Recombinant protein expression

Abstract

The present invention relates to inducible promoters, and to methods of protein production, wherein recombinant cells are cultivated in a cultivation medium comprising acetate. The invention further relates to formulations and compositions obtained with the method of the invention.

Description

[0001] RECOMBINANT PROTEIN EXPRESSION

[0002] Reference to a Sequence Listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] Background of the Invention

[0005] Field of the Invention

[0006] The present invention relates to inducible promoters, and to methods of protein production, wherein recombinant cells are cultivated in a cultivation medium comprising acetate. The invention further relates to formulations and compositions obtained with the method of the invention.

[0007] Description of the Related Art

[0008] Carbon capture and utilization technologies have gained significant attention for their potential to address environmental challenges associated with greenhouse gas emissions.

[0009] Capture and conversion of CO2 into different substrates, e.g., acetate, and the subsequent use of these substrates as carbon source for recombinant protein production, serves a dual purpose of (i) reducing greenhouse gas emissions, and (ii) providing low-cost protein to feed the earth’s growing population and the therewith associated protein demand.

[0010] Fungal systems are employed extensively in biotechnology for producing a variety of proteins, including pharmaceuticals, industrial enzymes, food / feed protein, and other biologically relevant molecules.

[0011] Current methodologies predominantly utilize glucose as the main carbon source for fungal fermentation processes. While effective, this approach is associated with several significant limitations. The reliance on glucose not only contributes to the consumption of agricultural resources but also has implications for global carbon emissions. As large-scale fermentation processes consume vast quantities of glucose, they indirectly exacerbate the environmental footprint of protein production.

[0012] Moreover, the extensive use of glucose in fermentation leads to an increased output of CO2, contributing further to the environmental challenges posed by anthropogenic greenhouse gas emissions. As humanity seeks sustainable solutions to mitigate climate change, there is a growing need to explore alternative carbon sources that can be derived from captured CO2, thereby turning a waste product into a valuable resource.

[0013] It is therefore an objective of the present disclosure to overcome the above limitations at least in part by developing a method contributing to a lowered CO2 footprint during product fermentation. Summary of the Invention

[0014] As provided herewith, acetate is employed as carbon substrate for the cultivation of fungal cells, offering a cost-effective and environmentally friendly alternative to traditional carbon sources, e.g., glucose and / or maltose. Fungal cells, known for their efficient protein synthesis machinery, are harnessed to produce recombinant proteins of interest in a highly controlled and scalable manner. Using acetate as carbon source for cultivating recombinant cells has previously been associated with low protein yield when compared to well-established protocols, e.g., using glucose as sole carbon source.

[0015] The present invention solves the problem of low protein expression by a novel method using acetate-inducible promoters. The inventors have identified at least 1479 Aspergillus promoter sequences which are associated with increased mRNA transcript levels when being cultivated on acetate (Example 7). The inducible promoters of the present invention may for example be utilized as part of a synthetic expression system (SES) which, as shown in the Examples, resulted in high protein expression during acetate-based fermentation. A schematic drawing showing the SES and its elements is shown in Figure 2. Alternatively, the inducible promoters of the invention may be operably linked to a polynucleotide encoding a polypeptide of interest (POI), for example in an expression system not relying on transcription factors or synthetic promoters. The use of acetate-inducible promoters can offer several advantages, including reduced metabolic burden on host cells, minimized catabolite repression, and potentially improved yield and efficiency of recombinant protein production.

[0016] Surprisingly, using acetate as carbon source the novel expression system provides protein yield which is comparable or increased relative to protein yield obtained by using glucose as carbon source (Examples 1 , 2, and 5). Even more surprising, some of the identified promoters achieve high protein expression both when using acetate as carbon source, and when using glucose as carbon source (Examples 1-2), allowing a high degree of flexibility depending on availabilities of each carbon source, and also allowing a mixing of the carbon sources if desired.

[0017] Furthermore, it was completely unexpected that fermentations using acetate as carbon source resulted in reduced viscosity of the fermentation broth compared to fermentations using maltose or glucose as carbon source (Example 3). Reduced viscosity during the fermentation of recombinant proteins using fungal cells offer several key advantages. Improved mass transfer ensures better delivery of oxygen and nutrients, promoting healthier cell growth and higher protein yields. It also facilitates downstream processing steps like filtration and centrifugation, reducing operational costs and time while preventing equipment clogging. Lower viscosity enhances scalability by maintaining efficient mixing and aeration in large bioreactors. Additionally, it minimizes shear stress on cells, improving cell viability and stability. Overall, reduced viscosity leads to a more efficient, cost-effective, and scalable production process for recombinant proteins. Also, it was totally unexpected that the SES and the promoters of the present invention can increase POI mRNA transcript levels so that up to around 28% of the cell’s total mRNA consist of the POI mRNA (Example 4). As also shown in Example 4, by a factor of circa 10-30, the promoters and SES of the invention outperform mRNA levels of the supposedly strong promoter Ptef which has become one of the de facto standards for POI expression in Aspergillus (Kitamoto et al. 1998, Appl Microbiol Biotechnol. 50:85-92; Rendsvig et al. 2019, Fungal Biol Biotechnol 6, 24).

[0018] It was observed that these high mRNA levels were stable throughout at least 144 hours of fermentation. Stable mRNA transcript levels of the product mRNA offer several advantages including consistent protein expression, enhanced protein yield, reduced variability, efficient resource utilization, and simplified process control.

[0019] Thus, in a 1staspect the invention relates to fungal host cells comprising in its genome: an acetate-inducible promoter operably linked to one or more first polynucleotide encoding one or more first polypeptide of interest (POI), wherein the one or more first polynucleotide is heterologous to the acetate-inducible promoter.

[0020] In a 2ndaspect the invention relates to fungal host cells comprising in its genome an inducible promoter operably linked to one or more first polynucleotide encoding one or more first POI, wherein the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide sequences of SEQ I D NO: 1.

[0021] In a 3rdaspect the invention relates to fungal host cells comprising in its genome an inducible promoter operably linked to one or more first polynucleotide encoding one or more first POI, wherein the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide sequences of SEQ ID NO: 2.

[0022] In a 4thaspect, the invention relates to a method of producing one or more POI wherein the cell according to any one of the 1st, 2nd, or 3rdaspect is cultivated under conditions conducive for the production of the one or more POI, preferably cultivated in a medium comprising at least 0.01 % v / v acetate.

[0023] In a 5thaspect, the invention relates to a whole broth formulation or cell culture composition comprising the cell according to any one of the 1st, 2nd, or 3rdaspect, or a whole broth formulation or cell culture composition obtained by the method of the 4thaspect.

[0024] In a 6thaspect the invention relates to the use of the cell according to any one of the 1st, 2nd, or 3rdaspect to produce a food or feed product and / or to produce the whole broth formulation or cell culture composition according to the 5thaspect.

[0025] In a 7thaspect, the invention relates to a polynucleotide comprising an inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the the polynucleotide sequence of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0026] In a 8thaspect, the invention relates to a nucleic acid construct or expression vector comprising the polynucleotide of the 7thaspect, wherein the inducible promoter is operably linked to one or more first polynucleotide encoding one or more first polypeptide of interest.

[0027] In a 9thaspect, the invention relates to a cell comprising in its genome the polynucleotide of the 7th, and / or the nucleic acid construct or expression vector of the 8thaspect.

[0028] Brief Description of the Drawings

[0029] Figure 1 shows an SDS gel of producing an endolase with the method of the invention.

[0030] Figure 2 shows a schematic drawing of the synthetic expression system (SES) in “OFF” configuration and in “ON” configuration.

[0031] Figure 3 shows pictures of reduced fermentation viscosities when using acetate as carbon source (left two reactors “1” and “2”), compared to when using maltose as carbon source (right two reactors “3” and “4”).

[0032] Figure 4 shows a SDS-PAGE gel for expression of 11 different POI using acetate as carbon source, compared to when using glucose as a carbon cource.

[0033] Definitions

[0034] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0035] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0036] Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0037] Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

[0038] Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0039] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0040] Extension: The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a TF, wherein the “extended” TF has DNA binding activity.

[0041] Fragment: The term “fragment” means a TF having one or more amino acids absent from the amino and / or carboxyl terminus of the mature TF, wherein the fragment has DNA binding activity.

[0042] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0043] Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0044] Acetate-inducible promoter: An acetate-inducible promoter in the context of the present invention refers to a specific DNA sequence that controls the initiation and regulation of transcription of an adjacent (operably linked) gene or set of genes in response to the presence of acetate in the environment. These promoters are characterized by their ability to become activated or significantly upregulated when acetate is available, leading to enhanced transcriptional activity and subsequent gene expression. In the context of recombinant protein production, an acetate-inducible promoter allows for the controlled expression of target genes in microbial or other host cells by utilizing acetate as the primary carbon source in the fermentation medium. This regulatory mechanism contrasts with traditional promoters that respond to common carbon sources such as glucose, maltose, or sucrose. Non-limiting examples for acetate-inducible promoters include the promoters of any one of SEQ ID NOs: 10, 1 , 2, and 11-1489.

[0045] Preferably, the acetate-inducible promoter refers to a segment of DNA that regulates the transcription of an mRNA transcript, where the promoter activity is increased in the presence of acetate compared to the promoter activity in the presence of glucose. Promoters classified as "acetate-inducible" include those promoters associated with mRNA transcripts that exhibit higher expression levels when cells are cultivated on acetate compared to their cultivation on glucose, as demonstrated by the differential gene expression analysis of Example 7.

[0046] In one embodiment the acetate-inducible promoter comprises or consist of the promoter from the acuD gene of Aspergillus nidulans (promoter of SEQ ID NO: 10), or the promoter from the acuE gene of Aspergillus nidulans (promoter of SEQ ID NO: 22).

[0047] In one embodiment the acetate-inducible promoter is induced by FacB, wherein FacB is encoded by the facB gene. FacB is a transcriptional activator of acetate utilization genes. FacB has a GAL4-type Zn(ll)2Cys6 zinc binuclear cluster DNA-binding domain which is required for DNA binding. For example, the acuD promoter of SEQ ID NO: 10 and the acuE promoter of SEQ ID NO 22 each comprise binding sites for FacB. FacB is also known as AcuA.

[0048] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0049] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

[0050] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and / or C-terminal processing (e.g., removal of signal peptide).

[0051] Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide.

[0052] Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0053] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0054] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

[0055] Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

[0056] Purified: The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0057] In one aspect, the term "purified" as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0058] Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0059] It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form ( / .e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0060] Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0061] Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g. depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheet or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solidliquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide. Sequence difference: The term "sequence difference", with respect to a polypeptide, means the percent of amino acid differences between a polypeptide and a given SEQ ID NO., and is calculated as follows:

[0062] (Different Residues x 100) / (Length of SEQ ID NO.) wherein the different residues comprise any substitution, deletion, or insertion (e.g., an extension at the N-terminus and / or C-terminus) in the sequence.

[0063] The term "sequence difference", with respect to a polynucleotide, means the percent of nucleic acid differences between a polynucleotide and a given SEQ ID NO., and is calculated as follows:

[0064] (Different Residues x 100) / (Length of SEQ ID NO.) wherein the different residues comprise any substitution, deletion, or insertion (e.g., an extension at the 5’-end and / or 3’-end) in the sequence.

[0065] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0066] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0067] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0068] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0069] (Identical Deoxyribonucleotides x 100) / (Length of Alignment- Total Number of Gaps in Alignment)

[0070] Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process. Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and / or 3' end of a mature polypeptide coding sequence.

[0071] Synthetic promoter: The term ’’synthetic promoter” or ”sTF-dependent promoter” means an engineered transcription factor-dependent promoter. sTF-dependent promoters are composed of a variable number of synthetic TF- binding sites linked to a core promoter. The number of binding sites in combination with a specific core promoter defines the level of expression of the target gene and it represents a significant improvement in expression level control compared to the systems which utilize host-specific promoters for the target gene expression. In the context of the invention, binding of the TF or synthetic TF to the synthetic promoter induces expression of the second polynucleotide encoding the polypeptide of interest.

[0072] Synthetic promoter refers to a region of DNA which functions as a eukaryotic promoter, but it is not a naturally occurring promoter of a host organism. It contains an upstream activation sequence (UAS) and a core promoter, wherein the UAS, or the core promoter, or both elements, are not native to the host organism. In the context of this invention, the synthetic promoter comprises (usually 1 -10, typically 1 , 2, 4 or 8) TF-specific binding sites (synthetic UAS - sUAS) linked to a core promoter.

[0073] Transcription factor: The term “Transcription factor” or “TF” refers to a protein that binds to specific DNA sequences present in the synthetic promoter, thereby controlling the rate of transcription, which is performed by RNA II polymerase. Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase to core promoters of genes. Synthetic transcription factor (sTF) refers to a protein which functions as a transcription factor, but is not a native protein of a host organism. In the context of this invention, the sTF is an artificial protein which typically comprises a DNA-binding protein of prokaryotic origin, a nuclear localization signal, and a transcription activation domain of viral origin.

[0074] Variant: The term “variant” means a polypeptide comprising a man-made mutation, i.e., a substitution, insertion (including extension), and / or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0075] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence). Detailed Description of the Invention

[0076] Host Cells

[0077] In some aspects, the present invention relates to recombinant host cells comprising one or more inducible promoters of the invention.

[0078] In a 1staspect the invention relates to fungal host cells comprising in its genome: an acetate-inducible promoter operably linked to one or more first polynucleotide encoding one or more first polypeptide of interest (POI), wherein the one or more first polynucleotide is heterologous to the acetate-inducible promoter.

[0079] In a 2ndaspect the invention relates to fungal host cells comprising in its genome an inducible promoter operably linked to one or more first polynucleotide encoding one or more first POI, wherein the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide sequences of SEQ ID NO:1.

[0080] In a 3rdaspect the invention relates to fungal host cells comprising in its genome an inducible promoter operably linked to one or more first polynucleotide encoding one or more first POI, wherein the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide sequences of SEQ ID NO: 2.

[0081] In one embodiment, the cell comprises in its genome synthetic expression system comprising:

[0082] (i) one or more first expression cassette comprising an inducible promoter operably linked to one or more first polynucleotide encoding one or transcription factor (TF), , and

[0083] (ii) one or more second expression cassette comprising one or more synthetic promoter, said one or more synthetic promoter being operably linked to one or more second polynucleotide encoding one or more protein of interest, wherein the one or more synthetic promoter comprises one or more transcription factor (TF)-specific binding site.

[0084] In one embodiment, the cell is comprising in its genome:

[0085] (i) an inducible promoter operably linked to one or more first polynucleotide encoding one or more first POI, wherein the inducible promoter is heterologous to the one or more first polynucleotide and comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 1-2.

[0086] In one embodiment, the cell is further comprising in its genome: ii) a synthetic promoter operably linked to one or more second polynucleotide encoding one or more second POI, said synthetic promoter comprising one or more transcription factor (TF)- specific binding site.

[0087] In one embodiment, the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0088] In one embodiment, the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 10.

[0089] In one embodiment, the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 1.

[0090] In one embodiment, the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 2.

[0091] In one embodiment, the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 11.

[0092] In one embodiment, the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 22.

[0093] In one embodiment, wherein the acetate inducible promoter is repressed, or partially repressed, by the presence of glucose.

[0094] In one embodiment, the acetate-inducible promoter comprises a FacB-binding.

[0095] In one embodiment, the acetate-inducible promoter is induced by FacB.

[0096] In one embodiment, the acetate-inducible promoter is associated with an increased mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using acetate as carbon source, relative to the mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using glucose as carbon source, the cells otherwise being cultivated in identical conditions.

[0097] In one embodiment, the polynucleotide located immediately downstream of the acetate-inducible promoter comprises or consists of the one or more polynucleotide.

[0098] In one embodiment, wherein the increased mRNA transcript level is associated with a Iog2-fold change of at least 0.1 , e.g., such as at least 0.14, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least

[0099] 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least

[0100] 6.5, at least 7.0, at least 7.5, or at least 8.0.

[0101] In one embodiment, the increased mRNA transcript level is associated with a Iog2-fold change of at least 4.0, and wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ ID NOs: 10-24.

[0102] In one embodiment, the increased mRNA transcript level is associated with a Iog2-fold change of at least 1 .0, and wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ ID NOs: 10-238.

[0103] In one embodiment, the increased mRNA transcript level is associated with a Iog2-fold change of at least 0.5, and wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ ID NOs: 10-814.

[0104] In one embodiment, the one or more first POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human betalactalbumin, or a bovine beta-lactalbumin, hydrolase, e.g., a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), or a glycosyl hydrolase 13 (GH13); isomerase, ligase, lyase, oxidoreductase, transferase, galactosidase, e.g., an alphagalactosidase, an alpha-galactosidase 3, or an alpha-galactosidase C, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, lipase, mannosidase, mutanase, ovalbumin, oxidase, pectinolytic enzyme, peroxidase, phosphatase, e.g., an alkaline phosphatase; phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, a transcription factor (TF), transglutaminase, or xylanase.

[0105] In one embodiment, the one or more first POI comprises one or more TF capable of binding to the TF-specific binding site of the synthetic promoter, preferably the one or more TF is a synthetic TF.

[0106] In one embodiment, the one or more TF comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 4.

[0107] In one embodiment, the one or more second POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human beta-lactalbumin, or a bovine beta-lactalbumin, a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), a glycosyl hydrolase 13 (GH13), an alpha-galactosidase, an alpha-galactosidase 3, or an alpha-galactosidase C, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, lipase, mannosidase, mutanase, ovalbumin, oxidase, pectinolytic enzyme, peroxidase, phosphatase, e.g., an alkaline phosphatase; phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0108] In one embodiment, the one or more first POI and / or the one or more second POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human beta-lactalbumin, or a bovine beta-lactalbumin, hydrolase, e.g., a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), or a glycosyl hydrolase 13 (GH13); an alpha-galactosidase, an alpha-galactosidase 3, or an alphagalactosidase C, alpha-glucosidase, endolase, beta-xylosidase, phosphodiesterase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, ovalbumin, phosphatase, e.g., an alkaline phosphatase; phytase.

[0109] In one embodiment, the synthetic promoter comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 7. In one embodiment, the one or more first POI is heterologous to the cell.

[0110] In one embodiment, the one or more second POI is heterologous to the cell.

[0111] In a preferred embodiment, both the one or more second POI and the one or more first POI are heterologous to the cell.

[0112] In one embodiment, the cell is a filamentous fungal cell.

[0113] In one embodiment, the cell is an Aspergillus cell.

[0114] In one embodiment, the cell is a filamentous fungal cell e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0115] In one embodiment, the cell is Aspergillus niger.

[0116] In one embodiment, cell is Aspergillus oryzae.

[0117] In one embodiment, the cell is Trichoderma reesei.

[0118] In one embodiment, the one or more inducible promoter, e.g. acetate-inducible promoter, is the only promoter regulating expression of the first POI, preferably the first POI consists of a TF.

[0119] In one embodiment, the one or more TF binds specifically to the TF-specific binding site of the synthetic promoter.

[0120] In one embodiment, the one or more first POI and / or the one or more second POI is secreted.

[0121] In one embodiment, the synthetic promoter comprises, essentially consist of, or consist of the polynucleotide of SEQ ID NO: 7. In one embodiment, the one or more inducible promoter is repressed by thiamine, e.g., repressed when thiamine is present in the cultivation medium.

[0122] In one embodiment, promoter activity of the inducible promoter is inversely proportional to thiamine concentration in the cultivation medium.

[0123] In one embodiment, the one or more inducible promoter comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 1 .

[0124] In one embodiment, the one or more inducible promoter comprises, essentially consists of, or consists of the polynucleotide of SEQ ID NO: 1.

[0125] In one embodiment, wherein the one or more inducible promoter is heterologous to the host cell.

[0126] In one embodiment, the acetate-inducible promoter is heterologous to the host cell.

[0127] In one embodiment, the acetate-inducible promoter is from Aspergillus nidulans.

[0128] In one embodiment, the acetate-inducible promoter is activated by acetate, e.g., activated when acetate is present in the cultivation medium, such as by adding acetic acid to the cultivation medium.

[0129] In one embodiment, promoter activity of the acetate-inducible promoter is proportional to acetate concentration in the cultivation medium.

[0130] In one embodiment, the one or more inducible promoter is a thiA promoter or variant thereof. In one embodiment, expression of the one or more polypeptide of interest is induced by cultivating the cell in a cultivation medium free of thiamine, or in a cultivation medium essentially free of thiamine.

[0131] In one embodiment, the one or more TF is a synthetic TF.

[0132] In one embodiment, the one or more TF comprises a transcription regulator, a nuclear localization signal (NLS), and a transcription activation domain.

[0133] In one embodiment, the one or more TF comprises one or more nuclear localisation signal NLS, e.g., two or more NLS, three or more NLS, or four or more NLS.

[0134] In one embodiment, the NLS comprises or consists of a SV40 NLS.

[0135] In one embodiment, the transcription regulator comprises or consists of a polypeptide from the TetR family.

[0136] In one embodiment, the transcription activation domain comprises or consists of a VP16 activation domain, and / or a VP64 activation domain.

[0137] In one embodiment, wherein the one or more second polynucleotide is encoding the one or more TF and comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 3. In one embodiment, the one or more TF comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 4.

[0138] In one embodiment, the one or more TF is heterologous to the host cell.

[0139] In one embodiment, the one or more polypeptide of interest is heterologous to the host cell. In one embodiment, the one or more polypeptide of interest is hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.

[0140] In one embodiment, the one or more polypeptide of interest is a hydrolase, isomerase, ligase, lyase, oxidoreductase, transferase, galactosidase, e.g., an alpha-galactosidase, alphaglucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0141] In one embodiment, the one or more polypeptide of interest is an endolase.

[0142] In one embodiment, the endolase is encoded by a first polynucleotide comprising, essentially consisting of, or consisting of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 5.

[0143] In one embodiment, the endolase comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 6.

[0144] In one embodiment, the one or more polypeptide of interest is a phosphodiesterase (PDE).

[0145] In one embodiment, the phosphodiesterase is encoded by a first polynucleotide comprising, essentially consisting of, or consisting of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 8.

[0146] In one embodiment, the phosphodiesterase comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 9.

[0147] In one embodiment, the one or more first POI and / or the one or more second POI is an inactivated enzyme.

[0148] In one embodiment, the one or more first POI and / or one or more second POI has reduced, substantially no, or no enzyme activity.

[0149] In one embodiment, the host cell comprises at least one undisrupted PrtT gene in its genome, e.g., where the transcription factor PrtT is expressed in its native form.

[0150] In one embodiment, the cell is isolated.

[0151] In one embodiment, the cell is purified.

[0152] A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The polypeptide can be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotides of the present invention.

[0153] The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a filamentous fungal cell.

[0154] The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0155] Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen et al., 1988, Bio / TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.

[0156] The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). For purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980). The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0157] Synthetic Expression System (SES)

[0158] The present invention also relates to a SES utilizing nucleic acid constructs comprising one or more synthetic promoter, one or more inducible promoter of the invention, and one or more TF, directing the expression of the polypeptide of interest in the fungal host cell under conditions compatible with the promoter sequences. A schematic drawing showing the SES and its elements is disclosed in Figure 2.

[0159] In one embodiment, the SES comprises:

[0160] (i) one or more first expression cassette comprising an inducible promoter, e.g. acetate-inducible promoter, operably linked to one or more first polynucleotide encoding one or more TF, wherein the inducible promoter is heterologous to the first polynucleotide encoding the TF, and

[0161] (ii) one or more second expression cassette comprising one or more synthetic promoter operably linked to one or more second polynucleotide encoding one or more POI , said synthetic promoter comprising one or more transcription factor (TF)-specific binding site. The one or more TF expressed under the control of the inducible promoter is capable of binding to the TF-specific binding site of the synthetic promoter and induces expression of the one or more POI. Preferably the one or more TF expressed under the control of the inducible promoter is a synthetic TF.

[0162] In a preferred embodiment, the one or more inducible promoter is the only promoter regulating expression of the one or more TF.

[0163] The polynucleotide encoding the inducible promoter may also be mutated by introduction of nucleotide substitutions.

[0164] The inducible promoter is a polynucleotide that is recognized by the host cell for expression of the one or more TF. The inducible promoter contains transcriptional control sequences that mediate the expression of the one or more TFs. The inducible promoter may be any polynucleotide that shows transcriptional activity in the host cell upon induction or repression of the inducible promoter.

[0165] Examples of regulatory sequences are those that cause expression of the one or more TF to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound, e.g., acetate, nitrate, or thiamine. The synthetic promoter may be manipulated in a variety of ways to provide for expression of the POI. The inducible promoter may be manipulated in a variety of ways to provide for expression of the TF. Manipulation of a promoter prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0166] Inducible promoters

[0167] The present invention also relates to polynucleotides encoding an inducible promoter. Such inducible promoter may be used as part of a SES, e.g., to express a TF which induces expression of a POI, or for direct expression of a POI (i.e. , not as part of any SES).

[0168] Thus, in a 7thaspect, the invention relates to a polynucleotide comprising an inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the the polynucleotide sequence of SEQ I D NOs: 10, 1 , 2, or 11-1489.

[0169] In one embodiment the polynucleotide comprises an inducible promoter operably linked to a polynucleotide encoding a POI, wherein the promoter is heterologous to the polynucleotide encoding the POI.

[0170] In one embodiment, the promoter comprises, essentially consists of, or consists of any of the polynucleotides of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0171] In one embodiment, the promoter is having at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2% or at most 1 % sequence differences to any of the polynucleotides of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0172] In one embodiment, the promoter differs from the polynucleotide of SEQ ID NOs: 10, 1 , 2, or 11-1489, by at most 10 nucleotides, such as at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0173] In one embodiment the polynucleotide comprises an inducible promoter operably linked to a polynucleotide encoding a TF, wherein the promoter is heterologous to the polynucleotide encoding the TF.

[0174] The polynucleotide encoding the inducible promoter or variant thereof may also be mutated by introduction of nucleotide substitutions.

[0175] The inducible promoter is a polynucleotide that is recognized by the host cell for expression of a polypeptide of interest. The inducible promoter contains transcriptional control sequences that mediate the expression of one or morey polypeptide of interest.

[0176] In one embodiment the polynucleotide is isolated.

[0177] In one embodiment the polynucleotide is purified. thiaA promoter In one embodiment, the inducible promoter is comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 1.

[0178] In one embodiment the inducible promoter comprises, essentially consists of, or consists of the polynucleotide of SEQ ID NO: 1.

[0179] In one embodiment, the inducible promoter differs from the polynucleotide of SEQ ID NO:1 , by at most 10 nucleotides, such as at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0180] In one embodiment the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and is induced by the absence of thiamine, e.g., induced by a low thiamine concentration in the cultivation medium.

[0181] In one embodiment the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and the promoter is repressed by the presence of thiamine in the cultivation medium.

[0182] In one embodiment the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and activity of the promoter is inversely proportional to the thiamine concentration in a cultivation medium.

[0183] In one embodiment the promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and is induced by the absence of thiamine, e.g., induced by a low thiamine concentration in the cultivation medium.

[0184] In one embodiment promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and the promoter is repressed by the presence of thiamine in the cultivation medium.

[0185] In one embodiment the promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and activity of the promoter is inversely proportional to the thiamine concentration in a cultivation medium. niaD promoter

[0186] In one embodiment, the inducible promoter is comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 2.

[0187] In one embodiment the inducible promoter comprises, essentially consists of, or consists of the polynucleotide of SEQ ID NO: 2. In one embodiment, the inducible promoter differs from the polynucleotide of SEQ ID NO:2, by at most 10 nucleotides, such as at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0188] In one embodiment the inducible promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 2 and is induced by nitrate, e.g., induced by adding nitrate to the cultivation medium.

[0189] Acetate-inducible promoters

[0190] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of any one of SEQ ID NQs:10, 1 , 2, or 11-1489.

[0191] In one embodiment the acetate-inducible promoter is induced by acetate, e.g., induced by adding acetate or acetic acid to the cultivation medium.

[0192] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 10.

[0193] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ I D NO: 1.

[0194] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:2.

[0195] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:22.

[0196] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:11.

[0197] In one embodiment, the inducible promoter is an acetate-inducible promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 11-1489.

[0198] In one embodiment the inducible promoter is an acetate-inducible promoter and comprises, essentially consists of, or consists of the polynucleotide of any one of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0199] In one embodiment, the inducible promoter is an acetate inducible promter and differs from the polynucleotide of SEQ ID NOs: 10, 1 , 2, or 11-1489, by at most 10 nucleotides, such as at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0200] In one embodiment the inducible promoter is an acetate-inducible promoter and comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of any one of SEQ ID NOs: 10, 1 , 2, or 11-1489 and is induced by acetate, e.g., induced by adding acetate or acetic acid to the cultivation medium.

[0201] In one embodiment the inducible promoter is an acetate-inducible promoter, wherein the promoter is derived from the acuD gene of Aspergillus nidulans.

[0202] In one embodiment the inducible promoter is an acetate-inducible promoter, wherein the promoter is derived from the acuE gene of Aspergillus nidulans.

[0203] In one embodiment, the acetate-inducible promoter comprises a FacB-binding site (AcuB- binding site).

[0204] In one embodiment, the acetate-inducible promoter is induced by FacB (AcuB).

[0205] In one embodiment the inducible promoter is an acetate-inducible promoter, wherein the promoter is derived from any gene, or gene ID, of Aspergillus nidulans listed in Table 4.

[0206] In one embodiment, the acetate inducible promoter is repressed, or partially repressed, by the presence of glucose.

[0207] In one embodiment the acetate-inducible promoter is associated with an increased mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using acetate as carbon source, relative to the mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using glucose as carbon source, the cells otherwise being cultivated in identical conditions.

[0208] In one embodiment, the polynucleotide located immediately downstream of the acetate- inducible promoter comprises or consists of the one or more polynucleotide. Synthetic promoter

[0209] The present invention also relates to polynucleotides encoding a synthetic promoter used in the SES, the synthetic promoter being operably linked to a second polynucleotide encoding one or more POI.

[0210] In one embodiment, the synthetic promoter comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 7.

[0211] In one embodiment, the synthetic promoter comprises, essentially consist of, or consist of the polynucleotide of SEQ ID NO: 7.

[0212] In an embodiment, the polynucleotide encoding the synthetic promoter is a subsequence comprising a TF-specific binding site.

[0213] The polynucleotide encoding the synthetic promoter may also be mutated by introduction of nucleotide substitutions.

[0214] The synthetic promoter is a polynucleotide that is recognized by a host cell for expression of the second polynucleotide encoding the polypeptide of interest. The synthetic promoter contains transcriptional control sequences that mediate the expression of the polypeptide of interest. The synthetic promoter may be any polynucleotide that shows transcriptional activity in the host cell upon binding of the TF to the synthetic promoter, wherein the TF is encoded by the first polynucleotide.

[0215] Transcription Factor

[0216] A transcription factor (TF) is a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase for expression of the polypeptide of interest. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. As part of the SES, the transcription factor used in the present invention regulates the expression of the polypeptide of interest directly, i.e., by activating the transcription of the second polynucleotide encoding the protein of interest by binding to the synthetic promoter located upstream of the second polynucleotide.

[0217] Alternatively, the transcription factor regulates the expression of the polypeptide of interest indirectly, i.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the polypeptide of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177.

[0218] In a preferred embodiment, the one or more TF binds specifically to the TF-specific binding site of the synthetic promoter.

[0219] In one embodiment, the one or more TF is a synthetic TF.

[0220] In one embodiment, the one or more TF comprises a transcription regulator, a nuclear localization signal (NLS), and a transcription activation domain.

[0221] In one embodiment, the one or more TF comprises one or more nuclear localisation signal NLS, e.g., two or more NLS, three or more NLS, or four or more NLS.

[0222] In one embodiment, the NLS comprises or consists of a SV40 NLS.

[0223] In one embodiment, the transcription regulator comprises or consists of a polypeptide from the TetR family.

[0224] In one embodiment, the transcription activation domain comprises or consists of a VP16 activation domain, and / or a VP64 activation domain.

[0225] In one embodiment, the one or more second polynucleotide encoding the one or more TF comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 3.

[0226] In one embodiment, the one or more TF comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 4.

[0227] In one embodiment, the one or more TF is heterologous to the host cell.

[0228] In one aspect, the TF is derived from SEQ ID NO: 4 by substitution, deletion or addition of one or several amino acids. In another aspect, the TF is derived from a mature polypeptide of SEQ ID NO: 4 by substitution, deletion or addition of one or several amino acids. In some embodiments, the TF is a variant of SEQ ID NO: 4 comprising a substitution, deletion, and / or insertion at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the TF of SEQ ID NO: 4 is up to 15, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the TF; small deletions, typically of 1-30 amino acids; small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.

[0229] Essential amino acids in a TF can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for DNA binding activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. Biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related TF, and / or be inferred from sequence homology and conserved catalytic machinery with a related TF or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally, or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of TFs. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0230] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman eta!., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a / ., 1988, DNA 7: 127).

[0231] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a TF.

[0232] Expression Vectors

[0233] The present invention also relates to recombinant expression vectors comprising an inducible promoter of the invention operably linked to the first polynucleotide.

[0234] The present invention also relates to recombinant expression vectors comprising the synthetic promoter operably linked to the second polynucleotide. In an 8thaspect, the present invention also relates to nucleic acid constructs or recombinant expression vectors comprising the polynucleotide of the 7thaspect operably linked to a polynucleotide encoding one or more polypeptide of interest.

[0235] In one embodiment, the vector or construct is isolated.

[0236] In one embodiment the vector or construct is purified.

[0237] In a 9thaspect, the invention relates to a cell comprising in its genome the polynucleotide of the 7thaspect, and / or the nucleic acid construct or expression vector of the 8thaspect.

[0238] The recombinant expression vectors may additionally comprise transcriptional and translational stop signals. The various nucleotide and control sequences (e.g., promoters) may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression of the TF or polypeptide of interest.

[0239] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the first and / or second polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0240] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0241] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0242] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0243] For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ). For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0244] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0245] Production method

[0246] In a 4thaspect, the invention relates to a method of producing one or more POI wherein the cell according to any one of the 1st, 2nd, or 3rdaspect is cultivated under conditions conducive for the production of the one or more POI.

[0247] In one embodiment, the cell is cultivated in a medium comprising at least 0.01 % v / v acetate.

[0248] In one embodiment, the cell is cultivated in a cultivation medium having a pH above pH 4.5, such as above pH 4.76, such as above pH 5.0, or above pH 5.5, such as above pH 6.0, or such as above pH 6.5.

[0249] In one embodiment, the method comprises recovering the one or more POI.

[0250] In one embodiment, the one or more POI is secreted into the cultivation medium.

[0251] In one embodiment, the cultivation medium comprises a pH in the range of 3-8, e.g., 4-8, 3-7, 4- 7, 3-6, 4-6, 5-6, 3-5, 4-5, 5-8, 5-7, or 5-6.

[0252] In one embodiment, the cultivation medium comprises a pH in the range of 4.5 - 8, e.g., 5 - 8, 4.5 - 7, 5 - 7, 4.5 - 6, 5 - 6, 5 - 6, or 4.5 - 5.

[0253] In one embodiment, the cultivation medium comprises at least 0.011% v / v acetate, such as e.g., at least 0.015% v / v acetate, at least 0.016% v / v acetate , at least 0.017% v / v acetate , at least 0.018% v / v acetate , at least 0.019% v / v acetate , at least 0.02% v / v acetate , at least 0.025% v / v acetate , at least 0.030% v / v acetate , at least 0.035% v / v acetate, at least 0.040% v / v acetate, at least 0.045% v / v acetate, at least 0.050% v / v acetate, at least 0.055% v / v acetate, at least 0.060% v / v acetate, at least 0.065% v / v acetate, at least 0.070% v / v acetate, at least 0.075% v / v acetate, at least 0.080% v / v acetate, at least 0.085% v / v acetate, at least 0.090% v / v acetate, at least 0.095% v / v acetate, at least 0.10% v / v acetate, at least 0.11% v / v, at least 0.12% v / v, at least 0.13% v / v, at least 0.14% v / v, at least 0.15% v / v, at least 0.16% v / v, at least 0.17% v / v, at least 0.18% v / v, at least 0.19% v / v, at least 0.20% v / v, at least 0.21 % v / v, at least 0.22% v / v, at least 0.23% v / v, at least 0.24% v / v, at least 0.25% v / v, at least 0.3% v / v, at least 0.4% v / v, or at least 0.5% v / v acetate.

[0254] In one embodiment, the cultivation medium comprises between 0.01 - 0.5% v / v acetate, e.g.,0.01 - 0.4% v / v acetate, 0.01 - 0.3% v / v acetate, 0.01 - 0.2% v / v acetate, 0.01 - 0.1% v / v acetate, 0.02 - 0.5% v / v acetate, 0.02 - 0.4% v / v acetate, 0.02 - 0.3% v / v acetate, 0.02 - 0.2% v / v acetate, 0.02 - 0.1 % v / v acetate, 0.03 - 0.5% v / v acetate, 0.03 - 0.4% v / v acetate, 0.03 - 0.3% v / v acetate, 0.03 - 0.2% v / v acetate, 0.03 - 0.1 % v / v acetate, 0.04- 0.5% v / v acetate, 0.04 - 0.4% v / v acetate, 0.04 - 0.3% v / v acetate, 0.04 - 0.2% v / v acetate, 0.04 - 0.1 % v / v acetate, 0.05 - 0.5 % v / v acetate, 0.05% - 0.4% v / v acetate, 0.05-0.3 % v / v acetate, 0.05-0.2% v / v acetate, or 0.05 - 0.1% v / v acetate, preferably between 0.01-0.5% v / v acetate.

[0255] In one embodiment, the cultivation medium comprises at least 0.02% v / v acetate.

[0256] In one embodiment, acetate is the sole carbon source of the cultivation medium.

[0257] In one embodiment, the acetate inducible promoter is repressed, or partially repressed by the presence of glucose.

[0258] In one embodiment, acetate is not the only carbon source of the cultivation medium.

[0259] In one embodiment, when acetate is not the only carbon source of the cultivation medium, the cultivation medium comprises acetate and glucose.

[0260] In one embodiment, when acetate is not the only carbon source of the cultivation medium, the cultivation medium comprises acetate and maltose.

[0261] In one embodiment, when acetate is not the only carbon source of the cultivation medium, the cultivation medium comprises acetate and sucrose.

[0262] In one embodiment, the cultivation medium comprises at most 2% v / v glucose, e.g., at most 1.5% v / v, at most 1.0% v / v, at most 0.5% v / v, at most 0.4% v / v, at most 0.3% v / v, at most 0.2% v / v, at most 0.1% v / v, at most 0.05% v / v, at most 0.04% v / v, at most 0.03% v / v, at most 0.02% v / v, or at most 0.01 % v / v glucose.

[0263] In one embodiment, the cultivation medium comprises at most 2% v / v maltose, e.g., at most 1 .5% v / v, at most 1.0% v / v, at most 0.5% v / v, at most 0.4% v / v, at most 0.3% v / v, at most 0.2% v / v, at most 0.1 % v / v, at most 0.05% v / v, at most 0.04% v / v, at most 0.03% v / v, at most 0.02% v / v, or at most 0.01% v / v maltose.

[0264] In one embodiment, the cultivation medium comprises at most 2% v / v sucrose, e.g., at most 1 .5% v / v, at most 1.0% v / v, at most 0.5% v / v, at most 0.4% v / v, at most 0.3% v / v, at most 0.2% v / v, at most 0.1 % v / v, at most 0.05% v / v, at most 0.04% v / v, at most 0.03% v / v, at most 0.02% v / v, or at most 0.01% v / v sucrose. In one embodiment, the acetate-inducible promoter is selected from the list of an A0X1 (alcohol oxidase 1) promoter, a PCK (phosphoenolpyruvate carboxykinase) promoter, a PDC (pyruvate decarboxylase) promoter, an ALD promoter, an amdS (acetamidase) promotor, an alcA (alcohol dehydrogenase 1) promotor, an acuL (succinate / fumarate mitochondrial transporter) promotor, an alcC (alcohol dehydrogenase 3) promotor, a facC (carnitine acetyl transferase) promotor, a coaT (acetyl-CoA hydrolase) promotor, an acuD (isocitrate lyase) promoter, or an acuE (malate synthase) promoter.

[0265] In one embodiment, the method is a fed-batch cultivation.

[0266] In one embodiment, the method is a batch cultivation.

[0267] In one embodiment, the method is a continuation cultivation.

[0268] In one embodiment, e.g., when the method is a fed-batch cultivation, the method comprises adding a feed to the cultivation medium, the feed comprising at least 10 % v / v acetic acid, e.g., such as at least 20 % v / v acetic acid, at least 30 % v / v acetic acid, at least 35 % v / v acetic acid, at least 40 % v / v acetic acid, at least 45 % v / v acetic acid, at least 50 % v / v acetic acid, at least 55 % v / v acetic acid, at least 60 % v / v acetic acid, at least 65 % v / v acetic acid, at least 70 % v / v acetic acid, at least 75 % v / v acetic acid, or at least 80 % v / v acetic acid.

[0269] The host cell is cultivated in a nutrient medium suitable for production of the polypeptide of interest using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and / or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0270] The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

[0271] The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered. In another aspect, a cell-free fermentation broth comprising the polypeptide is recovered. The polypeptide may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0272] In an alternative aspect, the polypeptide of interest is not recovered.

[0273] In a 5thaspect, the invention relates to a whole broth formulation or cell culture composition comprising the cell according to any one of the 1st, 2nd, or 3rdaspect, or a whole broth formulation or cell culture composition obtained by the method of the 4thaspect.

[0274] The fermentation broth formulation or the cell composition further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of interest which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and / or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and / or cell debris, and culture medium.

[0275] The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and / or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or nonviable microbial cells.

[0276] In some embodiments, the fermentation broth formulation or the cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and / or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and / or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

[0277] In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and / or cell debris. In some embodiments, the killed cells and / or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.

[0278] The fermentation broth formulation or cell composition may further comprise a preservative and / or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art. The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains the spent cell culture medium, extracellular enzymes, and killed fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0279] A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and / or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.

[0280] The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90 / 15861 or WO 2010 / 096673.

[0281] In a 6thaspect the invention relates to the use of the cell according to any one of the 1st, 2nd, or 3rdaspect to produce a food or feed product and / or to produce the whole broth formulation or cell culture composition according to the 5thaspect.

[0282] Advantageously, the high protein content of the whole broth formulation can be used to provide a protein-comprising food product, and / or a protein-comprising feed product. Such food or feed product contributes to the reduction of greenhouse gas emissions, as it is produced with acetate that has been converted from CO2.

[0283] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0284] Examples

[0285] Example 1 : niaD promoter allows POI expression both on glucose and / or acetate

[0286] For building the Aspergillus oryzae recipient strain expressing an endolase from a SES, four chromosomal loci were chosen for integration of the SES elements, which loci are present on four different chromosome arms to ensure genetic stability in the experiment. Integration took place according to the SPADE method disclosed in WO24218234A1. We chose the pepAb locus on chromosome 1 , the pepE locus on chromosome 2, the amdS locus on chromosome 6 and the pepAa locus on chromosome 8.

[0287] Four landing pads (LPs) were inserted at the four chromosomal loci using CRISPR guide RNAs targeting each of the four loci as well as four repair fragments, which contained the four LPs flanked by loci specific repair sequences. While integrating the LPs, the loci specific genes were deleted thereby removing the guide targeting sequence. The resulting Aspergillus oryzae recipient strain AT5885 containing the four LPs at the four different chromosomal loci was confirmed by genome sequencing. The recipient strain AT5885 is ApyrG and therefore requires supplementation of uridine to be able to grow. AT5885, comprising the four LPs, had the penicillin biosynthetic pathway removed resulting in strain AT6230. AT6230 was made pyrG+ resulting in strain AT6412.

[0288] The four LPs comprise the following protospacers:

[0289] - LP#1 : comprises the five protospacers PS-59, PS-73, PS-74, PS-75 and PS-76 for integration in the pepAb locus;

[0290] - LP#2: comprises the five protospacers PS-59, PS-73, PS-74, PS-75 and PS-11 for integration in the pepE locus;

[0291] - LP#3: comprises the five protospacers PS-59, PS-73, PS-48, PS-49 and PS-93 for integration in the amdS locus ;

[0292] - LP#4: comprises the five protospacers PS-59, PS-73, PS-48, PS-89 and PS-94 for integration in the pepAa locus

[0293] Following CRISPR guide RNA (gRNA) plasmids were built: pAT6656, which enables cutting PS-93 in LP#3 in the recipient strain. pAT6654, which enables cutting PS-75 in LP#1 and LP#2.

[0294] The guide RNA plasmids carry the bar gene for maintenance in the recipient strain upon transformation selecting on plates containing glyphosate.

[0295] A repair DNA construct, REPAI R1 , was built, which contained CASSETTE1 , encoding an endolase gene (SEQ ID NO: 5 encoding the endolase polypeptide of SEQ ID NO: 6) operably linked to a synthetic promotor (SEQ ID NO: 7) comprising a transcription factor (TF)-specific binding site. Moreover, the construct contained CASETTE2, encoding the TF (TF coding sequence of SEQ ID NO: 3 encoding the TF of SEQ ID NO: 4) operably linked to the niaD promotor with SEQ ID NO: 2. The niaD promoter is activated by nitrate. The TF binds specifically to the synthetic promoter which is operably linked to the endolase coding sequence. Thus, this synthetic expression system (SES) enables induction of the synthetic promoter, and thus expression of the endolase, when using nitrate as the sole nitrogen source. On the other hand, the SES enables repression of the synthetic promoter when using ammonia as the sole nitrogen source, preventing endolase expression when endolase expression is not desired (e.g., during generation of cell-bank, and related freeze and thaw cycles to prevent unfavorable phenotypes which are caused by high recombinant protein expression). CASSETTE1 and CASSETTE2 were flanked by the repair flanks SYN2L and SYN2R. A repair DNA construct, REPAI R2, was built, which contained CASSETTE2 in two copies, reading in opposite direction ensuring genetic stability. This construct therefore contained two copies of the endolase gene operably linked to the synthetic promotor.

[0296] Protoplasts of AT6412 were transformed with either: pAT6656 and REPAIR1 enabling one copy of the endolase under control of the synthetic promoter, and one copy of the TF under control of the niaD promoter.

[0297] Five strains comprising one copy of each of the endolase and the TF expression cassette were selected and named TR1 , TR3, TR5, TR6 and TR18. pAT6654 and REPAI R1 enabling two copies of the endolase under control of the synthetic promoter, and two copies of the TF under control of the niaD promoter. Two resulting strains were selected and named TR11 and TR12. pAT6654 and REPAIR1 and REPAIR2 enabling three copies of the endolase under control of the synthetic promoter, and one copy of the TF under control of the niaD promoter. Three resulting strains were selected and named TR22, TR24 and TR25.

[0298] Spores from above selected strains were inoculated in YP medium containing as carbon source either:

[0299] A) 2% (v / v) Acetate, or

[0300] B) 2% (v / v) Glucose.

[0301] The strains were grown for 4 days and samples were taken for SDS-PAGE. The expressed endolase has a molecular weight of 45946 Da and in the SDS-PAGE gel of Fig. 1 the band for the endolase is indicated with an arrow. As can be seen from the SDS-PAGE gel in Fig. 1 , all strains grown on acetate (A) strains grown on acetate left panel of Fig. 1) show endolase expression comparable to the same strains when grown on glucose as carbon source (B), right panel of Fig. 1).

[0302] Surprisingly, using acetate as carbon source in combination with the niaD promoter and SES resulted in recombinant protein expression with protein levels similar compared to protein expression levels when using glucose as sole carbon source. Thus, the niaD promoter of the invention can be utilized for efficient protein production either in fermentation using acetate as carbon source, using glucose as a carbon source, or a mix thereof.

[0303] Example 2: The novel thiA promoter allows POI expression both on glucose and / or acetate

[0304] One CRISPR guide RNA (gRNA) plasmid was built: pAT4873, which enables cutting PS-73 in LP#1 , LP#2, LP#3 and LP#4 in the recipient strain AT5885.

[0305] A repair DNA construct, REPAIRS, was built, which encodes a phosphodiesterase (PDE) (PDE coding sequnece is SEQ ID NO: 8, and PDE polypeptide sequence is SEQ ID NO: 9) operably linked to the synthetic promotor with SEQ ID NO: 7 comprising a TF-specific binding site.

[0306] Protoplasts of the recipient strain AT5885 were transformed with pAT4873 and REPAIRS.

[0307] Transformants were screened by ddPCR and one transformant AT6226 was selected having two copies of the REPAIR 3 construct at each locus, in total having eight gene copies of the REPAIR 3 construct.

[0308] Locus pepAc was chosen as the location of the cassette encoding the TF. A guide plasmid targeting pepAc was transformed together with a repair construct encoding the TF, wherein the TF coding sequence is operably linked to a promotor variant of the inducible thiA promotor of the invention shown in SEQ ID NO: 1. The inducible thiA promoter keeps a high degree of repression when grown in media comprising thiamine. The resulting strain was named AT6226. The TF is encoded by SEQ ID NO:3, and its polypeptide sequence is shown in SEQ ID NO: 4.

[0309] Spores were inoculated and grown in 2-liter fermentors using as a feed either:

[0310] A) 58% v / v acetic acid, or

[0311] B) 60% v / v glucose.

[0312] The strains were grown for 6 days and samples were taken for assay measurements after six days of cultivation. The relative PDE yields for using the two different feeds are shown in Table 1. As shown in Table 1 , using the thiA promoter of the present invention it was possible to achieve product yield when glucose was utilized as carbon source, but also when acetate was utilized as carbon source.

[0313] Table 1. Relative PDE yield.

[0314] Aspergillus oryzae strains expressing a lipase as POI were cultivated either using glucose or maltose as carbon source, or using acetate as carbon source. Unexpectedly, it was observed that cell cultures grown on acetate showed reduced viscosity levels compared to the viscosity of the same strains grown on glucose as sole carbon course (data not shown) or maltose as sole carbon source (Fig. 3). As shown in Fig. 3, bioreactors “1” and “2” (left two reactors) using acetate as carbon source show significantly reduced viscosity compared to bioreactors “3” and “4” (right two reactors) using maltose as sole carbon source.

[0315] Example 4: The acuD promoter achieves high and stable mRNA levels & product expression

[0316] Locus nprC was chosen as the location of the cassette encoding the TF. A guide plasmid targeting nprC was transformed together with a repair construct, REPAI R4, encoding the TF, wherein the TF coding sequence is operably linked to the acuD promotor from Aspergillus nidulans (SEQ ID NO: 10). The acuD promoter is inducible and keeps a high degree of repression when grown in media not comprising acetate as carbon source. Having acetate as the carbon source enables a high degree of activation of the promoter. The resulting strain is named A2F0225. The TF is encoded by SEQ ID NO:3, and its polypeptide sequence is shown in SEQ ID NO: 4.

[0317] One CRISPR guide RNA (gRNA) plasmid is built: pAT4848, which enables cutting PS-48 in LP#3 and LP#4 in the recipient strain AT6230.

[0318] A repair construct pA2F0213 was built, which encodes two copies of the gene encoding an alpha-Lactalbumin (ALA) in opposite direction operably linked to the synthetic promotor with SEQ ID NO: 7 comprising a TF-specific binding site. Protoplasts of the recipient strain AT6230 were transformed with guide pAT4848 and repair DNA pA2F0213 resulting in strain A2F0285 having four copies of the ALA gene being integrated in two of the landing pads, LP3 and LP4.

[0319] One CRISPR guide RNA (gRNA) plasmid is built: pAT4894, which enables cutting PS-94 in LP#4 in the recipient strain AT6230.

[0320] A repair construct pA2F0203 was built, which encodes two copies of the gene encoding ovalbumin (OA) in opposite direction operably linked to the synthetic promotor with SEQ ID NO: 7 comprising a TF-specific binding site. Protoplasts of the recipient strain AT6230 were transformed with guide pAT4894 and repair DNA pA2F0203 resulting in strain A2F0299 having two copies of the OA gene being integrated in one of the landing pads, LP4.

[0321] Spores were inoculated and grown in 2-liter fermentors using 60% v / v acetic acid as feed. Four tanks were run of each of the two strains. Samples were taken after 72 hours, 96 hours and 144 hours and mRNA samples were prepared and sequenced using illumina sequencing. For both strains the average relative abundance of product mRNA (OA mRNA, or ALA mRNA) was determined, and also the average relative abundance of TF mRNA was determined. Additionally, for both strains the average relative abundance of tef1 mRNA was determined. Transcription of tef1 is under the control of a native strong constitutive promoter Ptefl which has become one of the de facto standards for POI expression in Aspergillus (Kitamoto et al. 1998, Appl Microbiol Biotechnol. 50:85-92; Rendsvig et al. 2019, Fungal Biol Biotechnol 6, 24).

[0322] The mRNA abundance is shown in Table 2. As seen in Table 2, the acuD promoter as part of the SES is capable of inducing a constant and high mRNA level of POI mRNA transcripts already from 72 hours on and throughout the total fermentation time. In comparison, Ptef achieved only comparably low mRNA levels, which increased from 72 hours onwards to 144 hours, but never exceeded the POI mRNA levels. The promoters and SES of the invention outperform the supposedly strong Ptef at least 10-fold.

[0323] These results also show that the SES of the invention requires only a minimum of transcript levels for the synthetic TF, while transcript levels for the POI are significantly increased, highlighting the efficiency of the SES of the present invention even when using acetate as the carbon source.

[0324] Also, in recombinant protein production, maintaining stable mRNA transcript levels of the product mRNA offers several advantages:

[0325] 1) Consistent Protein Expression:

[0326] Stable mRNA levels ensure a continuous and steady production of the target protein, which is crucial for achieving high yield and quality.

[0327] 2) Enhanced Protein Yield:

[0328] With stable mRNA, cells can consistently translate the mRNA into protein, leading to higher overall yields compared to fluctuating mRNA levels.

[0329] 3) Reduced Variability:

[0330] Stability in mRNA levels helps minimize variability in protein production between different batches, contributing to more predictable and reproducible outcomes.

[0331] 4) Efficient Resource Utilization:

[0332] Cells can efficiently use their resources (such as ribosomes, amino acids, and energy) when mRNA levels are stable, improving the overall efficiency of the protein production process.

[0333] 5) Simplified Process Control:

[0334] Stable mRNA levels make it easier to control and optimize the fermentation or cell culture conditions, as the primary variable (mRNA level) is kept constant.

[0335] Table 2. relative mRNA abundance (%).

[0336] Example 5: Expression of different POIs using the acetate-inducible acuD promoter

[0337] For building the Aspergillus oryzae recipient strain expressing POI genes from a SES, induced by a TF expressed from the acuD promotor (SEQ ID NO: 10), four chromosomal loci were chosen for integration of the SES elements. Said integration loci are present on four different chromosome arms to ensure genetic stability in the experiment. Integration took place according to the SPADE method disclosed in WO24218234A1. We chose the pepAb locus on chromosome 1 , the pepE locus on chromosome 2, the amdS locus on chromosome 6 and the pepAa locus on chromosome 8.

[0338] Four landing pads (LPs) were inserted at the four chromosomal loci using CRISPR guide RNAs targeting each of the four loci as well as four repair fragments, which contained the four LPs flanked by loci specific repair sequences. While integrating the LPs, the loci specific genes were deleted thereby removing the guide targeting sequence. The resulting Aspergillus oryzae recipient strain A2F0241 containing the four LPs at the four different chromosomal loci was confirmed by genome sequencing. The recipient strain A2F0241 is ApyrG and therefore requires supplementation of uridine to be able to grow.

[0339] The four LPs comprise the following protospacers:

[0340] - LP#1A: comprises the five protospacers PS-59, PS-73, PS-74, PS-75 and PS-76 for integration in the pepAb locus;

[0341] - LP#2: comprises the five protospacers PS-59, PS-73, PS-74, PS-75 and PS-11 for integration in the pepE locus;

[0342] - LP#1 B: comprises the five protospacers PS-59, PS-73, PS-74, PS-75 and PS-76 for integration in the amdS locus ;

[0343] - LP#4: comprises the five protospacers PS-59, PS-73, PS-48, PS-89 and PS-94 for integration in the pepAa locus

[0344] Locus nprC was chosen as the location of the cassette encoding the TF. A guide plasmid targeting nprC was transformed together with a repair construct, REPAIRS (SEQ ID NO: 1490), encoding the TF, wherein the TF coding sequence is operably linked to the acuD promotor from Aspergillus nidulans of the invention shown in SEQ ID NO: 10. The inducible acuD promoter keeps a light degree of repression when grown in media not comprising acetate as the carbon source. Having acetate as the carbon source enables activation of the promoter. The resulting strain was named A2F0255. The TF is encoded by SEQ ID NO:3, and its polypeptide sequence is shown in SEQ ID NO: 4. A2F0255 was made pyrG+ by inserting the pyrG gene ffrom Aspergillus nidulans in the pepAc locus. The resulting strain was named A2F0343.

[0345] A total of 11 repair constructs were built, each containing one copy of a gene encoding a POI operably linked to the synthetic promotor with SEQ ID NO: 7 comprising a TF-specific binding site.

[0346] Repair construct pA2F0430 encodes Alkaline phosphatase (AP1).

[0347] Repair construct pA2F0431 encodes Alkaline phosphatase (AP2).

[0348] Repair construct pA2F0432 encodes Alkaline phosphatase (AP3).

[0349] Repair construct pA2F0434 encodes Saponin hydrolase (SA).

[0350] Repair construct pA2F0435 encodes alpha-galactosidase C (AGO).

[0351] Repair construct pA2F0436 encodes alpha-galactosidase 3 (AG3).

[0352] Repair construct pA2F0437 encodes a Glycosyl Hydrolase 71 (GH71).

[0353] Repair construct pA2F0439 encodes a Glycosyl Hydrolase 20 (GH20).

[0354] Repair construct pA2F0440 encodes a Glycosyl Hydrolase 13 (JA352).

[0355] Repair construct pA2F0441 encodes a Putative debranching beta-xylosidase (DBX). Repair construct pA2F0274 encodes a phytase (HP).

[0356] One CRISPR guide RNA (gRNA) plasmid was built: pAT6654, which enables cutting PS-75 in LP#1A, LP#1 B and LP#2 in the recipient strain A2F0343.

[0357] Protoplasts of the recipient strain A2F0343 were transformed with guide pAT6654 and each of the elleven repair DNAs individually resulting in strains having three copies of the genes integrated in LP1A, LP1 B and LP2. The eleven resulting strains are:

[0358] A2F0958 encoding three copies of AP1 (Repair pA2F0430). A2F0959 encoding three copies of AP2 (Repair pA2F0431). A2F0960 encoding three copies of AP3 (Repair pA2F0432). A2F0961 encoding three copies of SA (Repair pA2F0434). A2F0962 encoding three copies of AGO (Repair pA2F0435). A2F0963 encoding three copies of AG3(Repair pA2F0436). A2F0964 encoding three copies of GH71 (Repair pA2F0437). A2F0965 encoding three copies of GH20 (Repair pA2F0439). A2F0966 encoding three copies of JA352 (Repair pA2F0440). A2F0967 encoding three copies of DBX (Repair pA2F0441). A2F0968 encoding three copies of HP (Repair pA2F0274).

[0359] All eleven strains were grown in batch culture in 10ml scale using either acetate as the carbon source (0.1 % v / v acetate) or glucose as the carbon source (0.1 % v / v glucose). Equal c- mol amounts were added of each carbon source. An SDS-PAGE gel was run from each of the samples after five days of growth at 30 degrees Celsius. The SDS-PAGE gel is shown in Fig. 4, with lanes of the gel being described in Table 3. As shown in Fig. 4, each of the 11 POIs showed higher expression when acetate is used as carbon source (lanes 1 , 3, 5, 7, 9, 12, 14, 16, 18, 20,

[0360] 22), compared to when glucose is used as carbon source (lanes 2, 4, 6, 8, 10, 13, 15, 17, 19, 21 ,

[0361] 23). These results further show the advantages of the acetate-inducible acuD promoter allowing high POI expression whilst using acetate as the carbon source.

[0362] Table 3. Description of the lanes and POIs shown in the gel of Figure 4.

[0363] Lane Carbon POI strain source

[0364] 1 A (acetate) Alkaline phosphatase (AP1) A2F0958

[0365] 3 A Alkaline phosphatase (AP2) A2F0959

[0366] 5 A Alkaline phosphatase (AP3) A2F0960

[0367] 7 A Saponin hydrolase (SA) A2F0961

[0368] 9 A alpha-galactosidase C (AGO) A2F0962

[0369] 12 A alpha-galactosidase 3 (AG3) A2F0963

[0370] 14 A Glycosyl Hydrolase 71 (GH71) A2F0964

[0371] 16 A Glycosyl Hydrolase 20 (GH20) A2F0965

[0372] 18 A Glycosyl Hydrolase 13 (JA352). A2F0966 20 A Putative debranching beta-xylosidase (DBX) A2F0967

[0373] 22 A phytase (HP) A2F0968

[0374] Example 6: Acetatic acid feed protocol

[0375] In the context of optimizing fermentation processes, the selection of an appropriate carbon source is crucial for maximizing microbial growth and product yield. One effective strategy involves the use of acetic acid as a carbon source, with careful control of the fermentation pH to ensure its conversion into acetate.

[0376] Acetic acid (CH3COOH) and acetate (CH3COO“) represent two forms of the same molecule, distinguished by their state of ionization. The pKa of acetic acid is approximately 4.76, meaning that at pH levels above this value, acetic acid predominantly exists in its ionized form as acetate. Conversely, at pH levels below the pKa, acetic acid remains largely in its protonated form.

[0377] To leverage acetate as a carbon source effectively, it is advantageous to maintain the fermentation pH around or above the pKa of acetic acid. In such conditions, the acetic acid added to the feed is converted into acetate, which microorganisms can readily utilize. Acetate enters cellular metabolic pathways like the citric acid cycle, where it is metabolized to generate energy (ATP) and build biomass.

[0378] Maintaining the fermentation pH above the pKa offers several benefits. Firstly, acetate, being an ion, does not significantly lower the pH of the culture medium. This minimizes the risk of acid stress, which can inhibit microbial growth and adversely affect enzyme activities. Secondly, stable pH conditions contribute to a more consistent and controlled fermentation process, leading to higher efficiency and yield. Also, a higher pH allows a higher acetate concentration in the medium.

[0379] In summary, by controlling the fermentation pH to remain around or above the pKa of acetic acid, we ensured that the acetic acid in the feed is converted into acetate. This optimizes the carbon source utilization by the microbial cells and supports a more productive and stable fermentation environment. Details of the fermentation protocol used for above examples is shown below:

[0380] Aspergillus oryzae fermentation protocol Seed cultivation:

[0381] Spores from coveNgly agar slant were transferred to shake flask (glycerol 20g / L, yeast extract 18 g / L) and incubated for 2 days at 30 °C and 250 rpm.

[0382] Fed batch fermentation:

[0383] Tank medium (yeast extract 10 g / L, (NH^SCL 5 g / L, MgSO4 7H2O 2 g / L, K2SO42 g / L, citric acid 1 g / L, KH2PO42 g / L trace metal solution 0,5 ml / L) was adjusted to 32 °C. 5 g / L glacial acetic acid was added, and pH was adjusted to 6.8. Aeration was 1 vvm and pH was controlled at 6.8 using 10% NH4OH. Main medium was inoculated from seed cultivation. After 5 hours feeding (400 g / L glacial acetic acid) was started at a rate of 2 g / L / h. Stirrer speed was controlled to avoid too low (<20%) oxygen tension. The starting tank volume was 230 ml.

[0384] Example 7: Acetate-inducible promoters identified in A. nidulans

[0385] To identify acetate-inducible promoters, poly(A) mRNA transcriptomics was used in A. oryzae to find transcripts upregulated when acetate is used in the feed medium, compared to when glucose is used in the feed medium. To avoid homology-based recombination during transformation and growth of recombinant A. oryzae using any of the identified promoters in a POI expression cassette, we identified corresponding orthologous genes in A. nidulans FGSC A4 which can be used for POI expression in other fungal strains, e.g. in A. oryzae.

[0386] For promoter identification, we included the 1000 bp upstream sequence of the orthologous gene from A. nidulans FGSC A4. We identified 1479 acetate-inducible promoters which are shown in Table 4. In this table, the promoters are ranked in descending order from highest Iog2-fold change of the relative mRNA abundance of each related gene when using acetate as feed, to lowest log-fold change. Additional! , Table 4 also shows padj values (Benjamini- Hochberg adjusted p-values). Each of the identified A. nidulans promoters correspond to orthologous A. oryzae promoters that showed higher mRNA transcript levels when using acetate as feed, compared to when using glucose as feed. Promoters were only included with a minimum Iog2-fold change of 0.14. As shown in Table 4, the Iog2-fold change is around 8.07 for the highest ranked promoter (SEQ ID NO: 11). The top ranked 14 identified promoters (SEQ ID NOs: 11-24) all have a Iog2-fold change above 4.0, top ranked 228 promoters (SEQ ID NOs: 11-238) all have a Iog2-fold change of above 1.0, and the top 804 promoters (SEQ ID NOs: 11-814) all have a Iog2-fold change of above 0.50.

[0387] The acuD promoter of SEQ ID NO: 10 used in Examples 4-5 is part of the 1000 bp sequence shown as SEQ ID NO: 17 (gene ID AN5634), ranking at position 7. The acuD promoter is associated with a Iog2-fold change of 5.82 further indicating that it is strongly induced by acetate. Thus, at least any promoter of Table 4 which has a similarly high Iog2-fold change as the acuD promoter, can be used for high POI expression in fungal cells. For example, the acuE promoter which is comprised in the 1000 bp sequence shown as SEQ ID NO: 22 (gene ID AN6653), and ranked at position 12 of Table 4 with a Iog2-fold change of 4.20, is an additional strong promoter which is induced by acetate.

[0388] Any of the acetate-inducible promoters shown in Table 4 are beneficial for POI expression when changing from glucose-based cultivation to acetate-based cultivation.

[0389] The SEQ ID NO. for each identified promoter is shown in Table 4 including the 1000 bp upstream sequence from each gene and its start codon. Table 4 also shows the Aspergillus nidulans gene ID for the genes associated to the acetate-inducible promoters, which gene ID can be assessed in FungiDB (fungidb.org; https: / / pmc.ncbi.nlm.nih.gov / articles / PMC5872342 / ).

[0390] RNA extraction and sequencing

[0391] A total of 25 mg of biomass was harvested from the acetic acid and glucose fed-batch fermentations at 72, 96, and 144 hours, as described above. RNA was extracted using the Invitrogen™ PureLink™ RNA Mini Kit following the manufacturer's instructions. The RNA was eluted in 100 pL of RNase / DNase-free water, and its purity was assessed by spectrophotometry using a Nanodrop (Thermo Scientific). RNA concentration was determined using the RNA-specific Qubit fluorometric assay (Invitrogen). RNA integrity was assessed using the Agilent Bioanalyzer RNA Nano Chip. Poly(A)-enriched mRNA was isolated from total RNA and subsequently reverse transcribed into cDNA. The cDNA library was amplified by PCR, assessed for quality. Finally, the cDNA library was sequenced on a 150 bp paired-end NextSeq 1000 patterned flowcell.

[0392] RNA assembly and analysis:

[0393] RNA-seq fastq files were analyzed using the nf-core / RNA-seq pipeline (v3.17.0) implemented in Nextflow (v23.10.1) (Ewels et al.-, Nat Biotechnol 38, 276-278 (2020)). Reads were mapped to Aspergillus oryzae RIB40 genome ASM18445v3. The final count matrix, with mapped read counts scaled by gene length, was used for further analysis. Differential gene expression between acetate- (A / = 12) and glucose-fed ( / \ / =8) cultures was analyzed using DESeq2, with a Wald test identifying significantly upregulated mRNA transcripts on acetate relative to their mRNA transcript levels on glucose (Benjamini-Hochberg adjusted p-values (padj) < 0.01 , log fold changes > 0.05). Thus, a total of 1479 acetate-inducible promotors were identified.

[0394] Table 4. Aspergillus nidulans promoters ranked according to their activity using acetate as feed, relative to their activity when using glucose as feed.

[0395] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

[0396] The invention is further defined by the following numbered paragraphs:

[0397] 1 . A fungal host cell comprising in its genome: i) an acetate-inducible promoter operably linked to one or more first polynucleotide encoding one or more first polypeptide of interest (POI), wherein the one or more first polynucleotide is heterologous to the acetate-inducible promoter.

[0398] 2. A fungal host cell comprising in its genome:

[0399] (i) an inducible promoter operably linked to one or more first polynucleotide encoding one or more first POI, wherein the inducible promoter is heterologous to the one or more first polynucleotide and comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 1-2.

[0400] 3. The cell of any one of paragraphs 1- 2, further comprising in its genome: ii) a synthetic promoter operably linked to one or more second polynucleotide encoding one or more second POI, said synthetic promoter comprising one or more transcription factor (TF)- specific binding site. 4. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 10, 1, 2, or 11- 1489.

[0401] 4a. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 10.

[0402] 4b. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 1.

[0403] 4c. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 2.

[0404] 4d. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 11.

[0405] 4e. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 22.

[0406] 4f. The cell of any one of the preceding paragraphs, wherein the acetate inducible promoter is repressed, or partially repressed, by the presence of glucose.

[0407] 4g. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter comprises a facB-binding site (acuB-binding site).

[0408] 4h. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter is induced by facB (acuB).

[0409] 4i. The cell of any one of the preceding paragraphs, wherein the acetate-inducible promoter is associated with an increased mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using acetate as carbon source, relative to the mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using glucose as carbon source, the cells otherwise being cultivated in identical conditions.

[0410] 4j. The cell of paragraph 4i, wherein the polynucleotide located immediately downstream of the acetate-inducible promoter comprises or consists of the one or more polynucleotide.

[0411] 4k. The cell of any of paragraphs 4i-4j, wherein the increased mRNA transcript level is associated with a Iog2-fold change of at least 0.1 , e.g., such as at least 0.14, at least 0.2, at least 0.3, at least

[0412] 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least

[0413] 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least

[0414] 6.0, at least 6.5, at least 7.0, at least 7.5, or at least 8.0.

[0415] 4I. The cell of any of paragraphs 4i-4k, wherein the increased mRNA transcript level is associated with a Iog2-fold change of at least 4.0, and wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ ID NOs: 10-24.

[0416] 4m. The cell of any of paragraphs 4i-4l, wherein the increased mRNA transcript level is associated with a Iog2-fold change of at least 1.0, and wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ ID NOs: 10-238. 4n. The cell of any of paragraphs 4i-4m, wherein the increased mRNA transcript level is associated with a Iog2-fold change of at least 0.5, and wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ ID NOs: 10-814.

[0417] 5. The cell according to any one of the preceding paragraphs, wherein the one or more first POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human beta-lactalbumin, ora bovine beta-lactalbumin, hydrolase, e.g., a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), or a glycosyl hydrolase 13 (GH13); isomerase, ligase, lyase, oxidoreductase, transferase, galactosidase, e.g., an alpha-galactosidase, an alpha-galactosidase 3, or an alpha-galactosidase C, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta- xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, lipase, mannosidase, mutanase, ovalbumin, oxidase, pectinolytic enzyme, peroxidase, phosphatase, e.g., an alkaline phosphatase; phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, a transcription factor (TF), transglutaminase, or xylanase.

[0418] 6. The cell of paragraph 3, wherein the one or more first POI comprises one or more TF capable of binding to the TF-specific binding site of the synthetic promoter, preferably the one or more TF is a synthetic TF.

[0419] 7. The cell of paragraph 6, wherein the one or more TF comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 4.

[0420] 8. The cell of paragraph 6, wherein the one or more second POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; betalactalbumin, e.g., a human beta-lactalbumin, or a bovine beta-lactalbumin, a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), a glycosyl hydrolase 13 (GH13), an alpha-galactosidase, an alpha-galactosidase 3, or an alpha-galactosidase C, alphaglucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, lipase, mannosidase, mutanase, ovalbumin, oxidase, pectinolytic enzyme, peroxidase, phosphatase, e.g., an alkaline phosphatase; phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0421] 8a. The cell of any one of paragraphs 1-8, wherein the one or more first POI and / or the one or more second POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human beta-lactalbumin, or a bovine betalactalbumin, hydrolase, e.g., a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), or a glycosyl hydrolase 13 (GH13); an alpha-galactosidase, an alphagalactosidase 3, or an alpha-galactosidase C, alpha-glucosidase, endolase, beta-xylosidase, phosphodiesterase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, ovalbumin, phosphatase, e.g., an alkaline phosphatase; phytase.

[0422] 9. The cell of any one of paragraphs 3-8a, wherein the synthetic promoter comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 7.

[0423] 10. The cell of any one of paragraphs 1-9, wherein the one or more first POI is heterologous to the cell.

[0424] 11. The cell of any one of paragraphs 1-10, wherein the one or more second POI is heterologous to the cell.

[0425] 12. The cell of any one of the preceding paragraphs, wherein the cell is a filamentous fungal cell.

[0426] 13. The cell of any one of the preceding paragraphs, wherein the cell is an Aspergillus cell.

[0427] 14. The cell according to any one of the preceding paragraphs, wherein the cell is a filamentous fungal cell e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0428] 15. The cell according to any one of the preceding paragraphs, wherein the cell is Aspergillus niger.

[0429] 16. The cell according to any one of the preceding paragraphs, wherein the cell is Aspergillus oryzae.

[0430] 17. The cell according to any one of the preceding paragraphs, wherein the cell is Trichoderma reesei.

[0431] 18. The cell of any one of the preceding paragraphs, wherein the one or more inducible promoter, e.g. acetate-inducible promoter, is the only promoter regulating expression of the first POI, preferably the first POI consists of a TF.

[0432] 19. The cell according to any one of paragraphs 6-18, wherein the one or more TF binds specifically to the TF-specific binding site of the synthetic promoter.

[0433] 20. The cell according to any one of paragraphs 1-19, wherein the one or more polypeptide of interest is secreted. 21. The cell according to any one of paragraphs 3-20, wherein the synthetic promoter comprises, essentially consist of, or consist of the polynucleotide of SEQ ID NO: 7.

[0434] 22. The cell according to any one of paragraphs 1-21 , wherein the one or more inducible promoter is repressed by thiamine, e.g., repressed when thiamine is present in the cultivation medium.

[0435] 23. The cell according to any one of paragraphs 1-22, wherein promoter activity of the inducible promoter is inversely proportional to thiamine concentration in the cultivation medium.

[0436] 24. The cell according to any one of paragraphs 1-23, wherein the one or more inducible promoter comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 1.

[0437] 25. The cell according to any one of paragraphs 1-24, wherein the one or more inducible promoter comprises, essentially consists of, or consists of the polynucleotide of SEQ ID NO: 1.

[0438] 26. The cell according to any one of paragraphs 1-25, wherein the one or more inducible promoter is heterologous to the host cell.

[0439] 27. The cell according to any one of paragraphs 1-26, wherein the acetate-inducible promoter is heterologous to the host cell.

[0440] 28. The cell according to any one of paragraphs 1-27, wherein the acetate-inducible promoter is from Aspergillus nidulans.

[0441] 29. The cell according to any one of paragraphs 1-28, wherein the acetate-inducible promoter is activated by acetate, e.g., activated when acetate is present in the cultivation medium, such as by adding acetic acid to the cultivation medium.

[0442] 30. The cell according to any one of paragraphs 1 -29, wherein promoter activity of the acetate- inducible promoter is proportional to acetate concentration in the cultivation medium.

[0443] 31. The cell according to any one of paragraphs 1-30, wherein the one or more inducible promoter is a thiA promoter or variant thereof. 32. The cell according to any one of paragraphs 1-31 , wherein expression of the one or more polypeptide of interest is induced by cultivating the cell in a cultivation medium free of thiamine, or in a cultivation medium essentially free of thiamine.

[0444] 33. The cell according to any one of paragraphs 1-32, wherein the one or more TF is a synthetic TF.

[0445] 34. The cell according to any one of paragraphs 1-33, wherein the one or more TF comprises a transcription regulator, a nuclear localization signal (NLS), and a transcription activation domain.

[0446] 35. The cell according to any one of paragraphs 1-34, wherein the one or more TF comprises one or more nuclear localisation signal NLS, e.g., two or more NLS, three or more NLS, or four or more NLS.

[0447] 36. The cell according to any one of paragraphs 1-35, wherein the NLS comprises or consists of a SV40 NLS.

[0448] 37. The cell according to any one of paragraphs 1-36, wherein the transcription regulator comprises or consists of a polypeptide from the TetR family.

[0449] 38. The cell according to any one of paragraphs 1-37 wherein the transcription activation domain comprises or consists of a VP16 activation domain, and / or a VP64 activation domain.

[0450] 39. The cell according to any one of paragraphs 1-38, wherein the one or more second polynucleotide is encoding the one or more TF and comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 3.

[0451] 40. The cell according to any one of paragraphs 1-39, wherein the one or more TF comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 4. 41. The cell according to any one of paragraphs 1-40, wherein the one or more TF is heterologous to the host cell.

[0452] 42. The cell according to any one of paragraphs 1-41 , wherein the one or more polypeptide of interest is heterologous to the host cell.

[0453] 43. The cell according to any one of paragraphs 1-42, wherein the one or more polypeptide of interest is hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.

[0454] 44. The cell according to any one of paragraphs 1-43, wherein the one or more polypeptide of interest is a hydrolase, isomerase, ligase, lyase, oxidoreductase, transferase, galactosidase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0455] 45. The cell according to any one of paragraphs 1-44, wherein the one or more polypeptide of interest is an endolase.

[0456] 46. The cell according to any one of paragraphs 1-45, wherein the endolase is encoded by a first polynucleotide comprising, essentially consisting of, or consisting of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 5.

[0457] 47. The cell according to any one of paragraphs 1-46, wherein the endolase comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 6.

[0458] 48. The cell according to any one of paragraphs 1-44, wherein the one or more polypeptide of interest is a phosphodiesterase (PDE). 49. The cell according to any one of paragraphs 1-44 or 48, wherein the phosphodiesterase is encoded by a first polynucleotide comprising, essentially consisting of, or consisting of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ NO: 8.

[0459] 50. The cell according to any one of paragraphs 1-44 or 48-49, wherein the phosphodiesterase comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 9.

[0460] 51. The cell according to any one of paragraphs 1-50, wherein the one or more polypeptide of interest is an inactivated enzyme.

[0461] 52. The cell according to any one of paragraphs 1-51 , wherein the one or more polypeptide of interest has reduced, substantially no, or no enzyme activity.

[0462] 53. The cell according to any one of paragraphs 1-52, wherein the host cell comprises at least one undisrupted PrtT gene in its genome, e.g., where the transcription factor PrtT is expressed in its native form.

[0463] 54. The cell according to any one of paragraphs 1-43, which is isolated.

[0464] 55. The cell according to any one of paragraphs 1-54, which is purified.

[0465] 56. A method of producing one or more polypeptide of interest (POI), comprising cultivating the host cell of any one of paragraphs 1-55 under conditions conducive for the production of the one or more POI.

[0466] 57. The method of paragraph 56, wherein the cell is cultivated in a cultivation medium comprising at least 0.01 % v / v acetate.

[0467] 58. The method of any one of paragraphs 56-58, wherein the cell is cultivated in a cultivation medium having a pH above pH 4.5, such as above pH 4.76, such as above pH 5.0, or above pH 5.5, such as above pH 6.0, or such as above pH 6.5. 59. The method of any one of the preceding paragraphs, wherein the method comprises recovering the one or more POI.

[0468] 60. The method of any one of the preceding paragraphs, wherein the one or more POI is secreted into the cultivation medium.

[0469] 61. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises a pH in the range of 3-8, e.g., 4-8, 3-7, 4-7, 3-6, 4-6, 5-6, 3-5, 4-5, 5-8, 5-7, or 5-6.

[0470] 62. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises a pH in the range of 4.5 - 8, e.g., 5 - 8, 4.5 - 7, 5 - 7, 4.5 - 6, 5 - 6, 5 - 6, or 4.5 - 5.

[0471] 63. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises at least 0.011 % v / v acetate, such as e.g., at least 0.015% v / v acetate, at least 0.016% v / v acetate , at least 0.017% v / v acetate , at least 0.018% v / v acetate , at least 0.019% v / v acetate , at least 0.02% v / v acetate , at least 0.025% v / v acetate , at least 0.030% v / v acetate , at least 0.035% v / v acetate, at least 0.040% v / v acetate, at least 0.045% v / v acetate, at least 0.050% v / v acetate, at least 0.055% v / v acetate, at least 0.060% v / v acetate, at least 0.065% v / v acetate, at least 0.070% v / v acetate, at least 0.075% v / v acetate, at least 0.080% v / v acetate, at least 0.085% v / v acetate, at least 0.090% v / v acetate, at least 0.095% v / v acetate, at least 0.10% v / v acetate, at least 0.11 % v / v, at least 0.12% v / v, at least 0.13% v / v, at least 0.14% v / v, at least 0.15% v / v, at least 0.16% v / v, at least 0.17% v / v, at least 0.18% v / v, at least 0.19% v / v, at least 0.20% v / v, at least 0.21 % v / v, at least 0.22% v / v, at least 0.23% v / v, at least 0.24% v / v, at least 0.25% v / v, at least 0.3% v / v, at least 0.4% v / v, or at least 0.5% v / v acetate.

[0472] 64. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises between 0.01 - 0.5% v / v acetate, e.g. ,0.01 - 0.4% v / v acetate, 0.01 - 0.3% v / v acetate, 0.01 - 0.2% v / v acetate, 0.01 - 0.1 % v / v acetate, 0.02 - 0.5% v / v acetate, 0.02 - 0.4% v / v acetate, 0.02 - 0.3% v / v acetate, 0.02 - 0.2% v / v acetate, 0.02 - 0.1 % v / v acetate, 0.03 - 0.5% v / v acetate, 0.03 - 0.4% v / v acetate, 0.03 - 0.3% v / v acetate, 0.03 - 0.2% v / v acetate, 0.03 - 0.1 % v / v acetate, 0.04 - 0.5% v / v acetate, 0.04 - 0.4% v / v acetate, 0.04 - 0.3% v / v acetate, 0.04 - 0.2% v / v acetate, 0.04 - 0.1 % v / v acetate, 0.05 - 0.5 % v / v acetate, 0.05% - 0.4% v / v acetate, 0.05-0.3 % v / v acetate, 0.05-0.2% v / v acetate, or 0.05 - 0.1 % v / v acetate, preferably between 0.01-0.5% v / v acetate. 65. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises at least 0.02% v / v acetate.

[0473] 66. The method of any one fo the preceding paragraphs, wherein acetate is the sole carbon source of the cultivation medium.

[0474] 66a. The method of any one of the preceding paragraphs, wherein the acetate inducible promoter is repressed, or partially repressed by the presence of glucose.

[0475] 67. The method of any one of the preceding paragraphs, wherein acetate is not the only carbon source of the cultivation medium.

[0476] 68. The method of any one of the preceding paragraphs, wherein, when acetate is not the only carbon source of the cultivation medium, the cultivation medium comprises acetate and glucose.

[0477] 69. The method of any one of the preceding paragraphs, wherein, when acetate is not the only carbon source of the cultivation medium, the cultivation medium comprises acetate and maltose.

[0478] 70. The method of any one of the preceding paragraphs, wherein, when acetate is not the only carbon source of the cultivation medium, the cultivation medium comprises acetate and sucrose.

[0479] 71. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises at most 2% v / v glucose, e.g., at most 1.5% v / v, at most 1.0% v / v, at most 0.5% v / v, at most 0.4% v / v, at most 0.3% v / v, at most 0.2% v / v, at most 0.1% v / v, at most 0.05% v / v, at most 0.04% v / v, at most 0.03% v / v, at most 0.02% v / v, or at most 0.01% v / v glucose.

[0480] 72. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises at most 2% v / v maltose, e.g., at most 1.5% v / v, at most 1.0% v / v, at most 0.5% v / v, at most 0.4% v / v, at most 0.3% v / v, at most 0.2% v / v, at most 0.1% v / v, at most 0.05% v / v, at most 0.04% v / v, at most 0.03% v / v, at most 0.02% v / v, or at most 0.01% v / v maltose.

[0481] 73. The method of any one of the preceding paragraphs, wherein the cultivation medium comprises at most 2% v / v sucrose, e.g., at most 1.5% v / v, at most 1.0% v / v, at most 0.5% v / v, at most 0.4% v / v, at most 0.3% v / v, at most 0.2% v / v, at most 0.1% v / v, at most 0.05% v / v, at most 0.04% v / v, at most 0.03% v / v, at most 0.02% v / v, or at most 0.01 % v / v sucrose.

[0482] 74. The method of any one of the preceding paragraphs, wherein the acetate-inducible promoter is selected from the list of an AOX1 (alcohol oxidase 1) promoter, a PCK (phosphoenolpyruvate carboxykinase) promoter, a PDC (pyruvate decarboxylase) promoter, an ALD promoter, an amdS (acetamidase) promotor, an alcA (alcohol dehydrogenase 1) promotor, an acuL (succinate / fumarate mitochondrial transporter) promotor, an alcC (alcohol dehydrogenase 3) promotor, a facC (carnitine acetyl transferase) promotor, a coaT (acetyl-CoA hydrolase) promotor, an acuD (isocitrate lyase) promoter, or an acuE (malate synthase) promoter.

[0483] 75. The method of any one of the preceding paragraphs, wherein the method is a fed-batch cultivation.

[0484] 76. The method of any one of the preceding paragraphs, wherein the method is a batch cultivation.

[0485] 77. The method of any one of the preceding paragraphs, wherein the method is a continuation cultivation.

[0486] 77a. The method of any one of the preceding paragraphs, wherein the method comprises adding a feed to the cultivation medium, the feed comprising at least 10 % v / v acetic acid, e.g., such as at least 20 % v / v acetic acid, at least 30 % v / v acetic acid, at least 35 % v / v acetic acid, at least 40 % v / v acetic acid, at least 45 % v / v acetic acid, at least 50 % v / v acetic acid, at least 55 % v / v acetic acid, at least 60 % v / v acetic acid, at least 65 % v / v acetic acid, at least 70 % v / v acetic acid, at least 75 % v / v acetic acid, or at least 80 % v / v acetic acid.

[0487] 78. A whole broth formulation or cell culture composition obtained by the method of any one of the preceding paragraphs.

[0488] 79. A whole broth formulation or cell culture composition comprising the cell of any one of the preceding paragraphs.

[0489] 80. Use of the cell according to any one of the preceding paragraphs to produce a food or feed product and / or to produce the whole broth formulation or cell culture composition according to paragraphs 78-79.

[0490] 81. A polynucleotide comprising an inducible promoter operably linked to a polynucleotide encoding a POI, wherein the promoter is heterologous to the polynucleotide encoding the POI, the promoter comprising, essentially consisting of, or consisting of a polynucleotide having at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the polynucleotide sequences of SEQ NOs: 10, 1 , 2, or 11-1489.

[0491] 82. The polynucleotide according to paragraph 81 , wherein the promoter comprises, essentially consists of, or consists of any of the polynucleotides of SEQ ID NOs: 10, 1 , 2, or 11- 1489.

[0492] 83. The polynucleotide according to any one of paragraphs 81-82, wherein the promoter is having at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2% or at most 1 % sequence differences to any of the polynucleotides of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0493] 84. The polynucleotide according to any one of paragraphs 81-83, wherein the promoter differs from the polynucleotide of SEQ ID NOs: 10, 1 , 2, or 11-1489, by at most 10 nucleotides, such as at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0494] 85. The polynucleotide according to any one of paragraphs 81-84, wherein the promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and is induced by the absence of thiamine, e.g., induced by a low thiamine concentration in the cultivation medium.

[0495] 86. The polynucleotide according to any one of paragraphs 81-85, wherein the promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and the promoter is repressed by the presence of thiamine in the cultivation medium.

[0496] 87. The polynucleotide according to any one of paragraphs 81-86, wherein the promoter comprises or consists of a polynucleotide having at least 60% sequence identity to the polynucleotide of SEQ ID NO: 1 and activity of the promoter is inversely proportional to the thiamine concentration in a cultivation medium.

[0497] 88. The polynucleotide according to any one of paragraphs 81-84, wherein the promoter comprises or consists of a polynucleotide having at least 60% sequence identity to any of the polynucleotides of SEQ ID NOs: 10, 1 , 2, or 11-1489, and wherein the promoter is induced by acetate, e.g., induced by adding acetate or acetic acid to the cultivation medium.

[0498] 88a. The polynucleotide of paragraph 88, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 10, 1 , 2, or 11-1489.

[0499] 88b. The polynucleotide of paragraph 88, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 10.

[0500] 88c. The polynucleotide of paragraph 88, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 1.

[0501] 88d. The polynucleotide of paragraph 88, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 2.

[0502] 88e. The polynucleotide of paragraph 88, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 11.

[0503] 88f. The polynucleotide of paragraph 88, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 22. 88g. The polynucleotide of any one of paragraphs 88-88f, wherein the acetate inducible promoter is repressed, or partially repressed, by the presence of glucose.

[0504] 88h. The polynucleotide of any one of paragraphs 88-88f, wherein the acetate-inducible promoter comprises a FacB-binding site.

[0505] 88i. The polynucleotide of any one of paragraphs 88-88f, wherein the acetate-inducible promoter is induced by FacB.

[0506] 88j. The polynucleotide of any one of paragraphs 88-88i, wherein the acetate-inducible promoter is associated with an increased mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using acetate as carbon source, relative to the mRNA transcript level of the polynucleotide located immediately downstream of the acetate-inducible promoter when cultivated using glucose as carbon source, the cells otherwise being cultivated in identical conditions.

[0507] 88k. The polynucleotide of paragraph 88j, wherein the polynucleotide located immediately downstream of the acetate-inducible promoter comprises or consists of the one or more polynucleotide.

[0508] 89. A nucleic acid construct or expression vector comprising the polynucleotide of any one of paragraphs 81 -88k, wherein the promoter is operably linked to one or more polynucleotide sequences encoding a polypeptide of interest, e.g., a TF, and wherein the promoter directs the production of the polypeptide of interest.

[0509] 90. The polynucleotide, nucleic acid construct or expression vector according to any one of paragraphs 81-89, which is isolated.

[0510] 91. The polynucleotide, nucleic acid construct or expression vector according to any one of paragraphs 81-90, which is purified.

[0511] 92. A fungal cell comprising in its genome the polynucleotide according to any one of paragraphs 81 -88k, and / or the polynucleotide, nucleic acid construct or expression vector according to any one of paragraps 89-91 . 93. The cell of paragraph 92, wherein the cell comprises at least two copies, e.g., three, four, five, six, seven, or eight or more copies of the polynucleotide or vector or construct of any one of paragraphs 81-91.

Claims

CLAIMS1. A fungal host cell comprising in its genome: i) an acetate-inducible promoter operably linked to one or more first polynucleotide encoding one or more first polypeptide of interest (POI), wherein the one or more first polynucleotide is heterologous to the acetate-inducible promoter.

2. The cell of claim 1 , further comprising in its genome: ii) a synthetic promoter operably linked to one or more second polynucleotide encoding one or more second POI, said synthetic promoter comprising one or more transcription factor (TF)-specific binding site, wherein the one or more first POI comprises or consists of a TF capable of binding to the TF-specific binding site of the synthetic promoter.

3. The cell of claim 1 or 2, wherein the acetate-inducible promoter comprises or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 10, 1 , 2, or 11-1489.

4. The cell according to any one of claims 1-3, wherein the one or more first POI is selected from the list of alpha-lactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human beta-lactalbumin, or a bovine beta-lactalbumin, hydrolase, e.g., a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), or a glycosyl hydrolase 13 (GH13); isomerase, ligase, lyase, oxidoreductase, transferase, galactosidase, e.g., an alpha-galactosidase, an alpha-galactosidase 3, or an alpha-galactosidase C, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta- xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, lipase, mannosidase, mutanase, ovalbumin, oxidase, pectinolytic enzyme, peroxidase, phosphatase, e.g., an alkaline phosphatase; phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, a transcription factor (TF), transglutaminase, or xylanase.

5. The cell of claim 2, wherein the one or more first POI comprises or consists of one or more synthetic TF.

6. The cell of claim 5, wherein the one or more synthetic TF comprises, essentially consists of, or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ NO: 4.

7. The cell of claim 5, wherein the one or more second POI is selected from the list of alphalactalbumin, e.g., a human alpha-lactalbumin, or a bovine alpha-lactalbumin; beta-lactalbumin, e.g., a human beta-lactalbumin, or a bovine beta-lactalbumin, hydrolase, e.g., a saponin hydrolase, a glycosyl hydrolase 71 (GH71), a glycosyl hydrolase 20 (GH20), or a glycosyl hydrolase 13 (GH13); isomerase, ligase, lyase, oxidoreductase, transferase, galactosidase, e.g., an alpha-galactosidase, an alpha-galactosidase 3, or an alpha-galactosidase C, alphaglucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, endolase, esterase, glucoamylase, invertase, laccase, lactoferrin, e.g., a human lactoferrin, or a bovine lactoferrin, lipase, mannosidase, mutanase, ovalbumin, oxidase, pectinolytic enzyme, peroxidase, phosphatase, e.g., an alkaline phosphatase; phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

8. The cell of any one of claims 2-7, wherein the synthetic promoter comprises, essentially consists of, or consists of a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 7.

9. The cell of any one of claims 1-8, wherein the one or more first POI is heterologous to the cell.

10. The cell of any one of the preceding claims, wherein the cell is a filamentous fungal cell.

11. The cell of any one of the preceding claims, wherein the cell is an Aspergillus cell.

12. The cell of any one of the preceding claims, wherein the acetate-inducible promoter is heterologous to the cell.

13. A method of producing one or more polypeptide of interest (POI), comprising cultivating the host cell of any one of claims 1-12 in a cultivation medium comprising at least 0.01% v / v acetate, under conditions conducive for production of the one or more POI.

14. A whole broth formulation or cell culture composition comprising the cell of any one of claims 1-12, or a whole broth formulation or cell culture composition obtained by the method of claim 13.

15. Use of the cell according to any one of claims 1-12 to produce a food or feed product and / or to produce the whole broth formulation or cell culture composition according to claim 14.

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