia pastoris and affect the growth of Pichia pastoris (see Jiang B Argyros R, Bukowski J Nelson S, Sharkey N, Kim S, Copeland V Davidson RC, Chen R Zhuang J, Sethuraman N, Stadheim TA. 2015. Inactivation of a GAL4-LikeTranscription Factor Improves Cell Fitness and Product Yield in Glycoengineered Pichia pastoris Strains. Appl Environ Microbio 181:. https: / / doi.org / 10.1128 / AEM.02619-14, which is incorporated herein by reference in its entirety).

[0102] The term "GFP" or green fluorescent protein (GFP) used in the present disclosure is a commonly used reporter protein for gene expression. Arun et al., J. Pharmacol. Toxicol. Methods 51(1): 1-23, 2005.

[0103] The term "nucleic acid" or "nucleic acid sequence" used in the present disclosure refers to a polymeric form of a nucleotide, ribonucleotide or deoxynucleotide or a modified form of any type of nucleotide. These terms should also be understood to include analogs of RNA or DNA made from nucleotide analogs as equivalents, as well as single-stranded (such as sense or antisense) and double-stranded polynucleotides suitable for the described embodiments. The term "nucleic acid" or "nucleic acid sequence" can also encompass chains including non-natural nucleotides, modified nucleotides and / or non-nucleotide components (such as "nucleotide analogs") that can exhibit the same functions as natural nucleotides; in addition, the term "nucleic acid sequence" used herein refers to oligonucleotides, nucleotides or polynucleotides, and fragments or portions thereof, as well as genomic DNA or RA or DNA or RA of synthetic origin, which can be single-stranded or double-stranded, and represent sense or antisense strands. The terms "nucleic acid", "polynucleotide", "nucleotide sequence" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0104] The term "gene" as used in the present disclosure refers to a DNA sequence including control sequences and coding sequences for producing RAs with non-coding functions (e.g., ribosomal RAs or transport RAs), polypeptides, or precursors of any of the foregoing substances. RAs or polypeptides can be encoded by the full-length coding sequence or by any portion of the coding sequence, as long as the desired activity or function is retained. Therefore, "gene" refers to DNA or RNA or a portion thereof that encodes a polypeptide or RNA chain that plays a functional role in an organism. For the purposes of the present disclosure, a gene can be considered to include regions that regulate the production of a gene product, regardless of whether such regulatory sequences are adjacent to the coding sequence and / or the transcribed sequence. Therefore, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0105] When referring to a gene product, e.g., encoded by a coding sequence, the terms "protein" (if single-chain), "polypeptide" and "peptide" are used interchangeably herein. "Protein" may also refer to an association of one or more polypeptides. "Gene product" refers to a molecule produced as a result of transcription of a gene. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts. A "target product" herein is the product of a gene of interest.

[0106] The term "operably linked" as used in this disclosure is to be understood as the respective components being linked together in a manner that enables them to perform their functions (eg, binding DNA, catalyzing a reaction, or recruiting additional agents from within a cell) substantially unhindered.

[0107] The term "gene construct" used in the present disclosure refers to a single-stranded or double-stranded nucleic acid, such as a vector, plasmid, viral genome, etc. It is separated from a naturally occurring gene, or modified into a segment containing a nucleic acid in a manner that does not originally exist in nature, or is synthetic, and includes a "coding sequence" of a polypeptide or can be transcribed into a biologically active RA (e.g., antisense, bait, ribozyme, etc.) in other ways, can be transfected into a cell, for example, in certain embodiments, transfected into a yeast engineered bacterial cell, and can cause the coding sequence to be expressed in a cell transfected with the construct. A gene construct may include one or more regulatory elements operably linked to a coding sequence.

[0108] As used in this disclosure, the terms "non-naturally occurring," "engineered," and "synthetic" are used interchangeably and indicate the involvement of human effort. When referring to nucleic acid molecules or polypeptides, these terms refer to nucleic acid molecules or polypeptides that are at least substantially free of at least one other component with which they are naturally associated in nature and found in nature.

[0109] The gene editing technology used in the present disclosure may include homing endonucleases (MegNs) technology, zinc finger nucleases (ZFNs) technology, transcription activator-like effector nucleases (TALENs) technology, and CRISPR-related technologies, etc. This article describes the present disclosure in detail using the CRISPR-Cas9 system as an example.

[0110] The term "CRISPR-Cas9" used in this disclosure refers to a molecular biology technology that allows researchers to edit genes in cells and organisms using the CRISPR-Cas9 gene editing system, that is, to modify the genetic material in an organism through certain biological means and techniques.

[0111] Delivery vectors, such as mRNA or protein delivery systems based on nanoparticles and lipids, can be used during gene editing. Other examples of delivery vectors include lentiviral vectors, ribonucleoprotein (RP) complexes, lipid-based delivery systems, gene guns, fluid dynamics, electroporation or nuclear transfection microinjection and biolistics. Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1; 459 (1-2): 70-83) discuss various gene delivery methods in detail, incorporated herein by reference.

[0112] The term "vector" as used in this disclosure is a tool that allows or facilitates the transfer of an entity from one environment to another. Some vectors used in the present disclosure for recombinant nucleic acid technology allow entities, such as nucleic acid fragments (e.g., heterologous DNA fragments, such as heterologous cDNA fragments) to be transferred to cells. Vectors can be used for the following purposes: maintaining heterologous nucleic acids (DNA or RA) in cells, promoting the replication of vectors containing nucleic acid fragments, or promoting the expression of proteins encoded by nucleic acid fragments. Vectors can be non-viral or viral.

[0113] Vectors can be introduced into cells using various techniques known in the art, such as transfection, transformation, and transduction. When exogenous DNA, such as a recombinant expression vector, is introduced into a cell, the cell is "genetically modified," "transformed," or "transfected" by the exogenous DNA. The presence of exogenous DNA can result in permanent or temporary genetic changes. The transformed DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA can be maintained on an episomal element, such as a plasmid, maintained as an autonomously replicating sequence, or as an extrachromosomal element.

[0114] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes the process of delivering genes to cells using non-viral vectors. Typical transfection methods include electroporation, DNA gene guns, lipid-mediated transfection, compressed DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic surface amphiphiles (CFA) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

[0115] In the case of eukaryotic cells, stably transformed cells refer to cells in which the transforming DNA has been integrated into the chromosome and thus inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones that include a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or a common ancestor that is formed by mitosis. A "positive clone" refers to a population of cells that correctly expresses a gene of interest.

[0116] The term "integration site" as used in this disclosure refers to the location where a gene is inserted into a nucleic acid.

[0117] The term "neutral locus" used in the present disclosure refers to a position in the genome, and it is generally believed that genetic modification at this site will not affect cell viability or cause obvious phenotypic changes (Ng AH, BerlaBM, Pakrasi HB. Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC6803. Appl Environ Microbiol. 2015 Oct; 81 (19): 6857-63. doi: 10.1128 / AEM.01349-15. Epub 2015 Jul 24. PMID: 26209663; PMCID: PMC4561709). Therefore, the neutral site is considered to be an ideal gene expression cassette position.

[0118] The term "homologous recombination (HR)" as used in this disclosure refers to a form of recombination in which homologous sequences of two DNA molecules are directly exchanged. Homologous recombination does not rely on sequence specificity, but only on sequence homology. The homologous sequences exchanged may be exactly the same or very similar.

[0119] The term "knockout" used in the present disclosure refers to reducing, inhibiting or eliminating the expression of the corresponding gene sequence and / or reducing, inhibiting or eliminating the activity of the protein encoded by the gene. In addition to terminating the expression of a certain gene, gene knockout also includes the introduction of new genes and the introduction of site-directed mutations. It can be either using mutant genes or other genes to knock out the corresponding normal genes, or using normal genes to knock out the corresponding mutant genes. At present, gene knockout technology has derived more and more new methods from traditional homologous recombination strategies, such as the Red homologous recombination system, Cre / LoxP site-specific recombination, insertion mutation, RNA interference, zinc finger nuclease (ZFN) technology and transcription activator-like effector nuclease technology, CRISPR / Cas9 gene editing system, etc.

[0120] The term "gene silencing" as used in the present disclosure refers to the non-expression of a specific gene in an organism due to various reasons. Gene silencing can be achieved using gene knockout methods.

[0121] As used in this disclosure, the terms "gene of interest", "gene of interest", "target sequence" and "target sequence" are used interchangeably to refer to a specific gene that is intended to be studied or manipulated. A "gene of interest" can be endogenous or exogenous. As used in this disclosure, the terms "target product" and "product of interest" are used interchangeably to refer to the expression product of a gene of interest.

[0122] The term "promoter" used in the present disclosure refers to a site on a DNA template that is composed of a special nucleotide sequence and that RA polymerase specifically recognizes, binds to, and initiates transcription, and has directionality. The structure of the promoter affects its binding to RA polymerase, thereby controlling the start time and degree of gene expression (transcription). In addition, the end of the DNA template has a specific structure as a termination site, allowing transcription to proceed between the start and termination sites. The promoter is mainly composed of three parts: a core promoter, a proximal promoter, and a distal promoter.

[0123] The term "lysine-regulated promoter" as used in the present disclosure refers to a promoter sequence whose functional activity of initiating transcription is regulated by lysine, for example, a promoter sequence whose promoter function is inhibited by lysine.

[0124] The term "pLYS9" promoter used in the present disclosure refers to a DNA sequence that can be derived from the upstream regulatory sequence of the wild-type LYS9 gene and can promote the transcription of the wild-type LYS9 gene.

[0125] The term "transcription activator GAL4" used in the present disclosure refers to a transcription activator in the GAL regulatory system that can induce activation of downstream promoters such as pGAL1 promoter, pGAL2 promoter, pGAL3 promoter, pGAL4 promoter, pGAL7 promoter, pGAL10 promoter and pGAL80 promoter. The GAL4 protein has three functional regions, namely the N-terminal DNA binding region, the C-terminal GAL80 binding site which is also a transcription activation site, and the middle region which also affects the transcription activation level. GAL4 binding to DNA is a necessary condition for transcription initiation.

[0126] The term "exogenous" as used in this disclosure refers to something introduced from an external source into yeast.

[0127] The term "coding sequence" as used in the present disclosure means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are usually determined by an open reading frame that begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0128] The term "control sequence" used in the present disclosure means the nucleic acid sequence necessary for expressing the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence can be natural (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or natural or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At least, the control sequence includes a promoter, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that are conducive to connecting the control sequence to the coding region of the polynucleotide encoding the polypeptide, these control sequences can be provided with multiple joints.

[0129] The term "expression" as used in this disclosure includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0130] The term "gene leakage" or "leaky expression" used in the present disclosure refers to the leaky expression of genes, which refers to the phenomenon that the inserted gene is expressed before induction. At this time, if the induced recombinant protein has a certain cytotoxicity, it will cause the host bacteria to die prematurely.

[0131] In one aspect, the present disclosure provides a yeast engineered bacterium, wherein the yeast engineered bacterium comprises the following modifications:

[0132] (1) a modified endogenous GAL80 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL80 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL80 gene;

[0133] (2) a modified endogenous GAL4 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL4 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL4 gene;

[0134] (3) Introducing the GAL4 gene into the yeast engineering bacteria, wherein the GAL4 gene is operably linked to a lysine-regulated promoter, wherein the lysine-regulated promoter is derived from the upstream regulatory sequence of the LYS9 gene (pLYS9 promoter) and inhibits the expression of the GAL4 gene in the presence of lysine.

[0135] According to certain embodiments of the present disclosure, the modification is selected from deletion, substitution or knockout of nucleotides.

[0136] According to certain embodiments of the present disclosure, the pLYS9 promoter is a promoter located upstream of the LYS9 gene.

[0137] According to certain embodiments of the present disclosure, the pLYS9 promoter comprises the nucleotide sequence shown in SEQ ID NO:9.

[0138] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a sequence fragment or a combination of fragments of the pLYS9 promoter.

[0139] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a fragment of at least 300 bp at the 3' end of the sequence of the pLYS9 promoter. The fragment of 300 bp at the 3' end of the sequence of the pLYS9 promoter refers to a fragment of at least 300 bp upstream of the LYS9 gene in the 3' to 5' direction.

[0140] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:11.

[0141] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a fragment of at least 150 bp at the 5' end of the sequence of the pLYS9 promoter. The fragment of 150 bp at the 5' end of the sequence of the pLYS9 promoter refers to the nucleotide sequence from position 501 to position 650 and between the two in the 3' to 5' direction upstream of the LYS9 gene.

[0142] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises the sequence shown in SEQ ID NO:26.

[0143] According to certain embodiments of the present disclosure, a fragment of at least 150 bp at the 5' end of the sequence of the pLYS9 promoter is placed upstream of a fragment of at least 300 bp at the 3' end of the sequence of the pLYS promoter.

[0144] According to certain embodiments of the present disclosure, the amino acid-regulated promoter comprises the sequence shown in SEQ ID NO:19.

[0145] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a sequence as shown in any one of SEQ ID NOs: 27-30, or a combination thereof.

[0146] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises:

[0147] The combination of SEQ ID NOs: 28, 29 and 30;

[0148] The combination of SEQ ID NOs: 27, 29 and 30;

[0149] A combination of SEQ ID NOs: 27, 28 and 30; or

[0150] The combination of SEQ ID NOs: 27, 28 and 29.

[0151] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a sequence as shown in any one of SEQ ID NOs: 9-11 and SEQ ID NOs: 16-24; preferably comprises a sequence as shown in SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.

[0152] According to certain embodiments of the present disclosure, the modified endogenous GAL80 gene and / or its regulatory sequence is selected from a knocked-out endogenous GAL80 gene and / or its regulatory sequence, and a mutation-inactivated endogenous GAL80 gene and / or its regulatory sequence;

[0153] and / or

[0154] The modified endogenous GAL4 gene and / or its regulatory sequence is selected from a knocked-out endogenous GAL4 gene and / or its regulatory sequence, and a mutated inactivated endogenous GAL4 gene and / or its regulatory sequence.

[0155] According to certain embodiments of the present disclosure, the modified endogenous GAL80 gene and / or its regulatory sequence comprises knocking out the endogenous GAL80 gene and / or its regulatory sequence, and / or

[0156] The modified endogenous GA4 gene and / or its regulatory sequence includes replacing the promoter of the endogenous GAL4 gene with a terminator.

[0157] According to certain embodiments of the present disclosure, the GAL4 gene is expressed on the genome of the engineered yeast or expressed at a non-genomic location.

[0158] According to certain embodiments of the present disclosure, the GAL4 gene is expressed in the plasmid of the yeast engineering bacteria or integrated into the genome of the yeast engineering bacteria.

[0159] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the genome of the engineered yeast.

[0160] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into a neutral site of the genome of the engineered yeast.

[0161] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the downstream non-coding region of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene of the yeast genome.

[0162] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the region 0-2000 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene of the yeast genome.

[0163] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the region 0-1000 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene in the genome of the yeast engineered bacteria.

[0164] According to certain embodiments of the present disclosure, the GAL4 gene is integrated into the region 0-500 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene in the genome of the yeast engineered bacteria.

[0165] According to certain embodiments of the present disclosure, the GAL induction system is integrated into the genome of the engineered yeast.

[0166] According to certain embodiments of the present disclosure, the GAL inducible system is integrated into a neutral site in the genome of the engineered yeast.

[0167] According to certain embodiments of the present disclosure, the yeast engineering bacteria further comprises a yeast galactose inducible system (GAL inducible system), wherein the GAL inducible system comprises a GAL4-induced activated promoter, and a gene of interest operably linked to the GAL4-induced activated promoter.

[0168] According to certain embodiments of the present disclosure, the gene of interest is located at a neutral site in the genome of the engineered yeast.

[0169] According to certain embodiments of the present disclosure, the GAL4-induced activated promoter is selected from the group consisting of a pGAL 1 promoter, a pGAL2 promoter, a pGAL3 promoter, a pGAL4 promoter, a pGAL7 promoter, a pGAL 10 promoter, and a pGAL80 promoter.

[0170] According to certain embodiments of the present disclosure, the yeast of the present disclosure may be any yeast whose growth or metabolism is affected by the galactose regulatory system.

[0171] According to certain embodiments of the present disclosure, the yeast is selected from Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis; preferably, the yeast is Saccharomyces cerevisiae.

[0172] According to certain embodiments of the present disclosure, the GAL4 gene refers to a GAL4 gene exogenously introduced into yeast, which includes the sequence shown in SEQ ID NO:6.

[0173] In another aspect, the present disclosure provides use of engineered yeast in producing a target product encoded by the gene of interest.

[0174] In another aspect, the present disclosure provides a method for producing a target product, the method comprising:

[0175] 1) pre-culturing the yeast engineering bacteria of the present disclosure in a medium supplemented with lysine under conditions suitable for the growth of the yeast engineering bacteria;

[0176] 2) culturing the yeast engineering bacteria under conditions suitable for expressing the target product from the gene of interest; and

[0177] 3) Recovering the target product from the yeast engineered bacteria cells and / or culture medium.

[0178] According to certain embodiments of the present disclosure, the amount of lysine added to the culture medium in step 1) is about 0.2-1 g / L, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 g / L.

[0179] According to certain embodiments of the present disclosure, the amount of lysine added to the culture medium in step 1) is about 0.3-0.5 g / L.

[0180] In another aspect, the present disclosure provides a method for constructing an engineered yeast, comprising:

[0181] (1) modifying the endogenous GAL80 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL80 gene, and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL80 gene;

[0182] (2) modifying the endogenous GAL4 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL4 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL4 gene;

[0183] (3) Introducing the GAL4 gene and the lysine-regulated promoter into the yeast engineering bacteria, wherein the GAL4 gene is operably linked to the lysine-regulated promoter.

[0184] According to certain embodiments of the present disclosure, in the method for constructing an engineered yeast of the present disclosure, the modification or introduction in steps (1), (2) and / or (3) is achieved using the CRISPR / Cas9 system.

[0185] According to certain embodiments of the present disclosure, in the method for constructing a yeast engineering bacterium of the present disclosure, the Cas9 protein coding sequence of the CRISPR / Cas9 gene editing system is integrated into the genome of the yeast engineering bacterium.

[0186] According to certain embodiments of the present disclosure, the CRISPR / Cas9 gene is integrated into the yeast genome using the sequence shown in SEQ ID NO: 1.

[0187] According to certain embodiments of the present disclosure, the methods of the present disclosure include knocking out the endogenous GAL80 gene and / or its promoter.

[0188] According to certain embodiments of the present disclosure, the present disclosure uses homologous recombination to knock out the endogenous GAL80 gene.

[0189] According to certain embodiments of the present disclosure, the present disclosure uses the sequence shown in SEQ ID NO: 2 to knock out the endogenous GAL80 gene.

[0190] According to certain embodiments of the present disclosure, the methods of the present disclosure include knocking out the endogenous GAL4 gene and / or its promoter.

[0191] According to certain embodiments of the present disclosure, the method of the present disclosure comprises knocking out the promoter of the endogenous GAL4 gene using a homologous recombination method.

[0192] According to certain embodiments of the present disclosure, the method of the present disclosure comprises knocking out the promoter of the endogenous GAL4 gene using the sequence shown in SEQ ID NO:4.

[0193] According to certain embodiments of the present disclosure, the method of the present disclosure comprises introducing the GAL4 gene by homologous recombination.

[0194] According to certain embodiments of the present disclosure, the methods of the present disclosure include introducing a gene of interest using homologous recombination.

[0195] According to certain embodiments of the present disclosure, in the method for constructing an engineered yeast of the present disclosure, steps (1), (2) and (3) are performed in no particular order.

[0196] On the other hand, the present disclosure provides a lysine-regulated promoter, which is derived from the upstream regulatory sequence of the LYS9 gene (pLYS9 promoter), wherein the lysine-regulated promoter comprises a sequence fragment or a combination of fragments of the pLYS9 promoter, wherein the pLYS9 promoter comprises the nucleotide sequence shown in SEQ ID NO: 9.

[0197] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a fragment of at least 300 bp from the 3′ end of the sequence of the pLYS9 promoter;

[0198] Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:11.

[0199] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter;

[0200] Preferably, the lysine-regulated promoter further comprises the sequence shown in SEQ ID NO:26.

[0201] According to certain embodiments of the present disclosure, a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter is placed upstream of a fragment of at least 300 bp at the 3′ end of the sequence of the pLYS promoter;

[0202] Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:19.

[0203] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises a sequence as shown in any one of SEQ ID NOs: 27-30, or a combination thereof.

[0204] According to certain embodiments of the present disclosure, the lysine-regulated promoter further comprises:

[0205] The combination of SEQ ID NOs: 28, 29 and 30;

[0206] The combination of SEQ ID NOs: 27, 29 and 30;

[0207] A combination of SEQ ID NOs: 27, 28 and 30; or

[0208] The combination of SEQ ID NOs: 27, 28 and 29.

[0209] According to certain embodiments of the present disclosure, the lysine-regulated promoter comprises a sequence as shown in any one of SEQ ID NOs: 9-11 and SEQ ID NOs: 16-24;

[0210] Preferably, the lysine-regulated promoter comprises a sequence as shown in SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 19 or SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.

[0211] In the yeast engineering bacteria disclosed in the present invention, the endogenous GAL80 and endogenous GAL4 genes are prevented from being expressed, and the exogenous pLYS9 promoter is used to positively induce the expression of the GAL4 transcription activator, thereby activating the GAL promoter (such as pGAL1 promoter, pGAL2 promoter, pGAL3 promoter, pGAL4 promoter, pGAL7 promoter, pGAL10 promoter and pGAL80 promoter) to start the expression of downstream genes. During the fermentation process, lysine is artificially added to inhibit the pLYS9 promoter, so that the transcription activator GAL4 cannot be normally expressed, resulting in the GAL promoter (such as pGAL1 promoter, pGAL2 promoter, pGAL3 promoter, pGAL4 promoter, pGAL7 promoter, pGAL10 promoter and pGAL80 promoter) being unable to normally start the expression of downstream genes (such as genes of interest), thereby achieving the purpose of regulating gene expression with lysine.

[0212] In practical application, lysine is first added to inhibit the pLYS9 promoter, thereby inhibiting the expression of GLA4 and the gene of interest, which is conducive to the large-scale growth and reproduction of yeast cells. The stage of large-scale growth and reproduction of yeast cells is referred to as the growth stage herein; during the growth and reproduction of yeast cells, lysine is consumed to reduce the lysine concentration in the culture medium, and the inhibition of the pLYS9 promoter by low-concentration lysine is weakened, and GLA4 and the gene of interest begin to express. The stage of the gene of interest expressing and producing the target product is referred to as the production stage herein. The yeast engineering bacteria disclosed in the present invention can easily distinguish between the growth stage and the production stage under the regulation of lysine, which is conducive to increasing the yield of the target fermentation product. DETAILED DESCRIPTION

[0213] The technical solution of the present disclosure is further described below by way of specific implementation. It should be clear to those skilled in the art that the embodiments are only used to help understand the present disclosure and should not be regarded as specific limitations of the present disclosure.

[0214] The present invention discloses a new lysine-induced yeast engineering bacterium, which can use exogenous additive lysine to regulate expression through the yeast's galactose regulation mechanism, effectively avoiding the long-term consumption of galactose and reducing production costs. At the same time, the regulation of lysine can effectively distinguish the growth stage and production stage of the strain, and the addition of lysine in the early stage can inhibit the expression of downstream metabolism, promote the growth of bacterial cells, and pave the way for the accumulation of products in the later stage. The specific implementation plan is as follows.

[0215] Example 1 Construction of background strains

[0216] In order to develop a yeast engineered bacterium regulated by lysine, this example takes the Saccharomyces cerevisiae CEN.PK113-7D model strain (NCBI Taxon Id: 889517, from Euroscarf) as an example, and performs appropriate gene editing on Saccharomyces cerevisiae.

[0217] The technical flow chart of this embodiment is as follows Figure 1 As shown, the specific technical solution is as follows:

[0218] 1. Insert the CRISPR / Cas9 gene into the neutral locus of the Saccharomyces cerevisiae genome

[0219] In order to facilitate gene editing without affecting yeast metabolism, the CRISPR / Cas9 gene was inserted into the neutral locus of the Saccharomyces cerevisiae genome. In this embodiment, the integration site of the CRISPR / Cas9 gene is downstream of the ALG1 gene. The schematic diagram of the integration structure is shown in FIG. Figure 2 As shown, the nucleotide sequence of the integrated CRISPR / Cas9 gene (SEQ ID NO: 1) is:

[0220]

[0221]

[0222]

[0223] 2. Knockout of GAL80 gene using CRISPR / Cas9 system

[0224] The high cost of galactose makes it difficult to implement the galactose (GAL) regulatory mechanism in the practical application of Saccharomyces cerevisiae. The dependence of the galactose induction mechanism on galactose is solved by knocking out the GAL80 gene. Specifically, the CRISPR / Cas9 system is used to replace the GAL80 gene sequence with a short nucleotide sequence (SEQ ID NO: 25) to achieve the effect of knocking out the GAL80 gene. In this embodiment, homologous recombination is used to knock out the GAL80 gene. The nucleotide fragment used includes the upstream homologous arm sequence of the GAL80 gene, the short nucleotide sequence (SEQ ID NO: 25) and the downstream homologous arm sequence of the GAL80 gene. The nucleotide sequence (SEQ ID NO: 2) used is as follows:

[0225]

[0226]

[0227] The short nucleotide sequence (SEQ ID NO: 25) is as follows:

[0228]

[0229] 3. Integrate the gene of interest into the Saccharomyces cerevisiae genome using the CRISPR / Cas9 system

[0230] In this embodiment, GFP is taken as an example, and GFP is integrated into the genome of the yeast engineered bacteria as a reporter gene. By integrating the GFP gene into the neutral site of the genome MGA1, the lysine-regulated GAL promoter expression system is characterized, which facilitates the subsequent testing of the role of the lysine-regulated GAL promoter, and simultaneously detects the metabolic level of the yeast without affecting the metabolism of the yeast itself. Specifically, the GFP gene integration sequence (SEQ ID NO: 3) is integrated into the neutral site of the genome MGA1 using the CRISPR / Cas9 system. The GFP gene integration sequence (SEQ ID NO: 3) includes the MGA1 upstream homologous arm sequence, the GAL1 promoter, the GFP gene, the CYC1 terminator, and the MGA1 downstream homologous arm sequence, and its nucleotide sequence (SEQ ID NO: 3) is as follows:

[0231]

[0232]

[0233] GFP is used as a reporter gene, and the CRISPR / Cas9 system is used to integrate the GFP gene into the genome, and the expression is driven by the GAL1 promoter. In addition, in addition to the reporter gene GFP, it can also be any gene of interest or any gene of a target fermentation product, and the gene of interest is also driven by the GAL promoter.

[0234] In addition to the GAL1 promoter, other promoters such as the GAL2 promoter, the GAL7 promoter or the GAL10 promoter may be used as the promoter for promoting the expression of the GFP gene.

[0235] The expression of the GAL promoter is regulated by the transcription activator GAL4. Considering that in this embodiment, after knocking out the gene of the endogenous GAL80 negative regulatory protein, the inhibition of the GAL80 negative regulatory protein on the endogenous GAL4 transcription activator of the yeast engineering bacteria is released, if there is no intervention, the endogenous GAL4 of Saccharomyces cerevisiae will continue to be expressed. Knocking out the endogenous GAL4 gene of Saccharomyces cerevisiae or replacing the promoter of the endogenous GAL4 gene of Saccharomyces cerevisiae with a terminator makes the endogenous GAL4 gene of Saccharomyces cerevisiae not expressed or almost not expressed.

[0236] 4. Use the CRISPR / Cas9 system to replace the endogenous GAL4 gene promoter of Saccharomyces cerevisiae with a terminator.

[0237] The CRISPR / Cas9 system was used to replace the endogenous GAL4 gene promoter of Saccharomyces cerevisiae with the terminator tIDP1 to terminate the expression of endogenous GAL4, so as to remove the influence of the endogenously expressed GAL4 regulatory factor of Saccharomyces cerevisiae on the expression regulation of the downstream GAL promoter. The integration sequence (SEQ ID NO: 4) for terminating the expression of endogenous GAL4 in Saccharomyces cerevisiae consists of the upstream homology arm of the endogenous GAL4 in Saccharomyces cerevisiae, the terminator tIDP1 and the upstream homology arm of GAL4. The nucleotide sequence of the tIDP1 integration sequence (SEQ ID NO: 4) is as follows:

[0238]

[0239]

[0240] 5. Integrate the pLYS9 promoter and express GAL4 using the CRISPR / Cas9 system

[0241] In this example, the CRISPR / Cas9 system is used to integrate the pLYS9 promoter (such as the sequence shown in SEQ ID NO: 9) induced by lysine and the GAL4 that initiates expression thereof into the neutral site HPR1. pLYS9 can initiate the expression of the exogenous transcription activator GAL4, thereby activating the subsequent GAL promoter to initiate the expression of the gene of interest. Adding lysine inhibits the expression of the gene of interest regulated by the pLYS9 promoter. In subsequent examples, the effect of lysine regulation on the expression of the GAL4 transcription activator initiated by the pLYS9 promoter was tested.

[0242] The pLYS9 promoter and the GAL4 integration sequence that drives its expression are composed of the HPR1 upstream homology arm + pLYS9 promoter + GAL4 gene + GAL4 terminator + HPR1 downstream homology arm. Except for the pLYS9 promoter gene sequence, the gene sequences of the remaining fragments are as follows:

[0243] The nucleotide sequence of the upstream homology arm of HPR1 (SEQ ID NO: 5) is as follows:

[0244]

[0245] The nucleotide sequence of the GAL4 gene (SEQ ID NO: 6) is as follows:

[0246]

[0247]

[0248]

[0249] The nucleotide sequence of the GAL4 terminator (SEQ ID NO: 7) is as follows:

[0250]

[0251] The nucleotide sequence of the downstream homology arm of HPR1 (SEQ ID NO: 8) is as follows:

[0252]

[0253] The integration sequence of this embodiment uses a 650 bp pLYS9 promoter, whose sequence is shown in SEQ ID NO: 9. When testing the efficacy of pLYS9 promoters of different lengths and with different deletion fragments in subsequent embodiments, it is only necessary to replace the pLYS9 promoter sequence therein to form a new integration sequence, and the corresponding engineered bacteria can be constructed using the integration sequence. The specific sequences and effects of pLYS9 promoters of different lengths and with different deletion fragments are shown in Examples 2-4.

[0254] Example 2 Performance of pLYS9 promoters of different lengths in lysine-induced regulation

[0255] This example tests the performance of pLYS9 promoters of different lengths in lysine induction regulation. Under the same lysine induction, the effect of pLYS9 promoters of different lengths on the activation of the GAL4 gene is tested. It involves inserting pLYS9 promoters of different lengths into the HRP 1 downstream site of the Saccharomyces cerevisiae genome. The designs of the pLYS9 promoters of different lengths are as follows: Figure 3Specifically, this example tests the functions of pLYS9 promoters of different lengths, including 650 bp (SEQ ID NO: 9), 500 bp (500 bp at the 3' end of SEQ ID NO: 9, and the following fragments are all 3' end fragments of SEQ ID NO: 9), 300 bp, 220 bp, 160 bp, 115 bp, and 79 bp. The tested sequences are as follows:

[0256] The nucleotide sequence of pLYS9_650bp (SEQ ID NO: 9) is as follows:

[0257]

[0258] The nucleotide sequence of pLYS9_500bp (SEQ ID NO: 10) is as follows:

[0259]

[0260]

[0261] The nucleotide sequence of pLYS9_300bp (SEQ ID NO: 11) is as follows:

[0262]

[0263] The nucleotide sequence of pLYS9_220bp (SEQ ID NO: 12) is as follows:

[0264]

[0265] The nucleotide sequence of pLYS9_160bp (SEQ ID NO: 13) is as follows:

[0266]

[0267] The nucleotide sequence of pLYS9_115bp (SEQ ID NO: 14) is as follows:

[0268]

[0269] The nucleotide sequence of pLYS9_79bp (SEQ ID NO: 15) is as follows:

[0270]

[0271] Combined with the description in Example 1, the integration sequence containing the above promoter was inserted into the Saccharomyces cerevisiae genome using the CRISPR / Cas9 system to obtain different integrated expression strains. The positive strains were identified by colony PCR (cPCR). Four single clones of each bacterium were pre-cultured in MM medium containing 2% glucose and 2g / L lysine for 24 hours. The components and contents of the MM medium are shown in Table 1.

[0272] Table 1 Components and contents of MM medium

[0273]

[0274]

[0275] The pre-cultured strains were transferred to MM medium containing 2% glucose and MM medium containing 2% glucose and 1g / L lysine at an inoculum of 1%. The cultured bacterial cells were taken and the effect of gene expression of pLYS9 promoters of different lengths under lysine induction was evaluated by detecting the fluorescence intensity (RFU) of GFP in different groups. The fluorescence intensity of each group was detected at 8, 24, 32, and 48 hours, and the experimental results are shown in Figure 2. Figure 4-Figure 7 As can be seen from the figure, in the presence of lysine, during the period of 8-48 hours, 70%-90% of the expression can be inhibited by 300bp pLYS9, 20%-50% of the expression can be inhibited by 500bp pLYS9, and 60%-80% of the expression can be inhibited by 650bp pLYS9. Taking the 24-hour data as an example, the expression of GFP under the GAL1 promoter is as follows Figure 5 As shown in Figure 2, when the pLYS9 promoter length is less than 300 bp, the GAL1 promoter does not drive GFP expression; when the pLYS9 promoter length is greater than or equal to 300 bp, the GAL1 promoter drives GFP expression. Figure 5 It can be seen that when the length of the pLYS9 promoter is 300 bp, in the presence of lysine, the inhibitory effect of the GAL1 promoter driving GFP expression is most obvious, and its inhibition efficiency is as high as 80%.

[0276] In summary, in the lysine-regulated Saccharomyces cerevisiae engineered bacteria constructed in the present disclosure, when the pLYS9 promoter is greater than or equal to 300 bp in length, it can drive the downstream GAL promoter to further drive GFP expression. And when the pLYS9 promoter is 300 bp in length, in the presence of lysine, the effect of inhibiting the GAL1 promoter from driving GFP expression is obvious.

[0277] Example 3 Expression leakage of pLYS9 promoter with different deletion fragments under lysine regulation

[0278] This example tests the expression leakage of pLYS9 promoter sequences with different deletion fragments under lysine regulation. The pLYS9 promoter with different deletion fragments is designed as follows Figure 8 As shown. This embodiment tests a pLYS9 promoter including a 650 bp sequence (SEQ ID NO: 9), a 451-500 bp deletion sequence (a fragment of nucleotides 451-500 from the 3' end of SEQ ID NO: 9 is missing, the same below), a 401-500 bp deletion sequence, a 351-500 bp deletion sequence, a 301-500 bp deletion sequence, a 301-350 bp deletion sequence, a 301-400 bp deletion sequence, a 301-450 bp deletion sequence, a 401-450 bp deletion sequence, a 351-400 bp deletion sequence, and a sequence of nucleotide positions 1-300 (also referred to herein as 300 bp, recorded as pLYS9 (300)). ^ indicates a pLYS9 promoter sequence deletion, for example, a 451-500 bp deletion sequence is recorded as pLYS9_451-500 bp^.

[0279] The nucleotide sequence of pLYS9_650bp (SEQ ID NO: 9) is as follows:

[0280]

[0281] The nucleotide sequence of pLYS9_451-500 bp^ (SEQ ID NO: 16) is as follows:

[0282]

[0283] The nucleotide sequence of pLYS9_401-500 bp^ (SEQ ID NO: 17) is as follows:

[0284]

[0285]

[0286] The nucleotide sequence of pLYS9_351-500 bp^ (SEQ ID NO: 18) is as follows:

[0287]

[0288] The nucleotide sequence of pLYS9_301-500 bp^ (SEQ ID NO: 19) is as follows:

[0289]

[0290] The nucleotide sequence of pLYS9_301-350 bp^ (SEQ ID NO: 20) is as follows:

[0291]

[0292]

[0293] The nucleotide sequence of pLYS9_301-400 bp^ (SEQ ID NO: 21) is as follows:

[0294]

[0295] The nucleotide sequence of pLYS9_301-450 bp^ (SEQ ID NO: 22) is as follows:

[0296]

[0297] The nucleotide sequence of pLYS9_401-450 bp^ (SEQ ID NO: 23) is as follows:

[0298]

[0299]

[0300] The nucleotide sequence of pLYS9_351-400 bp^ (SEQ ID NO: 24) is as follows:

[0301]

[0302] The nucleotide sequence of pLYS9_300bp (SEQ ID NO: 11) is as follows

[0303]

[0304] The nucleotide sequence of pLYS9_501-650bp (SEQ ID NO: 26) is as follows:

[0305]

[0306] The nucleotide sequence of pLYS9_451-500bp (SEQ ID NO: 27) is as follows:

[0307]

[0308] The nucleotide sequence of pLYS9_401-450bp (SEQ ID NO: 28) is as follows:

[0309]

[0310] The nucleotide sequence of pLYS9_351-400bp (SEQ ID NO: 29) is as follows:

[0311]

[0312] The nucleotide sequence of pLYS9_301-350bp (SEQ ID NO: 30) is as follows:

[0313]

[0314] In combination with Example 1, the integration sequence containing the above promoter was inserted into the Saccharomyces cerevisiae genome using the CRISPR / Cas9 system to obtain different integrated expression strains. The obtained strains were identified as positive strains by cPCR. Four single clones of each bacterium were pre-cultured in MM medium containing 2% glucose and 1g / L lysine for 24 hours. The MM medium was the same as described in Example 2.

[0315] The pre-cultured strain was transferred to MM medium without glucose and MM medium containing 1 g / L lysine at an inoculum size of 1% for culture.

[0316] Take the cultured bacteria and detect the fluorescence intensity (RFU) of GFP in different groups to evaluate the gene expression leakage of pLYS9 promoter with different deletion fragments under lysine regulation. Detect the fluorescence intensity of each group for 48 hours. The experimental results are shown in Fig. 9 .Depend on Fig. 9 It can be seen that compared with the group without lysine, under the condition of 1g / L lysine, the pLYS9 promoter with different deletion fragments can inhibit the fluorescence expression of GFP. In addition, the expression rate of the leaky gene of the pLYS9 promoter with different deletion fragments at 48 hours is shown in Figure 2. Fig.10 Among them, pLYS9 with a 301-500bp sequence deletion can reduce the leaky expression of GFP driven by the GAL 1 promoter to 14%.

[0317] Example 4 Shake flask test of pLYS9 promoter regulating GAL4 expression

[0318] In order to better reflect the advantage of pLYS9 promoter regulating GAL4 expression, this example uses the superior strain constructed in the above example to conduct a shake flask test. The test medium is MM medium (the composition of MM medium is shown in Table 1). The test strains are shown in Table 2.

[0319] Table 2 Test strains

[0320]

[0321]

[0322] First, strain SD439-001 was used to test the effect of different concentrations of lysine (0 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 2 g / L, 5 g / L) on regulating the 300 bp pLYS9 promoter to induce GAL4 expression. Fig.11 As shown. Fig.11 It can be seen that when the lysine concentration is higher than 0.5g / L, it can inhibit the 300bp pLYS9 promoter from inducing the expression of GAL4. At the same time, it can be seen that when lysine is added above 0.5g / L, its leakage expression level is lower than 10% of the expression level of the group without lysine. The effect of regulating the expression of GAL4 induced by the 300bp pLYS9 promoter with different concentrations of lysine (0g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L) was further tested. The experimental results are shown in Fig.12 As shown. Fig.12 It can be seen that when the lysine concentration is higher than 0.3g / L, it can inhibit the 300bp pLYS9 promoter from inducing the expression of GAL4. Fig.12 It can be seen that under the condition of adding 0.2g / L lysine, the leakage expression increased when the fermentation time reached 36h. However, under the condition of adding 0.3g / L lysine, the leakage expression was not obvious as the fermentation experiment was prolonged. The above experimental data show that 0.3g / L lysine can be used as an added concentration for fermentation regulation, which can be used for the growth of bacterial cells in the early stage of fermentation, inhibit the expression of metabolism, and serve as an advantage for the accumulation of products in the later stage. The schematic diagram of lysine regulating gene expression during fermentation and distinguishing the growth stage from the production stage is shown in Fig.13 In the early stage of fermentation, the lysine concentration is high, which inhibits the expression of the gene of interest, allowing the strain to grow rapidly under conditions without metabolic pressure and suitable for growth and reproduction. As the fermentation time increases, the strain grows to the early stage of saturation, lysine is consumed to a low concentration, the inhibitory effect of lysine weakens, the expression of the gene of interest is turned on, and a large number of metabolites are produced, achieving the purpose of increasing production.

[0323] The strains in Table 2 were tested at a lysine concentration of 0.5 g / L. Fig.14 As shown. Fig.14 It can be seen that 0.5 g / L lysine can also regulate the expression of the pLYS9 promoter with a length of 300 bp, a sequence deletion of 50 bp, and a sequence deletion of 200 bp in Table 2. Fig.15 The leakage expression ratio of each strain in Table 2 after culturing for 48 hours under lysine regulation is shown. Fig.15It can be seen that the pLYS9 promoters with 300 bp, 50 bp sequence deletion and 200 bp sequence deletion have only 2-4% leakage under 0.5 g / L lysine conditions and can be used for amplified production.

[0324] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims

1. A yeast engineering bacterium, wherein the yeast engineering bacterium comprises the following modifications: (1) a modified endogenous GAL80 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL80 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL80 gene; (2) a modified endogenous GAL4 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL4 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL4 gene; (3) Introducing the GAL4 gene into the yeast engineering bacteria, wherein the GAL4 gene is operably linked to a lysine-regulated promoter, wherein the lysine-regulated promoter is derived from the upstream regulatory sequence of the LYS9 gene (pLYS9 promoter) and inhibits the expression of the GAL4 gene in the presence of lysine. 2 . The engineered yeast according to claim 1 , wherein the pLYS9 promoter comprises the nucleotide sequence shown in SEQ ID NO:

9.

3. The yeast engineering bacterium according to claim 1 or 2, wherein the lysine-regulated promoter comprises a sequence fragment or a combination of fragments of the pLYS9 promoter.

4. The yeast engineered bacterium according to any one of claims 1 to 3, wherein the lysine-regulated promoter comprises a fragment of at least 300 bp at the 3′ end of the sequence of the pLYS9 promoter; Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:

11.

5. The yeast engineering bacterium according to claim 4, wherein the lysine-regulated promoter further comprises a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter; Preferably, the lysine-regulated promoter further comprises the sequence shown in SEQ ID NO:

26.

6. The yeast engineering bacterium according to claim 5, wherein the fragment of at least 150 bp at the 5' end of the sequence of the pLYS9 promoter is placed upstream of the fragment of at least 300 bp at the 3' end of the sequence of the pLYS promoter; Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:

19.

7. The yeast engineered bacterium according to any one of claims 4 to 6, wherein the lysine-regulated promoter further comprises a sequence as shown in any one of SEQ ID NOs: 27 to 30, or a combination thereof.

8. The yeast engineering bacterium according to claim 7, wherein the lysine-regulated promoter further comprises: The combination of SEQ ID NOs: 28, 29 and 30; The combination of SEQ ID NOs: 27, 29 and 30; A combination of SEQ ID NOs: 27, 28 and 30; or The combination of SEQ ID NOs: 27, 28 and 29.

9. The yeast engineered bacterium according to any one of claims 1 to 3, wherein the lysine-regulated promoter comprises a sequence as shown in any one of SEQ ID NOs: 9 to 11 and SEQ ID NOs: 16 to 24; Preferably, the lysine-regulated promoter comprises a sequence as shown in SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:

24.

10. The engineered yeast according to any one of claims 1 to 9, wherein the modified endogenous GAL80 gene and / or its regulatory sequence is selected from a knocked-out endogenous GAL80 gene and / or its regulatory sequence, and a mutated inactivated endogenous GAL80 gene and / or its regulatory sequence; and / or The modified endogenous GAL4 gene and / or its regulatory sequence is selected from a knocked-out endogenous GAL4 gene and / or its regulatory sequence, and a mutated inactivated endogenous GAL4 gene and / or its regulatory sequence.

11. The engineered yeast according to any one of claims 1 to 10, wherein the modified endogenous GAL80 gene and / or its regulatory sequence comprises a knocked-out endogenous GAL80 gene and / or its regulatory sequence; and / or The modified endogenous GAL4 gene and / or its regulatory sequence includes the promoter of the endogenous GAL4 gene being replaced by a terminator.

12. The yeast engineering bacterium according to any one of claims 1 to 11, wherein the GAL4 gene is integrated into the genome of the yeast engineering bacterium; Preferably, the GAL4 gene is integrated into the downstream non-coding region of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene of the yeast engineering bacteria genome; Preferably, the GAL4 gene is integrated into the region 0-2000 bp downstream of the HPR1, ECM13, FRE4, GAT1, NOP4, SFM1, YKR104W or YPG1 gene of the yeast engineering bacteria genome; preferably the region 0-1000 bp; further preferably the region 0-500 bp.

13. The yeast engineering bacterium according to any one of claims 1 to 12, wherein the yeast engineering bacterium further comprises a yeast galactose induction system (GAL induction system), the GAL induction system comprising a GAL4-induced activated promoter, and a gene of interest operably linked to the GAL4-induced activated promoter.

14. The yeast engineering bacterium according to claim 13, wherein the GAL4-induced activated promoter is selected from the group consisting of pGAL1 promoter, pGAL2 promoter, pGAL3 promoter, pGAL4 promoter, pGAL7 promoter, pGAL10 promoter and pGAL80 promoter; Preferably, the GAL4-induced activated promoter is the pGAL1 promoter.

15. The yeast engineered bacteria according to any one of claims 1 to 14, wherein the yeast is selected from Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis; preferably, the yeast is Saccharomyces cerevisiae.

16. Use of the engineered yeast according to any one of claims 13 to 15 in producing a target product encoded by the gene of interest.

17. A method for producing a target product, the method comprising: 1) pre-culturing the yeast engineering bacteria according to any one of claims 13 to 15 in a medium supplemented with lysine under conditions suitable for the growth of the yeast engineering bacteria; 2) culturing the yeast engineering bacteria under conditions suitable for expressing the target product from the gene of interest; and 3) Recovering the target product from the yeast engineered bacteria cells and / or culture medium.

18. The method according to claim 17, wherein the amount of lysine added to the culture medium in step 1) is about 0.2-1 g / L; Preferably, the amount of lysine added is about 0.3-0.5 g / L.

19. A method for constructing the yeast engineering bacteria according to any one of claims 1 to 15, the method comprising: (1) modifying the endogenous GAL80 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL80 gene, and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL80 gene; (2) modifying the endogenous GAL4 gene and / or its regulatory sequence, wherein the modification reduces, inhibits or eliminates the expression of the endogenous GAL4 gene and / or reduces, inhibits or eliminates the activity of the protein encoded by the endogenous GAL4 gene; (3) Introducing the GAL4 gene and the lysine-regulated promoter into the yeast engineering bacteria, wherein the GAL4 gene is operably linked to the lysine-regulated promoter.

20. The method according to claim 19, wherein: The modification or introduction in steps (1), (2) and / or (3) is achieved using the CRISPR / Cas9 gene editing system.

21. The method according to claim 20, wherein the Cas9 protein coding sequence of the CRISPR / Cas9 gene editing system is integrated into the genome of the yeast engineered bacteria.

22. A lysine-regulated promoter, which is derived from an upstream regulatory sequence of a LYS9 gene (pLYS9 promoter), wherein the lysine-regulated promoter comprises a sequence fragment or a combination of fragments of the pLYS9 promoter, wherein the pLYS9 promoter comprises the nucleotide sequence shown in SEQ ID NO:

9.

23. The promoter according to claim 22, wherein the lysine-regulated promoter comprises a fragment of at least 300 bp at the 3′ end of the sequence of the pLYS9 promoter; Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:

11.

24. The promoter according to claim 23, wherein the lysine-regulated promoter further comprises a fragment of at least 150 bp at the 5′ end of the sequence of the pLYS9 promoter; Preferably, the lysine-regulated promoter further comprises the sequence shown in SEQ ID NO:

26.

25. The promoter according to claim 24, wherein a fragment of at least 150 bp at the 5' end of the sequence of the pLYS9 promoter is placed upstream of a fragment of at least 300 bp at the 3' end of the sequence of the pLYS promoter; Preferably, the lysine-regulated promoter comprises the sequence shown in SEQ ID NO:

19.

26. The promoter according to any one of claims 23-25, wherein the lysine-regulated promoter further comprises a sequence as shown in any one of SEQ ID NOs: 27-30, or a combination thereof.

27. The promoter of claim 26, wherein the lysine-regulated promoter further comprises: The combination of SEQ ID NOs: 28, 29 and 30; The combination of SEQ ID NOs: 27, 29 and 30; A combination of SEQ ID NOs: 27, 28 and 30; or The combination of SEQ ID NOs: 27, 28 and 29.

28. The promoter according to claim 22, wherein the lysine-regulated promoter comprises a sequence as shown in any one of SEQ ID NOs: 9-11 and SEQ ID NOs: 16-24; Preferably, the lysine-regulated promoter comprises a sequence as shown in SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 19 or SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.

Images