Truncated promoters for recombinant gene expression

Abstract

The present invention relates to a promoter with increased relative expression efficiency, in particular a promoter variant of a formate dehydrogenase promoter with increased relative expression efficiency, for use in recombinant protein production and a method for increasing the relative expression efficiency of a nucleotide sequence having promoter activity.

Description

Technical Field

[0001] The present invention relates to truncated promoters or variants thereof, methods for recombinantly producing target polypeptides, and methods for increasing the relative expression efficiency of nucleotide sequences having promoter activity. Background Art

[0002] For decades, the use of specific nucleotide sequences as promoters to promote recombinant gene expression and to achieve target protein production has been studied in the field of biotechnology. The promoters used for gene expression in prokaryotic hosts are usually relatively short in length, usually composed of less than 100 base pairs (bp), such as the promoter of T7 RNA polymerase promoter or the promoter of Escherichia coli lac operon. On the other hand, the promoters used for gene expression in eukaryotic hosts are usually 500 to 1000bp long, such as the FLD1 promoter or the AOX1 promoter of Pichia pastoris (Pichia pastoris), and even promoters exceeding 1kb (kilobase pairs) are also common.

[0003] Typical prokaryotic host organisms for recombinant gene expression are Escherichia coli or Bacillus subtilis. Examples of eukaryotic host organisms are Saccharomyces cerevisiae, Trichoderma reesei or Pichia pastoris. In recent years, strains of Pichia pastoris have been established as a platform for the recombinant production of industrially important proteins.

[0004] Well-known promoters for regulating recombinant gene expression in Pichia pastoris are the methanol-inducible promoter of the alcohol oxidase 1 gene (AOX1) and the constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP). Hartner et al. conducted promoter engineering studies on the Pichia pastoris alcohol oxidase 1 promoter to identify transcription factor binding sites or verify putative transcription factor binding sites by targeted short internal deletions in the Pichia pastoris alcohol oxidase 1 promoter nucleotide sequence (Hartner et al., Nucleid Acids Res. 2008. 36 (12: e76)). In addition, in recent years, several new promoter sequences with different regulation and expression strengths have been described (e.g., Vogl et al., ACS Synth Biol. 2016. 5 (2): 172-186). Despite these scientific advances, reasonable predictions of essential elements in eukaryotic promoters remain unreliable. Essential regulatory elements are usually separated from each other by hundreds or even thousands of base pairs on the primary nucleotide sequence, and may interact only due to secondary or tertiary nucleotide structures. Therefore, the effects of insertions or deletions of nucleotide sequence stretches cannot be predicted. Consequently, eukaryotic promoter engineering efforts still largely rely on intensive experiments with so far unpredictable outcomes.

[0005] In EP 0 299 108 A1 a nucleotide sequence comprising a control region and a structural gene encoding formate dehydrogenase is disclosed.

[0006] In WO 03 / 095653 A1, the nucleotide sequences of engineered variants of the promoter of the formate dehydrogenase gene (FMD) from Ogataea angusta are disclosed. The disclosed invention relates to promoter variants comprising a palindromic nucleotide sequence to achieve a higher transcription rate.

[0007] In WO 2017 / 109082 A1, variants of the formate dehydrogenase promoter for recombinant gene expression in Komagataella species yeast cells are disclosed, which are about 620 bp in length and are at least 90% identical to the unmutated wild-type FMD promoter of Ogataea angusta (also known as Ogataea polymorpha or Hansenula polymorpha). These variants illustrate the extent to which single point mutations or deletions or insertions can be introduced into the nucleotide sequence without negatively affecting its promoter function. It should be emphasized that the promoter shown can be induced not only by methanol, but also advantageously by so-called derepression. Induction by derepression is described as being achieved by depleting an inhibitory carbon source (e.g., glucose or glycerol) or by alternatively supplying a non-inhibiting carbon source (e.g., sorbitol). It is noteworthy that a particularly high induction of the wild-type FMD promoter is shown when glucose feeding is completely stopped.

[0008] In Song et al. (Bioechnol Lett. 2003. 25(23): 1999-2006), expression vectors comprising a 645 bp variant of the FMD promoter or the methanol oxidase promoter of Hansenula polymorpha are described for producing green fluorescent protein as a reporter protein in Hansenula polymorpha.

[0009] In Vogl et al. (AMB Express. 2020.10(1):38), it is described that the 623 bp wild-type FMD promoter of Hansenula polymorpha can be strongly derepressed in Pichia pastoris. It is particularly important to emphasize that when the inhibitory carbon source (e.g., glucose or glycerol) is depleted, derepression leads to strong activation of the HpFMD promoter in Pichia pastoris.

[0010] In Gellisen G et al.: "Heterologous gene expression in Hansnula polymorpha: efficient secretion of glucoamylase", Biotechnology. The international monthly for industrial biology, nature publishing Group, US, vo. 9, March 1, 1991 (1991-03-01), pages 291-295, the glucoamylase gene (GAM1) from Schwanniomyces occidntalis was introduced into the genome of the methylotrophic yeast Hansenula polymorpha ahs, and the potential of this organism as a host for high-level expression of heterologous genes encoding secreted proteins was studied. It was found that after insertion or transformation, colonies secreting active glucoamylase were isolated, and it was shown that several cycles of production produced mitotically stable strains carrying the integrated foreign DNA.

[0011] In the workflow required for achieving recombinant gene expression, a basic step is the amplification of expression plasmids and, in particular, expression cassettes. Plasmid amplification is usually performed in bacterial cells (e.g., E. coli). After separating the amplified plasmid from the cells, the expression cassette can be cut off from the circular plasmid by restriction enzyme digestion. Alternatively, the expression cassette can be obtained by DNA amplification of polymerase chain reaction (PCR). It is worth noting that both strategies rely on DNA replication by DNA polymerase, which tends to introduce undesirable mutations into the expression cassette. Although improvements have been made to the E. coli strains used for plasmid amplification and the polymerases used for PCR, a certain number of mutant expression cassette molecules must be considered in any amplification step, and therefore cannot be excluded. Statistically speaking, DNA polymerases produce erroneous nucleotide sequences after processing a certain number of nucleotides. Therefore, the longer the expression cassette, the higher the risk of introducing unwanted mutations during DNA replication by the polymerase. In addition, the yield of host cells successfully transformed with the expression cassette benefits from a higher number of molecules. However, it is known that when the nucleotide sequence to be amplified is longer, PCR produces fewer products, i.e., fewer molecules. Therefore, the increase of PCR product yield can be achieved by reducing the length of the nucleotide sequence to be amplified. Therefore, it is desirable to use an expression cassette as short as possible while still achieving its purpose. Generally, it is known that when the length of a single polynucleotide fragment to be assembled is shorter, the molecular assembly of a gene expression cassette or a plasmid is achieved with a higher success rate. Therefore, also at this point, it is expected that shorter fragments rather than longer fragments work. However, shortening the fragment by truncating a functional polynucleotide sequence (such as a promoter) cannot be expected to have a negative impact on the activity of such a functional sequence. In particular, up to now, the impact on the function of a eukaryotic promoter after omitting the nucleotide segment of the promoter sequence is unpredictable (Hartner et al., Nucleid Acids Res. 2008.36 (12: e76)).

[0012] Therefore, an object of the present invention is to provide a more efficient eukaryotic promoter having high expression intensity at a shorter length, ie, a eukaryotic promoter having increased relative expression efficiency.

[0013] This object is achieved by providing a promoter, wherein the promoter is truncated from the 5'-end of the nucleotide sequence of SEQ ID NO: 1 to a length of 140-610 base pairs, preferably truncated to a length of 140-560 base pairs, more preferably truncated to a length of 160-560 base pairs, most preferably truncated to a length of 160-500 base pairs; or by providing a variant thereof, i.e. a promoter variant, wherein the promoter variant has at least 80% identity, preferably at least 90% identity, preferably at least 95% identity, more preferably at least 97% identity, most preferably at least 99% identity with the promoter. A person skilled in the art will appreciate the following unexpected effect: despite the reduction in the overall length of the naturally occurring promoter having the nucleotide sequence of SEQ ID NO: 1 or its variant, the activity of the truncated promoter or promoter variant as described herein is not eliminated. By providing such truncated or shortened promoters or promoter variants, even short synthetic oligonucleotides can be used, such as primer oligonucleotides, which can be added to other recombinant nucleotide fragments in a simple PCR reaction, thus greatly speeding up the steps required for assembly of the recombinant gene expression cassette while reducing the risk of introducing point mutations (as would be the case when one must rely on assembly of larger polynucleotide fragments).

[0014] The convention in the art is to write deoxyribonucleic acid (DNA) sequences, particularly promoter sequences in the 5'-3' direction. The 5'-end refers to the 5'-carbon atom of the ribose moiety of the nucleotide, while the 3'-end refers to the 3'-carbon atom. To avoid any doubt, the 5'-end of the nucleotide sequence of the promoter is the distal end of the nucleotide sequence of the promoter. In other words, the 5'-end is determined by the nucleotide farthest from the start codon of the open reading frame, the transcription of which is regulated by the promoter. The 5'-end of the nucleotide sequence of the promoter is the most upstream nucleotide of the start codon of the open reading frame whose transcription is regulated by the promoter. In contrast, the 3'-end of the nucleotide sequence of the promoter is the closest nucleotide upstream of the start codon of the open reading frame whose transcription is regulated by the promoter. In other words, the 3'-end of the nucleotide sequence of the promoter is the last nucleotide of the promoter upstream of the start codon of the open reading frame whose transcription is regulated by the promoter.

[0015] Those skilled in the art are aware of the fact that a small number of single point mutations, single nucleotide insertions or single nucleotide deletions can be introduced into a promoter nucleotide sequence without substantially reducing the activity or function of the promoter nucleotide sequence. The activity or function of a promoter will be considered to be significantly reduced when one or more single point mutations, insertions or deletions will result in a functional change of at least 60% compared to the function of the unchanged promoter. Therefore, the present invention also includes mutant promoter variants having at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 97% identity, and most preferably at least 99% identity with the truncated promoter as described herein. For the sake of clarity only, it is noted that promoters or promoter variants truncated from the 5'-end of the nucleotide sequence of SEQ ID NO: 1 to a length of more than 610 base pairs (e.g., promoters or promoter variants having 611 bp, 612 bp, 620 bp or even longer) are not included in the present invention. Even if such longer promoters or promoter variants contain promoter sequences less than 610 bp, these promoters or promoter variants with a length of more than 610 base pairs are not included in the present invention because their relative expression efficiency is not considered to be increased sufficiently. In line with this, promoter variants having at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 97% identity, most preferably at least 99% identity with the truncated promoter as described herein and having a length of more than 610 base pairs are also not included in the present invention. Likewise, correspondingly, a non-truncated promoter having a nucleotide sequence of SEQ ID NO: 1 is also not included in the present invention, even if it contains a promoter with less than 610 bp. In other words, the promoter or promoter variant according to the present invention does not exist naturally, but requires at least one step of truncation by an experimenter. Therefore, the promoter or promoter variant according to the present invention only relates to non-natural or recombinant artificial molecules.

[0016] Those skilled in the art know methods for determining the degree of sequence identity between two or more nucleotide sequences expressed as a percentage (%). As an example, the Needleman-Wunsch algorithm (Needleman, Wunsch. J Mol Biol. 1970. 48 (3): 443-453) can be used. For example, the default algorithm parameters (match / mismatch score: 2, -3. Gap cost: existence: 5 extension: 2) are used to obtain the sequence identity from NCBI (National Center for Biotechnology Information; https: / / blast.ncbi.nlm.nih.gov / Blast.cgi? PAGE_TYPE=BlastSearch&PR OG_DEF=blastn&BLAST_PROG_DEF=blastn& BLAST_SPEC = Global Aln&LINK_LOC=BlastHomeLink) Appropriate tools are available online. It is assumed that using the default settings for the DNA input sequence, a global alignment algorithm will be used, such as the Clustal Omega tool from the European Bioinformatics Institute (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). When determining the degree of sequence identity between a promoter and a promoter variant comprising a point mutation and / or a point insertion and / or a point deletion using the Needleman-Wunsch algorithm or a comparable global alignment algorithm, only corresponding nucleotide sequences, i.e. nucleotide sequences of the same length, are compared. For example, if a promoter variant is truncated to a length of 160 bp from the 5'-end of the nucleotide sequence of SEQ ID NO: 1, and the promoter variant also comprises three point mutations, the corresponding promoter sequence to which the promoter variant will be aligned will also have a length of 160 bp. In other words, in order to allow the correct determination of the degree of sequence identity expressed as a percentage between the sequence segment contained in SEQ ID NO: 1 and one or more promoter variants, the length of the sequence segment of SEQ ID NO: 1 should be the same as the length of the promoter variant. Only in this way will the nucleotide sequence segment of SEQ ID NO: 1 be the correct corresponding nucleotide sequence segment of SEQ ID NO: 1, and therefore, only if the promoter variant does not contain any point mutations or insertions or deletions, will it be 100% identical to the corresponding nucleotide sequence segment of SEQ ID NO: 1 in the global alignment.

[0017] In a preferred embodiment of the present invention, the relative expression efficiency of the promoter or promoter variant as described herein is at least 5%, preferably at least 10% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO: 1. Thus, it can be achieved that the expression intensity of the truncated promoter or promoter variant is at least as high as the expression intensity of the non-truncated promoter in relation to its length.

[0018] As used herein, the term "relative expression efficiency" refers to the amount of target protein produced by a promoter nucleotide sequence / base pair. In the case where the target protein is an enzyme, "relative expression efficiency" (REE) may refer to the amount of enzyme activity of a promoter nucleotide sequence / base pair. For example, if the expression of a non-truncated promoter with 623 bp produces 80 units of enzyme activity / liter (U / L), its relative expression efficiency will be 80 / 623=0.128U / L / bp. If 83U / L is produced by the expression of a truncated promoter with 500 bp, its relative expression efficiency will be 83 / 500=0.166U / L / bp. The increase in the "relative expression efficiency" of a truncated promoter relative to a non-truncated promoter expressed as a percentage can be calculated by multiplying the relative expression efficiency of the truncated promoter variant by 100 and dividing by the relative expression efficiency of the non-truncated promoter: 0.166*100 / 0.128=129.3%. Therefore, the relative expression efficiency of the truncated promoter with 500 bp is 29.3% higher than that of the promoter with 623 bp. In other words, the relative expression efficiency of the truncated promoter with 500 bp will be 1.293 times higher than that of the promoter with 623 bp.

[0019] For the avoidance of doubt, a truncated promoter is considered to have a higher REE than a non-truncated promoter when the higher REE is determined by small-scale culture (e.g., culture in deep well plates) or medium-scale culture (e.g., culture in shake flasks) or large-scale culture (e.g., culture in bioreactors). It is well known in the art that transformation of yeast (e.g., Pichia pastoris) typically produces a large number of transformed strains that behave inconsistently, for example, Vogl et al. (Appl Environ Microbiol. 2018. 84 (6): e02712-17). Therefore, when comparing different expression strategies, such as when comparing different promoter variants, a preliminary screening of multiple transformants must be performed to exclude obvious bad strains or obvious poor performing strains. Without wishing to be bound by theory, it is believed that such bad strains or poor performing strains are the result of disruptive genomic integration, in which the expression cassette is integrated into a genomic locus that destroys or at least reduces the expression capacity of the expression cassette. Therefore, it is necessary to select only strains for comparison with each other that represent the respective expression strategy. In other words, it is important to note that only strains carrying the same amount of integrated expression cassettes are compared, and that at least 5, preferably at least 6, more preferably at least 7, even more preferably at least 8, and most preferably at least 9 transformed strains out of the 90 transformants from each expression strategy are compared.

[0020] The present invention also relates to a promoter or promoter variant as described herein, wherein the promoter or promoter variant is included in an expression cassette, wherein the expression cassette also includes a nucleotide sequence encoding a polypeptide and an optional transcription terminator sequence, and wherein the promoter or promoter variant is operably connected to the nucleotide sequence encoding the polypeptide. For clarity only, the optional transcription terminator sequence is also operably connected to the nucleotide sequence encoding the polypeptide. Surprisingly, by providing this expression cassette, recombinant gene expression at the same level as a non-truncated promoter can be achieved, while having a smaller molecular size due to the reduction in promoter length. Therefore, when a cell of a host organism is transformed with a certain amount of DNA of this expression cassette to produce a host organism, more expression cassette molecules can be introduced into the cell compared with the DNA of an expression cassette containing a non-truncated and thus longer promoter to produce a host organism. In a preferred embodiment of the present invention, a promoter or promoter variant as described herein is operably connected to a recombinant nucleotide sequence encoding a heterologous polypeptide. Therefore, proteins that are not usually produced by natural host cells can be produced. The present invention also relates to a promoter or a promoter variant as described herein, wherein the promoter or promoter variant is comprised in a plasmid, and wherein the promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide.

[0021] As used herein, the term "operably connected" is understood to mean that a promoter or a promoter variant is connected to a nucleotide sequence encoding a polypeptide in a certain direction, which allows the gene expression, i.e. transcription, of the nucleotide sequence encoding the polypeptide by the promoter or promoter variant to be promoted. In other words, the 3'-end of the promoter or promoter variant is connected to the 5'-end of the nucleotide sequence comprising the open reading frame encoding the polypeptide. The 5'-end of the open reading frame encoding the polypeptide is usually the start codon for the first amino acid of the encoded polypeptide. Usually, although not always necessary, the start codon has the sequence 5'-ATG-3', wherein the 5'-end of the open reading frame encoding the polypeptide is "A", i.e., the nucleotide deoxyadenosine monophosphate, and the start codon usually encodes the amino acid methionine. Usually, the 5'-end of the open reading frame encoding the polypeptide is located near the 3'-end of the promoter or promoter variant. The 5'-end of the open reading frame encoding the polypeptide can be directly connected to the 3'-end of the promoter or promoter variant. Alternatively, the 5'-end of the open reading frame encoding a polypeptide can be separated from the 3'-end of a promoter or a promoter variant by one or more nucleotides, without substantially changing the activity of the promoter or promoter variant on the expression of the open reading frame encoding a polypeptide operably connected to the promoter or promoter variant. It is believed that the one or more nucleotides separating the 3'-end of a promoter or a promoter variant from the 5'-end of the open reading frame encoding a polypeptide can be, for example, a nucleotide sequence recognized by one or more restriction enzymes. Preferably, the one or more nucleotides separating the 3'-end of a promoter or a promoter variant from the 5'-end of the open reading frame encoding a polypeptide are shorter than 100 nucleotides, more preferably shorter than 50 nucleotides, even more preferably shorter than 20 nucleotides, and most preferably shorter than 10 nucleotides. The terms "protein" and "polypeptide" are used interchangeably.

[0022] The present invention also relates to promoters or promoter variants as described herein, wherein the promoter or promoter variant is contained in a yeast cell, and wherein the promoter or promoter variant is operably connected to the nucleotide sequence of the encoded polypeptide. Therefore, biotechnology protein production can be achieved in a suitable host organism. In a preferred embodiment, the yeast cell is a cell selected from the genus Pichia, Komagataella, Ogataea, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Zygosaccharomyces and Yarrowia, preferably Pichia pastoris cells or Yarrowia lipolytica cells, most preferably Pichia pastoris cells. By selecting yeast cells from the genera Pichia, Komagataella, Ogataea, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Zygosaccharomyces and Yarrowia, and in particular by selecting yeast cells from the organisms Pichia pastoris or Yarrowia lipolytica, preferably Pichia pastoris, protein production can be achieved in a particularly efficient manner. The person skilled in the art is aware of the fact that the name "Pichia pastoris" as used herein refers to several yeast strains that are also classified into the Komagataella pastoris and Komagataella phaffii taxa (Yamada et al., Biosci Biotechnol Biochem. 1995. 59(3):439-444; Kurtzman. J Ind Microbiol Biotechnol. 2009. 35(11):1435-1438).Other synonymous, although less common, names for Pichia pastoris strains are Zygosaccharomyces pastoris Guilliermond, Zygosaccharomyces pastoriGuilliermond, Endomyces pastoris(Guilliermond), Endomyces pastori(Guilliermond), Saccharomyces pastoris(Guillliermond), Saccharomyces pastori(Guillliermond), Petasospora pastori(Guilliermond), Zymopichia pastoris(Guilliermond), Zymopichia pastori(Guilliermond) or Zygowilia pastori(Guilliermond) (Wolf et al., Springer-Verlag Berlin Heidelberg. 2003. Non-concentional yeasts in genetics, biochemistry and biotechnology. ISBN 978-3-540-44215-8: p. 489;. http: / / www.mycobank.org / BioloMICS.aspx?TableKey= 14682616000000067&Rec=107579&Fields=All Pichia pastoris platform strains of particular industrial relevance are especially Komagataella phaffii CBS 7435 (also deposited, for example, as ATCC 76273 or NRRL Y-11430), or Komagataella phaffii GS115 (also deposited, for example, as ATCC 20864), or Komagataella pastoris CBS 704 (also deposited, for example, as ATCC 28485 or NRRL Y-1603).

[0023] Another object of the present invention is to provide a method that allows high product titers by recombinant protein production. Therefore, another aspect of the present invention relates to a method for recombinantly producing a target polypeptide, comprising the steps of: introducing an expression cassette comprising a nucleotide sequence encoding the target polypeptide into at least one yeast cell; producing a yeast cell culture by propagating the at least one yeast cell; subjecting the yeast cell culture to conditions suitable for expressing the nucleotide sequence encoding the target polypeptide; and isolating the produced target protein from the yeast cell culture; wherein the expression cassette further comprises a promoter or a promoter variant as described herein, which is operably linked to the nucleotide sequence encoding the target polypeptide, and wherein the yeast cell is a Pichia pastoris cell or a Yarrowia lipolytica cell, preferably a Pichia pastoris cell. By applying this method, it was surprisingly found that particularly large amounts of the target polypeptide can be produced. Note that the method for recombinantly producing a target polypeptide according to the present invention can also be performed in such a way that the expression cassette comprises a promoter comprising a nucleotide sequence of SEQ ID NO: 1.

[0024] The expression cassette can be introduced into yeast cells by applying common yeast transformation protocols known to those skilled in the art, such as by electroporation (Lin-Cereghino et al., Biotechniques. 2005. 38 (1): 44-48). The expression cassette can be provided alone or as part of a larger polynucleotide construct (e.g., linear or circular plasmid). Yeast cell cultures are produced by breeding one or more yeast cells using culture protocols known in the art that are suitable for culturing yeast, such as Krainer et al. (Microb Cell Fact. 2012. 11: 22) or Cregg (Humana Press Inc. 2007. Pichia Protocols. ISBN 978-1-59745-456-8). In order to separate the target protein produced from the yeast cell culture, several options are available, including but not limited to separating cells from the liquid by centrifugation and collecting the supernatant containing the secreted target protein, or lysing the yeast cells and collecting the lysate containing the produced target protein. Additionally, further purification steps (such as fractional precipitation or chromatographic methods) may be applied to isolate the produced target protein.

[0025] In a preferred embodiment of the invention, the method as described herein is carried out in such a manner that the conditions suitable for expressing the target polypeptide are a fed-batch process. Preferably, the fed-batch process is operated with a glucose feed rate of at least 0.04 mmol glucose per gram of cell dry weight per hour (mmol Glu / g CDW / h) or a glycerol feed rate of at least 0.07 mmol glycerol per gram of cell dry weight per hour (mmol Gly / g CDW / h), preferably with a glucose feed rate of at least 0.11 mmol Glu / gCDW / h or a glycerol feed rate of at least 0.21 mmol Gly / g CDW / h, more preferably with a glucose feed rate of at least 0.18 mmol Glu / g CDW / h or a glycerol feed rate of at least 0.35 mmol Gly / g CDW / h. Contrary to current teachings in the prior art, it was found that rather than derepression by consumption of glucose or glycerol, active feeding with glucose or glycerol as described herein increases the production of a target polypeptide under the control of a HpFMD-based promoter, particularly when a glucose feed rate of at least 0.04 mmol Glu / g CDW / h or a glycerol feed rate of at least 0.07 mmol Gly / g CDW / h is applied, preferably at least 0.11 mmol Glu / g CDW / h or at least 0.21 mmol Gly / g CDW / h, more preferably at least 0.18 mmol Glu / g CDW / h or at least 0.35 mmol Gly / g CDW / h. In a preferred embodiment of the invention, the yeast cell is a Pichia pastoris cell or a Yarrowia lipolytica cell, preferably a Pichia pastoris cell. Thus, recombinant production can be performed by the methods described herein to produce particularly high product titers.

[0026] Another aspect of the present invention relates to a method for increasing the relative expression efficiency of a nucleotide sequence having promoter activity, the method comprising the step of providing a truncated promoter by truncating a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% identity to the nucleotide sequence of SEQ ID NO: 1 from the 5'-end to a length of 140-610 base pairs, preferably to a length of 140-560 base pairs, more preferably to a length of 160-560 base pairs, most preferably to a length of 160-500 base pairs. For the avoidance of doubt only, truncation is performed from the 5'-end only, while the 3'-end of the nucleotide sequence remains untruncated. Preferably, the relative expression efficiency of the truncated promoter is at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 50%, most preferably at least 100% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO: 1.

[0027] The present invention is also characterized by the following items:

[0028] 1. A promoter, wherein the promoter is truncated to a length of 160-500 base pairs from the 5'-end of the nucleotide sequence of SEQ ID NO: 1; or a variant thereof (promoter variant), wherein the promoter variant has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98%, and most preferably at least 99% identity with the promoter.

[0029] 2. The promoter according to item 1, wherein the promoter or promoter variant contains at least one non-natural substitution modification relative to SEQ ID NO: 1.

[0030] 3. A promoter, wherein the promoter is truncated from the 5'-end of any one of the nucleotide sequences of SEQ ID NOs: 1, 6-24 to a length of 560 base pairs, preferably 500 base pairs, more preferably 450 base pairs, more preferably 380 base pairs, more preferably 300 base pairs, more preferably 280 base pairs, more preferably 170 base pairs, and most preferably 160 base pairs.

[0031] 4. A promoter, wherein the promoter is truncated from the 5'-end of the nucleotide sequence of SEQ ID NO: 1 to a length of 560 base pairs, preferably 500 base pairs, more preferably 450 base pairs, more preferably 380 base pairs, more preferably 300 base pairs, more preferably 280 base pairs, more preferably 170 base pairs, most preferably 160 base pairs.

[0032] 5. The promoter according to item 4, wherein the promoter is truncated to a length of 160 base pairs from the 5'-end of the nucleotide sequence of SEQ ID NO: 1, and wherein the promoter has a nucleotide sequence of SEQ ID NO: 5.

[0033] 6. The promoter or promoter variant according to any one of items 1 to 5, wherein the relative expression efficiency of the promoter or promoter variant is at least 5% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO: 1, preferably at least 10%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, and most preferably at least 100%.

[0034] 7. An expression cassette comprising a promoter or promoter variant according to any one of items 1 to 6, and a nucleotide sequence encoding a polypeptide and optionally a transcription terminator sequence, wherein the promoter or promoter variant is operably linked to the nucleotide sequence encoding the polypeptide.

[0035] 8. The expression cassette according to item 7, wherein the nucleotide sequence encoding the polypeptide is a heterologous nucleic acid sequence.

[0036] 9. The expression cassette according to any one of items 7 and 8, wherein the nucleotide sequence encoding a polypeptide encodes a heterologous polypeptide.

[0037] 10. A plasmid comprising the promoter or promoter variant according to any one of items 1 to 6, or the expression cassette according to any one of items 7 to 9.

[0038] 11. A yeast cell comprising the promoter or promoter variant according to any one of items 1 to 6, or the expression cassette according to any one of items 7 to 9, or the plasmid according to item 10, wherein the yeast cell is a cell of the genus selected from the group consisting of Pichia, Komagataella, Ogataea, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Zygosaccharomyces and Yarrowia, preferably a Pichia pastoris cell or a Yarrowia lipolytica cell, most preferably a Pichia pastoris cell.

[0039] 12. A method for recombinantly producing a target polypeptide, comprising the steps of a) introducing an expression cassette comprising a nucleotide sequence encoding the target polypeptide into at least one yeast cell; b) producing a yeast cell culture by propagating the at least one yeast cell; c) subjecting the yeast cell culture to conditions suitable for expression of the nucleotide sequence encoding the target polypeptide; and d) isolating the produced target protein from the yeast cell culture, wherein the expression cassette comprises a promoter or promoter variant according to any one of items 1 to 6 operably linked to the nucleotide sequence encoding the target polypeptide.

[0040] 13. A method for recombinantly producing a target polypeptide, comprising the steps of a) introducing an expression cassette comprising a nucleotide sequence encoding the target polypeptide into at least one yeast cell; b) producing a yeast cell culture by propagating the at least one yeast cell; c) subjecting the yeast cell culture to conditions suitable for expression of the nucleotide sequence encoding the target polypeptide; and d) isolating the produced target protein from the yeast cell culture, wherein the expression cassette comprises a promoter comprising the nucleotide sequence of SEQ ID NO: 1 operably linked to the nucleotide sequence encoding the target polypeptide.

[0041] 14. The method according to any one of items 12 and 13, wherein the expression cassette further comprises a transcription terminator sequence.

[0042] 15. The method according to any one of items 12 to 14, wherein the conditions suitable for the expression of the nucleotide sequence encoding the target polypeptide are a fed-batch process.

[0043] 16. A method according to claim 15, wherein the fed-batch process is operated with a glucose feed rate of at least 0.04 mmol glucose per gram of cell dry weight per hour (mmol Glu / g CDW / h) or a glycerol feed rate of at least 0.07 mmol glycerol per gram of cell dry weight per hour (mmol Gly / g CDW / h), preferably with a glucose feed rate of at least 0.11 mmol Glu / gCDW / h or a glycerol feed rate of at least 0.21 mmol Gly / g CDW / h, more preferably with a glucose feed rate of at least 0.18 mmol Glu / g CDW / h or a glycerol feed rate of at least 0.35 mmol Gly / g CDW / h.

[0044] 17. The method according to any one of items 12 to 16, wherein the nucleotide sequence encoding the target polypeptide is a heterologous nucleic acid sequence.

[0045] 18. The method according to item 17, wherein the nucleotide sequence encoding the target polypeptide encodes a heterologous polypeptide.

[0046] 19. The method according to any one of items 12 to 18, wherein the yeast cell is a Pichia pastoris cell or a Yarrowia lipolytica cell, preferably a Pichia pastoris cell.

[0047] 20. A method for increasing the relative expression efficiency of a nucleotide sequence having promoter activity, the method comprising the following steps: providing a truncated promoter by truncating a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% identity to the nucleotide sequence of SEQ ID NO: 1 from the 5'-end to a length of 140-610 base pairs, preferably to a length of 140-560 base pairs, more preferably to a length of 160-560 base pairs, most preferably to a length of 160-500 base pairs.

[0048] 21. The method according to item 20, wherein the promoter or promoter variant contains at least one non-naturally occurring substitution modification relative to SEQ ID NO: 1.

[0049] 22. The method according to any one of items 20 and 21, wherein the relative expression efficiency of the truncated promoter is at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 50%, most preferably at least 100% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO: 1.

[0050] 23. The method according to any one of items 20 to 22, wherein the relative expression efficiency of the truncated promoter is increased in a yeast cell, wherein the yeast cell is a Pichia pastoris cell or a Yarrowia lipolytica cell, preferably a Pichia pastoris cell.

[0051] The solution of the present invention is further described below by means of non-limiting drawings and examples. Sequence Listing <110> elber ag <120> Truncated promoters for recombinant gene expression <130> P06005 <160> twenty four <170> BiSSAP 1.3.6 <210> 1 <211> 623 <212> DNA <213> Ogataea polymorpha <400> 1 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttc gctggcggcg 240 caacgacgaa tatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat atttcctaa aacatgcaat cggctgcccc gcaacggggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aatggcacta taataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc ​​623 <210> 2 <211> 1000 <212> DNA <213> Komagataella pastoris <400> 2 cgattgcccc tctacaggca taagggtgtg actttgtggg cttgaatttt acaccccctc 60 caacttttct cgcatcaatt gatcctgtta ccaatattgc atgcccggag gagacttgcc 120 ccctaatttc gcggcgtcgt cccggatcgc agggtgagac tgtagagacc ccacatagtg 180 acaatgatta tgtaagaaga ggggggtgat tcggccggct atcgaactct aacaactagg 240 ggggtgaaca atgcccagca gtcctcccca ctctttgaca aatcagtatc accgattaac 300 accccaaatc ttattctcaa cggtccctca tccttgcacc cctctttgga caaatggcag 360 ttagcattgg tgcactgact gactgcccaa ccttaaaccc aaatttctta gaaggggccc 420 atctagttag cgaggggtga aaaattcctc catcggagat gtattgaccg taagttgctg 480 cttaaaaaaa atcagttcag atagcgagac ttttttgatt tcgcaacggg agtgcctgtt 540 ccattcgatt gcaattctca ccccttctgc ccagtcctgc caattgccca tgaatctgct 600 aatttcgttg attcccaccc ccctttccaa ctccacaaat tgtccaatct cgttttccat 660 ttgggagaat ctgcatgtcg actacataaa gcgaccggtg tccgaaaaga tctgtgtagt 720 tttcaacatt ttgtgctccc cccgctgttt gaaaacgggg gtgagcgctc tccggggtgc 780 gaattcgtgc caattcctt tcaccctgcc tattgtagac gtcaacccgc atctggtgcg 840 aatatagcgc acccccaatg atcacaccaa caattggtcc acccctcccc aatctctaat 900 attcacaatt cacctcacta taatacccc tgtcctgctc ccaaattcttt ttttccttct 960 tccatcagct actagctttt atcttattta cttacgaaa 1000 <210> 3 <211> 1000 <212> DNA <213> Komagataella pastor <400> 3 atgttggtat tgtgaaatag acgcagatcg ggaacactga aaaataacag ttattattcg 60 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 120 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 180 tgcaaacgca ggacctccac tcctctctc ctcaacaccc acttttgcca tcgaaaaacc 240 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 300 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgtta 360 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 420 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 480 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 540 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 600 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 660 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacccgct 720 ttttggatga ttgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 780 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 840 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca tttagctt 900 actttcataa ttgcgactgg ttccaattga caagcttttg atttaacga cttttaacga 960 caacttgaga agatcaaaaa acaactaatt attcgaaacg 1000 <210> 4 <211> 1000 <212> DNA <213> Komagataella pastor <400> 4 aaatggcaga aggatcagcc tggacgaagc aaccagttcc aactgctaag taaagaagt 60 gctagaacgaa ggagacttca gaggtgaaaa gtttgcaaga agagagctgc gggagaataa 120 ttttcaattt areacttga gtgcgtccat attcgtgtac gtgtccaact gttttccatt 180 acctaagaaa aacaataaga ttaaaaagat aaacccaatc gggaaacttt agcgtgccgt 240 ttcggattcc gaaaaacttt tggagcgcca gatgactatg gaagaggag tgtaccaaaa 300 tggcaagtcg ggggctactc accggatagc caatacattc tctaggaacc agggaatgaat 360 ccaggttttt gttgtcacgg taggtcaagc attcacttct taggaatatc tcgttgaaag 420 ctacttgaaa tcccattggg tgcggaacca gcttctaatt aaatagttcg atgatgttct 480 ctaagtggga ctctacggct caaacttcta cacagcatca tcttagtagt cccttcccaa 540 aacaccattc taggtttcgg aacgtaacga aacaatgttc ctctcttcac attgggccgt 600 tactctagcc ttccgaagaa ccaataaaag ggaccggctg aaacgggtgt ggaaactcct 660 gtccagttta tggcaaaggc tacagaaatc ccaatcttgt cgggatgttg ctcctcccaa 720 acgccatatt gtactgcagt tggtgcgcat tttagggaaa atttacccca gatgtcctga 780 ttttcgaggg ctacccccaa ctccctgtgc ttatacttag tctaattcta ttcagtgtgc 840 tgacctacac gtaatgatgt cgtaacccag ttaaatggcc gaaaaactat ttaagtaagt 900 ttatttctcc tccagatgag actctccttc ttttctccgc tagttatcaa actataaacc 960 tattttacct caaatacctc caacatcacc cacttaaaca 1000 <210> 5 <211> 160 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 5 cggggtgtct cactggtgtc cgccaataag aggagccggc aggcacggag tctacatcaa 60 gctgtctccg atacactcga ctaccatccg ggtctctcag agaggggaat ggcactataa 120 ataccgcctc cttgcgctct ctgccttcat caatcaaatc 160 <210> 6 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 6 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagggggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 7 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 7 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccag ccgggtctct cagagagggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 8 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 8 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct ccgagagggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 9 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 9 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cacagagggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 10 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 10 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagcgagggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 11 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 11 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagacagggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 12 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic promoter <400> 12 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagcgggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 13 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 13 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagacggg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 14 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 14 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagcgg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 15 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 15 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagaggcg aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 16 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 16 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggc aatggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 17 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 17 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg catggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 18 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 18 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg actggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 19 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 19 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aacggcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 20 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 20 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aatcgcacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 21 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 21 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aatgccacta taaataccgc ctccttgcgc 600 tctctgcctt catcaatcaa atc 623 <210> 22 <211> 648 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 22 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aatggcacta tataaactgg tgataattcc 600 ttcgttctga gttccatctc atactcaaac tatattaaaa ctacaaca 648 <210> 23 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Promoter <400> 23 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aatggcacta taaatacaag acgagtgcgt 600 ccttttctag actcacccat aaacaaataa tcaataaat 639 <210> 24 <211> 623 <212> DNA <213> Artificial sequence <220> <223> Synthetic promoter <400> 24 aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60 agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120 cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180 ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240 caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300 cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360 aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420 ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480 gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540 cgactaccat ccgggtctct cagagagggg aatggcacta taaatactgc ctacttgtcc 600 tctattcctt catcaatcac atc 623 BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a bar graph showing the signal obtained from the reporter protein produced under the control of the promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated promoter variants.

[0053] Figure 2 is a bar graph showing the signal obtained from a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 2 and its truncated promoter variants as a comparative example with the signal obtained from a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated promoter variants.

[0054] Figure 3 is a bar graph showing the signal obtained from a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 3 and its truncated promoter variants, as a comparative example with the signal obtained from a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated promoter variants.

[0055] Figure 4 is a bar graph showing the signal obtained from a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 4 and its truncated promoter variants, as a comparative example with the signal obtained from a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated promoter variants.

[0056] Figure 5 is a bar graph showing the amount of volumetric enzyme activity as a reporter signal obtained from the recombinantly produced phosphatase (UniProtKB entry: P11491) under the control of the promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated promoter variants.

[0057] Figure 6is a bar graph showing the amount of volumetric enzyme activity as a reporter signal obtained from recombinantly produced alcohol dehydrogenase under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and truncated promoter variants thereof.

[0058] Figure 7 is a bar graph showing the amount of volumetric enzyme activity as a reporter signal obtained from recombinantly produced peroxidase (UniProtKB entry: P00433) under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated promoter variants.

[0059] Figure 8 is a graph showing the amount of volumetric enzyme activity as a reporter signal generated under the control of the promoter having the nucleotide sequence of SEQ ID NO: 1 throughout the culture process.

[0060] Fig. 9 is a graph showing the amount of volumetric enzyme activity produced as a reporter signal under the control of a truncated variant of a promoter having the nucleotide sequence of SEQ ID NO: 1 throughout the culture process.

[0061] Fig.10 is a graph showing the amount of bulk enzyme activity as a reporter signal produced by several strains, wherein the reporter is produced under the control of a truncated variant of a promoter having the nucleotide sequence of SEQ ID NO: 1 throughout the culture process. Detailed Description

[0062] refer to Figure 1-4 , shows a bar graph of the signal obtained from a reporter protein produced under the control of a promoter having a nucleotide sequence of any one of SEQ ID NOs: 1-4 and truncated variants thereof. Shown is the median with standard deviation obtained from at least nine different transformed strains for each promoter construct. Although it was surprisingly found that the promoter having a nucleotide sequence of SEQ ID NO: 1 tolerated a significant degree of truncation as short as 140 bp in length ( Figure 1 ), but the promoter of the Pichia pastoris ADH2 gene encoding alcohol dehydrogenase 2 of Pichia pastoris (SEQ ID NO: 2; Figure 2 ) and the promoter of the Pichia pastoris AOX1 gene encoding alcohol oxidase 1 of Pichia pastoris (SEQ ID NO: 3; Figure 3 ) and the promoter of the Pichia pastoris FDH1 gene encoding formate dehydrogenase 1 of Pichia pastoris (SEQ ID NO: 4; Figure 4) were unable to achieve this approach without significant loss of promoter activity. Thus, this tolerance of truncation of the promoter having the nucleotide sequence of SEQ ID NO: 1 appears to be the exception rather than the rule.

[0063] refer to Figure 5-7 , shows a bar graph of the amount of volumetric enzyme activity as a reporter signal from three different reporter enzymes produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and its truncated variants. Shown is a bar graph representing the average values ​​with standard deviations obtained from at least nine different transformed strains when cultured in 96-deep-well plates. For phosphatase ( Figure 5 )、Alcohol dehydrogenase( Figure 6 ) and peroxidase ( Figure 7 ), observed with esterase ( Figure 1 Based on these results, it can be concluded that the expression of the truncated variant of the promoter having the nucleotide sequence of SEQ ID NO: 1 occurs independently of the protein encoded in the open reading frame whose expression is regulated by the promoter.

[0064] refer to Figure 8-10 , showing that as under the control of a promoter having a nucleotide sequence of SEQ ID NO: 1 ( Figure 8 ) or under the control of a truncated variant thereof ( Fig. 9 and 10 ) is a graph of the volumetric enzyme activity of the reporter signal generated throughout the bioreactor culture. The strain carrying the expression cassette containing the 280 bp long truncated promoter having the nucleotide sequence of SEQ ID NO: 1 ( Fig. 9 ) and several different strains carrying an expression cassette comprising a 160 bp long truncated promoter having the nucleotide sequence of SEQ ID NO: 1 ( Fig.10 ) was found to produce at least the same amount of target enzyme as a strain carrying an expression cassette comprising a 623 bp long non-truncated promoter having the nucleotide sequence of SEQ ID NO: 1. Example

[0065] Example 1: Expression studies using truncated eukaryotic promoters in Pichia pastoris

[0066] To assess whether eukaryotic promoters can be truncated without substantial loss of promoter activity, full-length polynucleotide fragments of selected promoters were either ordered as synthetically produced fragments or amplified directly from the respective eukaryotic organisms. In particular, the Ogataea polymorpha FMD promoter (SEQ ID NO: 1), the Pichia pastoris ADH2 promoter (SEQ ID NO: 2), the Pichia pastoris AOX1 promoter (SEQ ID NO: 3), and the Pichia pastoris FDH1 promoter (SEQ ID NO: 4) were studied. To obtain truncated variants of these promoters, oligonucleotide primers were designed for amplification by polymerase chain reaction (PCR), wherein a reverse primer was designed to bind to the 3'-end of the promoter and a forward primer was designed to bind within the full-length promoter sequence, depending on the desired final length of the PCR product. For Ogataea polymorpha FMD promoter, truncated promoters with 612, 610, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 158, 156, 154, 152, 150, 150, 130, 120, 110, 100 bp were tested. For Pichia pastoris ADH2 promoter, Pichia pastoris AOX1 promoter and Pichia pastoris FDH1 promoter, variants with 1000, 500, 350, 280, 160, 140 bp were tested. Full-length promoters or their truncated variants were cloned into expression cassettes for recombinant gene expression in Pichia pastoris using standard cloning methods of the prior art (e.g., JM Cregg, Pichia Protocols, 2nd edition, ISBN-10: 1588294293, 2007; J. Sambrook et al., 2012. Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor). These expression cassettes include full-length promoters or their truncated variants, open reading frames encoding secretory reporter proteins, transcription terminators (Pichia pastoris AOX1 transcription terminators) and expression cassettes of antibiotic selection markers. To quantify the amount of target protein produced by the corresponding full-length promoter or its truncated variants, esterase (UniProtKB entry: D2D3B6) was used as reporter protein.Esterase secretion from transformed cells was achieved using the prepro peptide of Saccharomyces cerevisiae mating factor alpha fused to the N-terminus of the esterase, which replaced the first 47 amino acids of the full-length esterase polypeptide.

[0067] By electroporation, basically follow the protocol of Lin-Cereghino et al. (Biotechniques. 2005. 38 (1): 44-48), use any one of the generated expression cassettes to transform the Pichia pastoris wild-type strain. However, any alternative protocol for transforming Pichia pastoris is also applicable. After transformation, the cells are incubated on agar plates containing appropriate antibiotics to select for transformed strains.

[0068] In 96 deep well plates (Hamilton 2.2mL PP deep well plates), the microculture of at least 9 transformed strains for each transformed expression cassette was performed with a scheme similar to that described by Weis et al. (FEMS Yeast Res. 2004.5 (2): 179-189). Cells were grown in Kuhner ISF1-X Climo-Shaker (throw 50mm) at 28°C, 320rpm, 90% humidity for approximately 60 hours in 450 μL YPD (1% w / v yeast extract, 2% w / v peptone, 2% w / v glucose) per well. In the case of strains transformed with expression cassettes comprising AOX1 promoter or FDH1 promoter, expression was induced by adding 100 μL YPM0.5% (1% w / v yeast extract, 2% w / v peptone, 0.5% w / v methanol) per well 60 hours after inoculation. In the case of strains transformed with expression cassettes containing the FMD promoter or the ADH1 promoter, 100 μL of YPD was added to each well 60 hours after inoculation. After about 120 hours of cultivation, the 96-deep well plates were centrifuged to separate the biomass from the supernatant. The supernatant was used to analyze the secreted target reporter protein produced. In order to make the plates comparable, a stable esterase-producing strain was also cultured in at least four wells of each plate as a positive control and calibration strain.

[0069] The amount of target reporter protein secreted into the supernatant was determined by quantifying the amount of volumetric esterase activity in the supernatant: the enzyme activity was quantified by measuring the hydrolysis of the esterase substrate 4-nitrophenyl 2-(trimethylsilyl)ethyl carbonate, which was measured photometrically by following the increase in absorbance at 405 nm as described by Ruth et al. (Microb Cell Fact. 2014. 13: 120). For the non-truncated promoters of FMD, ADH2, AOX1 and FDH1 and their truncated promoter variants, the amount of signal of the reporter protein generated by the tested expression cassettes is shown in Figure 1 , 2, 3 and 4. It is well known in the art that Pichia pastoris transformants tend to show clonal variation (e.g., Vogl et al., Appl Environ Microbiol. 2018. 84 (6): e02712-17). Compared with other expression systems (e.g., E. coli), it is therefore necessary to test not only one transformed strain, but also multiple strains, as described herein. It is noteworthy that when the promoter is shorter, clonal variation is found to be more pronounced, as reflected by the higher standard deviation, and as seen, for example, for truncated variants of the FMD promoter with 160 bp or 150 bp compared to longer variants with 600 bp. Without wishing to be bound by theory, it is believed that this effect is due to the greater influence of the integration site of the shorter promoter.

[0070] Use the median value of the enzymatic activity produced by at least nine transformed strains for each expression cassette to calculate the relative expression efficiency of corresponding promoter or promoter variant. Relative expression efficiency is calculated by dividing the median value of the enzymatic activity produced by the length of the promoter for controlling the expression of the target reporter gene. In the case of the FMD promotor, it is found that the promoter variant with the truncation of a length of 140 to 610bp has at least 5% increase of relative expression efficiency compared with the full-length promoter, see Table 1 below. No matter which pichia pastoris strain (K.phaffii CBS 7435, K.phaffii GS115 or K.pastoris CBS 704) is transformed, substantially the same result is obtained.

[0071] Table 1: Relative expression efficiency (REE) of the full-length FMD promoter (623 bp) and its truncated variants.

[0072]

[0073] To demonstrate the mutational stability of the truncated promoter variants, mutant variants having at least 80% sequence identity with a promoter truncated to a length of 160 bp from the 5'-end of the nucleotide sequence of SEQ ID NO: 1 were tested. The mutant variants were truncated to a length of 160 bp from the 5'-end of any one of the nucleotide sequences of SEQ ID NO: 6-24, whereby these truncated mutant variants had 80.74% to 99.38% sequence identity with a promoter truncated to a length of 160 bp from the 5'-end of the nucleotide sequence of SEQ ID NO: 1. For all mutant variants, the same effects were observed as those observed for the truncated variants of the promoter of the nucleotide sequence of SEQ ID NO: 1. Despite being nearly a quarter the length, the reporter signal obtained was comparable to that of the non-truncated promoter, and thus the new truncated promoter achieved at least a 5% increase in relative expression efficiency.

[0074] In contrast, truncated variants of any of the promoters of ADH2, AOX1 or FDH1 did not result in increased relative expression efficiency compared to the respective full-length promoters (each 1000 bp in length), see Table 2 below.

[0075] Table 2: Relative expression efficiency (REE) of the full-length promoters (1000 bp each) of ADH2, AOX1 or FDH1 and their truncated variants.

[0076]

[0077] In conclusion, it was surprisingly found that only certain truncated variants of the O. polymorpha FMD promoter, but not truncated variants of any other tested eukaryotic promoter, resulted in increased relative expression efficiency in Pichia pastoris compared to the non-truncated full-length promoter.

[0078] Example 2: Recombinant production of different reporter proteins using promoters with increased relative expression efficiency in small-scale cultures

[0079] The regulation of expression by the promoter occurs at the DNA level, in particular at the level of DNA upstream of the open reading frame whose expression is regulated by the promoter. Therefore, one skilled in the art would not expect substantial differences in promoter activity when the expression of different open reading frames is regulated. In other words, it would not be expected that the production of a protein different from the esterase described in Example 1 would produce substantially different results in terms of promoter activity or functionality compared to the production of the esterase described in Example 1. However, in order to experimentally verify the general applicability of truncated promoter variants with increased relative expression efficiency for the expression of any recombinant target gene, other expression cassettes carrying open reading frames encoding phosphatases, alcohol dehydrogenases or peroxidases were generated.

[0080] The expression of genes containing open reading frames encoding phosphatases, alcohol dehydrogenases or peroxidases was designed to be regulated by the full-length O. polymorpha FMD promoter (623 bp) or truncated variants thereof of 500 bp, 350 bp, 280 bp, 160 bp or 140 bp in length. The transformation of Pichia pastoris and cultivation in 96 deep-well plates were performed essentially as described in Example 1 for this promoter.

[0081] To determine the amount of phosphatase produced, a p-nitrophenol (pNPP) assay was performed essentially as described in Takai and Mieskes (Biochem J. 1991. 275 (1): 233-239), wherein the assay solution contained 10 mM pNPP, 1 mM MgCl2, 0.1 mM ZnCl2, 40 mM Tris-HCl, pH 8.1. To determine the amount of alcohol dehydrogenase produced, the enzyme activity was determined essentially as described in WO2016154640A1. A Pichia pastoris strain producing alcohol dehydrogenase was cultured in YPD containing an additional 1 mM pyrroloquinoline quinone and 1 mM calcium chloride. To determine the amount of alcohol dehydrogenase produced, the enzyme activity was determined by mixing 5 μL of the clarified culture supernatant containing the produced enzyme with 280 μL of assay buffer (20 mM potassium ferrocyanide (III), 50 mM Tris-HCl, pH 7.5, 50 ppm deoxynivalenol). The assay reactions were incubated at 30°C for 60 minutes. 50 μL samples were periodically withdrawn from the reactions and analyzed by HPLC. To determine the amount of peroxidase produced, the substrate ABTS (CAS No.: 30931-67-0) was exchanged with TMB (3,3',5,5'-tetramethylbenzidine; CAS No.: 54827-17-7) similarly to the measurement of enzyme activity by Krainer et al. (J Biotechnol. 201. 233: 181-189); product formation was followed at 653 nm. For phosphatase, alcohol dehydrogenase and peroxidase, the amount of reporter enzyme signal generated by the tested promoter variants is shown at Figure 5 , 6 and 7.

[0082] The relative expression efficiency was determined in a similar manner to that described in Example 1 and is shown in Table 3.

[0083] Table 3: Relative expression efficiency (REE) of phosphatases, alcohol dehydrogenases or peroxidases produced under the control of the FMD full-length promoter (623 bp) or its truncated variants (500 bp, 350 bp, 280 bp, 160 bp, 140 bp).

[0084]

[0085] In conclusion, the functionality and applicability of truncated promoter variants of the FMD wild-type promoter were found to be not limited to a single open reading frame but rather well suited to modulate the expression of any target gene.

[0086] Example 3: Recombinant protein production using promoters with increased relative expression efficiency in bioreactor culture

[0087] It is known that small-scale cultivation of microorganisms, for example in 24 or 96 well plates or in shake bottles, tends to produce different data compared to the cultivation carried out in bioreactors. Without wishing to be bound by theory, it is believed that these differences arise from different conditions between the cultivation types regarding nutrient supply and pH and temperature control, oxygen supply and stirring. Although small-scale cultivation is very suitable for, for example, testing the general concept of scientific hypotheses and screening a large number of microbial transformants for producing bacterial strains, bioreactor cultivation is the first choice for evaluating production performance data.

[0088] In order to demonstrate the applicability of the truncated variants of the O. polymorpha FMD promoter not only in small-scale cultivation but also in large-scale cultivation, a Pichia pastoris strain carrying an expression cassette containing a truncated FMD promoter variant was cultivated in a bioreactor and compared with a strain carrying an expression cassette containing the non-truncated FMD promoter as a benchmark. As a reporter enzyme, the esterase of Example 1 was used.

[0089] The pre-culture was cultured in a 500 mL baffled shake flask at 180 rpm and 28 ° C for 24 h in YPG medium containing 10 g / kg yeast extract, 20 g / kg soy peptone, 0.3 mL / kg defoamer Struktol J650 and 40 g / kg glycerol. Before inoculation, 500 mL batch culture medium as reported by Krainer et al. (Microb Cell Fact. 2015.14:4) was sterilized in a 1 L glass bioreactor container (DASGIP, Switzerland). A 12-hour batch phase was performed at 28 ° C, and the minimum saturation of 30% of dissolved oxygen during the entire culture process was controlled by the cascade setting of the agitator speed, air flow rate and oxygen flow rate. 13 hours after inoculation, the glycerol feed rate that started to increase linearly at 6.85 g / L / h continued for 17 hours to the maximum glycerol feed rate of 8.72 g / L / h at the end. Then, the feed rate is set to promote a constant growth rate of 0.014 / h, and the temperature is set to 24°C. This feed rate continues until 120 hours after inoculation. The feed medium contains 696g / L glycerol, 4.4g / L trace element solution (Krainer et al., Microb Cell Fact. 2015.14:4), 200ppm biotin and 0.3mL / kg defoamer Struktol J650. Supernatant is sampled regularly by centrifuging culture broth at 4000rpm in a desktop centrifuge (Eppendorf Centrifuge 5810R, Eppendorf, Germany), and further used for enzyme activity determination.

[0090] Figure 8The amount of reporter signal produced by the strain carrying the expression cassette containing the non-truncated FMD promoter (i.e., the promoter having the nucleotide sequence of SEQ ID NO: 1) during the entire culture process is shown in FIG. After about 119 h of culture, the volumetric enzyme activity obtained was set to 100% as the reporter signal. The strain carrying the expression cassette containing the promoter truncated to a length of 280 bp from the 5'-end of the nucleotide sequence of SEQ ID NO: 1 produced 115% of the reporter signal after about 119 h of culture ( Fig. 9 ). Several different strains carrying an expression cassette comprising a promoter truncated to 160 bp in length from the 5'-end of the nucleotide sequence of SEQ ID NO: 1 were cultured and produced reporter molecule signals of 112.6%, 160.4%, 139.1%, 149.2%, 107.0%, 110.1% and 134.5% after about 119 h of culture ( Fig.10 ). It was concluded that the relative expression efficiency (REE) of the 623 bp promoter was 0.161% / bp, the REE of the 280 bp promoter was 0.411% / bp, and the REE of the 160 bp promoter was 0.699 to 1.003% / bp. Thus, the REE of the 280 bp promoter was found to be 2.56 times that of the non-truncated 623 bp promoter, and the REE of the 160 bp promoter was found to be 4.16-6.25 times that of the REE of the 623 bp promoter. Surprisingly, all strains carrying the truncated promoters were found to produce more target enzyme than the non-truncated promoter.

[0091] Example 4: Promoter induction by glycerol or glucose feeding

[0092] In WO2017 / 109082A1 and Vogl et al. (AMB Express.2020.10(1):38), it is taught that in Pichia pastoris, expression from the 623bp wild-type HpFMD promoter is activated when glucose or glycerol is exhausted, i.e. induced. In order to study whether the truncated promoter described herein will follow this doctrine, after 17 hours of a linearly increasing glycerol feed rate starting with 6.85g / L / h as described in Example 3, the actual consumption of glucose / glycerol without feeding was tested 30 hours after the start of culture, and different constant glucose or glycerol feed rates of 0.07mmol glycerol / g CDW / h or 0.04mmol glucose / g CDW / h to 0.48mmol glycerol / g CDW / h or 0.25mmol glucose / g CDW / h. Otherwise, culture was carried out substantially as described in Example 3. In addition, these conditions were also tested for the untruncated wild-type FMD promoter. As the reporter enzyme, the esterase of Example 1 was used.

[0093] The reporter molecule signals from the produced reporter enzymes obtained from different glucose or glycerol feed rates as described above are shown in Table 4 as examples, where the expression was regulated by the truncated FMD promoter with 280 bp.

[0094] Table 4: Volumetric enzyme activity obtained after approximately 60 hours of cultivation, where promoter induction was promoted by depletion of glucose / glycerol (ie 0.00 mmol / g CDW / h) or at different rates of feeding of glucose or glycerol. The reporter signal obtained under glucose depletion was set as 100%.

[0095]

[0096]

[0097] Surprisingly, even a positive correlation between feed rate and specific production rate can be observed: higher feed rates lead to higher volumes and specific activities. The same trend was found when expression was regulated by the 623 bp promoter, and when expression was regulated by promoter variants with 500 bp, 350 bp, 280 bp or 160 bp truncations. Based on this, it was concluded that surprisingly and in contrast to previous publications, the best mode for operating the wild-type FMD promoter or its truncated variants in Pichia pastoris is not activation by depletion of glucose or glycerol, but by feeding at least 0.07 mmol Gly / g CDW / h or 0.04 mmol Glu / g CDW / h.

[0098] Example 5: Recombinant gene expression with a truncated eukaryotic promoter in Yarrowia lipolytica

[0099] To test whether expression by the truncated FMD promoter is limited to Pichia pastoris strains (as found for K. phaffii CBS 7435, K. phaffii GS115 and K. pastoris CBS 704), Yarrowia lipolytica was tested. Transformation of the Yarrowia lipolytica CBS7504 strain using the expression cassette described in Example 1 was performed by electroporation according to the protocol of Lin-Cereghino et al. (Biotechniques. 2005. 38 (1): 44-48). Nevertheless, alternative protocols for transforming Yarrowia lipolytica can be used to transform this yeast with the expression cassette. Subsequently, cells were cultured and selected on agar plates containing appropriate antibiotics for selecting positive transformants. Essentially as described by Weis et al. (FEMS Yeast Res. 2004. 5 (2): 179-189), cultivation of at least 50 positive transformants was performed in 96-well plates: cell growth was achieved in 450 μL YPD (1% w / v yeast extract, 2% w / v peptone, 2% w / v glucose) per well at 28° C., 320 rpm, 90% humidity in a Kuhner ISF1-X Climo-Shaker for 60 hours, similar to Example 1. After 60 hours of cultivation at 320 rpm and 28° C., cells were fed twice with 100 μL YPD per well, 60 hours and 85 hours after inoculation, and then cultivated for another 24 hours before being harvested by centrifugation. The reporter protein level in the supernatant was determined by monitoring the volumetric esterase activity of the hydrolysis of 4-nitrophenyl 2-(trimethylsilyl)ethyl carbonate. After the hydrolysis reaction, the increase in absorbance at 405 nm was measured photometrically, essentially as described by Ruth et al. (Microb Cell Fact. 2014.13:120). Similar to the recombinant production controlled by the truncated FMD promoter in the Pichia pastoris strain, when these promoters were used to regulate the production of recombinant proteins in Yarrowia lipolytica, essentially the same increase in REE was found. It was found that not only Pichia pastoris, but also Yarrowia lipolytica also showed the ability to produce and secrete the target reporter protein, whose production was controlled by the truncated FMD promoter.

Claims

1. A promoter or promoter variant, characterized in that The promoter is truncated to a length of 140-610 base pairs from the 5'-end of the nucleotide sequence of SEQ ID NO: 1; The promoter variant has at least 80% identity with the promoter, and the promoter variant is truncated to a length of 140-610 base pairs from the 5'-end of the nucleotide sequence of any one of SEQ ID NOs: 6-24.

2. The promoter or promoter variant of claim 1, wherein the promoter is truncated to a length of 140-560 base pairs from the 5'-end of the nucleotide sequence of SEQ ID NO: 1, wherein the promoter variant has at least 80% identity with the promoter.

3. The promoter or promoter variant of claim 1, wherein the promoter is truncated to a length of 160-560 base pairs from the 5'-end of the nucleotide sequence of SEQ ID NO: 1, wherein the promoter variant has at least 80% identity with the promoter.

4. The promoter or promoter variant of claim 1, wherein the promoter is truncated to a length of 160-500 base pairs from the 5'-end of the nucleotide sequence of SEQ ID NO: 1, wherein the promoter variant has at least 80% identity with the promoter.

5. The promoter or promoter variant of any one of claims 1-4, wherein the promoter variant is at least 90% identical to the promoter.

6. The promoter or promoter variant of any one of claims 1-4, wherein the promoter variant is at least 95% identical to the promoter.

7. The promoter or promoter variant of any one of claims 1-4, wherein the promoter variant is at least 97% identical to the promoter.

8. The promoter or promoter variant of any one of claims 1-4, wherein the promoter variant is at least 99% identical to the promoter.

9. The promoter or promoter variant of claim 1, wherein the relative expression efficiency of the promoter or promoter variant is at least 5% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO:

1.

10. The promoter or promoter variant of claim 1, wherein the relative expression efficiency of the promoter or promoter variant is at least 10% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO:

1.

11. The promoter or promoter variant of any one of claims 1 and 9, wherein the promoter or promoter variant is contained in an expression cassette, wherein the expression cassette further comprises a nucleotide sequence encoding a polypeptide and optionally a transcription terminator sequence, and wherein the promoter or promoter variant is operably linked to the nucleotide sequence encoding the polypeptide.

12. The promoter or promoter variant of any one of claims 1 and 9, wherein the promoter or promoter variant is contained in a plasmid, and wherein the promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide.

13. The promoter or promoter variant of any one of claims 1 and 9, wherein the promoter or promoter variant is contained in a yeast cell, and wherein the promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide, and the yeast cell is a Pichia pastoris cell or a Yarrowia lipolytica cell.

14. The promoter or promoter variant of claim 13, wherein the yeast cell is a Pichia pastoris cell.

15. A method for recombinantly producing a target polypeptide, comprising the steps of: a) introducing an expression cassette comprising a nucleotide sequence encoding the target polypeptide into at least one yeast cell; b) producing a yeast cell culture by propagating the at least one yeast cell; c) subjecting the yeast cell culture to conditions suitable for expression of the nucleotide sequence encoding the target polypeptide; and d) isolating the produced target protein from the yeast cell culture, Features The expression cassette further comprises the promoter or promoter variant of any one of claims 1 or 9 operably linked to the nucleotide sequence encoding the target polypeptide, and The yeast cell is a Pichia pastoris cell or a Yarrowia lipolytica cell.

16. The method of claim 15, wherein the yeast cell is a Pichia pastoris cell.

17. The method of claim 15, characterized in that Suitable conditions for the expression of the nucleotide sequence encoding the target polypeptide are a fed-batch process.

18. The method of claim 17, characterized in that The fed-batch process is operated with a glucose feed rate of at least 0.04 mmol glucose per gram cell dry weight per hour (mmol Glu / g CDW / h) or a glycerol feed rate of at least 0.07 mmol glycerol per gram cell dry weight per hour (mmol Gly / g CDW / h).

19. The method of claim 18, wherein the fed-batch process is operated with a glucose feed rate of at least 0.11 mmol Glu / g CDW / h or a glycerol feed rate of at least 0.21 mmol Gly / g CDW / h.

20. The method of claim 18, wherein the fed-batch process is operated with a glucose feed rate of at least 0.18 mmol Glu / g CDW / h or a glycerol feed rate of at least 0.35 mmol Gly / g CDW / h.

21. A method for increasing the relative expression efficiency of a nucleotide sequence having promoter activity, The method comprises the following steps: A truncated promoter is provided by truncating a nucleotide sequence selected from any one of SEQ ID NOs: 1, 6 to 24 from the 5'-end to a length of 140 to 610 base pairs.

22. The method of claim 21, wherein the method comprises the following steps: A truncated promoter is provided by truncating a nucleotide sequence selected from any one of SEQ ID NOs: 1, 6 to 24 from the 5'-end to a length of 140 to 560 base pairs.

23. The method of claim 21, wherein the method comprises the steps of: A truncated promoter is provided by truncating a nucleotide sequence selected from any one of SEQ ID NOs: 1, 6 to 24 from the 5'-end to a length of 160 to 560 base pairs.

24. The method of claim 21, wherein the method comprises the steps of: A truncated promoter is provided by truncating a nucleotide sequence selected from any one of SEQ ID NOs: 1, 6 to 24 from the 5'-end to a length of 160 to 500 base pairs.

25. The method of claim 21, wherein the relative expression efficiency of the truncated promoter is at least 5% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO:

1.

26. The method of claim 21, wherein the relative expression efficiency of the truncated promoter is at least 10% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO:

1.

27. The method of claim 21, wherein the relative expression efficiency of the truncated promoter is at least 20% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO:

1.

28. The method of claim 21, wherein the relative expression efficiency of the truncated promoter is at least 50% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO:

1.

29. The method of claim 21, wherein the relative expression efficiency of the truncated promoter is at least 100% higher than the relative expression efficiency of the promoter having the nucleotide sequence of SEQ ID NO: 1.

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