constitutive promoter

Abstract

This invention relates to an isolated nucleic acid sequence comprising a promoter, which is a natural Pichia pastoris sequence comprising the nucleic acid sequence of pCS1 shown in SEQ ID 1 or a functionally active variant thereof, the variant being a length variant, a mutant, or a heterozygous sequence of SEQ ID 1, or a combination thereof, relating to expression constructs and recombinant cells comprising said promoter, and also to a method for producing a protein of interest under the control of said promoter. The invention further relates to a method for identifying constitutive promoters from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter that, when operably linked to a nucleotide sequence encoding a POI, directs its expression level in host cells to be higher than the expression level under the control of the natural pGAP promoter at high and low growth rates.

Description

Technical Field

[0001] This invention relates to an isolated nucleic acid sequence comprising a strong constitutive promoter and a method for producing a protein of interest in a eukaryotic cell culture medium under the control of the promoter. Background Technology

[0002] Recombinant proteins have been successfully produced using eukaryotic hosts. The most prominent examples are yeasts such as *Saccharomyces cerevisiae*, *Pichia pastoris*, or *Hansenula polymorpha*, filamentous fungi such as *Aspergillus awamori* or *Trichoderma reesei*, or mammalian cells such as Chinese hamster ovary (CHO) cells. While some proteins have achieved high-rate production, many others are only available in relatively low yields.

[0003] Heterologous gene expression in a host organism requires a vector capable of stable transformation within the host cell. This vector provides a gene with a functional promoter located immediately adjacent to the 5′ end of the coding sequence. This promoter sequence then regulates and initiates transcription. Most promoters currently used are derived from genes encoding proteins that are typically present in high concentrations within the cell.

[0004] EP0103409A2 discloses the use of yeast promoters associated with the expression of specific enzymes in the glycolysis pathway, such as promoters involving pyruvate kinase, triose phosphate isomerase, glucose phosphate isomerase, phosphoglycerate mutase, hexokinase 1 and 2, glucoskinase, phosphofructokinase, aldolase, and glycolysis regulatory genes.

[0005] WO 97 / 44470 describes the transcription elongation factor 1 (TEF1) protein derived from Yarrowia lipolytica and a yeast promoter suitable for expressing the ribosomal protein S7 in yeast, while EP1951877A1 describes the use of the Pichia pastoris TEF1 promoter for producing heterologous proteins.

[0006] WO2005003310 provides a method for expressing coding sequences of interest in yeast using glyceraldehyde-3-phosphate dehydrogenase or glycerol phosphate mutase promoters from the oleaginous yeast Yarrowia lipolytica.

[0007] Promoter sequences derived from genes involved in the methanol metabolism pathway in Pichia pastoris have been published in US4808537 and US4855231 (alcohol oxidases AOX1 and AOX2) and US6730499B1 (formaldehyde dehydrogenase FLD1). US20080153126A1 includes a mutant promoter sequence based on the AOX1 promoter.

[0008] The AOX1 promoter is only induced by methanol and repressed by other carbon sources such as glucose or ethanol. Methanol is unsuitable for the production of certain products due to its potential toxicity and flammability. Therefore, we sought alternatives to the AOX1 promoter.

[0009] Vassileva et al. (J. Biotechnol. (2001) 88: 21-35) described the use of a multi-copy expression cassette as an alternative to the AOX1 promoter to express hepatitis B surface antigen (HBsAg) in Pichia pastoris using the GAP promoter. They proposed a system for maintaining continuous cell culture in mid-log phase.

[0010] Promoters used in Pichia pastoris are either tightly regulated (like pAOX or pFLD) and active against specific substrates such as methanol, or constitutively active under many different conditions, media, and substrates. Among these constitutive promoters, GAP and TEF promoters, in particular, have been reported to have strong and useful initiation effects for the production of recombinant proteins.

[0011] However, the results showed that neither constitutive promoter was consistently strongly activated during fed-batch production. In particular, the promoter activity decreased in the later stages of the process when cell growth rate was low, thus limiting the expression and production of the gene of interest (GOI) (Stadlmayr et al., 2010. J Biotechnol. 150: 519-529).

[0012] Even highly abundant glycolytic enzymes such as enolase (ENO), triose phosphate isomerase (TPI), or glucose-6-phosphate isomerase (PGI) do not have promoters as strong as pGAP and pTEF. Therefore, choosing a suitable promoter is not based on intuition but on rational consideration. (Stadlmayr et al. 2010. J Biotechnol. 150: 519-529; Gasser et al. 2010. Metabolic Engineering 12: 573-580).

[0013] Qin et al. (Applied and Environmental Microbiology (2011) 3600-3608) reported a GAP promoter library and various promoters with different activities.

[0014] WO2007 / 015178A2 reports a transcriptional fusion mate and a Pichia pastoris cDNA library that can induce the secretion of recombinant proteins.

[0015] CN102180954A reports a cell surface display system that uses Pichia pastoris cell wall protein GCW14 as an anchoring protein.

[0016] What is needed is to provide improved recombinant eukaryotic cell lines for the production of fermentation products that can be isolated in high yields. Therefore, the object of this invention is to provide a simple and efficient selectable control element suitable for recombinant production methods. Summary of the Invention

[0017] The objective of the invention is achieved through the subject matter of the claims.

[0018] The present invention provides an isolated nucleic acid sequence containing a promoter, the promoter being a natural Pichia pastoris sequence, comprising or consisting of the pCS1 nucleic acid sequence shown in SEQ ID 1, or a functionally active variant thereof, which is a length variant, a mutant, or a combination thereof.

[0019] According to one particular aspect, the present invention provides an isolated nucleic acid sequence comprising a promoter, the promoter being a natural Pichia pastoris sequence comprising or consisting of the pCS1 nucleic acid sequence shown in SEQ ID 1, or a functionally active variant thereof, the functionally active variant being a length variant, a mutant or heterozygous sequence of SEQ ID 1, or a combination thereof, wherein the functionally active variant exhibits substantially the same activity as pCS1, particularly the pCS1 nucleic acid sequence shown in SEQ ID 1.

[0020] Specifically, the pCS1 nucleic acid sequence is identical to that of SEQ ID 1.

[0021] Preferably, the nucleic acid sequence of the present invention is not the same as the nucleic acid sequence of SEQ ID 87 (i.e., SEQ ID 24 of this application) in the list of WO2007 / 015178 A2.

[0022] Specifically, the functionally active variant is

[0023] a) A length variant of pCS1 shown in SEQ ID 1, preferably comprising or consisting of a nucleic acid sequence selected from or composed of the group consisting of SEQ ID 2, 3, 4, 5, 6, 7 and 8;

[0024] b) A mutant of pCS1 shown in SEQ ID 1, or a mutant of the length variant of a), wherein the mutant has at least 60% homology with the sequence SEQ ID 1 or the length variant;

[0025] c) A heterozygous sequence containing

[0026] —A sequence selected from the group consisting of pCS1 as shown in SEQ ID 1, a length variant of a), and a mutant of b); and

[0027] —At least one further sequence selected from the group consisting of pCS1 shown in SEQ ID 1, a length variant of a), a mutant of b), and a heterologous sequence; or

[0028] d) A sequence that hybridizes under stringent conditions with a variant of any length, or with a mutant nucleic acid sequence of a) or b).

[0029] Preferably, the length variation of pCS1 shown in SEQ ID 1 consists of any one of SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 or SEQ ID 6.

[0030] According to a particular embodiment, the functionally active variant is selected from the group consisting of sequences homologous to the following sequences:

[0031] i) At least approximately 60% nucleotide sequence identity;

[0032] ii) Homologous sequences obtained by modifying the nucleotide sequence of pCS1 shown in SEQ ID 1 or variations thereof, or by inserting, deleting, or replacing one or more nucleotides within or at one or both ends of the sequence, preferably having a nucleotide sequence of 80 bp to 1500 bp, or 200 bp to 1500 bp, more preferably having a nucleotide sequence of at least 200 bp; and

[0033] iii) Analogs derived from species other than Pichia pastoris.

[0034] In particular, the functionally active variant of the present invention has the same promoter activity as pCS1.

[0035] According to a particular embodiment, the nucleic acid sequence is operatively linked to a nucleotide sequence encoding a POI, the nucleic acid and the nucleotide sequence encoding the POI not being naturally related.

[0036] According to one particular aspect, the nucleic acid sequence further includes a signal peptide gene for realizing POI secretion, preferably wherein the signal peptide gene is located adjacent to the 5′ end of the nucleotide sequence encoding the POI.

[0037] Accordingly, the present invention particularly relates to a nucleic acid sequence that further comprises a nucleic acid sequence encoding a signal peptide that enables POI secretion, preferably wherein the nucleic acid sequence encoding the signal peptide is located at the 5′ end of the nucleotide sequence encoding the POI.

[0038] The present invention further provides an expression construct comprising the nucleic acid sequence described herein, preferably, a self-replicating vector or plasmid, or a construct that integrates into the chromosomal DNA of a host cell.

[0039] The present invention further provides a recombinant host cell comprising the nucleic acid sequence described in the present invention or the expression construct of the present invention, preferably, a eukaryotic cell, more preferably, a yeast or filamentous fungal cell, and even more preferably, a yeast cell of the genera *Saccharomyces cerevisiae* or *Pichia pastoris*.

[0040] According to one particular aspect, the recombinant host cell contains multiple copies of the nucleic acid sequence and / or multiple copies of the expression construct. For example, the recombinant cell contains 2, 3, 4, or 5 copies (gene copy number GCN).

[0041] Specifically, the recombinant host cell is selected from the group consisting of mammals, insects, yeast, filamentous fungi, and plant cells, preferably yeast, and preferably any one of Pichia pastoris strains CBS 704, CBS 2612, CBS 7435, CBS 9173-9189, DSMZ70877, X-33, GS115, KM71, and SMD1 168. In some embodiments, the recombinant host cell is a Pichia pastoris strain other than X-33.

[0042] The present invention further provides a stable culture of the cells of the present invention.

[0043] The present invention further provides a method for producing POI by culturing recombinant host cells, the recombinant host cells comprising the nucleic acid sequence or promoter of the present invention or the expression construct of the present invention, and nucleic acid encoding POI under the transcriptional control of said promoter, comprising the steps of:

[0044] a) Culture the cell line under conditions expressing the POI, and

[0045] b) Recover the POI.

[0046] Specifically, the POI is expressed under growth-restricted conditions, such as culturing cell lines at rates below the maximum growth rate, typically below 90% of the maximum cell growth rate, preferably below 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, or 0.2% of the maximum growth rate. Typically, the maximum growth rate is determined individually for a specific host cell.

[0047] According to one particular embodiment, the cell line is cultured under batch culture, fed-batch culture, or continuous culture conditions, and / or in a culture medium containing a limiting carbon source.

[0048] Specifically, the cultivation is carried out in a bioreactor, starting with a batch culture period followed by a fed-batch culture period or a continuous culture period.

[0049] Specifically, the host cells are grown in a carbon-rich medium during high-rate growth (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or up to the maximum growth rate) and produce the POI during low-rate growth (e.g., below 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, or 0.2% of the maximum growth rate), for example, while preferably, the carbon source is limited by supplementing a carbon-limiting basal medium. Specifically, the carbon-limiting basal medium does not contain a carbon source that induces transcription.

[0050] Specifically, the POI is a heterologous protein, preferably a therapeutic protein, including antibodies or fragments thereof, enzymes and peptides, protein antibodies, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, growth factors, hormones and cytokines, or metabolites of the POI, particularly including recombinant cell culture metabolites expressing genes of interest under the transcriptional control of the promoters of the present invention.

[0051] The present invention further provides a method for recognizing constitutive promoters from eukaryotic cells, comprising the steps of:

[0052] a) Culture eukaryotic cells at a high growth rate;

[0053] b) Further culture the eukaryotic cells at a low growth rate;

[0054] c) Provide samples of the cell cultures from steps a) and b);

[0055] d) Perform transcriptional analysis in the sample and compare the transcriptional levels with those of the cell's natural pGAP promoter; and

[0056] f) Select a constitutive promoter with higher transcriptional strength compared to natural pGAP promoters with high and low growth rates, preferably by determining the transcriptional level of the identified constitutive promoter, which is at least 1.1 times, preferably at least 1.2 times, preferably at least 1.3 times, preferably at least 1.4 times, preferably at least 1.5 times, preferably at least 1.6 times, preferably at least 1.7 times, preferably at least 1.8 times, preferably at least 1.9 times, preferably at least 2 times, preferably at least 3 times, preferably at least 4 times, preferably at least 5 times, preferably at least 10 times, or at least 15 times.

[0057] Specifically, in the range of 0.01–0.2 h -1 Within this range, 0.015–0.15h is preferred. -1 Transcriptional levels can be determined, for example, by examining at least two samples from a cell culture at both high and low growth rates, with the first sample representing a high growth rate, such as at least 0.05 h. -1 Preferably at least 0.06h -1 or at least 0.07h -1 At least 0.08h -1 At least 0.09h -1 At least 0.1h -1 For example, in 0.15h -1 The first sample represents the highest growth rate, while the second sample represents a lower growth rate, for example, lower than the growth rate of the first sample, for example, less than 0.05 h. -1 Preferably, less than 0.04h -1 Below 0.03h -1 or less than 0.02h -1 For example, in 0.015h -1The growth rate. In particular, for example, according to Example 11 below, the transcription level was determined by analyzing gene expression patterns using a DNA microarray.

[0058] The present invention further provides the use of a promoter comprising or composed of a separate nucleic acid sequence, which, when operatively linked to a nucleotide sequence encoding a POI, directs the expression of the POI in a host cell at a level higher than that controlled by a natural pGAP promoter at both high and low growth rates. The use is preferably in a method for producing the POI by culturing host cells transformed with the nucleic acid sequence, wherein the culture is carried out in a bioreactor, beginning with a batch culture period followed by a fed-batch culture period or a continuous culture period.

[0059] Specifically, the expression level is between 0.01 and 0.2 h. -1 Within the range of high and low growth rates, the preferred range is 0.015–0.15 h. -1 The range is determined by examining at least two samples of a cell culture, the first sample representing a high growth rate, for example, at least 0.05 h. -1 Preferably at least 0.06h -1 or at least 0.07h -1 At least 0.08h -1 At least 0.09h -1 At least 0.1h -1 For example, in 0.15h -1 The first sample represents a growth rate of 0.05 h, while the second sample represents a low growth rate, for example, lower than the growth rate of the first sample, for example, lower than 0.05 h. -1 Preferably, less than 0.04h -1 Below 0.03h -1 or less than 0.02h -1 For example, in 0.015h -1 The growth rate. In particular, the expression level is determined under growth-restricted conditions. Example 10 provides an example of determining expression intensity at high and low growth rates under growth-restricted conditions, such as glucose-restricted chemostat cultures.

[0060] Specifically, the expression level compared to the pGAP promoter is at least 1.1 times, preferably at least 1.2 times, preferably at least 1.3 times, preferably at least 1.4 times, preferably at least 1.5 times, preferably at least 1.6 times, preferably at least 1.7 times, preferably at least 1.8 times, preferably at least 1.9 times, preferably at least 2 times, preferably at least 10 times, preferably at least 4 times, preferably at least 5 times, preferably at least 10 times, or at least 15 times.

[0061] According to a particular aspect, the isolated nucleic acid sequence of the present invention or the expression construct of the present invention can be used in a method for producing POI by culturing host cells transformed with the nucleic acid sequence and / or the expression construct, preferably wherein the culture is carried out in a bioreactor, starting from a batch culture period followed by a fed-batch culture period or a continuous culture period. Attached Figure Description

[0062] Figure 1 The nucleic acid sequence pCS1 (985 bp, SEQ ID 1) and promoter sequence of Pichia pastoris, which is a DNA sequence including pCS1 and additional nucleotides at the 5′ end, or a pCS1 fragment that promotes the expression of CS1 in Pichia pastoris, comprising 1488 bp (SEQ ID 2), 767 bp (SEQ ID 3), 500 bp (SEQ ID 4), 344 bp (SEQ ID 5), 234 bp (SEQ ID 6), 138 bp (SEQ ID 7) and 85 bp (SEQ ID 8).

[0063] Figure 2 The CS1 encoding nucleotide and amino acid sequences of the following strains are as follows.

[0064] i) Strains GS115, CBS7435 and CBS2612 (PAS_chr1-4_0586), coding sequence (SEQ ID 9), translated sequence (XM_002490678.1, SEQ ID 10); and

[0065] ii) Strain DSMZ70382 (PIPA02805), coding sequence (SEQ ID 11), translation sequence (SEQ ID 12).

[0066] Figure 3 : The natural pGAP promoter sequence of Pichia pastoris (GS115) (SEQ ID 13). Detailed Implementation

[0067] The specific terms used throughout the instruction manual have the following meanings:

[0068] As used herein, the term "carbon source" or "carbon source compound" refers to a fermentation carbon source, typically a source carbohydrate suitable as a microbial energy source, such as a carbon source that can be metabolized by the host microorganism or the production cell line, particularly a carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and alcohols including glycerol, in purified form, provided in a basal culture medium or as a raw material, such as a complex nutrient. According to the invention, the carbon source can be used as a single carbon source or as a mixture of different carbon sources.

[0069] The term "carbon-rich conditions" as used herein specifically refers to a carbon source suitable for cell growth, such as the type and quantity of nutrients suitable for eukaryotic cell growth. This carbon source can be a culture medium, such as a basal medium or a compound medium, but can also be provided in the form of a (chemically) limiting medium containing a purified carbon source.

[0070] The carbon source used in this article for the growth phase of the cell culture process is referred to as the “basal carbon source” and is generally provided in an amount suitable for providing cell growth, such as obtaining at least 5 g / L of cell dry matter, preferably at least 10 g / L of cell dry matter, a cell density of at least 15 g / L of cell dry matter, and exhibiting, for example, more than 90%, preferably more than 95%, viability during a standard secondary culture step.

[0071] During the growth period, the carbon source is often used in excess or surplus, which can generally be understood as, for example, providing excess energy to increase biomass during the cultivation of cells with a high specific growth rate.

[0072] Specifically, the excess amount exceeds the limit of the carbon source used under growth-limiting conditions to obtain an excess concentration in the fermentation broth, which is measurable and is typically at least 10 times higher than the concentration obtained by culturing cells in a medium containing the limiting carbon source substrate, preferably at least 50 times or at least 100 times higher.

[0073] The terms "carbon source limiting conditions" or "limiting carbon source" in this invention also refer to "supplementary carbon source," for example, as used according to the invention, to the type and quantity of carbon source that facilitates the production of fermentation products by producing cell lines, especially in culture processes at controlled growth rates below the maximum growth rate. In particular, the growth period, such as batch culture, fed-batch culture, and continuous culture processes, is followed by the production period.

[0074] In summary, cell culture processes can be categorized into batch culture, continuous culture, and fed-batch culture. Batch culture involves adding a small amount of seed culture medium to the culture medium, without adding feed or draining the culture medium during cell growth. Continuous culture involves continuously adding and draining the culture medium during the culture period. Continuous culture also includes perfusion culture. Fed-batch culture, also known as semi-batch culture, is an intermediate method between batch and continuous culture. It involves continuously adding or draining the culture medium during the culture period, but unlike continuous culture, the culture medium is not continuously drained.

[0075] A particularly preferred method is a fed-batch culture method based on the addition of growth-limiting factors to the culture medium. This fed-batch scheme, including individual fed-batch fermentations and repeated fed-batch fermentations, is typically used in bioindustry processes to achieve high cell densities in bioreactors. The controlled addition of carbon sources directly affects culture growth and helps avoid spillover metabolism or the formation of unwanted metabolic byproducts. Under carbon-limited conditions, specifically, the carbon source is contained within the feed during the fed-batch culture process. Therefore, the amount of carbon source provided is limited.

[0076] In the chemostat or continuous culture described in this invention, the growth rate can be strictly controlled.

[0077] In this invention, the “limiting amount” of carbon source can be understood as the amount of carbon source required to maintain the cell line under growth-limiting conditions, such as during the growth phase or in production mode. Such a limiting amount can be used in fed-batch culture processes, where the carbon source is contained in the feed medium and added to the culture at a low rate to maintain energy supply, for example, to generate POI, while maintaining biomass at a low, specific growth rate. Typically, the feed medium is added to the fermentation broth during the production phase of cell culture.

[0078] For example, the limiting amount of carbon source can be determined by the residual amount of carbon source in the cell culture medium, below a predetermined threshold or even below the detection limit measured by a standard (carbohydrate) detection method. Typically, this residual amount is determined in the fermentation broth after harvesting the fermentation product.

[0079] The limiting amount of carbon source can also be determined by limiting the average feed rate of carbon source added to the fermenter, for example, by the amount added in batches during the entire culture process, such as each fermentation time, thus determining the calculated average amount of carbon source added per batch. This average feed rate is kept low to ensure that the cell culture makes full use of the supplemented carbon source, for example, at 0.6 g / L. -1 h -1 (g carbon source / L initial fermentation volume, h time) and 25 g / L -1 h -1 The preferred concentration is between 1.6 g / L -1 h -1 -20g / L -1 h -1 .

[0080] The limiting quantity of this carbon source can also be determined by a specific growth rate, which is maintained at a low rate during production, such as below the maximum specific growth rate, for example, within a predetermined range, such as 0.001 h. -1 -0.20h -1 or 0.02h -1 -O.20h-1 0.02h is preferred -1 or ~0.15h -1 .

[0081] Any type of organic carbon source suitable for eukaryotic cell culture can be used. According to a particular embodiment, the carbon source is a hexose, such as glucose, fructose, galactose, or mannose; a disaccharide, such as sucrose; an alcohol, such as glycerol or ethanol; or a mixture thereof.

[0082] According to a particularly preferred embodiment, the basic carbon source is selected from the group consisting of glucose, glycerol, ethanol, or mixtures thereof, and complex nutrients. According to a preferred embodiment, the basic carbon source is glycerol.

[0083] According to a further specific embodiment, a supplementary carbon source is a hexose, such as glucose, fructose, galactose, and mannose; a disaccharide, such as sucrose; or an alcohol, such as glycerol, ethanol, or mixtures thereof. According to a preferred embodiment, the supplementary carbon source is glucose.

[0084] Specifically, the method may use glycerol as the basic carbon source and glucose as a supplementary carbon source.

[0085] In particular, the feeding culture medium used in this invention has a clearly defined chemical composition and is methanol-free.

[0086] Regarding cell culture media, such as basal or feed media in batch-feed processes, the terms "chemically defined" or "defined" refer to a medium suitable for in vitro cell culture to produce cell lines, wherein all chemical components and (poly)peptides are known. Typically, chemically defined media are completely free of animal-derived components and represent a pure and consistent cell culture environment.

[0087] As used in this invention, the term "cell line" refers to a clone of a specific cell type that has been established and acquired long-term proliferative capacity. The term "host cell line" refers to a cell line used to express endogenous or recombinant genes or products of metabolic pathways to produce polypeptides or metabolites regulated by such polypeptides. "Production host cell line" or "production cell line" is generally understood to be a cell line readily available for culture in a bioreactor to obtain a product of a production process, such as a point of interest (POI). The term "eukaryotic host" or "eukaryotic cell line" refers to any eukaryotic cell or organism that can be cultured to produce a POI or host cell metabolites. It is well known that the terminology of this invention does not include humans.

[0088] According to the particularly preferred eukaryotic host cell of the present invention, the cell or cell line is selected from the group consisting of mammalian, insect, yeast, filamentous fungi and plant cell lines, with yeast being preferred.

[0089] Specifically, the yeast is selected from the genera *Pichia*, *Candida*, *Globosa*, *Lactobacillus*, *Hansenula*, *Yersinia*, *Kluyveromyces*, *Saccharomyces*, and *Pichia*, with methane-nutritive yeasts being preferred.

[0090] The preferred yeasts are Pichia pastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris.

[0091] Regarding host cell lines, the term “cell culture” or “culture”, also known as “fermentation,” refers to the maintenance of cell viability or quiescent state in an artificial, such as in vitro, environment under conditions favorable to growth, differentiation, or sustained activity, particularly in a controlled bioreactor, according to industrially known methods.

[0092] When cells are cultured using the culture medium of the present invention, the cell culture is brought into contact with the culture medium or with the substrate under conditions suitable for supporting cell culture in a culture vessel. In certain embodiments, the culture medium of the present invention is used to culture cells according to standard cell culture techniques known in the art. In other aspects of the invention, the provided culture medium can be used for the growth of eukaryotic cells, particularly yeast or filamentous fungi.

[0093] Cell culture media can provide the nutrients necessary for cell growth in controlled, artificial, and in vitro environments. The characteristics and chemical composition of cell culture media vary depending on the specific needs of the cells. Important parameters include molar osmotic pressure concentration, pH value, and nutrient solution formulation. Nutrients can be added continuously or discontinuously according to methods known in the art. The culture media used in this invention are particularly useful for the production of recombinant proteins.

[0094] Batch culture is a culture mode in which all the nutrients required for cell culture are contained in the initial culture medium. During fermentation, no further nutrients are supplied during the feed-batch phase. After the batch phase, a fed-batch phase begins, during which one or more nutrients are supplied to the culture medium via feeding. The purpose of nutrient feeding is to increase biomass, thereby also increasing the amount of recombinant protein. Although the feeding mode is critical and important in most culture processes, this invention, employing the promoter of this invention, is not limited to any particular culture mode.

[0095] In some embodiments of the invention, a fed-batch method is employed. Specifically, host cells transformed with a nucleic acid construct encoding the desired POI are cultured in a growth-phase medium and then transferred to a production-phase medium to produce the desired recombinant POI.

[0096] The supplemental carbon source added to the culture medium can be in liquid or other alternative forms, such as solid (e.g., tablets or other sustained-release methods) or gas (e.g., carbon dioxide). However, according to a preferred embodiment, the limiting amount of supplemental carbon source added to the cell culture medium can even be zero. Preferably, under the limiting carbon source condition, the concentration of the supplemental carbon source in the culture medium is 0–1 g / L, preferably less than 0.6 g / L, more preferably less than 0.3 g / L, more preferably less than 0.1 g / L, preferably 1–50 mg / L, more preferably 1–10 mg / L, particularly preferably 1 mg / L or even lower, for example, below the detection limit determined by a suitable standard detection method, for example, as the residual concentration after consumption by cell culture in the culture medium.

[0097] In a preferred method, the limiting amount of carbon source determines the residual amount in cell culture below the end of production or the output period of the fermentation process, preferably the detection limit determined in the fermentation culture broth after harvesting the fermentation product.

[0098] Preferably, the limiting amount of supplemental carbon source is growth-limiting, thereby maintaining a specific growth rate below a maximum specific growth rate, for example, at 0.001 h. -1 ~0.20h -1 or 0.02h -1 ~0.20h -1 0.02h is preferred -1 ~0.15h -1 Within the range.

[0099] In another embodiment, the host cells of the present invention are cultured continuously (e.g., using a chemostat). The continuous fermentation process is characterized by the addition of fresh culture medium to the bioreactor at a defined, constant, and continuous rate, thereby simultaneously removing the culture medium from the bioreactor at a restricted, constant, and continuous rate. By maintaining the culture medium, the feed rate and removal rate are kept at the same constant level, and the culture parameters and conditions of the bioreactor are also kept constant.

[0100] Specifically, the stable cells described in this invention can be understood as cell cultures that maintain their genetic characteristics, particularly high POI yields, even after 20 generations, preferably at least 30 generations, more preferably 40 generations, and most preferably 50 generations. In particular, stable recombinant host cell lines considered most advantageous for industrial-scale production are provided.

[0101] The cell culture techniques (e.g., effective volume and technology system) of the present invention, combined with nutrient-feed-based culture modes, particularly fed-batch culture or batch culture processes, or continuous or semi-continuous processes (e.g., chemostats), are particularly advantageous for industrial-scale methods.

[0102] The terms "expression," "expression system," or "expression cassette" refer to a operatively linked nucleic acid molecule containing a desired coding sequence and control sequences, so that a host transformed or transfected with these sequences can produce a protein-coding protein or a host metabolite. For transformation, the expression system may be incorporated into a vector; however, the relevant DNA may also be integrated into the host chromosome. Expression can be applied to secreted or non-secretory expression products, including peptides or metabolites.

[0103] The term "expression construct," "vector," or "plasmid" as used in this invention refers to the DNA sequence required for the transcription of a cloned recombinant nucleotide sequence, such as the transcription of a recombinant gene and the translation of its mRNA in a suitable host organism. Expression vectors or plasmids typically contain an autonomous replication initiation site in the cell, selection markers (e.g., amino acid synthesis genes or genes resistant to antibiotics such as bleomycin, kanamycin, G418, or hygromycin), multiple restriction endonuclease sites, a suitable promoter sequence, and a transcription terminator, these components being operatively linked together. The terms "plasmid" and "vector" as used in this invention include autonomously replicating nucleic acid sequences as well as genome-integrated nucleic acid sequences.

[0104] In particular, the expression constructs of the present invention contain a promoter operatively linked to a nucleotide sequence encoding a POI under the transcriptional control of the promoter, the promoter being not naturally associated with the coding sequence of the POI.

[0105] The term "heterologous" as used in this invention with respect to nucleotides or proteins refers to a nucleotide that is foreign to a given host cell, e.g., "exogenous," as it is not naturally found, or naturally found in a given host cell, e.g., "endogenous," but is "exogenous" in the context of a heterologous construct, e.g., when using a heterologous nucleic acid. Endogenously found heterologous nucleotides can also be non-naturally produced, e.g., in greater quantities than expected or greater than naturally found in cells. The heterologous nucleotide sequence, or the nucleic acid containing the heterologous nucleotide sequence, may differ from the endogenous nucleotide sequence but encode the same protein as the endogenously found one. In particular, a heterologous nucleotide sequence is one that has not been found to have an essentially identical relationship with the host cell. Any recombinant or artificial nucleotide can be understood as heterologous. As described in this invention, an example of a heterologous polynucleotide is, for example, to obtain a heterozygous promoter, or operatively linked to a nucleotide sequence whose coding sequence is not naturally associated with the promoter. As a result, a heterozygous or chimeric polynucleotide can be obtained. A further embodiment of the heterologous compound is operatively linked to a transcriptional control element, such as the POI-encoded polynucleotide of the promoter of the present invention, where the endogenous, naturally occurring POI-encoded sequence is not normally operatively linked to the promoter.

[0106] In the context of this invention, the term "variant" specifically refers to any sequence derived from a parental sequence, for example, obtained through length variation, such as elongation or breakage, mutation, hybridization (including sequence binding), or having a particular degree of homology, or similarly.

[0107] The present invention provides a promoter that is a wild-type promoter, such as that of Pichia pastoris, or a variant having its functional activity, such as being able to control the transcription of a specific gene in wild-type or recombinant eukaryotic cells.

[0108] As used in the context of this invention, the term "natural" specifically refers to an individual structure or component of an organism that is naturally associated with its environment. However, it is well known that natural structures or components can be isolated from their naturally associated environment and can be presented as isolated natural structures or components. Such isolated natural structures or components can also be of artificial or synthetic origin and still possess the same characteristics as those of natural origin.

[0109] Although some embodiments of the invention refer to natural structures or components, such as isolated forms, it is well known that the materials, methods, and uses of the invention, for example, particularly isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, and recombinant proteins, are “artificial” and therefore not considered as results of “natural laws”.

[0110] The functionally active variant promoter, for example derived from the promoter sequence pCS1 (SEQ ID 1) generated through mutation, is used as a “parental” sequence to produce a suitable promoter sequence for recombinant cell lines. Such variant promoters can be derived from a library of mutant sequences (pCS1) by selecting library members with predetermined characteristics. Variant promoters can possess the same or even improved characteristics, for example, improved promoter strength supporting POI production, maintaining substantially the same promoter function and strength at both high and low growth rates, which is specifically understood herein as “growth rate-dependent function.”

[0111] The variant promoter may also be derived from similar sequences, for example from eukaryotic species other than Pichia pastoris, or from genera other than Pichia pastoris (e.g., from K. lactis, Z. rouxii, P. stipitis, H. polymorpha). In particular, similar promoter sequences naturally associated with similar genes to the corresponding Pichia pastoris genes can be used as the sequence or as parental sequences to produce functionally active variants. Specifically, a pCS1-like promoter is characterized by being naturally associated with a CS1-like gene (see amino acid sequence SEQ ID 9 or 11), and the characteristics of such similar promoter sequences or their functionally active variants can be determined using standard techniques.

[0112] In particular, the “functionally active” variant of the nucleotide or promoter sequence used in this invention refers to a mutant sequence, for example, generated by inserting, deleting, or replacing one or more nucleotides in the sequence within the sequence or at one or both ends of the sequence, and such modification does not affect (in particular impair) the activity of the sequence.

[0113] Specifically, the functionally active variants of the promoter sequence described in this invention are selected from the group consisting of the following sequences:

[0114] —Having at least 60% nucleotide sequence identity with the parental sequence, preferably at least 70%, at least 80%, or at least 90% homology or sequence identity; and / or

[0115] —A homolog obtained by modifying the parental nucleotide sequence, such as the pCS1 sequence or a length variant thereof, which can serve as a template to provide mutations, for example by inserting, deleting, or replacing one or more nucleotides within the sequence or at one or both ends of the sequence. The homolog preferably has (i.e., contains or constitutes thereof) a nucleotide sequence of 80 bp to 1500 bp, 200 bp to 1500 bp, or 234 bp to 1488 bp, preferably at least 100 bp, at least 200 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.

[0116] — Analogs derived from species other than Pichia pastoris.

[0117] In particular, preferred functionally active variants are those derived from the promoter of the present invention, obtained through modification, extension, and / or multiple fragments of the promoter, having (i.e. comprising or consisting of) a nucleotide sequence of at least 80 bp, preferably at least 100 bp, preferably at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp, preferably up to 1500 bp.

[0118] Specifically, functionally active variants of the parental promoter sequence described in this invention can be generated through mutation. As used in the context of this invention, the term "mutation generation" refers to a method of providing nucleotide sequence mutants, for example by inserting, deleting, and / or replacing one or more nucleotides, thereby obtaining variants with at least one alteration in a non-coding or coding region. Mutations can be formed through random, semi-random, or site-directed mutations. Typically, large random gene pools are generated through high genetic diversity, which can be selected according to particularly desired genes or gene phenotypes.

[0119] Certain preferred functionally active variants of the promoter described in this invention are length variants, or particularly pCS1 fragments, preferably those containing the 3' end of the promoter nucleotide sequence, for example, nucleotide sequences derived from a promoter nucleotide sequence having a particular length and 5' end insertion or deletion, such as 5' end nucleotide sequence extension or termination, thereby obtaining a specific length having a 5' end ranging from 3' to varying lengths, for example, nucleotide sequence lengths of at least 80 bp, preferably at least 100 bp, preferably at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.

[0120] The extended length variant of the present invention preferably includes one or more additional nucleotides at the 5′ end of the pCS1 sequence, such as those pCS1 sequences naturally associated with the wild-type pCS1 sequence at the origin of cell replication.

[0121] For example, a functionally active variant of pCS1 may comprise a nucleotide sequence, or be composed of a nucleotide sequence selected from the group consisting of pCS1 a (SEQ ID 2), pCS1 b (SEQ ID 3), pCS1 c (SEQ ID 4), pCS1 d (SEQ ID 5), pCS1e (SEQ ID 6), pCS1f (SEQ ID 7), and pCS1 g (SEQ ID 8), and thus comprise a nucleotide sequence with a length ranging from 80 to 1500 bp. Preferably, the functionally active variant of pCS1 comprises a nucleotide sequence selected from the group consisting of pCS1a (SEQ ID 2), pCS1 b (SEQ ID 3), pCS1 c (SEQ ID 4), pCS1 d (SEQ ID 5), and pCS1 e (SEQ ID 6), or is composed of these sequences.

[0122] The functionally active variant of the promoter of the present invention can also be understood as comprising a heterozygous sequence of said pCS1 and any functionally active variant thereof, particularly any parental length variant or fragment sequence, for example, generated by binding with one or more of any sequence called pCS1 or a functionally active variant thereof, for example at least two such parental sequences, at least three, at least four, or at least five such parental sequences, for example, two or more pCS1 extension sequences or fragments selected from the group consisting of pCS1 a, pCS1 b, pCS1 c, pCS1d, pCS1e, pCS1f, and pCS1g, preferably, fragments selected from the group consisting of pCS1 a, pCS1b, pCS1 c, pCS1d, and pCS1e. In another embodiment, the heterozygosity consists of at least one sequence selected from pCS1 or any functionally active variant thereof, particularly any length variant or fragment sequence, and, for example, a heterologous sequence not naturally associated with the pCS1 sequence of Pichia pastoris.

[0123] The functionally active variant of the promoter of the present invention can be further understood as a nucleotide sequence that hybridizes with the pCS1 promoter or its functionally active length variant or fragment under stringent conditions, or any of the mutant or hybrid nucleic acid sequences thereof.

[0124] As used in this invention, the term "hybridization" or "crossover" refers to a process in which two nucleic acid sequences anneal each other under appropriate conditions, forming stable, specific hydrogen bonds to create a double strand. The hybridization of two complementary or fully complementary sequences depends on the operating conditions used, particularly the stringency. The stringency can be understood as indicating homology; higher stringency indicates a higher percentage of homology between sequences. This stringency can be specifically defined as the base composition of the two nucleic acid sequences and / or the degree of mismatch between them. By varying conditions, such as salt concentration and temperature, a given nucleic acid sequence can be made to hybridize only with its exact complementary sequence (high stringency) or with any related sequence (low stringency). Increasing the temperature or decreasing the salt concentration tends to increase the selectivity of the hybridization reaction.

[0125] As used in this invention, the term "hybridization under stringent hybridization conditions" is preferably understood to refer to hybridization under certain stringent conditions. In a preferred embodiment, the "stringent hybridization conditions" are conditions in which the homology of the two nucleic acid sequences is at least 70%, preferably at least 80%, and preferably at least 90%, i.e., hybridization can only occur if the double strands obtained during hybridization contain preferably at least 70%, preferably at least 80%, and preferably at least 90% of the AT and CG bonds.

[0126] The stringent conditions depend on reaction parameters such as the concentration and type of ions present in the hybridization solution, the nature and concentration of the denaturant, and / or the hybridization temperature. Suitable conditions can be determined by those skilled in the art as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989).

[0127] In particular, the characteristics of the functional active variant of the present invention can be determined by exhibiting essentially the same activity as pCS1.

[0128] As used in this invention, the term "substantially identical activity" specifically refers to a substantially identical or improved promoter strength, particularly the expression or transcription strength of the promoter, and substantially identical or improved characteristics related to promoter strength, particularly determined independent of the host cell's growth rate, such as expression or transcription strength substantially identical to pCS1 as determined by a suitable testing system, for example, + / - 20% or + / - 10%, and / or higher than that of the native pGAP promoter in the native host cell, for example, at least 1 relative to the pGAP promoter strength. An increase of 1-fold, or at least 1.2-fold, preferably at least 1.3-fold, preferably at least 1.4-fold, preferably at least 1.5-fold, preferably at least 1.6-fold, at least preferably 1.7-fold, at least preferably 1.8-fold, at least preferably 1.9-fold, and at least preferably 2.0-fold, or even higher, such as at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least up to 15-fold, of activity, the improvement in this property is determined using a suitable assay system employing the same host cell type, the same culture conditions, and the same nucleic acid encoding the expression product, such as POI.

[0129] The term "homology" refers to the presence of identical or conserved base pairs at corresponding positions in two or more nucleotide sequences to a degree approaching 100%. A homologous sequence of the present invention typically has at least 60% nucleotide identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably 98% or 99%.

[0130] Preferably, the homologous promoter sequence of the present invention has at least some homology in a specific portion of the nucleotide sequence with any one of the promoter nucleotide sequences of *Pichia pastoris* pCS1, pCS1a, pCS1b, pCS1c, pCS1d, pCS1e, pCS1f, and pCS1g, and with analogues derived from other species besides *Pichia pastoris*. For example, it includes a 3' portion of the individual promoter nucleotide sequence, preferably a portion reaching a specific length of the 3' of the individual promoter nucleotide sequence, for example, a length of 80 bp to 1500 bp, preferably 200 bp to 1500 bp, more preferably 234 bp to 1488 bp, preferably at least 100 bp, preferably at least 200 bp, preferably at least 300 bp, preferably at least 400 bp, preferably at least 500 bp, preferably at least 600 bp, preferably at least 700 bp, preferably at least 800 bp, preferably at least 900 bp, or at least 1000 bp. In particular, at least those portions are preferably homologous within a 300–1000 bp range of the 3' end sequence, which includes the individual promoter nucleotide sequence.

[0131] Similar sequences typically originate from other species or strains. It is understood that any similar promoter sequence of the present invention derived from species other than Pichia pastoris may contain homologous sequences, such as sequences having a certain homology described in the present invention. Therefore, the term "homologous" may also include similar sequences. On the other hand, it is understood that the present invention also refers to similar sequences containing a certain homology and their homologous sequences.

[0132] The "percentage of homology" for a gene's nucleotide sequence is defined as the percentage of nucleotides in a candidate DNA sequence that is nucleotide-identical to the stated DNA sequence, after sequence alignment and gap introduction, to obtain the maximum percentage of sequence identity, if necessary, without considering any conserved substitutions as part of the sequence identity. Sequence alignment performed to determine the percentage of nucleotide sequence identity can be carried out using methods skilled in the art, such as publicly available computer software. Those skilled in the art can determine suitable parameters for determining the alignment, including any algorithms necessary to obtain the maximum alignment of the full length of the sequences being compared.

[0133] As used in this invention, the term "isolated" or "separated" in relation to nucleic acids, POIs, or other compounds refers to a compound that has been sufficiently isolated from its natural environment in order to exist in a "substantially pure" form. "Isolated" does not necessarily mean artificially excluded or a mixture synthesized with other compounds or materials, or, for example, impurities present due to incomplete purification that do not interfere with the essential activity. Specifically, the isolated nucleic acid molecules of this invention also include those chemically synthesized nucleic acid molecules. Regarding the nucleic acids of this invention, the terms "isolated nucleic acid" or "isolated nucleic acid sequence" are sometimes used. When applied to DNA, this term refers to a DNA molecule isolated from a sequence that is most closely related to the sequence in the genome of the organism of its origin, which naturally exists within it. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or viral vector, or a sequence integrated into the genomic DNA of a prokaryotic or eukaryotic cell or a host microorganism. Specifically, according to the invention, the term "isolated nucleic acid" excludes the pCS1 sequence linked to a nucleic acid sequence encoding the CS1 protein. An “isolated nucleic acid” (which also includes DNA or RNA) can further represent molecules that are directly produced by biological or synthetic methods and isolated from other components present during production.

[0134] As used in this invention, the term "operable link" refers to the linking of nucleotide sequences on a single nucleic acid molecule, such as a vector, in a manner in which one or more nucleotide sequences are influenced by at least one other nucleotide sequence present in said nucleic acid molecule. For example, when expression of a coding sequence is possible, a promoter is operably linked to the coding sequence of a recombinant gene. As a further embodiment, when expression of a secreted form of a protein, such as a precursor or mature protein of a mature protein, is possible, a nucleic acid encoding a single peptide is operably linked to a nucleic acid sequence encoding a POI. In particular, such interoperable links can be directly linked, i.e., without any further elements or nucleic acid sequences between the nucleic acid encoding the signal peptide and the nucleic acid encoding the POI.

[0135] As used in this invention, the term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Promoter activity can be determined by transcriptional efficiency. This activity can be measured by determining the amount of mRNA transcribed from the promoter, for example, directly by Northern hybridization, or indirectly by determining the amount of gene product expressed from the promoter.

[0136] The promoter of this invention can specifically initiate, regulate, or conversely mediate or control the expression of coding DNA. The promoter DNA and coding DNA may originate from the same gene or different genes, and may come from the same or different organisms.

[0137] In particular, the promoters of the present invention can be understood as constitutive promoters, for example, those that do not require induction or are impossible to repressively control expression. Therefore, they exhibit sustained and stable expression at a certain level. Since the host cell has a unique function at high promoter strength across all growth phases and growth rates, the constitutive promoters of the present invention are particularly useful for fed-batch culture of host cell lines. Prior art constitutive promoters have the disadvantage of low strength implementations under growth-restricted conditions, such as in fed-batch processes, or for maintaining host cells at high growth rates, such as the intermediate logarithmic phase.

[0138] The promoter strength of this invention specifically refers to transcription strength, expressed as the efficiency of transcription initiation occurring at a high or low frequency in the promoter. Higher transcription strength corresponds to a higher transcription frequency in the promoter. Promoter strength is quite important because it determines the frequency of effective transcription of a given mRNA sequence, giving certain genes higher transcriptional priority than others, resulting in higher transcriptional concentrations. For example, genes encoding large quantities of desired proteins typically have fairly strong promoters. RNA polymerases can only perform transcription once per period of time; therefore, their efficiency must be prioritized. This priority can be achieved selectively through differences in promoter strength. According to the invention, for example, a promoter is fairly strong if it exhibits a state of maximum activity, particularly a constant level, regardless of the host cell's metabolism or growth rate, and especially regardless of the carbon source, in both high and low growth rate conditions of cell culture.

[0139] The strength of a promoter is typically determined based on a standard promoter, such as the specific pGAP promoter of the cell used as the host cell. Transcription frequency is generally understood as the transcription rate, determined, for example, by employing appropriate analytical methods, such as RT-PCR or Northern blotting. Promoter strength expressing the gene of interest is generally understood as expression intensity or the ability to support high expression levels / rates. For example, the expression and / or transcription strength of the promoters of the present invention can be determined in a host cell (which is Pichia pastoris) and compared with the native pGAP promoter of Pichia pastoris.

[0140] Transcription rate can be determined by measuring transcription intensity using gene chips or by using real-time quantitative PGR (qRT-PCR). Here, gene chip or qRT-PCR data show differences in expression levels and signal intensity under conditions of high and low growth rates compared to the natural pGAP promoter, or under conditions of different culture medium components. A suitable assay system is specifically described in Example 11 below.

[0141] Compared to the natural pGAP promoter in the host cell, sometimes referred to as the "homogeneous pGAP promoter," a transcription rate or transcription intensity of at least 110% reflects that the promoter of the present invention exhibits a considerably high transcription intensity. Preferably, for example, compared to the natural pGAP promoter, such as in a host cell selected for POI production, for example under carbon-rich conditions such as batch culture, or under carbon-limited conditions such as chemostat or fed-batch culture, the transcription rate or transcription intensity is at least 110%, preferably at least 120%, or at least 130%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 3 times, at least 4 times, at least about 5 times, at least about 10 times, at least about 15 times, or even higher.

[0142] Preferably, the transcriptional analysis is quantitative or semi-quantitative using qRT-PCR, DNA microarray, RNA sequencing, and transcriptome analysis.

[0143] For example, the expression rate can be determined by the expression level of a reporter gene, such as GFP as described in the following examples section, with the testing system specifically described in Example 10. Results from culturing clones in solution and comparing them to the natural pGAP promoter showed that the pCS1 promoter had a considerably high transcription rate (at least 110%).

[0144] The expression level of the gene of interest reflects that the promoter of the present invention exhibits a considerably high expression intensity, at least 110% compared to the natural pGAP promoter in the host cell, sometimes referred to as a "homologous promoter." Preferably, for example, determined in eukaryotic cells selected as host cells for POI production, such as under carbon-rich conditions (e.g., batch culture) or under carbon-limited conditions (e.g., chemostat or fed-batch culture), the expression intensity is at least 110%, preferably at least 120%, or at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, or even higher.

[0145] The natural pGAP promoter initiates the Gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutive promoter present in most living microorganisms. GAPDH (EC 1 / 2 / 1 / 12), a key enzyme in glycolysis and gluconeogenesis, plays a crucial role in the catabolism and anabolism of carbohydrates. Therefore, while the pGAP promoter is understood as a constitutive promoter (e.g., not induced by supplementation with a specific carbon source), it is a metabolic promoter whose strength increases with increasing growth rate. Consequently, the pGAP promoter is largely unsuitable for efficient production processes during cell line culture at low growth rates.

[0146] Conversely, the promoter of the present invention surprisingly maintains its high promoter strength (essentially) at a constant high transcriptional level throughout the entire growth period of the host cell culture.

[0147] Specifically, the natural pGAP promoter is active in recombinant eukaryotic cells in a similar manner to that in natural eukaryotic cells of the same species or strain, including unmodified (non-recombinant) or recombinant eukaryotic cells. Therefore, such a natural pGAP promoter can generally be understood as an endogenous promoter homologous to eukaryotic cells, serving as a standard or reference promoter for comparison purposes.

[0148] For example, a natural pGAP promoter of Pichia pastoris is an unmodified endogenous promoter sequence of Pichia pastoris used to control GAPDH expression in Pichia pastoris, for example, having Figure 3 The sequence shown is the natural pGAP promoter sequence (GS115) of Pichia pastoris (SEQ ID 13). According to the present invention, if Pichia pastoris is used as a host for POI production, the transcription strength or transcription rate of the promoter according to the present invention will be compared with this natural GAP promoter of Pichia pastoris.

[0149] As another embodiment, such as in the expression control of *Saccharomyces cerevisiae*, a natural pGAP promoter of *Saccharomyces cerevisiae* is an unmodified endogenous promoter sequence of *Saccharomyces cerevisiae*. According to the invention, if *Saccharomyces cerevisiae* is used as a host for POI production, the transcriptional strength or transcription rate of the promoter according to the invention is compared with that of this natural pGAP promoter of *Saccharomyces cerevisiae*.

[0150] Therefore, the expression or transcriptional intensity of the promoters of the present invention is generally compared with the natural pGAP promoters of the same species or strains used as hosts for POI production.

[0151] In particular, it can be understood that the promoter of the present invention is preferably a non-metabolic promoter, such as a promoter that is naturally operatively linked to a gene that encodes a fully glycolytic enzyme or gluconeogenesis enzyme, a ribosomal protein, or an enzyme such as an intracellular protease or a protease secreted from a host cell.

[0152] Particularly preferred is a promoter of the present invention having an expression intensity or transcription intensity of at least pCS1. The promoter intensity can be determined by standard methods, such as by measuring the amount of expression product or transcription, for example using gene chips, Northern blotting, RNA sequencing, or qRT-PCR, or other methods in cell culture, such as by measuring the amount of individual gene expression products in recombinant cells, using the pGAP promoter as a reference for comparison. The examples section explains the experimental determinations.

[0153] As used in this invention, the term "protein of interest (POI)" refers to a polypeptide or protein produced by recombinant technology in a host cell. More specifically, the protein can be a polypeptide not naturally present in the host cell, i.e., a homologous protein of the host cell, or, for example, produced by transformation using a self-replicating vector containing a nucleic acid sequence encoding the POI, or by integrating one or more copies of the nucleic acid sequence encoding the POI into the genome of the host cell via recombinant technology, or by recombinantly modifying one or more regulatory sequences, such as promoter sequences, that control the expression of the gene encoding the POI. In some instances, the term POI as used in this invention also refers to any metabolite of the host cell regulated by a recombinantly expressed protein.

[0154] According to a particular embodiment, in particular, if the promoter of the present invention is naturally associated with any nucleic acid sequence encoding a CS1 protein, the term POI will not include the CS1 protein of Pichia pastoris, for example, through Figure 2 The characteristics are determined by any amino acid sequence.

[0155] In particular, the POI described in this invention is a eukaryotic protein, preferably a mammalian protein, and especially a heterologous protein of a host cell.

[0156] The POI generated according to the present invention can be an oligomeric protein, preferably a dimer or a tetramer.

[0157] According to one aspect of the invention, the POI is a recombinant or heterologous protein, preferably selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibodies, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, growth factors, hormones and cytokines, or metabolites of the POI.

[0158] A specific point of interest (POI) is an antigen that binds to a molecule such as an antibody or a fragment thereof. In specific POIs, the antibody is, for example, a monoclonal antibody (mAbs), an immunoglobulin (Ig) or a class G immunoglobulin (IgG), a heavy chain antibody (HcAbs), or a fragment thereof, such as an antigen-binding fragment (Fab), Fd, a single-chain variant fragment (scFv), or an engineered variant thereof such as an Fv dimer (biantibody), an Fv trimer (triantibody), an Fv tetramer (tetraantibody), or a microantibody, or a single-domain antibody class VH or VHH or V-NAR.

[0159] According to a particular embodiment, a fermentation product is produced using POI, a metabolite, or a derivative thereof.

[0160] The POI can be specifically isolated from cell cultures in a purified form, for example, substantially pure.

[0161] As used in this invention, the terms "substantially pure" or "pure" refer to a compound containing at least 50% by mass, preferably at least 60%, 70%, 80%, 90%, or 95%, such as a nucleic acid molecule or a point of interest (POI). Purity is determined by a method suitable for the compound (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0162] As used in this invention, the term "recombinant" means "prepared by or the result of genetic engineering." Therefore, a "recombinant microorganism" comprises at least one "recombinant nucleic acid." Specifically, a recombinant microorganism comprises an expression vector or cloning vector, or is genetically engineered to contain a recombinant nucleic acid sequence. A "recombinant protein" is produced by expressing an individual recombinant nucleic acid in a host. As described in this invention, a "recombinant promoter" is a non-coding nucleotide sequence obtained through genetic engineering that is suitable for use as a functionally active promoter.

[0163] Therefore, the new promoter can be identified using unique functional characteristics. Unexpectedly, by analyzing transcriptional intensity under production process conditions, PAS_chr1-4 (referred to as the CS1 gene in this invention, SEQ ID 9) was identified as the most potent transcription gene in Pichia pastoris. The encoded CS1 protein (SEQ ID 10) is not a glycolytic enzyme or protein, but we predicted its localization to the cell surface using GPI anchoring. Although the 9.43 Mbp genome sequence of Pichia pastoris strain GS1 15 has been determined and disclosed in US20110021378A1, promoter characteristics, such as the promoter sequence, have not yet been specifically studied.

[0164] Surprisingly, according to the present invention, such promoters can be used effectively. Existing Pichia pastoris promoters, such as those used for industrial-scale POI production, are primarily derived from the methanol metabolism pathway, requiring the addition of methanol to induce POI production, which is generally unsatisfactory. The promoters and methods of the present invention have the advantage of providing increased yields through enhanced expression, particularly when using a chemically defined methanol-free carbon source, while reducing the risk of contamination from specific promoter regulation.

[0165] The results demonstrate that the promoter of this invention exhibits enhanced activity primarily independent of suitable carbon source amounts and specific culture media. As an example, *Pichia pastoris* can be successfully cultured under industrial production conditions. First, batch culture is performed on a basal carbon source, such as glycerol, followed by supplemental carbon source supplementation, such as glucose. Samples are then delivered to the end of the first batch phase under limiting growth conditions, for example, using a limiting amount of supplemental carbon source. Transcriptome analysis using DNA microarrays revealed specific genes exhibiting strong activity on supplemental carbon sources and in the presence of excess carbon sources, such as an excess of the basal carbon source.

[0166] Surprisingly, the pCS1 promoter sequence was identified as a strong promoter at both high and low growth rates. Comparable pGAP promoters in the prior art are significantly weaker.

[0167] The fermentation process can verify the characteristics of strong recombinant gene expression on a basic carbon source and strong expression on a restricted supplemental carbon source.

[0168] The nucleotide sequences that can be used as constitutive promoter sequences according to the present invention will provide increased recombinant protein yields, and can be from a variety of sources. The promoters of the present invention are preferably derived from yeast cells, most preferably from methyltrophic yeasts, such as those from the genus *Pichia* or species *Pichia*, whose promoters can be used as parental sequences to generate suitable variants, such as mutants or analogs.

[0169] We considered that a range of yeast cells, particularly Pichia pastoris strains, could be suitable for obtaining different types of individual promoter sequences or analogs.

[0170] Identified variants of Pichia pastoris promoters include functionally active variants, such as homologs or analogs that can be obtained using standard techniques. For example, promoters can be modified to produce promoter variants with altered expression levels and regulatory properties.

[0171] For example, according to the present invention, a promoter library can be prepared by mutagenesis of the promoter sequence. This promoter library can serve as a parental molecule, for example, by analyzing the expression of variants under different fermentation schemes and selecting suitable variants to fine-tune gene expression in eukaryotic cells. A variant synthesis library can be used, for example, selecting a required-match promoter to produce a selective POI. Such promoters exhibit enhanced expression efficiency in eukaryotic host cells and are highly efficient in both carbon-rich and carbon-limited environments.

[0172] Different fermentation schemes can distinguish between the growth and production phases. Growth and / or production can occur appropriately in batch, fed-batch, or continuous modes. Any suitable bioreactor can be used, including batch, fed-batch, continuous, stirred tank reactors, or airlift reactors.

[0173] Providing fermentation processes for laboratory or industrial scale is beneficial. For industrial-scale methods, a volume of at least 10 L is preferred, particularly at least 50 L, and preferably at least 1 m³. 3 Preferably at least 10m 3 The optimal length is at least 100m. 3 .

[0174] Preferred production conditions for industrial scale are, for example, 100L to 10m³. 3Alternatively, fed-batch culture can be performed in larger reactors with a typical processing time of several days, or in fermentation vessels of approximately 50–1000 L or larger, with a simultaneous dilution rate of 0.02–0.15 h. -1 Continuous process.

[0175] Suitable culture techniques include starting with a batch culture period, followed by a short, exponentially high-specific-growth-rate fed-batch period, and then a low-specific-growth-rate fed-batch period. Another suitable culture technique may include a batch culture period followed by a continuous culture period with a low dilution rate.

[0176] A preferred embodiment of the present invention includes batch culture to provide biomass followed by fed-batch culture to provide high-yield POI production.

[0177] According to the invention, it is preferable to obtain a cell concentration of at least 1 g / L cell dry weight, more preferably at least 10 g / L cell dry weight, and preferably at least 20 g / L cell dry weight, in a bioreactor under growth conditions while culturing the host cell line. Providing the yield of this biomass production on a laboratory or industrial scale is advantageous.

[0178] Growth media that enable biomass accumulation, particularly, or basic growth media, typically contain a carbon source, a nitrogen source, a sulfur source, and a phosphorus source. Typically, such a medium contains additional trace elements and vitamins, and further includes amino acids, pentoses, or yeast isolates.

[0179] Preferred nitrogen sources include NH4H2PO4, or NH3 or (NH4)2SO4.

[0180] Preferred sulfur sources include MgSO4, (NH4)2SO4, or K2SO4;

[0181] Preferred phosphorus sources include NH4H2PO4, or H3PO4, or NaH2PO, KH2PO4, Na2HPO4, or K2HPO4;

[0182] Further typical culture medium components include KCl, CaCl2, and trace elements such as Fe, Co, Cu, Ni, Zn, Mo, Mn, I, and B.

[0183] Preferably, the culture medium is supplemented with vitamin B7;

[0184] Typical growth media for Pichia pastoris include glycerol, sorbitol or glucose, NH4H2PO4, MgSO4, KCl, CaCl2, biotin, and trace elements.

[0185] During production, a production medium is specifically used in conjunction with a single, limited quantity of supplemental carbon source.

[0186] Preferably, the host cell line is cultured on a mineral culture medium supplemented with a suitable carbon source, thus further and significantly simplifying the isolation process. One preferred embodiment of the mineral culture medium contains an available carbon source (e.g., glucose, glycerol, sorbitol, methanol), macroelements (potassium, magnesium, calcium, ammonium, chlorine, sulfur, phosphorus) and microelements (copper, manganese, iodine, molybdenum, cobalt, zinc, and iron salts and boric acid), and optional vitamins or amino acids, for example, for supplementing auxotrophic media.

[0187] The cells are cultured under conditions suitable for the point of interest (POI) required to achieve expression. The protein can be purified from the cells or culture medium depending on the expression system and the properties of the expressed protein, such as whether the protein is fused to a signal peptide and whether the protein is soluble or membrane-bound. As those skilled in the art will soon understand, the culture conditions will vary depending on factors including the host cell type and, in particular, the expression vector used.

[0188] According to the present invention, by selecting a suitable promoter sequence, selectively binding with a preferred regulatory sequence, under comparative conditions, a natural or pGAP isolated from Pichia pastoris can provide the same, or at least about 1.1 times, or at least about 1.2 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, or at least up to 15 times the activity, for example, under growth-limited conditions in a fed-batch process, as expressed by promoter activity or transcriptional strength, or regulated by promoter strength.

[0189] A typical production culture medium contains a supplemental carbon source, and further NH4H2PO4, MgSO4, KCl, CaCl2, biotin, and trace elements.

[0190] For example, the feedstock added to the fermentation broth as a supplementary carbon source can contain up to 50% available sugars. A low feedstock rate will limit the inhibitory effect of products or byproducts on cell growth; therefore, high production rates based on carbon source supply are possible.

[0191] Fermentation is preferably carried out in the pH range of 3 to 7.5.

[0192] The typical fermentation time is 24 to 120 hours, and the temperature range used is 20℃ to 35℃, preferably 22 to 30℃.

[0193] In summary, the recombinant nucleic acids or organisms mentioned in this invention can be generated using recombination techniques well known to those skilled in the art. According to the invention, conventional molecular biology, microbiology, and recombinant DNA techniques are available in the prior art. These techniques are well explained in the literature. See, for example, Maniatis, Fritsch & C., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, (1982).

[0194] According to a preferred embodiment of the present invention, the recombinant construct is obtained by linking a promoter and related genes into a vector or expression construct. These genes can be stably integrated into the host cells by transforming them using such a vector or expression construct.

[0195] Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. The preferred expression vectors used in this invention can be any expression vector suitable for expressing recombinant genes in host cells, and can be selected according to the host organism. The recombinant expression vector can be any vector capable of replicating or integrating into the host organism's genome, also known as a host vector.

[0196] Suitable expression vectors typically contain further regulatory sequences suitable for expressing DNA encoding a POI in eukaryotic host cells. Examples of regulatory sequences include operons, enhancers, ribosome binding sites, and sequences controlling the initiation and termination of transcription and translation. These regulatory sequences are operatively linked to the expression sequence.

[0197] According to the present invention, to achieve the expression of recombinant nucleotide sequences in host cells, the expression vector may provide an upstream promoter adjacent to the 5' end of the coding sequence, such as GOI, or a signal peptide gene that enables POI secretion. Therefore, the transcription is regulated and initiated by this promoter sequence.

[0198] Signal peptides can be heterologous signal peptides or hybrids of natural and heterologous signal peptides, and can be heterologous or homologous to the host organism that produces the protein. The function of the signal peptide is to facilitate the secretion of the POI into the endoplasmic reticulum. The signal peptide is typically a short polypeptide chain (3–60 amino acids long) that guides protein transport to the extracellular space, thus facilitating the isolation and purification of heterologous proteins. After protein transport, certain signal peptides are cleaved by signal peptidases.

[0199] Typical signal peptides include α-pairing factor pretreatment peptides from Saccharomyces cerevisiae and signal peptides from the Pichia pastoris acid phosphatase gene (PHO1).

[0200] If a promoter controls transcription of a coding sequence, the promoter sequence can be understood as being associated with the operation of the coding sequence. If the promoter sequence is not naturally associated with the coding sequence, its transcription is not controlled by the natural (wild-type) cell promoter, or the sequence recombines with different neighboring sequences.

[0201] To demonstrate the function of the relevant sequences, expression vectors containing one or more regulatory elements can be constructed to drive POI expression, and the expression yield can be compared with constructs containing conventional regulatory elements. The following examples describe the experimental procedures in detail. The identified gene was amplified from *Pichia pastoris* using specific nucleotide primers via PCR, cloned into an expression vector, and transformed into eukaryotic cell lines, such as yeast vectors and *Pichia pastoris* strains producing various different POIs at high levels. To estimate the effect of the promoter of the present invention on the yield of recombinant POIs, eukaryotic cell lines can be cultured in batches in shake-flask experiments or fermented in a chemostat, and compared with strains containing conventional constitutive promoters, such as growth-dependent promoters, for example, the standard pGAP promoter in individual cells. In particular, promoter selection has a significant impact on the production of recombinant proteins.

[0202] POIs can be produced by culturing transformants using recombinant cell lines, thereby obtaining them in a suitable culture medium. The expressed products or metabolites can be isolated from the culture medium, and the POIs can be selectively purified using a suitable method.

[0203] According to the present invention, the transformant can be obtained by introducing such vector DNA, such as plasmid DNA, into the host and selecting transformants that express the POI or host metabolites in high quantities. The host cell can be treated with conventional methods for transforming eukaryotic cells, such as electroporation, protoplast transformation, lithium acetate transformation, and improved methods thereof, to integrate the exogenous DNA. Pichia pastoris transformation is preferred. Preferred methods for the transformation microorganism to absorb the recombinant DNA fragment include chemical transformation, electroporation, or protoplast transformation. The transformant of the present invention can be obtained by introducing such vector DNA, such as plasmid DNA, into the host and selecting transformants that express the relevant protein or host metabolite in high quantities.

[0204] According to the present invention, several different methods for producing POIs are preferred. The substance can be expressed, processed, and selectively secreted by transforming eukaryotic host cells with an expression vector containing recombinant DNA encoding a related protein and at least one regulatory element described in the present invention, allowing the culture to grow, inducing transcription and POI production, and recovering the products of the fermentation process.

[0205] According to the present invention, the expression capacity or yield of host cells is preferably determined by the following experiments: enzyme-linked immunosorbent assay (ELISA), enzyme activity detection, high performance liquid chromatography (HPLC) or other suitable tests.

[0206] The POI is preferably expressed under conditions where the production yield is at least 1 mg / L, preferably at least 10 mg / L, preferably at least 100 mg / L, and more preferably at least 1 g / L.

[0207] It is understood that the method disclosed in this invention may further include culturing the recombinant host cells under conditions that allow for POI expression, preferably expressing POI in a secretory form or other intracellular product form. The recombinantly produced POI or host cell metabolites can then be isolated from the cell culture medium and further purified using techniques known to those skilled in the art.

[0208] According to the present invention, typically, existing techniques may be used to separate and purify the generated POI by increasing the concentration of the desired POI and / or decreasing the concentration of at least one impurity.

[0209] If the POI is secreted from cells, it can be isolated and purified from cell culture using existing techniques. Because the product is recovered from the culture suspension rather than from a protein complex mixture (obtained when yeast cells are disrupted to release intracellular proteins), secretion of the recombinant expression product from the host cell is generally beneficial for facilitating the purification process.

[0210] The cultured transformant cells can also be disrupted by ultrasonic or mechanical, enzymatic or chemical methods to obtain a cell isolate containing the desired POI, from which the POI can be isolated and purified.

[0211] Methods for separating and purifying recombinant peptides or protein products include, for example, methods using differences in solubility, such as salting out and solvent precipitation; methods using differences in molecular weight, such as ultrafiltration and gel chromatography; methods using differences in charge, such as ion exchange chromatography; methods using specific affinity, such as affinity chromatography; methods using differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods using differences in isoelectric point, such as isoelectric focusing.

[0212] The highly purified product is essentially a contamination-free protein, preferably with a purity of at least 90%, more preferably at least 95%, or even at least 98%, up to 100%. The purified product can be obtained by purifying cell culture suspensions or other materials from cell debris.

[0213] The following standard methods are preferred as separation and purification methods: cell disruption (if the POI is obtained from intracellular sources), separation and washing of cells (fragments) by microfiltration or tangential flow filtration (TFF) or centrifugation, purification of POI by precipitation or heat treatment, activation of POI by enzymatic digestion, purification of POI by chromatography such as ion exchange (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography, molecular sieve (SEC), or high performance liquid chromatography (HPLC), and concentration and washing of POI precipitate by ultrafiltration.

[0214] The POI can be separated and purified by conventional methods such as immunoblotting, HPLC, enzyme activity assay or ELISA.

[0215] The POI can be any eukaryotic, prokaryotic, or synthetic polypeptide. It can be a secreted protein or an intracellular protein. This invention also provides functional homologs of naturally occurring proteins, recombinant products of functionally equivalent variants, derivatives, and biologically active fragments. Preferably, the functional homolog is identical to or corresponds to a certain sequence and possesses its functional characteristics.

[0216] In this invention, POI can refer to a product that is homologous to or heterologous to eukaryotic host cells, preferably for therapeutic, preventive, diagnostic, analytical or industrial use.

[0217] Preferably, the POI is a heterologous recombinant polypeptide or protein produced in eukaryotic cells, preferably in yeast cells, and more preferably a secreted protein. Preferred examples of the producing protein are immunoglobulins, immunoglobulin fragments, aprotinins, tissue factor pathway inhibitors or other protease inhibitors, and insulin or insulin precursors, insulin analogs, growth hormone, interleukins, tissue plasminogen activator, transforming growth factor a or b, glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), GRPP, factor VII, factor VIII, factor XIII, platelet-derived growth factor 1, serum albumin, enzymes such as lipases or proteases, or functionally equivalent variants, or bioactive fragments with similar functions to natural proteins. The POI can be structurally similar to a natural protein and derived from it by adding one or more amino acids to the C-terminus and N-terminus or side chain of the natural protein, replacing one or more amino acids at one or more sites in the natural amino acid sequence, deleting one or more amino acids at one or both ends or at one or more sites in the natural amino acid sequence, or inserting one or more amino acids at one or more sites in the natural amino acid sequence. Such modifications are well known for the aforementioned proteins.

[0218] A POI may also be selected from those that provide a biological reaction substrate, enzyme, inhibitor, or cofactor in the host cell, with the aim of obtaining the product of the biochemical reaction or a cascade of multiple reactions, such as obtaining metabolites of the host cell. According to the invention, exemplary products may be vitamins, such as riboflavin, organic acids, and alcohols, which may yield increased production after expression of the recombinant protein or POI.

[0219] In summary, the host cell expressing the recombinant product can be any eukaryotic cell suitable for expressing the POI.

[0220] Preferred mammalian cell examples include young hamster kidney cells (BHK), CHO (CHO-DG44, CHO-DUXB11, CHO-DUKX, CHO-K1, CHOK1SV, CHO-S), HeLa, human embryonic kidney (HEK293), canine kidney passaged cells (MDCK), NIH3T3, NSO, PER.C6, SP2 / 0, and VERO cells.

[0221] According to the present invention, preferred yeast cells for use as host cells include, but are not limited to, yeast genera (e.g., Pichia pastoris or Pichia methylbenzyl), Pichia pastoris (K. pastoris, K. psydopastoris or K. phaffii), Hansenula polymorpha or Kiuyveromyces lactis.

[0222] Updated literature classifies and renames Pichia pastoris to Komagataella pastoris, Komagataella phaffii, and Komagataellapseudopastoris. In this invention, Pichia pastoris is used as a synonym for all Pichia pastoris, Komagataella phaffii, and Komagataellapseudopastoris.

[0223] Preferred yeast cells are derived from methanol-nutritive yeasts, such as those from Pichia pastoris or Komagataella pastoris. Examples of hosts include yeasts such as Pichia pastoris. Examples of Pichia pastoris strains include CBS 704 (=NRRL Y-1603=DSMZ 70382), CBS2612 (=NRRL Y-7556), CBS 7435 (=NRRL Y-11430), CBS 9173-9189 (CBS strains: CBS-KNAW Center for Fungal Biodiversity (Central bureau voor Schimmel-cultures, Utrecht, The Netheds), and DSMZ 70877 (German Collection of Micro-organisms and Cell Cultures), as well as strains derived from Invitrogen, such as X-33, GS115, KM71, and SMD1168. Examples of Saccharomyces cerevisiae strains include W303, CEN.PK, and the BY-series (collected by EUROSCARF). All of the above strains have been successfully used to generate transformants and express heterologous genes.

[0224] According to the invention, the preferred yeast host cell, such as Pichia pastoris or Saccharomyces cerevisiae host cell, contains a heterologous or recombinant promoter sequence derived from a P. pastoris or Saccharomyces cerevisiae strain different from the production host. According to other particular embodiments of the invention, the host cell contains a recombinant construct comprising a promoter derived from a genus, species, or strain of the same origin as the host cell.

[0225] The promoter of the present invention is preferably derived from a gene encoding a protein homologous to the host cell.

[0226] For example, one promoter of the present invention may be derived from yeast, such as a *Saccharomyces cerevisiae* strain, and for expressing POI. According to the present invention, a particularly preferred embodiment relates to a *Pichia pastoris* promoter derived from a method for producing recombinant POI in a *Pichia pastoris* production host cell line. Homologous origin of the nucleotide sequence facilitates its integration into host cells of the same genus or species, thus achieving stable POI production while potentially increasing yield in industrial production processes. Functionally active variants of promoters derived from other suitable yeasts or other fungi, or from other organisms such as vertebrates or plants, may also be used.

[0227] If the POI is a homologous protein of the host cell, i.e. a protein naturally present in the host cell, the expression of the POI in the host cell can be regulated by exchanging its natural promoter sequence with the promoter sequence of the present invention.

[0228] For example, this goal is achieved by transforming host cells with a recombinant DNA molecule containing a homologous sequence of the target gene, allowing site-specific recombination, wherein the promoter sequence and selectable markers are adapted to the host cell. Site-specific recombination can be performed to operatively link the promoter sequence to the nucleotide sequence encoding the POI. This results in the expression of the POI from the promoter sequence of the present invention, rather than from a natural promoter sequence.

[0229] In a particularly preferred embodiment of the invention, the promoter sequence has enhanced promoter activity compared to the natural POI promoter sequence.

[0230] According to the present invention, a Pichia pastoris host cell comprising the promoter sequence of the present invention is preferably provided, wherein the promoter sequence is operatively linked to a nucleotide sequence encoding POI.

[0231] The present invention may also provide a wildcard vector or host cell according to the invention, comprising a promoter according to the invention, and capable of instantaneously integrating a gene of interest encoding a POI. Thus, this wildcard cell line is a formed host cell line characterized by its expression capacity. This follows an innovative “wildcard” platform scheme for generating cell lines for POI production, such as using site-specific recombinase-mediated cassette exchange. Such novel host cells facilitate the cloning of the gene of interest (GOI) into a pre-defined genomic expression hotspot within days to obtain a reproducible, highly efficient production cell line.

[0232] According to a preferred embodiment, the method of the present invention employs a recombinant nucleotide sequence encoding a POI, which is provided on a plasmid suitable for integration into a host cell line in single or multiple copies per cell. The recombinant nucleotide sequence encoding the POI may also be provided in single or multiple copies per cell on an autonomously replicating plasmid.

[0233] The preferred method of this invention employs a plasmid, which is a eukaryotic expression vector, preferably a yeast expression vector. The expression vector may include, but is not limited to, cloning vectors, modified cloning vectors, and specially designed plasmids. The preferred expression vector used in this invention can be any expression vector suitable for expressing recombinant genes in host cells, and can be selected according to the host organism. The recombinant expression vector can be any vector capable of replicating or integrating into the host organism's genome, also known as a host vector, such as a yeast vector carrying the DNA construct according to this invention. A preferred yeast expression vector is used for expression in yeasts selected from the group consisting of *Hansenula*, *Pichia*, *Candida*, and *Gnaphalium*.

[0234] In this invention, plasmids derived from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis, or pPUZZLE are preferably used as vectors.

[0235] According to a preferred embodiment of the invention, a recombinant construct is obtained by linking relevant genes into a vector. These genes can be stably integrated into host cells by transforming them using this vector. The recombinant host cell line can be used to culture transformants on a suitable culture medium, from which expressed POIs can be isolated, and then purified using methods suitable for product expression, particularly isolating POIs from contaminating proteins, to produce polypeptides encoded by these genes.

[0236] Expression vectors may contain one or more optional phenotypic markers, such as a gene encoding a protein that provides antibiotic resistance or provides autotrophic requirements. Yeast vectors typically contain an origin of replication from the yeast chromosome, an autonomous replication sequence (ARS), or optionally, a sequence for integration into the host genome, a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and optional markers.

[0237] Procedures for linking DNA sequences, such as those encoding precursor sequences and / or POIs, promoters and terminators, and then inserting them into a suitable vector containing information necessary for integration or host replication, are well known to those skilled in the art, for example, as described by Sambrook et al. (A Laboratory Manual, Cold Spring Harbor, 1989).

[0238] It is understood that the vector using the regulatory element and / or POI according to the invention as an integration target can be constructed either by preparing a DNA construct containing the entire DNA sequence encoding the regulatory element and / or POI, and then inserting the fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information suitable for individual elements, and then ligating them.

[0239] According to the present invention, a polyclonal vector having a multiple cloning site can be used, wherein a desired heterologous gene can be bound to the multiple cloning site to provide an expression vector. In the expression vector, the promoter is located upstream of the POI gene and regulates the expression of that gene. In the case of the polyclonal vector, since the POI gene is introduced at the multiple cloning site, the promoter is located upstream of the multiple cloning site.

[0240] The DNA constructs for obtaining recombinant host cells according to this invention can be prepared by established standard methods, such as the phosphoramidic acid method. The DNA constructs can also be of genomic or cDNA origin, for example, by preparing a genomic or cDNA library and using synthetic oligonucleotide probes conforming to standard techniques, and by screening for DNA sequences encoding all or part of the polypeptides of this invention through hybridization (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989). Finally, according to standard techniques, the DNA constructs can be a mixture of synthetic and genomic origins, a mixture of synthetic and cDNA origins, or a mixture of genomic and cDNA origins, prepared by annealing synthetic, genomic, or cDNA-derived fragments, which, if applicable, correspond to various parts of the whole genomic construct.

[0241] In another preferred embodiment, for example, the yeast expression vector can be stably integrated into the host genome via homologous recombination.

[0242] The host cells of the present invention, obtained by transforming host cells using regulatory elements and / or the POI gene, are preferably first cultured under conditions that allow for efficient growth to a large number of cells. When the cell line is prepared for POI expression, a culture technique is selected to produce the expression product.

[0243] A particular embodiment relates to a Pichia pastoris cell line for the recombinant production of reporter proteins via fed-batch fermentation, using glycerol-fed and glucose-fed-batch media. Comparative promoter activity studies have demonstrated that the promoters of this invention can be successfully used for recombinant protein production.

[0244] According to a further embodiment, human serum albumin (HSA) is produced as a point of interest (POI) under glucose restriction conditions, and the HSA yield and gene copy number are determined.

[0245] According to another embodiment, a Pichia pastoris strain expressing HSA was cultured in batches under promoter control conditions of the present invention.

[0246] Further embodiments involve the expression of porcine carboxypeptidase B as a model protein under the transcriptional control of the pCS1 promoter.

[0247] Furthermore, a further embodiment involves expressing an antibody fragment under the transcriptional control of pCS1.

[0248] A further embodiment relates to a size or length variant of the promoter according to the invention, such as an extended pCS1 sequence pCS1 a (SEQ ID 2), which comprises a pCS1 sequence and an extension at the 5′ end, or a pCS1 fragment in the range of 80bp to 800bp in length.

[0249] The foregoing description can be fully understood by referring to the following embodiments. However, these embodiments are merely representative of one or more implementations of the present invention and are not intended to limit the scope of the invention.

[0250] Example

[0251] The following examples illustrate the materials and methods used to identify new promoters and analyze their expression characteristics in Pichia pastoris.

[0252] Example 1: Identification of strong expression promoters in Pichia pastoris

[0253] To identify strong genes and specific promoters in Pichia pastoris, its gene expression profile was analyzed using DNA microarrays. The growth of Pichia pastoris cells under glycerol batching and glucose restriction (chemostat) conditions was also analyzed.

[0254] a) strain

[0255] Wild-type Pichia pastoris strains capable of growing on basal media without supplementation (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) were used.

[0256] b) Pichia pastoris culture

[0257] Fermentation was carried out using a small laboratory fermenter (Infors-HT, Switzerland), with a final effective volume of 2.5L.

[0258] Use the following culture media:

[0259] Each liter of PTM1 trace salt stock solution contains:

[0260] 6.0g CuSO4·5H2O, 0.08g NaI, 3.36g MnSO4·H2O, 0.2g Na2MoO4·2H2O, 0.02g H3BO3, 0.82g CoCl2, 20.0g ZnCl2, 65.0g FeSO4·7H2O, 0.2g biotin, and 5.0ml H2SO4 (95%-98%)

[0261] Each liter of glycerol fractionated culture medium contains: 2 g citric acid monohydrate (C6H8O7·H2O), 39.2 g glycerol, 20.8 g NH4H2PO4, 0.5 g MgSO4·7H2O, 1.6 g KCl, 0.022 g CaCl2·2H2O, 0.8 mg biotin, and 4.6 ml PTM1 trace element salt stock solution. HCl is added to adjust the pH to 5.

[0262] Each liter of glycerol fractionated culture medium contains:

[0263] 632g glycerol, 8g MgSO4·7H2O, 22g KCl, and 0.058g CaCl2·2H2O.

[0264] Each liter of chemiluminescent medium contains:

[0265] 2g citric acid monohydrate (C6H8O7·H2O), 99.42g glucose monohydrate, 22g NH4H2PO4, 1.3g MgSO4·7H2O, 3.4g KCl, 0.02g CaCl2·2H2O, 0.4mg biotin, and 3.2ml PTM1 trace element salt stock solution. Add HCl to adjust the pH to 5.

[0266] Dissolved oxygen was controlled at DO = 20% using a stirring speed of 500–1250 rpm. The aeration rate was 60 L / h of air, the temperature was controlled at 25 °C, and the pH was controlled at 5 by adding NH4OH (25%).

[0267] 1.5 L of sterile batch culture medium was filtered and transferred to the fermenter. Fermentation was initiated by inoculating 1 L of Pichia pastoris with an optical density (OD600) of 1 after an overnight pre-culture at YPG, 180 rpm, and 28°C. After approximately 25 h of batch culture, a dry matter concentration (DMC) of approximately 20 g / L was reached. This was followed by a 10 h fed-batch culture on glucose medium, producing a DMC of approximately 50 g / L. The culture volume was then reduced to 1.5 L, and chemostat culture was initiated at a feed / harvest ratio of 0.15 L / h, achieving a constant growth rate (μ = 0.1). Fermentation was terminated 50 h after the start of chemostat culture.

[0268] This fermentation was carried out three times to obtain the biological samples necessary for reproducible microarray analysis.

[0269] The carbon source limitation conditions (not measurable residual glucose) during the chemostat period can be verified by HPLC analysis of the culture suspension.

[0270] c) Sampling

[0271] Sampling was performed at the end of the glycerol batching period and under stable conditions using a glucose chemostat. Routine sampling was conducted at the fermenter side for each fermentation to determine OD or yeast dry matter, qualitative microscopy, and cell viability analysis. Sampling and processing for microarray analysis were performed as follows: For photoquenching, 9 ml of cell culture medium was directly mixed with 4.5 ml of ice-cold 5% phenol (Sigma) solution (dissolved in ethanol, absolute value) and aliquoted. Each aliquot was centrifuged (13200 rpm for 1 min) in a pre-chilled collection tube (GE Healthcare, NJ) to completely remove the suspension. The collection tubes were stored at -80°C until RNA purification was performed.

[0272] d) Sample preparation for RNA purification and microarray hybridization

[0273] RNA was isolated using TR1 reagent according to the supplier's (Ambion, US) instructions. Cell pellets were resuspended in TR1 reagent and incubated with FastPrep 24 glass beads (MP. Biomedicals, CA) for 5 minutes. -1 Homogenize the cell pellets for 40 seconds. After adding chloroform, centrifuge the sample and precipitate total RNA from the aqueous phase by adding isopropanol. Wash the cell pellets with 70% ethanol, dry, and resuspend in RNase-free water. OD was measured using a Nanodrop 1000 spectrophotometer (NanoDrop product, DE). 260RNA concentration was determined. Residual DNA in the sample was removed using a DNA-free kit (Ambion, CA). The sample volume equivalent to 10 g of RNA was diluted to 50 μL in RNase-free water, and then DNase buffer I and rDNase I were added. The sample was incubated at 37°C for 30 minutes. After adding DNase inactivator, the sample was centrifuged, and the suspension was transferred to a new tube. RNA concentration was determined again as described above. RNA integrity was also analyzed using an RNA nanoarray (Agilent). To monitor the amplification and hybridization labeling process from the sample, a Spike In Kit (Agilent, Product Nr.: 5188-5279) was used as a positive control. This control contains 10 different polyadenylated transcripts from adenovirus, which can be amplified, labeled, and co-hybridized with their own RNA sample. The sample was labeled with Cy3 and Cy5 using the Quick Amp Labelling Kit (Agilent, Prod. Nr.: 5190-0444). Therefore, dilute 500 ng of purified sample RNA in 8.3 μL of RNase-free water, add 2 μL of Spike A or B, and 1.2 μL of LT7 promoter primer. Denature the mixture at 65 °C for 10 min and hold on ice for 5 min. Then, add 8.5 μL of cDNA series (per sample: 4 μL 5x first-strand buffer, 2 μL 0.1 M dithiothreitol (DTT), 1 μL 10 mM dNTP mixture, 1 μL MMLV-RT, 0.5 μL RNase out), incubate at 40 °C for 2 h, then transfer to 65 °C for 15 min and hold on ice for 5 min. Prepare a transcription series (each sample contains: 15.3 μL nuclease-free water, 20 μL transcription buffer, 6 μL 0.1 M DTT, 6.4 μL 50% polyethylene glycol (PEG), 0.5 μL RNase inhibitor, 0.6 μL inorganic phosphophosphatase, 0.8 μL T7 RNA polymerase, and 2.4 μL anthocyanin 3 or anthocyanin 5) and add it to each tube, incubating at 40 °C for 2 hours. Purify the obtained labeled cDNA using the RNeasy Mini Kit (Qiagen, Cat. No. 74104). Store samples at -80 °C.

[0274] The concentration and labeling efficiency of cRNA were quantified using a Nanodrop spectrophotometer.

[0275] e) Microarray chip analysis

[0276] Gene expression hybridization kits (Agilent, Cat. No. 5188-5242) were used for hybridization of labeled sample cRNA. To prepare hybridization samples, 300 ng of cRNA (Cy3 and Cy5) per sample and 6 μL of 10-fold blocking agent were diluted to a final volume of 24 μL with nuclease-free water. After adding 1 μL of 25-fold fragmentation buffer, the mixture was incubated at 60°C for 30 min. The reaction was then terminated by adding 25 μL of GEx hybridization buffer HI-RPM. After centrifugation at 13,200 rpm, the samples were cooled on ice and used immediately for hybridization. In-house designed oligonucleotide arrays specific to Pichia pastoris (AMAD-ID: 026594, 8x15K custom arrays, Agilent) were used. Gene array hybridization was performed according to the Microarray Hybridisation Chamber User Guide (Agilent G2534A). First, open the backing slide and place the sample tube on the sample chamber base with the Agilent label facing up. Load the sample (40 μL per chip) into the center of each of the eight squares. Then, carefully place the gene chip slide on the backing slide (Agilent label facing down), close the sample chamber lid, and secure it to the tube clamp. Hybridize in a hybridization oven at 65°C for 17 hours. Before scanning, wash the gene chip. Therefore, when removing the sample chamber and immersing it in Wash Buffer 1, the three layers of slides will separate from each other. Transfer the gene chip directly to another dish containing Wash Buffer 1, wash for 1 minute, transfer to Buffer 2 (temperature at least 30°C), and wash for another 1 minute. After drying the gene chip slide by contacting the edges of the slide with the tissue, place it in the slide holder (Agilent label facing up), place the slide holder on the conveyor belt, and begin scanning.

[0277] f) Data acquisition and statistical evaluation of gene chip data

[0278] Images obtained using an Agilent G2565AA gene chip scanner at 50nm resolution were input into Agilent Feature Extraction 9.5 software. Agilent Feature Extraction 9.5 was used for point intensity quantification. The raw average point intensity data were then input into the open-source software R for further standardization and data analysis.

[0279] The limma package, VSN, and m-array were used for data preprocessing and standardization. The intensity data were not background corrected and were standardized using VSN.

[0280] To access high signal intensity data, gene chip data under both states were reviewed to identify strongly expressed constitutive genes. Table 1 shows the strongest transcribed genes with the strongest signal intensity under both states. Data for pGAP and pTEF were added as references. On average, under both conditions (glycerol batch culture and glucose-restricted chemostat culture), pCS1 exceeded pGAP by approximately 30%, while pTEF was approximately 12% weaker than pGAP.

[0281]

[0282] Table 1: Gene chip data, with promoters selected for further characterization, and pGAP and pTEF data used as controls.

[0283] 1 During the glycerol batch culture period (average of two channels)

[0284] 2 In a glucose-limited chemistostat (average of two channels)

[0285] Example 2: Comparative promoter activity study of the newly identified Pichia pastoris promoter pCS1 using green fluorescent protein (eGFP) as an intracellular reporter gene.

[0286] To analyze the characteristics of the newly identified promoters, the following shake-flask screening was performed: pre-culture in a rich medium containing glycerol as the carbon source for 24 hours—simulating the batch culture period of the entire process—followed by master culture in basal medium and glucose feed beads—simulating the glucose-limited feeder period of the entire process. Green fluorescent protein (eGFP) was used as the intracellular reporter gene for promoter activity.

[0287] a) Strains & Expression Vectors

[0288] A wild-type Pichia pastoris strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Centralbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with an internally designed vector named pPUZZLE (Stadlmayr et al. J. Biotechnol 2010; 150(4): 519-29), which contained an Escherichia coli (pUC19) replication origin, an antibiotic resistance cassette for selection between E. coli and yeast (the Sh ble gene providing bleomycin resistance), an expression cassette for the gene of interest (GOI), an expression cassette consisting of a multiple cloning site and a Saccharomyces cerevisiae CYC1 transcription terminator, and a locus (3AOX1 region) integrated into the Pichia pastoris genome.

[0289] b) The newly identified promoter pCS1 was amplified and cloned into the pPUZZLE expression vector containing eGFP as GOI.

[0290] The pCS1 promoter (SEQ ID) contains a 985 bp 5' untranslated region of the CS1 gene (see Example 1) ascending to the start codon ATG and amplified from Pichia pastoris genomic DNA by PCR (hybrid polymerase, New England Biolabs) using the primers shown in Table 2. This sequence was cloned into the pPUZZLE expression vector pPM1aZ10_eGFP, digested with Apal and Sbfl enzymes to produce pPM1aZ10_pCS1_eGFP. Additionally, the vector pPM1aZ10_pGAP_eGFP, containing the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (Pichia pastoris pGAP, referred to in this invention as SEQ ID 13), was used as a reference. This promoter was inserted upstream of the start codon of the eGFP gene using Apal and Sbfl restriction endonuclease sites (see Tables 2 and 3). The correctness of the promoter sequence was verified by Sanger sequencing.

[0291]

[0292] Cloning enzymes promoter 5' primer 3' primer 5’ 3‘ length pCS1 pCS1 - Forward pCS1-Reverse Apal Sbfl 985

[0293] Table 3: Lengths of amplification primers, cloning enzymes, and cloning promoters

[0294] c) Expressing eGFP in Pichia pastoris for analyzing promoter activity.

[0295] The entire plasmid was linearized using Ascl within the 3′AOX genome integration region and then injected into competent Pichia pastoris cells via electroporation (2kV, 4ms, GenePulser, BioRad).

[0296] Positive transformants were selected on yeast extract peptone-glucose (YPD) plates containing 25 μg / mL bleomycin (Invivogen, CA) (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar). Colony PCR was performed to ensure the presence of the transformation plasmid. Therefore, genomic DNA was obtained by cooling and freezing Pichia pastoris clones for 5 minutes and used directly for PCR with appropriate primers. For expression screening, single colonies were pre-cultured in liquid YPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 g glycerol, and 25 mg bleomycin). Approximately 24 hours later, the preculture was used to inoculate the master culture with an OD600 of 0.1 into 2 mL of synthetic selection medium (per liter containing: 22 g glucose monohydrate, 22 g citric acid, 3.15 g (NH4)2HPO4, 0.027 g CaCl2·2H2O, 0.9 g KCl, 0.5 g MgSO4·7H2O, 2 mL 500x biotin, and 1.47 mL of trace salt stock solution [per liter containing: 6 g CuSO4·5H2O, 0.08 g NaI, 3 g MnSO4·H2O, 0.2 g Na2MoO4·2H2O, 0.02 g H3BO3, 0.5 g CoCl2, 20 g ZnCl2, 5 g FeSO4·7H2O, and 5 mL H2SO4]; pH adjusted to 5 with 5 MkOH; filtered and sterilized) and two of the four glucose feedbeads. (A second feed bead was added after 24 hours; Kuhner, CH). Due to the slow glucose release kinetics of these feed beads as described in the following equation, glucose-limited growth conditions can be achieved: (glucose) = 1.63 * t0.74 [mg / culture dish]. Samples were taken at the end of each pre-culture, and at 24 and 48 hours after inoculation of the master culture. Cell density was determined by measuring OD600, and eGFP expression was analyzed by flow cytometry as described by Stadlmayr et al. (J. Biotechnology 2010 Dec; 150(4): 519-29). 10,000 cells were analyzed for each sample. The autofluorescence of Pichia pastoris was measured using unconverted wild-type cells, and the signal was subtracted. The relevant eGFP expression level (fluorescence intensity relative to cell size) is shown as the percentage of eGFP expression level in clones expressing eGFP under constitutive pGAP control.

[0297] The results are shown in Table 4. At the end of the pre-culture (batch culture), the expression of clonal expression under the control of the pCS1 promoter exceeded that of pGAP by 38%, and at the end of the master culture (feed-batch culture), the GFP expression level was 4-fold higher.

[0298]

[0299]

[0300] Table 4: Mean GFP fluorescence per cell size of Pichia pastoris clones expressing eGFP under the control of the novel pCS1 promoter. Data show correlation with pGAP at the same time points.

[0301] d) Example 2C: Determination of the copy number (GCN) of the eGFP gene in the Pichia pastoris clone.

[0302] GCN represents the number of reporter expression cassettes integrated into the Pichia pastoris genome. The GCN of clones expressing eGFP under pCS1 or pGAP control is determined as described in Further Example 5 below. eGFP expression level analysis is as in Example 2c. As an example, Table 5 shows the results for one clone for each promoter. In selection cultures, clones expressing eGFP under the new pCS1 promoter control produced twice the amount of eGFP compared to clones expressing eGFP under the control of the pGAP promoter with the same GCN.

[0303]

[0304] Table 5: eGFP expression in screening cultures controlled by pGAP or pCS1 associated with GCNs. The amount of eGFP expressed by the pCS1 clone was twice that of the pGAP clone for each GCN.

[0305] e) Analysis of pCS1 promoter strength in fed-batch fermentation

[0306] To evaluate pCS1 promoter activity under similar production conditions, one pCS1 clone (pCS1_eGFP#4) and one pGAP clone (pGAP_eGFP#2) were cultured in fed-batch batches, each containing one copy of the eGFP expression cassette (see Example 2d).

[0307] Fed-batch fermentation with a final volume of 1.0 L was carried out in a DASGIP reactor.

[0308] The following culture media were used:

[0309] PTM1 trace element salt mother liquor, containing per liter:

[0310] 6.0g CuSO4·5H2O, 0.08g NaI, 3.36g MnSO4·H2O, 0.2g Na2MoO4·2H2O, 0.02g H3BO3, 0.82g CoCl2, 20.0g ZnCl2, 65.0g FeSO4·7H2O, 0.2g biotin, and 5.0ml H2SO4 (95%-98%).

[0311] Glycerol fractionated culture medium contains: per liter

[0312] 2g citric acid monohydrate (C6H8O7·H2O), 39.2g glycerol, 12.6g NH4H2PO4, 0.5g MgSO4·7H2O, 0.9g KCl, 0.022g CaCl2·2H2O, 0.4mg biotin, and 4.6ml PTM1 trace element salt stock solution. Adjust the pH to 5 by adding HCl.

[0313] Glucose fed-batch culture medium, containing:

[0314] 464g glucose monohydrate, 5.2g MgSO4·7H2O, 8.4g KCl, 0.28g CaCl2·2H2O, 0.34mg biotin and 10.1mL PTM1 trace element salt mother liquor.

[0315] Dissolved oxygen was controlled at DO = 20% using a stirring speed of 400-1200 rpm. The air flow rate was 24 L / h, the temperature was controlled at 25℃, and the pH was adjusted to 5 by adding NH4OH (25%).

[0316] To initiate fermentation, 400 ml of batch culture medium was sterilized, filtered into the fermenter, and inoculated (from the pre-culture) with the Pichia pastoris clone pCS1_eGFP#1, starting with an OD of 1 (OD600). A batch culture period of approximately 25 hours was established (reaching a dry matter concentration of approximately 20 g / L), followed by glucose-restricted fed-batch fermentation (starting with exponential feeding for 7 hours, followed by a constant feeding rate of 15 g / L for 13 hours, resulting in a final dry matter concentration of approximately 110 g / L). Samples were taken during both the batch and fed-batch cultures, and eGFP expression was analyzed using a plate reader (Infinite 200, Tecan, CH). Therefore, samples were diluted to OD600 = 5. Fermentation was performed in duplicate. Results are shown in Table 6 as relative fluorescence values ​​(FL / r) for each bioreactor. Clonal expression under the control of the pCS1 promoter showed an average eGFP expression 4.2-fold compared to pGAP throughout the fermentation process.

[0317]

[0318] Table 6: Relative fluorescence of two different Pichia pastoris clones expressing eGFP in each bioreactor in optimized fed-batch culture, t represents the feeding time.

[0319] f) Promoter activity of pCS1 under different growth conditions and substrates.

[0320] To obtain more information about the promoter activity of pCS1 under different culture and growth conditions, strain pCS1_eGFP#4 was cultured in YP medium with different carbon sources and different pH values, and in synthetic basal medium. Samples were taken 24 hours and 48 hours after inoculation of the master culture and analyzed by flow cytometry as described in Example 2c.

[0321] As per reference, single colonies of pCS1-eGFP#4 or pGAP-eGFP#2 were inoculated into liquid YPG-Zeo medium (containing 20 g peptone, 10 g yeast extract, 12.6 g glycerol, and 25 mg bleomycin per liter) as a pre-culture medium. After approximately 24 hours of pre-culture, the pre-culture was used to inoculate 2 mL of the main culture (OD600 0.1) into the main medium. The main medium composition was as follows: YP medium containing 20 g peptone, 10 g yeast extract, pH 7.0-7.5 per liter; YPD: YP + 2% glucose. YPG: YP + 2% glycerol, YPM: YP + 1% methanol, YPE: YP + 1% ethanol, YP feed beads: YP + 1 glucose feed bead (Kuhner, CH, 6mm diameter); YPD pH 4.5: YPD pH adjusted to 4.5 with HCl; SCD: Synthetic screening medium (per liter contains: 22g glucose monohydrate, 22g citric acid, 3.15g (NH4)2HPO4, 0.027g CaCl2·2H2O, 0.9g KCl, 0.5g MgSO4·7H2O, 2mL 500X biotin and 1.47mL trace element salt stock solution [per liter contains: 6g CuSO4·5H2O, 0.08g NaI, 3g MnSO4·H2O, 0.2g Na2MoO4·2H2O, 0.02g H3BO3, 0.5g CoCl2, 20g... ZnCl2, 5g FeSO4·7H2O and 5mL H2SO4; pH adjusted to 5 with 5M KOH; filtered and sterilized). Except for cultures using fed beads, all other cultures were cultured with their respective carbon sources at 5% after 19 hours and 43 hours. The results of eGFP fluorescence for each cell size are shown in Table 7.

[0322] clone Main culture medium 24 hours 48 hours pGAP-eGFP#2 YPD 291.0 392.9 pGAP-eGFP#2 YPG 194.8 393.0 pGAP-eGFP#4 YPD 598.7±6.4 890.2±52.2 pGAP-eGFP#4 YPG 770.8±17.5 1521.4±77.2 pGAP-eGFP#4 YP+ Glucose Feeding Beads 724.3±4.6 1559.3±28.3 pGAP-eGFP#4 YPM 844.0±25.4 1526.4±65.3 pGAP-eGFP#4 YPE 873.1±16.0 1658.0±344.4 pGAP-eGFP#4 YPD, pH 4.5 742.1±62.8 1938.8±62.9 pGAP-eGFP#4 SCD, pH 5.0 382.4±11.5 1072.2±167.1

[0323] Table 7: eGFP fluorescence values ​​per cell size for Pichia pastoris clones pGAP_eGFP#2 or pCS1-eGFP#4 after culturing for 24 and 48 hours on different selection media. The average values ​​and SD for both cultures are given here.

[0324] Example 3: Comparative promoter activity study of the newly identified Pichia pastoris promoter pCS1 using human serum albumin (HSA) as an extracellular reporter gene.

[0325] To analyze the characteristics of newly identified promoters expressing secreted reporter proteins, the following shake-flask screening was performed: pre-culture for 24 hours in a rich medium containing glycerol as the carbon source—simulating the batch phase of the entire process—followed by master culture in a buffered rich medium (2% glucose). Master culture was then fed with 0.5% glucose every 12 hours.

[0326] a) Strains & Expression Vectors

[0327] The wild-type strain of *Pichia pastoris* (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Centralbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. Transformation was performed using an internally designed vector named pPUZZLE (Stadlmayr et ai. J. Biotechnol 2010 Dec; 150(4): 519-29), and positive transformants were selected based on bleomycin resistance. For secretory expression of human serum albumin (HSA), its natural secretory precursor was used.

[0328] b) The newly identified promoter pCS1 was amplified and cloned into the expression vector.

[0329] The promoter amplified in Example 2b was cloned into the pPUZZLE expression vector pPM1 aZ10_HSA, generating pPM1aZ10_pCS1_HSA. Additionally, the vector pPM1aZ10_pGAP_HSA, containing the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP), was used as a reference. This promoter was inserted upstream of the start codon of the HSA gene using the Apal and SbfI restriction sites (see Table 3). The correctness of the promoter sequence was verified by Sanger sequencing.

[0330] c) Expression of HSA in Pichia pastoris under the control of the newly identified promoter pCS1

[0331] The entire plasmid was linearized using Ascl restriction endonuclease and then introduced into *P. pastoris* via electroporation. Positive transformants were selected on YPD plates (containing: 10 g yeast extract, 20 g peptone, 20 g glucose, and 20 g agarose per liter for selecting positive transformants), with the plates containing 25 μg / mL bleomycin. Colony PCR was used to ensure the presence of the transformant plasmid described in Example 2c.

[0332] clone HSA (mg / L) 48-hour master culture pGAP-HSA#3(1GCN) 32.8 pCS1-HSA 76.5+ / -5.0

[0333] Table 8: Quantification of HSA levels secreted by Pichia pastoris clones expressing HSA under pGAP and pCS1 control in suspension after 48 hours of screening culture.

[0334] Example 4: Feed-batch culture of Pichia pastoris expressing HSA under the control of the pCS1 promoter

[0335] To analyze the ability of pCS1 to drive HSA expression under production process conditions, a pCS1 clone (pCS1_HSA#1) carrying a 1-copy HSA expression cassette (see Example 3) was cultured in batches.

[0336] Fermentation was carried out in a DASGIP bioreactor with an effective volume of 1.0 L. Two Pichia pastoris strains expressing HSA were cultured under the control of different pGAP (pGAP_HSA#3 with one copy of the HSA gene, as described by Prielhofer et al., 2013. Microb. Cell. Fact. 12: 5, and pGAP_HSA#4 with two copies of the HSA gene) according to the reference.

[0337] The following culture medium was used:

[0338] Each liter of PTM1 trace element salt mother liquor contains:

[0339] 6.0g CuSO4·5H2O, 0.08g NaI, 3.36g MnSO4·H2O, 0.2g Na2MoO4·2H2O, 0.02g H3BO3, 0.82g CoCl2, 20.0g ZnCl2, 65.0g FeSO4·7H2O, 0.2g biotin, and 5.0ml H2SO4 (95%-98%).

[0340] Each liter of glycerol batch fermentation medium contains:

[0341] 39.2g glycerol, 27.9g H3PO4 (85%), 7.8g MgSO4·7H2O, 2.6g KOH, 9.5g K2SO4, 0.6g CaSO4·2H2O, 0.4mg biotin, and 4.6mL PTM1 trace element salt mother liquor. After sterilization and filtration, the mixture was introduced into the fermenter and the pH was adjusted to 5.85.

[0342] Each liter of glucose fed-batch medium contains:

[0343] 550g glucose monohydrate, 6.5g MgSO4·7H2O, 10g KCl, 0.35g CaCl2·2H2O, 0.4mg biotin and 12mL PTM1 trace element salt mother liquor.

[0344] Dissolved oxygen was controlled at DO = 20% using a stirring speed of 400–1200 rpm. The aeration rate was 24 L / h of air, the temperature was controlled at 25 °C, and the pH was adjusted to 5.85 by adding NH4OH (25%).

[0345] To initiate fermentation, 400 ml of batch culture medium was sterilized, filtered into the fermenter, and Pichia pastoris clones were inoculated (from the pre-culture) with an initial OD of OD600 = 1. A batch culture period of approximately 25 hours was established (reaching a dry matter concentration of approximately 20 g / L), followed by a fed-batch glucose culture for 100 hours (constant feed rate of 2 g / L, producing a final dry matter concentration of approximately 100 g / L). The pH was maintained at 5.85 during the batch culture and throughout the fermentation process. Samples were taken during both the batch and fed-batch cultures. HSA concentration was quantified using a human serum albumin ELISA kit (Bethyl, Cat. No. E80-129) as described in Example 3c.

[0346] As previously shown, pGAP clones with two copies of HSA secreted twice the amount secreted by pGAP clones with one copy (Prielhofer et al., 2013. Microb. Cell. Fact. 12: 5). Compared with single-copy GAP clones, the two clones secreting HSA under pCS1 control secreted more than 4 times the HSA titer at the end of batch and fed-batch cultures (results shown in Table 9). In terms of biomass and GCN, the amount of HAS secreted by the pCS1 clone was equivalent to 390% of that secreted by the pGAP clone with the same gene copy number.

[0347]

[0348] Table 9: Yeast dry matter concentration and HSA titer in suspension at the end of batch culture and fed-batch culture, and HSA titer / per dry matter of Pichia pastoris clones expressing HSA under pCS1 or pGAP control at the end of fed-batch culture in bioreactor.

[0349] Example 5: Determination of the gene copy number (GCN) of the selected clone

[0350] Expression intensity is generally correlated with the number of expression cassettes integrated into the Pichia pastoris genome. Therefore, the gene copy number of the selected clones was determined. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Quiagen, Cat. No. 69504). Gene copy number was determined by quantitative PCR. Therefore, the SensiMix SYBR Kit (Bioline, QT605-05) was used. The fluorescent dye Sensi Mix SYBR was mixed with primers (Prielhofer et al., 2013. Microb. Cell. Fact. 12: 5) and samples for real-time PCR analysis (Rotor Gene, Qiagen). All samples were analyzed in triplicate or quadruplicate. Data analysis was performed using Rotor Gene software.

[0351] Example 6: Comparative study of promoter activity of the newly identified Pichia pastoris promoter pCS1 using porcine carboxypeptidase gene B (CpB) as an extracellular reporter gene.

[0352] To analyze the characteristics of the newly identified promoters, the following shake-flask screening was performed: pre-cultured in a rich medium containing glycerol as the carbon source for 24 hours, followed by master culture in a rich medium.

[0353] a) Strains & Expression Vectors

[0354] Wild-type Pichia pastoris strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Centralbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with an internally designed vector named pPUZZLE (Stadlmayr et al. J. Biotechnol 2010; Dec; 150(4): 519-29), and positive transformants were selected based on bleomycin resistance. Secretory expression of porcine carboxypeptidase B (CpB) was performed using the yeast α-mating factor leader peptide.

[0355] b) Amplification and cloning of the newly identified promoter pCS1 in an internally designed expression vector.

[0356] The promoter amplified in Example 2b was cloned into the pPUZZLE expression vector pPM1aZ30_aMF_CpB, generating pPM1aZ30_pCS1_aMF_CpB. Additionally, the vector pPM1dZ30_pGAP_CpB, containing the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP), was used as a reference. This promoter was inserted upstream of the start codon of the CpB gene using the Apal and Sbfl restriction sites. The correctness of the promoter sequence was verified by Sanger sequencing.

[0357] c) Pichia pastoris expression of CpB under the control of the newly identified glucose-restricted inducible promoter.

[0358] The plasmid was linearized using the Ascl restriction endonuclease and then introduced into Pichia pastoris using electroporation (using a standard transformation method suitable for Pichia pastoris). Positive transformants were selected on YPD plates containing the following components per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar, and 25 μg / mL bleomycin. The presence of the transformant plasmid as described in Example 2c was confirmed using colony PCR.

[0359] Single colonies were inoculated into liquid YPG-Zeo (containing 20 g peptone, 10 g yeast extract, 12.6 g glycerol, and 25 mg bleomycin per liter) as a pre-culture for CpB expression screening. Approximately 24 hours later, the pre-culture was used to inoculate the master culture (OD600 = 1) into YPD medium (containing 20 g peptone, 10 g yeast extract, and 20 g glucose per liter). 0.5% glucose was added to the master culture every 12 hours. Samples were taken at the end of the pre-culture and at 24 and 48 hours after master culture inoculation. Biomass concentration was determined by measuring OD600 and cell wet weight. The CpB concentration in the culture suspension was quantified by enzymatic analysis based on the conversion of hippuryl-L-arginine to hippuric acid in CpB. Reaction kinetics were measured at 25°C and 254 nm using a Hitachi U-2910 spectrophotometer at the start of the reaction. Samples and standards were buffered with analytical buffer (25 mM Tris, 100 mM HCl, pH 7.65) and activated with activation buffer (0.01 mg / L trypsin, 300 mM Tris, 1 μM ZnCl2, pH 7.65). Trypsin-free activation buffer was used as a negative control instead of the sample. The reaction was initiated by adding substrate solution (1 mM hippuryl-L-arginine dissolved in analytical buffer).

[0360] d) Fed-batch culture of Pichia pastoris strain expressing CpB under the control of the pCS1 promoter. The fed-batch fermentation described in Example 4 was carried out using the culture medium as described in Example 2d.

[0361] Example 7: Comparative promoter activity study of the pCS1 promoter in newly identified Pichia pastoris multicopy clones using human serum albumin (HSA) as an extracellular reporter gene.

[0362] To investigate whether higher GCN levels could further increase HSA yield, P-type HSA-expressing vectors were amplified using the post-transformation vector amplification method described by Marx et al. (2009). CS1 Cloning. A vector is generated by homologous recombination and integration into the rDNA locus. Amplification is selectively achieved by progressively increasing antibody concentrations.

[0363] a) Strains & Expression Vectors

[0364] A wild-type Pichia pastoris strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Centralbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. A variant of the vector pPUZZLE was transformed using the NTS region containing the ribosomal DNA locus as the integration site (Marx et al., 2009. FEMS Yeast Res. 9(8): 1260-70.). Positive transformants were selected based on bleomycin resistance. Human serum albumin (HSA) was expressed using its native secretory leader peptide.

[0365] The pCS1 promoter amplified in Example 2b was cloned into the pPUZZLE expression vector pPM1 nZ30_HSA to generate pPM1nZ30_pCS1_HSA.

[0366] The promoter was inserted upstream of the start codon of the HSA gene using the Apal and Sbfl restriction sites. The correctness of the promoter sequence was verified by Sanger sequencing.

[0367] c) Transformation vector amplification and HSA expression in Pichia pastoris under the control of the newly identified promoter pCS1.

[0368] The plasmid was linearized with Spel restriction endonuclease and then introduced into Pichia pastoris using electroporation (using a standard transformation method suitable for Pichia pastoris). Positive transformants were selected on YPD plates containing the following components per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar, and 25 μg / mL bleomycin. Colony PCR was performed to ensure the presence of the transformant plasmid as described in Example 2c. Gene copy number amplification was performed by streaking clones on YPD agar plates containing higher concentrations of bleomycin (50, 100, and 500 μg / mL bleomycin), as described by Marx et al. (FEMS Yeast Res. 2009 Dec; 9(8): 1260-70).

[0369] HSA expression screening and product quantification were performed using HSA ELISA as described in Example 3c. GCNs of certain clones were identified using the highest HSA secretion levels as described in Example 5. P-amplification of GCNs was performed. Cs1 Cloning and P with known GCNs (1 or 2) GAP and P cs1 The clones were used as controls and cultured on BM medium for 48 hours.

[0370] clone HSA(μg) / WCW(g) GCN HSA(μg) / WCW(g) / GCN pGAP-HSA#3 291.0 1 291.0 pGAP-HSA#4 476.2 2 238.1 pGAP-HSA#1 540.7 1 540.7 pCS1-HSA#1-25-100#7 1464，1 3 488.0 pCS1-HSA#1-50-100#5 1178.3 4 294.6 pCS1-HSA#1-50-100#9 1580.7 3 526.9

[0371] Table 10: Screening results of HSA expression in single and multiple copy clones under pGAP or pCS1 control. GCNs and titers per GCN are shown.

[0372] Example 8: Comparative study of promoter activity of the newly identified Pichia pastoris promoter pCS1 using antibody fragment (Fab) as an extracellular reporter gene.

[0373] To analyze the characteristics of the newly identified promoters, the following shake-flask screening was performed: pre-cultured in a rich medium containing glycerol as the carbon source for 24 hours, followed by master culture in a rich medium.

[0374] a) Strains & Expression Vectors

[0375] A wild-type Pichia pastoris strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Centralbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The pCS1 promoter amplified in Example 2b was cloned into an LC vector containing the HyHEL antibody or a pPUZZLE expression vector containing Fab-HC as the GOI. The promoter was inserted upstream of the start codon of the Fab gene using Apal and Sbfl restriction sites. After sequencing verification, the expression cassettes of both strands were bound to a single vector using the compatible restriction endonucleases Mrel and Agel.

[0376] b) Expression of Fab in Pichia pastoris under the control of the newly identified pCS1 promoter.

[0377] The plasmid was linearized using the Ascl restriction endonuclease and then introduced into Pichia pastoris using electroporation (using a standard transformation method suitable for Pichia pastoris). Positive transformants were selected on YPD plates containing the following components per liter (10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar), with the plates containing 25 μg / mL bleomycin. Colony PCR was performed to ensure the presence of the transformant plasmid as described in Example 2c.

[0378] The expression screening for Fab was similar to the HSA expression screening described in Example 3c. The amount of intact Fab was quantitatively detected by ELISA using an anti-human IgG antibody (Abeamab7497) as the coating antibody (1:1000) and a goat anti-human Kappa light chain (bound and free)-alkaline phosphatase-bound antibody (Sigma A3813) as the detection antibody (1:1000). Human Fab / Kappa, IgG fragment (Bethyl P80-115) was used as a standard at an initial concentration of 50 ng / mL. Suspension culture samples were diluted accordingly. Detection was performed using pNPP substrate (Sigma S0942). The coating, dilution, and washing buffers were prepared on PBS (2 mM KH2PO4, 10 mM Na2HPO4·2H2O, 2.7 mM g KCl, 8 mM NaCl, pH 7.4), and respectively, with BSA (1% (w / v)) and / or Tween 20 (0.1% (v / v)).

[0379] Genomic DNA was isolated from selected clones, and GCNs for the heavy chain (HC) and light chain (LC) were determined as described in Example 5. The GCNs were associated with clone PCS1#5, thus the GCNs for HC and LC were set. Fab expression yields (μg Fab / gWCW), associated GCNs, and Fab yield per GCN are shown in Table 11. Regarding other pattern proteins, compared to expression clones under pGAP control, the Fab yield per GCN of each clone expressed under pCS1 control was approximately twice that of the former (average 35.4 μg Fab / gWCW).

[0380]

[0381]

[0382] Table 11: Screening results of Fab expression under pGAP or pCS1 control. GCNs and Fab yield for each GCN are shown on the right.

[0383] c) Feed-batch culture of Pichia pastoris strains expressing Fab under the control of the pCS1 promoter.

[0384] Following similar conditions as described in Example 4, fed-batch culture fermentation was carried out using the culture medium described in Example 2d.

[0385] Compared with pGAP, Fab expression under pCS1 control in bioreactor culture yielded a 3.0-fold specific yield (qP / GCN) (see Table 12).

[0386]

[0387] Table 12: Results of bioreactor culture of Fab expression under pGAP or pCS1 control. Fab yield at the end of fed-batch culture, mean specific yield qP (mean Fab yield per unit biomass (DCW) throughout the fed-batch culture), associated GCN for each clone, and mean qP / GCN are shown.

[0388] Example 9: Comparison of pCS1 variants

[0389] As shown in Example 2a, length variations of the pCS1 promoter were cloned, and similar variations were screened as described in Example 2c. Cloned expression controlled by pCS1 (standard length) and pGAP was used as controls. The forward primers and lengths of pCS1 and their variations are listed in Table 13.

[0390]

[0391]

[0392] Table 13: pCS1 and its variants: forward primers and lengths of the length variant of pCS1 (SEQ ID 1).

[0393] clone 24-hour FL / cell FL / cells for 48 hours pGAP-eGFP#2 15.39 17.59 pGAP-eGFP#4 25.30 37.77 pCS1-85pool -0.15 0.04 pCS1-138pool 7.70 6.30 pCS1-234pool 18.57 19.71 pCS1-344pool 25.19 27.63 pCS1-500pool 28.50 38.82 pCS1-767pool 26.88 36.47 pCS1-1488pool 30.59 44.40

[0394] Table 14: Screening results of eGFP expression under pGAP, pCS1, or pCS1 variant control (relative fluorescence)

[0395] Example 10: Validation of the expression intensity of the promoter in clones expressing eGFP under growth restriction conditions of high and low growth rates.

[0396] a) strain

[0397] The expression levels were compared between a host strain (e.g., Pichia pastoris) that expressed eGFP under the control of a promoter of interest and a strain that expressed eGFP under the control of pGAP.

[0398] b) Culture of eGFP-expressing strains used for promoter comparison.

[0399] These strains were cultured in a 1.0 L effective volume DASGIP bioreactor in a chemostat at two fixed special growth rates (adjusted to one high and one low special growth rate by setting dilution ratios).

[0400] Use the following culture media:

[0401] Each liter of PTM1 trace element salt mother liquor contains:

[0402] 6.0g CuSO4·5H2O, 0.08g NaI, 3.36g MnSO4·H2O, 0.2g Na2MoO4·2H2O, 0.02g H3BO3, 0.82g CoCl2, 20.0g ZnCl2, 65.0g FeSO4·7H2O, 0.2g biotin, and 5.0ml H2SO4 (95%-98%).

[0403] Each liter of glycerol batch fermentation medium contains:

[0404] 2g citric acid monohydrate (C6H8O7·H2O), 39.2g glycerol, 12.6g NH4H2PO4, 0.5g MgSO4·7H2O, 0.9g KCl, 0.022g CaCl2·2H2O, 0.4mg biotin, and 4.6ml PTM1 trace element salt stock solution. Adjust the pH to 5 by adding HCl.

[0405] Each liter of chemistostat medium contains:

[0406] 2.5g citric acid monohydrate (C6H8O7·H2O), 55.0g glucose monohydrate, 21.75g ​​(NH4)2HPO4, 1.0g MgSO4·7H2O, 2.5g KCl, 0.04g CaCl2·2H2O, 0.4mg biotin, and 2.43mL PTM1 trace element salt stock solution. Adjust the pH to 5 by adding HCl.

[0407] Dissolved oxygen was controlled at DO = 20% using a stirring speed (400-1200 rpm). Aeration was maintained at 24 L / h, temperature at 25°C, and pH was adjusted to 5 by adding NH4OH (25%). To initiate fermentation, 400 ml of sterilized batch culture medium was filtered into the fermenter and inoculated with Pichia pastoris clones (from the pre-culture) with an initial OD of OD600 = 1. After approximately 25 hours of batching (reaching a dry matter concentration of approximately 20 g / L), a glucose-limited chemostat fermentation culture (20 g / L) was initiated. The chemostat feed rate and harvest rate were used to maintain the required constant specific growth rate. During culture, to ensure a constant growth rate, the culture volume and cell dry weight were kept constant. Cell cultures were conducted at high and low cell growth rates of 0.15 and 0.015 / h, respectively. Therefore, the feed / harvest rate was controlled at 150 mL / h. -1 L -1 (the number of mL of chemiluminescent medium added per liter of culture medium per hour) and 15 mL h -1 5L -1 .

[0408] c) Sampling

[0409] Samples were taken at a steady state (at least 5 volumes exchanged) and eGFP expression was analyzed using a plate reader (Infinite 200, Tecan, CH). Therefore, samples were diluted to OD600 = 5. Fermentation cultures were performed in duplicate. Relative fluorescence expression data were calculated for each bioreactor as described in Example 2d.

[0410] Example 11: Determination of Pichia pastoris promoters for high-speed transcription under specific high and low growth rates

[0411] To identify Pichia pastoris promoters that enable rapid transcription at specific high and low growth rates, gene expression profiles were analyzed using DNA microarrays. High-speed transcription genes at specific high and low growth rates were selected from the transcriptome data. Therefore, Pichia pastoris cells were cultured in the chemostat described in Example 10b for 0.15 and 0.015 hours, respectively. -1The RNA was cultured at both high and low specific growth rates. Sampling, RNA purification, preparation of gene chip hybridization samples, gene chip analysis, data acquisition, and statistical evaluation were performed as described in Examples 1c), 1d), 1e), and 1f). Genes and specific promoters that transcribed rapidly at both high and low growth rates were identified by reviewing gene chip data showing high signal intensity under both low and high growth rate conditions. As a second criterion, the signal intensity should be higher than that of glyceraldehyde-3-phosphate dehydrogenase (GAP, also known as GAPDH and TDH3) genes under both conditions. To isolate the promoters, a nucleic acid fragment approximately 1000 bp upstream of the start codon ATG of the corresponding gene was amplified.

Claims

1. An isolated pCS1 promoter nucleic acid sequence, comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

2. The nucleic acid sequence according to claim 1, wherein it consists of the pCS1 nucleic acid sequence identified as SEQ ID NO:

1.

3. The nucleic acid sequence according to claim 1, wherein, The pCS1 promoter nucleic acid sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

4. The nucleic acid sequence of claim 1, wherein the nucleic acid is not naturally associated with the nucleotide sequence encoding the protein of interest by means of operably linking to a nucleotide sequence encoding the protein of interest.

5. The nucleic acid sequence according to claim 4, further comprising a nucleic acid sequence encoding a signal peptide that generates the secretion of the protein of interest.

6. An expression construct comprising the nucleic acid sequence of claim 1.

7. The expression construct according to claim 6, wherein it is an autonomously replicating vector or plasmid, or an expression construct integrated into the chromosomal DNA of a host cell.

8. A recombinant host cell comprising the nucleic acid sequence of claim 1 or the expression construct of claim 6 or 7.

9. The recombinant host cell according to claim 8 is a eukaryotic cell.

10. The recombinant host cell according to claim 8, wherein it is a yeast or filamentous fungal cell.

11. The recombinant host cell according to claim 8, wherein it is a yeast cell of the genus *Pichia pastoris* or *Pichia pastoris*.

12. The recombinant host cell of claim 8, comprising multiple copies of the nucleic acid sequence and / or multiple copies of the expression construct.

13. The recombinant host cell according to claim 8, wherein the recombinant host cell is selected from the group consisting of mammals, insects, yeast, filamentous fungi and plant cells.

14. The recombinant host cell according to claim 13, wherein it is any one of Pichia pastoris strains CBS 704, CBS2612, CBS7435, CBS 9173-9189, DSMZ 70877, X-33, GS115, KM71 and SMD1168.

15. A multi-cell stable culture of recombinant host cells according to any one of claims 8-14.

16. A method for producing a protein of interest by culturing a recombinant host cell line comprising a promoter according to any one of claims 1-5 or an expression construct according to claim 6 or 7 and containing nucleic acid encoding the protein of interest under transcriptional control conditions of said promoter, or by culturing recombinant host cells according to any one of claims 8-15, comprising the steps of: a) Culture the cell line under conditions expressing the protein of interest, and b) Recover the protein of interest.

17. The method according to claim 16, wherein, The protein of interest was expressed under growth-restricted conditions.

18. The method according to claim 16, wherein, The cell lines are cultured under batch culture, fed-batch culture, or continuous culture conditions, and / or in a medium containing a limiting carbon source.

19. The method according to claim 18, wherein, The cultivation is carried out in a bioreactor, starting with a batch culture period, followed by a fed-batch culture period or a continuous culture period.

20. The method according to any one of claims 16 to 19, wherein, The protein of interest is a heterologous protein.

21. The method according to claim 20, wherein, The heterologous protein is selected from therapeutic proteins, antibodies or fragments thereof, enzymes, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine proteins, growth factors, hormones, and cytokines.

22. The method according to claim 20, wherein, The heterologous protein is selected from polypeptides.

23. The method according to claim 20, wherein, The heterologous protein is selected from processing enzymes.

24. The use of the isolated nucleic acid sequence according to any one of claims 1 to 5 or the expression construct according to claim 6 or 7 in a method for producing a protein of interest by culturing host cells transformed with said nucleic acid sequence and / or said expression construct.

25. The application according to claim 24, wherein the culture is carried out in a bioreactor, starting with a batch culture period followed by a fed-batch culture period or a continuous culture period.

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