WO2024120767A1

WO2024120767A1 - Modified rna polymerase activities

Info

Publication number: WO2024120767A1
Application number: PCT/EP2023/081808
Authority: WO
Inventors: Takashi Nakanishi
Original assignee: Novozymes A/S
Priority date: 2022-12-05
Filing date: 2023-11-15
Publication date: 2024-06-13

Abstract

The present invention relates to mutant host cells with reduced RNA polymerase subunit expression, polynucleotides and expression vectors for reduced expression of RNA polymerase subunit in a host cell, and host cells and methods for producing a polypeptide of interest.

Description

MODIFIED RNA POLYMERASE ACTIVITIES

Reference to a Sequence Listing

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

Background of the Invention

Field of the Invention

Description of the Related Art

Recombinant gene expression in recombinant host cells, such as bacterial host cells or fungal host cells, is a common method for recombinant protein production. Recombinant proteins produced in such systems are enzymes and other valuable proteins. In industrial and commercial purposes, the productivity of the applied cell systems, i.e. , the production of total protein per fermentation unit, is an important factor of production costs. Traditionally, yield increases have been achieved through mutagenesis, signal peptide optimation, and screening large number of mutants for increased production of proteins of interest. However, this approach is mainly only useful for the overproduction of endogenous proteins in isolates containing the enzymes of interest. Therefore, for each new protein or enzyme product, a lengthy strain and process development program is required to achieve improved productivities.

For the overexpression of heterologous proteins in recombinant host cell systems, the production process is recognized as a complex multi-phase and multi-component process. Cell growth and product formation are determined by a wide range of parameters, including the composition of the culture medium, fermentation pH, fermentation temperature, dissolved oxygen tension, shear stress, and bacterial morphology.

Various approaches to improve transcription have been used in bacteria. For the expression of heterologous genes, codon-optimized, synthetic genes can improve the transcription rate (WO9923211 , Novozymes A/S). To obtain high-level expression of a particular gene, a well-established procedure is targeting multiple copies of the recombinant gene constructs to the locus of a highly expressed endogenous gene.

However, multi-copy strains often reach the host cell's expression limits, whereafter integration of additional copies of the recombinant gene does not further improve recombinant yields.

On a molecular level, expression of a protein of interest can be divided into the following steps: i) transcription of the gene of interest from DNA into RNA, ii) translation of the RNA into a polypeptide, and iii) maturation and secretion of the polypeptide of interest. During step i), RNA in all cellular organisms is synthesized by a complex molecular machine, the DNA-dependent RNA polymerase (RNAP, or Rpo). In its simplest bacterial form, the enzyme comprises at least four subunits with a total molecular mass of around 400 kDa. The eukaryotic enzymes comprise upwards of a dozen subunits, with a total molecular mass of around 500 kDa. The catalytically competent bacterial core Rpo (subunit composition: 2x RpoA (a), 1x RpoB (p), 1x RpoB’ (p’), and 1x RpoZ (w)) is evolutionary conserved in sequence, structure and fuction from bacteria to eukaryots including fungal cells and mammalian cells (Borukhov & Nudler, Trends in Microbiology, 16(3), 2008, 126-134).

Despite the presented approaches, it is of continuous interest to further improve recombinant protein production in genetically modified host cells. The object of the present invention is to provide a modified host cell and a method of protein production with increased productivity and/or yield of recombinant protein.

Summary of the Invention

As disclosed herein, the inventors of the present invention have identified that for increasing yields during recombinant protein production, one bottleneck can be described as a sub-optimal transcription capability of the host cell. In other words, transcript (RNA) formation for the polypeptide of interest has to compete with all other transcription processes taking place in the host cell, i.e., all other transcribed genes in a host cell.

Surprisingly, the inventors have shown that for recombinant cells producing a recombinant protein of interest, reduced expression of native RNA polymerase subunits improves secretion and/or yield of the recombinant protein of interest. After reducing expression of the RNA polymerase subunit alpha (RpoA) recombinant protein yield was improved significantly, i.e., by 12 %, when compared to protein yield from host cells with unmodified RpoA expression, and biomass formation was reduced by 18% when compared to biomass formation of host cells with unmodified RpoA expression. As described in the Examples, the inventors have identified that reduced expression of the RNAP subunit surprisingly resulted in increased yield of different classes of proteins of interest (amylases and proteases). Therefore, we expect that these findings also apply for other proteins of interest, such as other enzymes, and in particular to other heterologous proteins. In addition, reduced RNA polymerase subunit expression resulted in decreased biomass formation (18% decrease) which is beneficial in terms of fermentation, e.g. a more efficient downstream processing and product formulation since less biomass per polypeptide product has to be removed, which was totally unexpected.

Without wishing to being bound to any theory, reduced expression of RNA polymerase subunits may resolve the bottleneck of transcription and/or translation of the polypeptide of interest, in particular, when the RNA transcript (mRNA) of the polypeptide of interest has good stability and/or when several copies of the gene of interest are integrated into the host cell genome, and thus provides increased yield of the polypeptide of interest. Fine tuning of the expression of RNA polymerase subunits may ensure that, proportionally to the mRNA formation of other host cell genes, more RNA transcipt of the gene of interest can be generated, which leads to an overall increased yield of the polypeptide of interest. Furthermore, reduced RNA polymerase subunit expression can surprisingly be used as a tool to decrease biomass formation without compromising product yield or cell viability, but actually increasing product yield at the same time.

Accordingly, in a first aspect the present invention relates to a mutant cell comprising in its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptide, wherein expression of the one or more Rpo subunit polypeptide is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell.

In a second aspect, the invention relates to methods for producing one or more polypeptides of interest, the method comprising, a) providing a mutant cell according to the first aspect, b) cultivating said cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally recovering the one or more polypeptide of interest.

In a third aspect, the invention relates to nucleic acid constructs comprising a heterologous promoter and/or a mutated Shine-Dalgarno sequence operably linked to a second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptide.

In a fourth aspect, the invention relates to expression vectors comprising the nucleic acid construct according to the third aspect.

Definitions

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.

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.

Amylase: The term “amylase” means a polypeptide having amylase activity, such as an alphaamylase (EC 3.2.1.1) that catalyzes the hydrolyzation of the 1 ,4-a-glucosidic linkages in amylose and amylopectin. A non-limiting example for an amylase is the alpha-amylase shown in SEQ ID NO:7.

Amylase Activity: Amylase activity can be determined in various ways known to the skilled person. For example, amylase activity can be measured by the assay described under the section ’’Enzymatic Assays” in the Examples.

Bacterial RNA polymerase subunit: The term “Bacterial RNA polymerase subunit” means any bacterial RNA polymerase subunit polypeptide selected from the list of subunit beta (p), subunit beta’ (p’), subunit alpha (a), and subunit omega (w).

Biomass: In the context of the present invention, the term "biomass" means the accumulation of cells during cultivation. For fermentation of bacterial cells, the term ’’biomass” also includes spores and other cellular structures. For fermentation of fungal cells, the term ’’biomass” also includes hyphae and other cellular structures. Biomass is typically measured as dry weight or wet weight of a plurality of fungal cells. Additionally, or alternatively, biomass can be measured by determining the optical density of the cultivation broth at a specific wavelength, e.g., at 650 nm wavelength for bacterial cell cultures. 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.

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.

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. A non-limitng example for a promoter is shown by the P3 promoter with SEQ ID NO: 38. 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.

Eukaryotic RNA polymerase: The term “eukaryotic RNA polymerase” means any eukaryotic polymerase including RNA polymerase I, RNA polymerase II or RNA polymerase III , and also includes any subunit from a eukaryotic RNA polymerase.

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.

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.

Extension: The term “extension” means an addition of one or more amino acids to the amino and/or carboxyl terminus of a RNA polymerase subunit polypeptide, wherein the “extended” subunit polypeptide modifies RNA polymerase activity, e.g, decreases RNA polymerase activity. Fragment: The term “fragment” means a polypeptide having one or more amino acids absent from the amino and/or carboxyl terminus of the mature RNA polymeras subunit polypeptide, wherein the fragment modifies RNA polymerase activity, e.g., decrases RNA polymerase activity.

Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Gene: The term ’’gene”, such as ”rpoA gene”, means a polynucleotide sequence comprising a polynucleotide sequence encoding a polypeptide product / PCI (protein of interest), a promoter sequence and a shine-dalgarno sequence (ribosome binding site, RBS) upstream of the polynucleotide sequence encoding the PCI. Transcription of a gene and/or translation of a gene product may, for example, be modified by using different promoters and/or by using altered shine-dalgarno sequences, respectively. As a non-limiting example, transcription of the rpoA gene can be modified by replacing the native promoter with a heterologous promoter. As a further non-limiting example, translation of the RpoA polypeptide can be modified by providing a mutated shine-dalgarno sequence in the rpoA gene comprising one or more nucleic acid substitutions in the native shine-dalgarno sequence of ’’AAGGAGG”.

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.

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.

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. Isogenic cell: The term “isogenic” refers, with respect to a host cell, to a parent or clonal host cell with an essentially identical genotype, e.g. a parent host cell having essentially identical background mutations as the daughter cell, yet with specific differences due to a later on introduced additional mutation or polynucleotide to the daughter cell resulting in a daughter cell with the additional mutation and/or polynucleotide but the daughter cell otherwise being isogenic to the parent cell.

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.

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). In one aspect, the mature polypeptide is SEQ ID NO: 3.

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

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.

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.

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. In another example, a SD sequence is operably linked to a coding sequence such that both sequences will be transcribed into one mRNA.

Protease: The term “protease” means a polypeptide having protease activity, catalyzing the hydrolytic degradation of proteins or polypeptides to smaller amino acid polymers (EC 3.4.21 .-). A nonlimiting example for a protease is the polypeptide shown in SEQ ID NO:9.

Protease Activity: Protease activity can be determined in various ways known to the skilled person. For example, protease activity can be measured by the assay described under the section ’’Enzymatic Assays” in the Examples.

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.

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.

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.

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 (i.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.

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”.

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 sheed 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 solid-liquid 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.

RNA polymerase activity: The term “RNAP activity” or “RNA polymerase activity” means the capability of synthesizing RNA molecules from a template of DNA through the process of transcription.

As a non-limiting example, RNAP activity can be determined with a Rifampicin dilution assay, wherein RNAP activity is assessed by determining Rifampicin-resistance, i.e. cell survival in the presence of Rifampicin is related to increased RNAP activity, whereas cell death in the presence of Rifampicin is related to decreased RNAP activity. Such assay is shown in Example 6.

RNA polymerase subunit polypeptide: The term “RNA polymerase subunit polypeptide” or “RNAP subunit” or “Rpo subunit” means any subunit of an RNA polymerase across the animal kingdom. RNA polymerase, comprising several subunits, is involved in catalyzating the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. This reaction is also known as EC:2.7.7.6. RNA in all cellular organisms is synthesized by a complex molecular machine, the DNA-dependent RNA polymerase (RNAP, or Rpo). In its simplest bacterial form, the enzyme comprises at least four subunits with a total molecular mass of around 400 kDa. The eukaryotic enzymes comprise upwards of a dozen subunits, with a total molecular mass of around 500 kDa. The catalytically competent bacterial core Rpo (subunit composition: 2x RpoA (a), 1x RpoB (p), 1x RpoB’ (p’), and 1x RpoZ (w)) is evolutionary conserved in sequence, structure and function from bacteria to eukaryots including fungal cells and mammalian cells, see Table 1 (Borukhov & Nudler, Trends in Microbiology, 16(3), 2008, 126-134).

Table 1 : The table shows a comparative scheme of the RNA polymerase subunits aligned according to sequence and/or functional homology (Barba-Aliaga et al., Front. Mol. Biosci. 21 April 2021).

RpoA polypeptide: The term “RpoA polypeptide” or “RNAP subunit alpha” or “RpoA subunit” means a DNA-directed RNA polymerase subunit alpha which is involved in catalyzating the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. This reaction is also known as EC:2.7.7.6. A non-limiting example for a RpoA polypeptide is the Bacillus licheniformis RpoA polypeptide shown in SEQ ID NO:2. rpoA gene: The term "rpoA gene” means a polynucleotide sequence encoding a a RNA polymerase subunit alpha polypeptide RpoA, such as an DNA-directed RNA polymerase subunit alpha (EC 2.7.7.6) also known as RNAP subunit alpha. RpoA I RNAP subunit alpha is a DNA-dependent RNA polymerase which catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. A non-limiting example for a rpoA gene is the rpoA gene from Bacillus licheniformis shown in SEQ ID NO: 3, which includes a 7 nucleotide long Shine Dalgarno sequence ” AAGGAGG” at its 5’ end at positions 1-7 of SEQ ID NO: 3. A non-limiting example for a RpoA polypeptide is the RpoA polypeptide from Bacillus licheniformis shown in SEQ ID NO: 2.

The term "rpoA gene” includes the polynucleotide sequence upstream of the start codon ”ATG”, comprising the Shine-Dalgarno (SD) sequence ’’AAGGAGG”, as shown in SEQ ID NO: 3. During transcription of the rpoA gene, said SD sequence is transcribed onto the mRNA together with the polynucleotide sequence encoding RpoA. The SD sequence is a ribosomal binding site and generally located 5-9 bases upstream of the start codon AUG. The SD RNA sequence helps to recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon. Mutations in the SD sequence can reduce or increase translation in host cells, resulting in reduced or increased polypepitde levels, respectively (Velaquez et al., Journal of Bacteriology, May 1991 , p. 3261 - 3264). Thus, modification of the SD sequence in the rpoA gene can reduce or increase RpoA polypeptide levels. This change is due to a reduced or increased rpoA mRNA pairing efficiency with the ribosomes. A non-limiting example for a mutated SD sequence is the mutated SD sequence in the B. licheniformis rpoA gene shown by the nucleotide sequence of SEQ ID NO: 4, where G is substituted by A at a position corresponding to position 3 ”G3A” of SEQ ID NO:3 (native SD + rpoA sequence). During translation, although such mutation affects the mRNA-ribosome pairing efficiency, said mutation does not impair the sequence of the matured RpoA polypeptide.

Homologs of RpoA in eukaryotic cells include RNA polymerase I subunits RPAC40 and RPAC19, RNA polymerase II subunits RPB3 and RPB11 , and RNA polymerase III subunits RPAC40 and RPAC19 (see Table 1).

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

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: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

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 NUC4.4) 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:

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

Shine-Dalgarno sequence: The term “Shine-Dalgarno sequence” or “SD sequence” means a ribosomal binding site on an RNA sequence which, in bacterial and archeal cells, is generally located 5-9 bases upstream of the start codon AUG. The SD RNA sequence helps to recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon. Mutations in the SD sequence can reduce or increase translation in host cells, resulting in reduced or increased polypepitde levels, respectively (Velaquez et al., Journal of Bacteriology, May 1991 , p. 3261 - 3264). Thus, modification of the SD sequence in a rpo subunit gene can reduce or increase RPO subunit polypeptide levels. This change of RPO subunit levels is due to a reduced or increased rpo subunit mRNA pairing efficiency with the ribosomes.

Mutated Shine-Dalgarno sequence: The term “mutated Shine-Dalgarno sequence” or “mutated SD sequence” means a SD sequence comprising one or more nucleic acid modifications, such as a nucleic acid substitution, nucleic acid deletion, or nucleic acid insertion. As described above, modification of the SD sequence can, depending on the mutation, reduce or increase translation of the gene located downstream of the modified or mutated SD sequence.

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 RNA polymerase subunit polypeptide coding sequence; wherein the subsequence encodes a fragment of a RNA polymerase subunit which does not reduce RNA polymerase activity.

Therapeutic polypeptide: The term “therapeutic polypeptide” means any polypeptide or protein, or variant thereof, which is suitable for use in the therapy of human diseases or conditions, or for use in veterinary medicine. Non-limiting examples for therapeutic polypeptides are antibody-based drugs, Fc fusion proteins, an anticoagulant, a blood factors, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a hormone, an interferon (e.g. an interferon alpha-2b), an interleukin, a lactoferrin, an alpha-lactalbumin, a beta-lactalbumin, an ovomucoid, an ovostatin, a cytokine, an obestatin, a human galactosidase (e.g. human alpha-galactosidase A) and a thrombolytic.

Variant: The term “variant” means a RNA polymerase subunit polypeptide having RNA polymerase activity 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.

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

Overview of Sequence information

Reduced expression of RNA polymerase subunit

Reduction of RNA polymerase activity in host cells In one aspect, the present invention relates to decreased transcription and/or translation of one or more RNA polymerase subunits, which effects in a decrease of overall RNA polymerase (RNAP) activity. Decreased transcription and/or translation has been shown to increase yield of the polypeptide of interest in recombinant host cells, while also reducing biomass formation.

Decreased RNAP activity, and/or decreased transcription and/or decreased translation of one or more RNA polymerase subunits can, for example, be achieved by: a) operably linking the second polynucleotide encoding the RNAP subunit to a second heterologous promoter which is weaker than the native promoter of the RNAP subunit encoding gene, e.g. by replacing the native promoter with the weaker second heterologous promoter, b) operably linking the second polynucleotide to a mutated Shine-Dalgarno sequence comprising one or more nucleic acid modifications compared to the native SD sequence of the RNAP subunit encoding gene, wherein the mutated SD sequence results in a weaker ribosome binding of the RNA during translation of the RNAP subunit, c) using CRISPRi, RNAi or other interference techniques known to the skilled person to target the coding sequence of the RNAP subunit gene or its transcript during transcription or translation, respectively, d) deleting one or more genes encoding a RNAP subunit, and/or e) introducing one or more mutation in one or more of the RNAP subunit genes to provide a mutated RNAP subunit which reduces RNAP activity.

In a particular embodiment, the second polynucleotide is operably to a second heterologous promoter, wherein the second heterologous promoter constitutes a decrease of transcription, relative to the transcription of the native promoter of the RNAP subunit encoding gene, and wherein the SD sequence upstream of the RNAP subunit encoding gene is not mutated.

The present invention relates to reduced expression of one or more RNA polymerse (RNAP) subunit polypeptides. In one aspect, the invention relates to reduced expression of one or more RNAP subunit polypeptides, selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40;

(b) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO:39, or the cDNA sequence thereof;

(c) a polypeptide derived from SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40, by substitution, deletion or addition of one or several amino acids;

(d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and/or C- terminal end has been extended by the addition of one or more amino acids; and (e) a fragment of the polypeptide of (a), (b), (c), or (d).

In an aspect, the one or more RNAP subunit polypeptide has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% to SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40.

The one or more RNAP subunit polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40, or a mature polypeptide thereof.

The polypeptide may have an N-terminal and/or C-terminal extension of one or more amino acids, e.g., 1-5 amino acids.

In some embodiments, the second polynucleotide encoding the one or more RNA subunit polypeptide has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86% , at least 87%, at least 88%, at least 89%, 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% to the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO:39, or the cDNA sequence thereof.

The second polynucleotide preferably comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO:39.

In another aspect, the RNAP subunit polypeptide is derived from of SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40by substitution, deletion or addition of one or several amino acids. I n another aspect, the polypeptide is derived from a mature polypeptide of of SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40by substitution, deletion or addition of one or several amino acids. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into the polypeptide of of SEQ ID NO: 2, SEQ ID NO: 11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:40is 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 protein; 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.

Essential amino acids in a polypeptide 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 RNA polymerase 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. The active site of the enzyme or other 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 polypeptide, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide 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 polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

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 et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ef a/., 1988, DNA 7: 127).

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 polypeptide.

The RNAP subunit polypeptide may be a fusion polypeptide.

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. 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 of interest and its source. The polypeptide of interest 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 first polynucleotide encoding the polypeptide of interest. Additionally or alternatively, one or more control sequences may be operably linked to the second polynucleotide, preferably being heterologous to the second polynucleotide. The recombinant host cell may comprise a single copy, or at least two copies, e.g., at least three, at least four, at least five, at least six, or more copies of the first polynucleotide encoding the polypeptide of interest. Additionally or alternatively, the host cell may comprise a single copy, or at least two copies, e.g., at least three, at least four, at least five, at least six, or more copies of the second polynucleotide encoding the RNAP subunit polypeptide.

The host cell may be any microbial cell useful in the recombinant production of a polypeptide of interest, e.g., a prokaryotic cell or a fungal cell.

According to a first aspect, the invention relates to a mutant cell comprising in its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptide, wherein expression of the one or more Rpo subunit polypeptide is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell.

In one embodiment, the second polynucleotide is operably linked to a second heterologous promoter.

In one embodiment, the second polynucleotide is operably linked to a mutated Shine-Dalgarno sequence derived from a parent Shine-Dalgarno sequence.

In another embodiment, the second polynucleotide comprises one or more nucleic acid insertion, deletion, or substitution.

In one embodiment, expression of the second polynucleotide is decreased by a CRISPR inhibition construct.

In another embodiment, expression of the second polynucleotide is decreased by RNA interference.

In one embodiment, the parent Shine-Dalgarno sequence has a sequence identity of 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% to the polynucleotide sequence of AAGGAGG or to SEQ ID NO: 58.

In one embodiment, the second polynucleotide is native to the cell.

In one embodiment, the cell comprises at least two second polynucleotides, e.g., at least three, or at least four second polynucleotides, each second polynucleotide encoding a RNA polymerase subunit polypeptide. In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more bacterial RNA polymerase subunit polypeptide selected from the list of subunit beta (p), subunit alpha (a), and subunit omega (w).

In one embodiment, the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA.

In one embodiment, the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit beta (p) RpoB.

In one embodiment, the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit beta’ (p’) RpoB’.

In one embodiment, the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

In one embodiment, the one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA, and one or more secondary second polynucleotides encodes one or more bacterial RNA polymerase subunit beta (p) RpoB and/or (p’) RpoB’.

In one embodiment, the one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA, and one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

In one embodiment, the one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit beta (p) RpoB, and one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

In one embodiment, the one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit beta’ (p’) RpoB’, and one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

In one embodiment, the one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA, one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit beta (p) RpoB and/or (p’) RpoB’, and one or more tertiary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoA polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoA polypeptide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 1.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 10.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB’ polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 40.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB’ polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 39.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoZ polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 13.

In one embodiment, the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoZ polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 12.

In one embodiment, the second polynucleotide is heterologous to the cell.

In one embodiment, the first polynucleotide is operably linked to one or more promoter that direct the production of the polypeptide of interest, preferably the promoter is heterologous to the first polynucleotide.

In one embodiment, the heterologous promoter comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 38.

In one embodiment, the cell comprises at least two copies, e.g., at least three, at least four, or at least five, or at least six, or more copies of the first polynucleotide in its genome.

In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more Archaea RNA polymerase subunit polypeptide selected from the list of Rpo1 , Rpo2, Rpo3, Rpo11 , Rpo4, Rpo5, Rpo6, Rpo8, Rpo10, Rpo12, Rpo7, or Rpo13. In one embodiment, the one or more RNA polymerase subunit polypeptide is a subunit polypeptide of a eukaryotic RNA polymerase I, RNA polymerase II, and/or RNA polymerase III.

In one embodiment, the the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase I subunit polypeptide selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase subunit polypeptide selected from the list of RPAC40 (AC40), RPAC19 (AC19), RPO3, RPO1 1 , RPB3, and RPB11 .

In one embodiment, the second polynucleotide encodes a RPAC40 (AC40) polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18.

In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase I subunit polypeptide selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

In one embodiment, the second polynucleotide encodes a RPAC40 (AC40) polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

In one embodiment, the second polynucleotide encodes a RPAC19 (AC19) polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

In one embodiment, the second polynucleotide encodes a RPAC19 (AC19) polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.

In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase II subunit polypeptide selected from the list of RPB1 , RPB2, RPB3, RPB11 , RPB6, RPB5, RPB8, RPB10, RPB12, RPB4, RPB7, RPB9, TFIIFa, and TFIlFp.

In one embodiment, the second polynucleotide encodes a RPB3 polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30. In one embodiment, the second polynucleotide encodes a RPB3 polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31 .

In one embodiment, the second polynucleotide encodes a RPB11 polypeptide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.

In one embodiment, the second polynucleotide encodes a RPB11 polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.

In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase III subunit polypeptide selected from the list of RPC160, RPC128, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPC17, RPC25, RPC11 , RPC53, RPC37, RPC82, RPC34, and RPC31.

In one embodiment, the one or more RNA polymerase subunit polypeptide is one or more yeast RNA polymerase subunit polypeptide selected from the list of Rpb5 (ABC27), Rpb6 (ABC23, or Rpo26), Rpb8 (ABC14.5), Rpb10 (ABC10p), and Rpb12 (ABC10a).

In one embodiment, the one or more RNA polymerase subunit polypeptide comprises an N- terminal extension and/or C-terminal extension of 1-10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably and extension of 1 -6 amino acid residues in the N- terminus and/or 1 -6 amino acids in the C-terminus, such as 1 -5, or 1-4, or 1-3, or 1-2 amino acids, and wherein the extended polypeptide has RNA polymerase activity.

In one embodiment, the cell is a eukaryotic cell.

In one embodiment, the cell is a mammalian cell.

In one embodiment, the cell is a prokaryotic cell.

In one embodiment, the cell is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

In one embodiment, the cell is a filamentous fungal recombinant host 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.

In one embodiment, the cell is an Aspergillus cell.

In one embodiment, the cell is an Aspergillus niger cell.

In one embodiment, the cell is an Aspergillus oryzae cell.

In one embodiment, the cell is a Trichoderma cell.

In one embodiment, the cell is a Trichoderma reesei cell.

In one embodiment, the cell is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

In one embodiment, the cell is a Bacillus cell.

In one embodiment, the cell is a Bacillus licheniformis cell.

In one embodiment, the cell is a Bacillus subtilis cell.

In one embodiment, the cell is isolated.

In one embodiment, the cell is purified.

In one embodiment, the second heterologous promoter operably linked to the second polynucleotide results in decreased transcription of the second polynucleotide, relative to the transcription of the second polynucleotide when being operably linked to its native or endogenous promoter.

In one embodiment, the mutated Shine-Dalgarno sequence operably linked to the second polynucleotide results in decreased transcription of the second polynucleotide, relative to the transcription of the second polynucleotide when being operably linked to its native or endogenous Shine-Dalgarno sequence.

In one embodiment, the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase; even more preferably the one or more polypeptide of interest comprises an amylase or a protease.

In one embodiment, the polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of an antibody, an antibody fragment, an antibody-based drug, a Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a blood clotting factor, a hormone, an interferon (such as an interferon alpha-2b), an interleukin, a lactoferrin, an alpha-lactalbumin, a beta-lactalbumin, an ovomucoid, an ovostatin, a cytokine, an obestatin, a human galactosidase (such as an human alpha-galactosidase A), a vaccine, a protein vaccine, and a thrombolytic.

In one embodiment, the polypeptide of interest comprises a nanobody (Nb), preferably the nanobody consists of a single variable light chain (VL).

In one embodiment, the first polynucleotide encodes a polypeptide having amylase activity and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 6.

In one embodiment, the polypeptide of interest is an amylase, such as an amylase which comprises, or consists of the mature polypeptide having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 7.

In one embodiment, the first polynucleotide encodes a polypeptide having protease activity and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 8.

In one embodiment, the polypeptide of interest is a protease, such as a protease which comprises, or consists of the mature polypeptide having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 9. In one embodiment, expression of the one or more Rpo subunit polypeptide is decreased by at least 10%, e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to expression of the one or more Rpo subunit polypeptide of the parent cell when cultivated under identical conditions.

In one embodiment the mutated Shine-Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 5 of the parent Shine Dalgarno sequence with the nucleic acid sequence of SEQ ID NO: 58 with Adenine (A), G5A, with Cytosine (C), G5C, or with Thymine (T), G5T.

In one embodiment, the mutated Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and/or deletion at one or more positions of the nucleotides corresponding to positions 1 to 7 of the nucleic acid sequence “AAGGAGG”, or of the nucleic acid sequence at positions 1 - 7 of SEQ ID NO: 3.

In one embodiment, the mutated Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and/or deletion at one or more positions of the nucleotides of the nucleic acid sequence, “GAGGGGTG”, “AAGGGAG”, or “GGAGGTTG”.

In one embodiment, the mutated Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and/or deletion at a position corresponding to position 3 of the nucleic acid sequence “AAGGAGG”, or of the nucleic acid sequence at positions 1 -7 of SEQ ID NO: 3.

In one embodiment, the the mutated Shine-Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 3 the nucleic acid sequence “AAGGAGG” with Adenine (A), G3A, with Cytosine (C), G3C, or with Thymine (T), G3T.

In one embodiment, the mutated Shine-Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 3 of the nucleic acid sequence “AAGGAGG” with Adenine (A), G3A.

In one embodiment, the mutated Shine-Dalgarno sequence comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of “AAAGAGG”, or to the nucleic acid sequence at positions 1 -7 of SEQ ID NO: 4.

In one embodiment, the mutated Shine-Dalgarno sequence comprises or consists of the nucleic acid sequence of “AAAGAGG”, or of the nucleic acid sequence of positions 1 -7 of SEQ ID NO: 4.

In one embodiment, the second polynucleotide is operably linked to a mutated Shine-Dalgarno sequence forming a coding nucleic acid sequence comprising or consisting of the a coding nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of “AAGGAGG” or “AAAGAGG”, or to the nucleic acid sequence at positions 1 - 7 of SEQ ID NO: 3 or 4, or to the nucleic acid sequence of “GAGGGGTG”, “AAGGGAG”, or “GGAGGTTG”.

In one embodiment, the transcription and/or translation of the second polynucleotide is decreased compared to the parent cell when cultivated under identical conditions.

In one embodiment, the transcription and/or translation of the second polynucleotide (RNAP subunit) is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least 52%, at least

53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least

76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%, relative to the transcription and/or translation of the RNAP subunit of the parent cell.

In one embodiment, the transcription and/or translation of the second polynucleotide (RNAP subunit) is decreased relative to the transcription and/or translation of the parent cell, after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

In one embodiment, the yield of the polypeptide of interest is increased compared to the parent cell when cultivated under identical conditions,.

In one embodiment, the yield of the polypeptide of interest is increased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, or at least 35% relative to the yield of the parent cell.

In one embodiment the yield of the polypeptide of interest is increased at least 12% relative to the yield of the parent cell.

In one embodiment, the the yield of the polypeptide of interest is increased relative to the yield of the parent cell, after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

In one embodiment, during cultivation of the cell, biomass is decreased relative to the biomass during cultivation of the parent cell when cultivated under identical conditions.

In one embodiment, the biomass is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 1 1 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least

20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, or at least

35% relative to the biomass of the parent cell.

In one embodiment the biomass formation is decreased at least 18% relative to the biomass formation of the parent cell. In one embodiment, the biomass is decreased relative to the biomass of the parent cell, after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

In one embodiment, the cultivation is a fed-batch, batch or continuous cultivation process, preferably a fed-batch cultivation process.

The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.

For purposes of this invention, Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.

The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes , Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al. , 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods 64: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612-2620.

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).

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/TechnologyB'. 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.

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 yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii).

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.

The filamentous fungal host cell may be 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 a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

For example, the filamentous fungal host cell may be 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.

In an aspect, the host cell is isolated. In another aspect, the host cell is purified.

Methods of Production

In a 2nd aspect, the invention relates to methods of producing one or more polypeptides of interest, the method comprising, a) providing a mutant cell according to the first aspect, b) cultivating said cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally, recovering the one or more polypeptide of interest.

The host cell is cultivated in a nutrient medium suitable for production of the polypeptide 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 microcarrierbased 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.

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.

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).

In an alternative aspect, the polypeptide is not recovered.

Polynucleotides

The present invention also relates to one or more second polynucleotides encoding a RNAP subunit polypeptide of the present invention, as described herein.

The second polynucleotide may be operably linked to a second heterologous promoter.

Additionally or alternatively, the second polynucleotide may be operably linked to a mutated SD sequence.

The mutated SD sequence and/or second heterologous promoter are located upstream of the second polynucleotide.

The second polynucleotide, second heterologous promoter, and/or mutated SD sequence may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof. The second polynucleotide and/or mutated SD sequence may be cloned from a strain of Bacillus, Trichoderma,

Aspergillus, or a related organism.

In one embodiment the second polynucleotide and/or mutated SD sequence of the present invention is isolated from a Bacillus licheniformis cell.

In one embodiment the second polynucleotide and/or mutated SD sequence of the present invention is isolated from a Bacillus subtilis cell.

In one embodiment the second polynucleotide and/or mutated SD sequence of the present invention is isolated from a Aspergillus niger cell.

In one embodiment the second polynucleotide and/or mutated SD sequence of the present invention is isolated from a Aspergillus oryzae cell.

In one embodiment the second polynucleotide and/or mutated SD sequence of the present invention is isolated from a Trichoderma reesei cell.

The mutated SD sequence is mutated by introduction of nucleotide substitutions, insertions or deletions, that do not result in a change in the amino acid sequence of the RNAP subunit polypeptide.

Additionally or alternatively, the second polynucleotide sequence is mutated by introduction of nucleotide substitutions, insertions or deletions, that do not result in a change in the amino acid sequence of the RNAP subunit polypeptide, but which correspond to the codon usage of the host organism intended for production of the polypeptide of interest, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

In an aspect, the polynucleotide is isolated.

In another aspect, the polynucleotide is purified.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the RNAP subunit coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to provide for expression of the RNAP subunit polypeptide. Manipulation of the polynucleotide 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.

Promoters

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of the first polynucleotide encoding the polypeptide of interest. The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of the second polynucleotide encoding the RNAP subunit polypeptide. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, supra, and Song et al., 2016, PLOS One 11 (7): e0158447.

Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma'. Biology and Applications”, and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

For expression in a yeast host, examples of useful promoters are described by Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae), and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

Terminators

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma'. Biology and Applications”, and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488. mRNA Stabilizers

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene encoding the polypeptide of interest.

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177: 3465-3471). Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.

Leader Sequences

The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the first polynucleotide encoding the polypeptide of interest, and/or to the second polynucleotide encoding the RNAP subunit . Any leader that is functional in the host cell may be used.

Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.

Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

Polyadenylation Sequences

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the first polynucleotide and/or second polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alphaglucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

Signal Peptides

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the polypeptide of interest and directs the polypeptide of interest into the cell’s secretory pathway. The 5’-end of the coding sequence of the first polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide of interest. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide of interest. Any signal peptide coding sequence that directs the expressed polypeptide of interest into the secretory pathway of a host cell may be used. Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Freudl, 2018, Microbial Cell Factories 17: 52.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

Propeptides

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Additionally or alternatively, when both signal peptide and propeptide sequences are present, the polypeptide may comprise only a part of the signal peptide sequence and/or only a part of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of mature polypeptides and polypeptides which comprise, either partly or in full length, a propeptide sequence and/or a signal peptide sequence.

Regulatory Sequences

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide of interest relative to the growth of the host cell. It may also be desirable to add regulatory sequences that regulate expression of the RNAP subunit polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.

Shine-Dalqarno sequences

The control sequence may also be a Shine-Dalgarno (SD) sequence. SD sequences are ribosomal binding sites on an RNA sequence which are generally located 5-9 bases upstream of the start codon AUG. On DNA-level the SD sequence is located upstream of the start codon ATG. The SD RNA sequence helps to recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon. Mutations in the SD sequence can reduce or increase translation in host cells, resulting in reduced or increased polypepitde levels, respectively (Velaquez et al., Journal of Bacteriology, May 1991 , p. 3261 - 3264). Thus, modification of the SD sequence in a rpo subunit/rnap subunit gene can reduce or increase RNAP subunit polypeptide levels. This change of RNAP subunit levels is due to a reduced or increased map subunit mRNA pairing efficiency with the ribosomes.

A mutated Shine-Dalgarno sequence may be obtained by one or more nucleic acid modifications, such as a nucleic acid substitution, nucleic acid deletion, or nucleic acid insertion. As described above, modification of the SD sequence can, depending on the mutation, reduce or increase translation of the gene located downstream of the modified or mutated SD sequence.

In a 3rd aspect, the invention relates to a nucleic acid construct comprising a second heterologous promoter and/or a mutated Shine-Dalgarno sequence operably linked to the second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptide.

In one embodiment, the nucleic acid construct is isolated.

In one embodiment, the nucleic acid construct is purified.

Expression Vectors

In a 4th aspect, the invention relates to an expression vector comprising the nucleic acid construct according to the 3rd aspect.

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences 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 the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. 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.

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 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.

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.

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.

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.

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.

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.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulation or a cell composition comprising live or killed cells of the present invention. The fermentation broth formulation or the cell composition further comprises additional ingredients used in the fermentation process, such as, for example, 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.

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.

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.

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.

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 filamentous 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.

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.

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.

Removal or Reduction of RNAP Activity

The present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting, modifying, substituting, or deleting a promoter, or a portion thereof, regulating the transcripton of one or more RNAP subunit polypeptides, which results in the mutant cell comprising less RNAP activity than the parent cell when cultivated under the same conditions. The present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting, modifying, substituting, or deleting a SD sequence, or a portion thereof, upstream of a RNAP subunit encoding gene, which results in a decreased ribosome-binding strength of the RNAP subunit mRNA during translation. Thus, mutation of the SD sequence results in the mutant cell comprising less RNAP activity than the parent cell when cultivated under the same conditions. For expression of polypeptides of interest (product) which are toxic to the cell, a reduced RNAP activity can be advantageous, leading to a more balanced cell growth and product expression, thereby increasing overall product yield.

The present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting, modifying, substituting, or deleting a second polynucleotide, or a portion thereof, encoding one or more RNAP subunit polypeptide, which results in the mutant cell comprising less RNAP activity than the parent cell when cultivated under the same conditions.

Decreased transcription and/or translation of one or more RNA polymerase subunits, which decreases overall RNA polymerase (RNAP) activity, has been shown to increase the polypeptide of interest yield in recombinant host cells, while also reducing the formation of biomass.

Decreased RNAP activity, and/or decreased transcription and/or decreased translation of one or more RNA polymerase subunits can, for example, be achieved by: a) operably linking the second polynucleotide encoding the RNAP subunit to a second heterologous promoter which is weaker than the native promoter of the RNAP subunit encoding gene, e.g. by replacing the native promoter with a weaker heterologous promoter, b) operably linking the second polynucleotide to a mutated Shine-Dalgarno sequence comprising one or more nucleic acid modifications compared to the native SD sequence of the RNAP subunit encoding gene, wherein the mutated SD sequence results in a weaker ribosome binding of the RNA during translation of the RNAP subunit, c) using CRISPRi, RNAi or other interference techniques known to the skilled person to target the coding sequence of the RNAP subunit gene or its transcript during transcription or translation, respectively, and/or d) deleting or mutating one or more of the RNAP subunit genes.

The mutant cell may be constructed by reducing or eliminating expression of the second polynucleotide using methods well known in the art, for example, one or more nucleotide insertions, one or more gene disruptions, one or more nucleotide replacements, or one or more nucleotide deletions.

The second polynucleotide to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory or control element required for expression of the coding region, e.g., a functional part of a promoter sequence, and/or a regulatory or control element required for the transcription or translation of the polynucleotide. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator. Modification or inactivation of the second polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the second polynucleotide has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

Examples of a physical or chemical mutagenizing agent include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues (see J. L. Bose, Springer Protocols 2016, Methods in Molecular Biology, The Genetic Manipulation of Staphylococci).

Additionally or alternatively, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art, or by targeted gene editing using one or more nucleases, e.g., zinc-finger nucleases or CRISPR-associated nucleases. Additionally or alternatively, the modification or inactivation may be achieved by gene silencing, genetic repression, genetic activation, and/or post-translational mutagenesis, e.g., by methods employing non-coding RNA, RNAi, siRNA, miRNA, ribozymes, catalytically inactive nucleases, CRISPRi, nucleotide methylation, and/or histone acetylation. A suitable method for reducing rpo subunit polypeptide expression is CRISPR inhibition (CRISRPi), e.g. as disclosed in WO18009520. The modification may be transient and/or reversible, irreversible and/or stable, or the modification may be dependent on chemical inducers or dependent on cultivation conditions, such as the cultivation temperature.

The modification may be performed in vivo, i.e., directly on the cell expressing the second polynucleotide, or the modification be performed in vitro.

An example of a convenient way to modify expression of the second polynucleotide is shown in Example 1 .

Another convenient way of reducing or eliminating RNAP activity in the cell is the use of RNA polymerase directed antimycobacterials (antibiotics), such as Rifampicin. This is particularly useful when the polypeptide of interest is translated using a T7 RNA-polymerase dependent expression system. Using an antibiotic will decrease or eliminate the native RNAP activity of the host cell while not interfere with the expression of the polypeptide of interest. Suitable antibiotic concentrations will be known to the skilled person. A non-limiting example for an antibiotic concentration during cultivation is between 0.01 - 5.0 ng/ul, e.g. a concentration of about 0.2 ng/ul. The concentration is chosen to i) inactivate eliminate native RNAP activity effectively, and ii) not significantly interfere with cell viability. A too high concentration will interfere significantly with cell viability, while a too low concentration will not effectively decrease native RNAP activities.

Further examples of a convenient way to eliminate or reduce expression of a polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the polynucleotide has been modified or destroyed. In an aspect, the polynucleotide is disrupted with a selectable marker such as those described herein.

The present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of the second polynucleotide or a control sequence thereof or a silenced gene encoding the RNAP subunit polypeptide, which results in the mutant cell producing less of the RNAP subunit polypeptide or no RNAP subunit polypeptide compared to the parent cell.

The mutant cells with decreased RNAP subunit polypeptide levels are useful as host cells for expression of native and heterologous polypeptides of interest, as said mutants have at least two advantages: i) increased yield of the polypeptide of interest, and ii) decreased biomass formation.

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

Examples

Strains

AEB1517: This is a B. subtilis donor strain for conjugation of B. licheniformis as described previously (see US5695976, US5733753, US5843720, US5882888, and W02006042548). The strain contains pLS20 and the methylase gene M.blil 904II (US20130177942) expressed from a triple promoter at the amyE locus, the pBC16-derived orf beta and the B. subtilis comS gene (and a kanamycin resistance gene) are expressed from a triple promoter at the air locus (making the strain D-alanine requiring).

PP3724: AEB1517 derivative where a second gene cassette consisting of the comS gene expressed from a triple promoter is inserted at the pel locus (pectate lyase) (see US20190276855).

SJ1904: A derivative of B. licheniformis Ca63, described in WO 2008/066931 .

AN865: SJ1904 derivative with amylase genes under triple promoter.

JA4468: SJ1904 derivative with protease genes under triple promoter.

BN01 : SJ1904 derivative with protease genes under triple promoter.

BN02: SJ1904 derivative with protease genes under triple promoter.

Halo-9: SJ1904 derivative with protease genes under triple promoter.

Plasmids pMDT411 : a derivative of pMDT454, see US2021021670 pMDT417: a derivative of pMDT452, see US2021021670 pEB-prsA: A derivative of pMDT411 with prsA expression cassette driven by a triple promoter. pTNA634: A derivative of pEB-prsA with GFP expression cassette, driven by a constitutive amyL promoter (PamyL4199) and amyL RBS (ribosome binding sequences). pTNA635: A derivative of pTNA634 with wildtype rpoA RBS instead of amyL RBS. pTNA636: A derivative of pTNA634 with mutated rpoA RBS instead of amyL RBS. pTNA637: A derivative of pTNA634 without RBS instead of amyL RBS.

Media and Solutions

LB: 10 g/l tryptone, 5 g/l yeast extract, 5 g/l sodium chloride, adjusted to pH 7.0.

LB-agar: LB with 15 g/l Bacto-agar

TY: 20 g/L Tryptone, 5 g/L yeast extract, 7 mg/L FeCI2, 1 mg/LMnCI2, 15 mg/L MgCI2

TY-agar: TY with 15 g/l Bacto-agar

M-9 buffer: di-sodiumhydrogenphosphate, 2H2O 8.8g/l; potassiumdihydrogenphosphate 3 g/l; sodium chloride 4 g/l; magnesium sulphate, 7H2O 0.2 g/l

PRK-50: 110 g/l soy grits; di-sodiumhydrogenphosphate, 2H2O 5 g/l; Antifoam (Struktol SB2121 ; Schill & Seilacher, Hamburg, Germany) 1 ml/l, pH adjusted to 8.0 with NaOH/H2PO 4 before sterilization.

Make-up medium: Tryptone (Casein hydrolystae from Difco (BactoTM Tryptone pancreatic Digest of Casein 211699) 30 g/l; magnesium sulphate, 7H2O 4 g/l; di-potassiumdihydrogenphosphate 7 g/l; di- sodiumhydrogenphosphate, 2H2O 7 g/l; di-ammoniumsulphate 4 g/l; citric acid 0.78 g/l; vitamins (thiamin- dichlorid 34.2 mg/l; riboflavin 2.9 mg/l; nicotinic acid 23 mg/l; calcium Dpantothenate 28.5 mg/l; pyridoxai- HCI 5.7 mg/l; D-biotin 1.1 mg/l; folic acid 2.9 mg/l); trace metals (MnSO4, H2O 39.2 mg/l, FeSO4, 7H2O 157 mg/l, CuSO 4, 5H2O 15.6 mg/l; ZnCI2 15.6 mg/l); Antifoam (Struktol SB2121 ; Schill & Seilacher, Hamburg, Germany) 1.25 ml/l; pH adjusted to 6.0 with NaOH/H2PO4 before sterilization.

Feed-medium: Glucose, 1 H2O 820 g/l

Cultivations

Bacillus strains were grown on LB- or TY-agar plates or in LB or TY liquid medium. To select for erythromycin resistance, agar and liquid media were supplemented with 5 pg/ml erythromycin. To select for tetracycline resistance, agar and liquid media were supplemented with 15 pg/ml tetracycline. Growth media for strains carrying the air gene disruption was supplemented with D-alanine to a final concentration of 0.1 mg/mL. Transformation of Bacilli was in Spizizen I medium which consists of 1 x Spizizen salts (6 g/L KH2PO4, 14 g/L K2HPO4, 2 g/L (NH4)2SO4, 1 g/L sodium citrate, 0.2 g/L MgSO4 pH7.0), 0.5% glucose, 0.1 % yeast extract and 0.02% casein hydrolysate. Enzymatic Assays

Amylase assay: Amylase activity was measured in culture broth using the Pureauto S AMY-G7 (Sekisui Medical). Culture broth was diluted in dissolution buffer (0.03 M CaCI2; 0.0025% Brij L23; 6.67 M urea) firstly, and then dilution buffer (0.03 M CaCI2; 0.0025% Brij L23) was used for subsequent sample dilution. Enzyme activity measurement was performed with the Gallery Plus automated photometric analyzer. Sixteen pL of diluted sample was mixed with 200 pL of Reagent 1 of the Pureauto kit, and then 20 pL of reagent 2 was added there. After 3 min incubation at 37C, absorbance was measured over time at 405 nm for 2 min. An amylase standard was included from the final activity value, KNU(T)/g, was determined.

Protease assay: The serine endopeptidase hydrolyses the substrate N-Succinyl-Ala-Ala-Pro-Phe p- nitroanilide. The reaction was performed at 37C at pH 9.0. The release of pNA results in an increase of absorbance at 405 nm and this increase is proportional to the enzymatic activity measured against a standard.

In vivo GFP assay: The in vivo GFP expression levels were measured as follows. First, Bacillus strains with GFP expression plasmids were grown overnight in LB liquid medium supplemented with 100 ug/ml D- Ala and 5 ug/ml erythromycin. 25 uL of broth was transferred to 96-well black plates and mixed with 75 uL of fresh LB medium for dilution. The diluted broth was subjected to GFP intensity measurement by Synergy2 spectrophotometer (BioTek Instruments, Inc.) with Ex 485nm/Em 528nm.

Molecular biological methods

DNA manipulations and transformations were performed by standard molecular biology methods as described in:

Sambrook et al. (1989): Molecular cloning: A laboratory manual. Cold Spring Harbor laboratory, Cold Spring Harbor, NY.

Ausubel et al. (eds) (1995): Current protocols in Molecular Biology. John Wiley and Sons.

Harwood and Cutting (eds) (1990): Molecular Biological Methods for Bacillus. John Wiley and Sons.

Competent cells and transformation of B. subtilis was obtained as described in Yasbin et al. (1975, Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 121 , 296-304). Conjugation of B. licheniformis was performed essentially as described in WO1996/029418.

Genomic DNAwas prepared by using the commercially available QIAamp DNA Blood Kit (Qiagen). The respective DNA fragments were amplified by PCR using the PrimeStar GXL DNA Polymerase system (TaKaRa). PCR amplification reaction mixtures contained 1 pL of template DNA, 2 pL of sense primer (20 pmol/pL), 2 pL of anti-sense primer (20 pmol/pL), 10 pL of 5X PCR buffer, 4 pL of dNTP mix, 30 pL water, and 1 pL DNA polymerase. A thermocycler was used to amplify the fragment. The PCR products were purified from a 1.0% agarose gel with 1x TAE buffer using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions.

The condition for POE-PCR is as follows: purified PCR products were used in a subsequent PCR reaction to create a single fragment using splice overlapping extension PCR (SOE) using the PrimeStar GXL DNA Polymerase system (TaKaRa) as follows. The very 5’ end fragment and the very 3’ end fragment have complementary end which will allow the SOE to concatemer into the POE PCR product. The PCR amplification reaction mixture contained 50 ng of each of the gel purified PCR products. POE PCR was performed as described in You, C et al., (2017) Methods Mol. Biol. 116, 183-92.

Procedure for fed-batch fermentation with a lab tank fermenter

1 . inoculum steps a) Grow the strain on LB-agar plates overnight at 37°C. b) Wash the agar with M-9 buffer and collect the cell suspension. Measure OD650 by photometer. c) Inoculate PRK-50 shake flask (OD650 x ml cell suspension = 1). d) Incubate the shake flask at 220 rpm overnight at 37°C. e) Start the main fermenter by adding the growing shake flask culture (10% of make-up media, i.e. 80 ml to 800ml).

2. Fermenter equipment

Standard lab fermenters equipped with a temperature control system, pH control with ammonia-water and phosphoric acid, dissolved oxygen electrode to measure >20% oxygen saturation through the entire fermentation.

3. Fermentation parameters

Temperature: 37°C.

Keep pH between 6.8 and 7.2 using ammonia-water and phosphoric acid.

Aeration: 1.5 L/min/kg broth weight

Agitation: 1500 rpm.

Example 1. Construction of plasmid DNAs for introduction of mutations in rpoA gene.

The purpose of this experiment is to prepare the plasmid DNAs for introducing single-nucleotide mutation(s) into native rpoA gene to alter its ribosome binding sequences (=SD, Shine-Dalgarno sequence) in B. licheniformis strains. Hereafter, this mutation is referred to as rpoA SD mutation. A plasmid DNA pMDT41 1 has an expression cassette of single guide RNA (sgRNA) to recruit Mad7 nuclease to the sgRNA-complementary region on the genome. To edit the ribosome binding region of rpoA gene, one protospacer was designed and cloned into pMDT41 1 (see Table 2).

Table 2.

The oligo DNAs for cloning were listed in Table 2. The protospacer and homology regions with desired rpoA SD mutation were inserted into pMDT411 by PoE PCR. First, each PCR fragment was amplified and purified by gel extraction method with a QIAquick Gel Extraction Kit (Qiagen). Table 3 shows used primer pairs. Purified fragments were then combined by PoE PCR as described in method section. PoE PCR products were then directly used for transformation of B. subtilis host PP3724 (hereafter PP3724- pMDT41 1-rpoA), which is D-Alanine auxotroph. Transformants were spread onto TY plus Erythromycin and D-Alanine agar-plates and incubated at 34C for 1 -2 days. Plasmid DNA was purified from several transformants using a QIAGEN mini-prep kit. The plasmid DNA was screened for proper ligation by sanger sequencing. Similarly, the plasmid DNA pMDT417, comprising expression cassette of Mad7 nuclease, was transformed into B. subtilis host PP3724 (Ohereafter PP3724-pMDT417). Transformants were spread onto TY plus tetracycline and D-Alanine agar-plates and incubated at 34C for 1 -2 days.

Table 3.

Example 2. Transformation of B. licheniformis strains AN865 and JA4468 for integration of rpoA SD mutation The purpose of this experiment was to generate the desired rpoA mutants of B. licheniformis strains. First, to transfer the plasmid DNAs, the B. subtilis donor strain, PP3724-pMDT411-rpoA, was conjugated with B. licheniformis recipient strains, AN865 or JA4468. Conjugants were spread onto TY plus erythromycin agar-plates and incubated at 34C for 1-2 days. The correct conjugants of B. licheniformis were selected by the resistance of erythromycin and no D-Alanine auxotroph phenotypes. Next, erythromycin resistant B. licheniformis strains were conjugated with PP3724-pMDT417. Conjugants were spread onto TY plus erythromycin and tetracycline agar-plates and incubated at 34C for 2-3 days. The correct conjugants of B. licheniformis were selected by the double resistance of erythromycin and tetracycline.

Colonies on double selection plates were then transferred to LB-liquid medium with tetracycline and erythromycin for cultivation at 34C overnight. To isolate a single colony from the liquid culture, culture broth was serially diluted and spread on TY plus erythromycin and tetracycline agar-plates and incubated at 34C for 2-3 days. Single colonies were then screened for existence of the desired rpoA mutation on the genome by genomic PCR and sanger sequencing. We found desired rpoA mutants from AN865 and JA4468, hereafter AN865-rpoA-5 and JA4468-rpoA-3-3, respectively. The plasmid DNAs used for genome editing were removed by cultivating these strains at 50C overnight in LB-liquid medium. Finally, erythromycin and tetracycline double sensitive clones were selected and stocked in glycerol.

Example 3. Increased amylase productivity after introducing rpoA SD mutation

The purpose of this experiment was to test if the introduced rpoA mutation affects the amylase productivity of B. licheniformis strain AN865 in Shake flasks (SFs). First, 100 ul of frozen stocks of AN865 (wildtype rpoA) and AN865-rpoA-5 (mutated rpoA) were added to 100 ml of PRK-50 medium in 500 ml SFs. SFs were incubated at 37C overnight, at 220 rpm of rotation speed with SF shaker. Ten ml of the culture broths were then inoculated to 100 ml of 10R-av-30CG medium in 500 ml SFs. SFs were incubated at 37C for 3 days at 220 rpm. In the end, amylase activity in the culture broth was measured as described in the [Enzymatic assays] section. Relative amylase activity is shown in Table 4.

Table 4.

‘The average amylase activity from 3 replicates from each strain was calculated. The average amylase yield from AN865 (wildtype SD) is normalized to 1 .00.

The above data showed the rpoA SD mutation clearly improved the productivity of amylase expression in B. licheniformis strain AN865 by 12%.

Example 4. Increased protease expression after introducing rpoA SD mutation The purpose of this experiment was to test if the introduced rpoA mutation affects the protease productivity of B. licheniformis strain JA4468 in lab tank fermenters with the process described in the method section. In the end, protease activity in the culture broth was measured as described in the [Enzymatic assays] section. Relative protease activity is shown in Table 5.

Table 5.

‘The average protease activity from 2 replicates from JA4468 (wildtype SD), or single batch from JA4468- rpoA-3-3 (mutated rpoA SD) was calculated. The average protease yield from JA4468 is normalized to 1.00.

The above data showed the rpoA SD mutation clearly improved the productivity of protease expression in B. licheniformis strain JA4468 by 12%. Together with example 3, the rpoA SD mutation was shown to have positive impact on recombinant production of different protein products in B. licheniformis strains.

Example 5. Reduced biomass formation after introducing rpoA SD mutation

The purpose of this experiment was to see the rpoA SD mutation effect on not only enzyme productivity but also cell growth during fermentation. The amylase strains, AN865 and AN865-rpoA-5, and protease strains, JA4468 and JA4468-rpoA-3-3 were cultivated with 100ml of LB-liquid medium in 500 ml SFs. Cultivation was done at 37C overnight at 220 rpm. After 24 hours of fermentation, 5 ul of culture broths were mixed with 195 ul of deionized water for dilution, then OD650 value was measured by a photometer. Results are summarized in Table 6.

Table 6.

The above data showed the rpoA SD mutation clearly decreased the cell density (18% decrease) at the end of fermentation. Similar effect was also seen in the fermentations done in example 3 and 4. Reducing biomass after fermentation is highly beneficial feature for bio-production since it decreased biomass lowers the product purification and formulation costs. Example 6: The rpoA SD mutation results in increased Rifampicin-sensitivity

The purpose of this experiment was to investigate the rpoA SD mutation effect at molecular level. As the SD (Shine-Dalgarno) region functions as ribosome binding sequences, the rpoA SD mutation may change the translation level of RpoA proteins in cells. Bacterial RNA polymerase consists of 2x alpha (encoded by rpoA gene), beta, beta prime and omega subunits. The RNA polymerase complex is inhibited by an antibiotic Rifampicin (a reference: EA Campbell et al., “Structural mechanism for rifampicin inhibition of bacterial rna polymerase”, Cell, 2001). We hypothesized that rpoA SD mutants are more sensitive to Rifampicin if cellular RpoA protein level is lower in the rpoA SD mutants than in the wildtype strains.

To test this hypothesis, the protease strains listed in Table 7 were spread on Rifampicin (Rif) containing agar plates to see the sensitivity to Rif of each strain. The JA4468 strain showed decrease of survival ratio with increasing dosage of Rif in the TY-based agar plate. However, BN02, having a known rpoB (encodes beta subunit) mutation (A478D) conferring Rif resistance, did not show any decrease of survival ratio. This indicates this Rif sensitivity assay is reasonable. Then, protease strains with or without the rpoA SD mutation (halo-9 and BN01 , respectively) were assayed. As shown in Table 7, halo-9, having the rpoA SD mutation, showed lower survival ratio than BN01 , indicating halo-9 was more sensitive to Rif. Consequently, it is concluded that the identified rpoA SD mutation decreases RNAP activities in the host cell, e.g. by reduced rpoA expression.

Table 7.

Example 7: Construction of plasmid DNAs for GFP expression tethered with various SD sequences

The purpose of this experiment was to confirm that the SD mutation causes a decrease in RpoA expression and thus a decrease in RNAP activity. To do so, we prepared plasmid DNAs for investigating the effect of different SD sequences on expression of GFP. As shown in Example 6, rpoA SD mutation should lower the RpoA protein expression level. To further investigate this in a quantitative manner, a series of GFP expression plasmid DNAs were made.

A plasmid DNA pEB-prsA, a derivative of pMDT411 , has an overexpression cassette of prsA driven by a triple promoter with multiple RBSs in form of SD sequences. To make a simple expression cassette to compare the effect of SD sequences, a single amyL promoter (PamyL4199) was chosen instead of the triple promoter. Then, PamyL4199 and various RBSs (comprising amyL SD, wildtype rpoA with wildtype SD, wildtype rpoA with mutated SD, or no SD) with GFP CDS were cloned into pEB-prsA backbone by PoE PCR, resulting in pTNA634 to 637, respectively. Transformation of these constructs into B. subtilis host PP3724 was done as written in Example 1. The oligo DNAs for cloning were listed in Table 8. The SD sequences used in this study were summarized in Table 9, annotated as ribosomal binding sites (RBS).

Table 8.

Table 9.

*The mutated nucleotide in rpoA SD (RBS) is capitalized.

Example 8: The rpoA SD mutation results in decreased GFP expression

The purpose of this experiment was to compare the in vivo GFP expression level between wildtype rpoA SD sequence and mutated rpoA SD sequence. The single colonies of PP3724 transformants of pTNA634 to 637 were cultured in LB liquid medium supplemented with 100 ug/ml D-Ala and 5 ug/ml erythromycin. After overnight cultivation at 32°C, culture broth was collected to measure OD650 and GFP intensity, as written in Assay section. The results are summarized in Table 10. Table 10.

GFP intensity and OD650 shown in Table 10 are average values of two biological replicates.

As shown in Table 10, the transformants of pTNA634 to 637 showed various levels of GFP expressions, while that of negative control (pEB-prsA) did not show any GFP expressions. Since the measured GFP intensity^(a) should be affected by cell mass in the broth, GFP intensity was normalized by OD650 (GFP/OD650^(b), hereafter normalized GFP expression). Relative values of normalized GFP expression were calculated using pTNA635 as reference (see very right column of Table 10). pTNA635 encodes the GFP cassette with wildtype rpoA SD. As can be seen from Table 10, pTNA636 (mutated rpoA SD) resulted in a 85% reduction of GFP expression, relative to GFP expression of pTNA635. This is well aligned to what was seen in Example 6, Rif sensitivity experiments. In conclusion, these examples show that rpoA SD mutation resulted in decreased RpoA expression of circa 80-90%. In addition, we have demonstrated that rpoA SD mutation results in increased yield of recombinant protein (examples 3-4) and reduced biomass formation (example 5).

Thus, the reduced rpoA expression is directly linked to the observed biomass reduction and to the increased product yields.

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.

The invention is further defined by the following numbered paragraphs:

1 . A mutant cell comprising in its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptide, wherein expression of the one or more Rpo subunit polypeptide is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell. 2. The mutant cell according to paragraph 1 , wherein a) the second polynucleotide is operably linked to a second heterologous promoter, b) the second polynucleotide is operably linked to a mutated Shine-Dalgarno sequence derived from a parent Shine-Dalgarno sequence, c) the second polynucleotide comprises one or more nucleic acid insertion, deletion, or substitution, d) expression of the second polynucleotide is decreased by a CRISPR inhibition construct, and/or e) expression of the second polynucleotide is decreased by RNA interference.

3. The cell according to any one ofthe preceding paragraphs, wherein expression of the one or more Rpo subunit polypeptide is decreased by at least 10%, e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to expression of the one or more Rpo subunit polypeptide of the parent cell when cultivated under identical conditions.

4. The cell according to any one of paragraphs 1 -3, wherein the second polynucleotide is native to the cell.

5. The cell according to any previous paragraph, wherein the second heterologous promoter is heterologous to the second polynucleotide.

6. The cell according to any previous paragraph, wherein the second heterologous promoter results in decreased expression of the Rpo subunit polypeptide encoded by the second polynucleotide, compared to Rpo subunit polypeptide expression controlled by the native promoter of the second polynucleotide in the parent cell when cultivated under identical conditions.

7. The cell according to any previous paragraph, wherein the parent Shine-Dalgarno sequence has a sequence identity of 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% to the polynucleotide sequence of AAGGAGG or to SEQ ID NO: 58.

8. The cell according to any one of the proceeding paragraphs, wherein the cell comprises at least two second polynucleotides, e.g., at least three, or at least four second polynucleotides, each second polynucleotide encoding a RNA polymerase subunit polypeptide.

9. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more bacterial RNA polymerase subunit polypeptide selected from the list of subunit beta (p), subunit alpha (a), and subunit omega (w).

10. The cell according to any one of the proceeding paragraphs, wherein the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA.

11. The cell according to any one of the proceeding paragraphs, wherein the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit beta (p) RpoB.

12. The cell according to any one of the proceeding paragraphs, wherein the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit beta’ (p’) RpoB’.

13. The cell according to any one of the proceeding paragraphs, wherein the one or more second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ. 14. The cell according to any one of the proceeding paragraphs, wherein one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA, and one or more secondary second polynucleotides encodes one or more bacterial RNA polymerase subunit beta (p) RpoB and/or (p’) RpoB’.

15. The cell according to any one of the proceeding paragraphs, wherein one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA, and one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

16. The cell according to any one of the proceeding paragraphs, wherein one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit beta (p) RpoB, and one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

17. The cell according to any one of the proceeding paragraphs, wherein one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit beta’ (p’) RpoB’, and one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

18. The cell according to any one of the proceeding paragraphs, wherein one or more primary second polynucleotide encodes one or more bacterial RNA polymerase subunit alpha (a) RpoA, one or more secondary second polynucleotide encodes one or more bacterial RNA polymerase subunit beta (p) RpoB and/or (p’) RpoB’, and one or more tertiary second polynucleotide encodes one or more bacterial RNA polymerase subunit omega (w) RpoZ.

19. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoA polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 2.

20. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoA polypeptide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 1.

21. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 1 1 .

22. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 10.

23. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB’ polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 40.

24. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoB’ polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 39.

25. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoZ polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 13.

26. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide, e.g. the primary, secondary or tertiary second polynucleotide, encodes a RpoZ polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 12.

27. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide is heterologous to the cell.

28. The cell according to any one of the proceeding paragraphs, wherein the first polynucleotide is operably linked to one or more first promoter that direct the production of the polypeptide of interest, preferably the first promoter is heterologous to the first polynucleotide.

29. The cell according to any one of the proceeding paragraphs, wherein the first heterologous promoter comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 38.

30. The cell according to any one of the proceeding paragraphs, wherein the cell comprises at least two copies, e.g., at least three, at least four, or at least five, or at least six, or more copies of the first polynucleotide in its genome. 31. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more Archaea RNA polymerase subunit polypeptide selected from the list of Rpo1 , Rpo2, Rpo3, Rpo11 , Rpo4, Rpo5, Rpo6, Rpo8, Rpo10, Rpo12, Rpo7, or Rpo13.

32. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is a subunit polypeptide of a eukaryotic RNA polymerase I, RNA polymerase II, and/or RNA polymerase III.

33. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase I subunit polypeptide selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

34. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase subunit polypeptide selected from the list of RPAC40 (AC40), RPAC19 (AC19), RPO3, RPO11 , RPB3, and RPB11 .

35. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPAC40 (AC40) polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18.

36. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase I subunit polypeptide selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

37. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPAC40 (AC40) polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

38. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPAC19 (AC19) polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

39. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPAC19 (AC19) polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.

40. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase II subunit polypeptide selected from the list of RPB1 , RPB2, RPB3, RPB1 1 , RPB6, RPB5, RPB8, RPB10, RPB12, RPB4, RPB7, RPB9, TFIIFa, and TFHFp.

41. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPB3 polypetide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.

42. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPB3 polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31 .

43. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPB11 polypeptide and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.

44. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide encodes a RPB11 polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.

45. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more eukaryotic RNA polymerase III subunit polypeptide selected from the list of RPC160, RPC128, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPC17, RPC25, RPC11 , RPC53, RPC37, RPC82, RPC34, and RPC31.

46. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide is one or more yeast RNA polymerase subunit polypeptide selected from the list of Rpb5 (ABC27), Rpb6 (ABC23, or Rpo26), Rpb8 (ABC14.5), Rpb10 (ABC10P), and Rpb12 (ABC10a).

47. The cell according to any one of the proceeding paragraphs, wherein the one or more RNA polymerase subunit polypeptide comprises an N-terminal extension and/or C-terminal extension of 1-10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably and extension of 1 -6 amino acid residues in the N- terminus and/or 1 -6 amino acids in the C-terminus, such as 1 -5, or 1 -4, or 1 -3, or 1 -2 amino acids, and wherein the extended polypeptide has RNA polymerase activity.

48. The cell according to any one of the proceeding paragraphs, wherein the cell is a eukaryotic cell.

49. The cell according to any one of the proceeding paragraphs, wherein the cell is a mammalian cell.

50. The cell according to any one of the proceeding paragraphs, wherein the cell is a prokaryotic cell.

51. The cell according to any one of the proceeding paragraphs, which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

52. The cell according to any one of the proceeding paragraphs, which is a filamentous fungal recombinant host 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 rose urn, 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.

53. The cell according to any one of the proceeding paragraphs, wherein the cell is an Aspergillus cell.

54. The cell according to any one of the proceeding paragraphs, wherein the cell is an Aspergillus niger cell.

55. The cell according to any one of the proceeding paragraphs, wherein the cell is an Aspergillus oryzae cell.

56. The cell according to any one of the proceeding paragraphs, wherein the cell is a Trichoderma cell. 57. The cell according to any one of the proceeding paragraphs, wherein the cell is a Trichoderma reesei cell.

58. The cell according to any one of the proceeding paragraphs, which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

59. The cell according to any one of the proceeding paragraphs, wherein the cell is a Bacillus cell.

60. The cell according to any one of the proceeding paragraphs, wherein the cell is a Bacillus licheniformis cell.

61. The cell according to any one of the proceeding paragraphs, wherein the cell is a Bacillus subtilis cell.

62. The cell according to any one of the proceeding paragraphs, which is isolated.

63. The cell according to any one of the proceeding paragraphs, which is purified.

64. The cell according to any one of the proceeding paragraphs, wherein the second heterologous promoter operably linked to the second polynucleotide results in decreased transcription of the second polynucleotide, relative to the transcription of the second polynucleotide when being operably linked to its native or endogenous promoter.

65. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence operably linked to the second polynucleotide results in decreased transcription of the second polynucleotide, relative to the transcription of the second polynucleotide when being operably linked to its native or endogenous Shine-Dalgarno sequence.

66. The cell according to any one of the proceeding paragraphs, wherein the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alphagalactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase; even more preferably the one or more polypeptide of interest comprises an amylase or a protease. The cell according to any one of the proceeding paragraphs, wherein the polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of an antibody, an antibody fragment, an antibody-based drug, a Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a blood clotting factor, a hormone, an interferon (such as an interferon alpha-2b), an interleukin, a lactoferrin, an alpha-lactalbumin, a beta-lactalbumin, an ovomucoid, an ovostatin, a cytokine, an obestatin, a human galactosidase (such as an human alpha-galactosidase A), a vaccine, a protein vaccine, and a thrombolytic. The cell according to any one of the proceeding paragraphs, wherein the polypeptide of interest comprises a nanobody (Nb), preferably the nanobody consists of a single variable light chain (VL). The cell according to any one of the proceeding paragraphs, wherein the first polynucleotide encodes a polypeptide having amylase activity and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 6. The cell according to any one of the proceeding paragraphs, wherein the polypeptide of interest is an amylase, such as an amylase which comprises, or consists of the mature polypeptide having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 7. The cell according to any one of the proceeding paragraphs, wherein the first polynucleotide encodes a polypeptide having protease activity and comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of SEQ ID NO: 8. The cell according to any one of the proceeding paragraphs, wherein the polypeptide of interest is a protease, such as a protease which comprises, or consists of the mature polypeptide having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 9. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and/or deletion at one or more positions of the nucleotides:

- corresponding to positions 1 to 7 of the nucleic acid sequence “AAGGAGG”, or of the nucleic acid sequence at positions 1 -7 of SEQ ID NO: 3,

- corresponding to positions 1 -8 of the nucleid acid sequence “GAGGGGTG”,

- corresponding to positions 1-7 of the nucleic acid sequence “AAGGGAG”, or - corresponding to positions 1-8 of the nucleic acid sequence “GGAGGTTG”.

74. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and/or deletion at a position corresponding to position 3 of the parent SD sequence with the nucleic acid sequence “AAGGAGG”, or of the nucleic acid sequence at positions 1 -7 of SEQ ID NO: 3.

75. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 3 of the parent SD sequence with the nucleic acid sequence “AAGGAGG” with Adenine (A), G3A, with Cytosine (C), G3C, or with Thymine (T), G3T.

76. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 3 of the parent SD sequence with the nucleic acid sequence “AAGGAGG” with Adenine (A), G3A.

77. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises or consists of a nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of “AAAGAGG”, or to the nucleic acid sequence at positions 1 -7 of SEQ ID NO: 4.

78. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises or consists of the nucleic acid sequence of “AAAGAGG”, or of the nucleic acid sequence of positions 1 -7 of SEQ ID NO: 4.

79. The cell according to any one of the proceeding paragraphs, wherein the second polynucleotide is operably linked to a mutated Shine-Dalgarno sequence forming a coding nucleic acid sequence comprising or consisting of the a coding nucleic acid sequence having a sequence identity of 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% to the nucleic acid sequence of “AAGGAGG” or “AAAGAGG”, or to the nucleic acid sequence at positions 1 - 7 of SEQ ID NO: 3 or 4, or to the nucleic acid sequence of “GAGGGGTG”, “AAGGGAG”, or “GGAGGTTG”.

80. The cell according to any one of the proceeding paragraphs, wherein the mutated Shine-Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 5 of SEQ ID NO: 58 with Adenine (A), G5A, with Cytosine (C), G5C, or with Thymine (T), G5T.

81. The cell according to any one of the proceeding paragraphs, wherein transcription and/or translation of the second polynucleotide is decreased compared to a parent cell when cultivated under identical conditions, the parent cell not comprising any of a) a heterologous promoter being operably linked to the second polynucleotide, or b) a mutated Shine-Dalgarno sequence being operably linked to the second polynucleotide, the parent cell otherwise being isogenic to the mutant cell.

82. The cell according to any one of the proceeding paragraphs, wherein transcription and/or translation of the second polynucleotide (RNAP subunit) is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least

17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least

24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least

31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least

38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least 44%, at least

45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least

52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least

59%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least

66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least

73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least

80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

87%, at least 88%, at least 89%, 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%, or at least 99%, relative to the transcription and/or translation of the RNAP subunit of the parent cell.

83. The cell according to any one of the proceeding paragraphs, wherein the transcription and/or translation of the second polynucleotide (RNAP subunit) is decreased relative to the transcription and/or translation of the parent cell, after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

84. The cell according to any one of the proceeding paragraphs, wherein yield of the polypeptide of interest is increased compared to the parent cell when cultivated under identical conditions.

85. The cell according to any one of the proceeding paragraphs, wherein yield of the polypeptide of interest is increased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, or at least 35% relative to the yield of the parent cell, preferably increased at least 12% relative to the yield of the parent cell.

86. The cell according to any one of the proceeding paragraphs, wherein the yield of the polypeptide of interest is increased relative to the yield of the parent cell, after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

87. The cell according to any one of the proceeding paragraphs, wherein, during cultivation of the cell, biomass is decreased relative to the biomass during cultivation of the parent cell when cultivated under identical conditions.

88. The cell according to any one of the proceeding paragraphs, wherein the biomass is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, or at least 35% relative to the biomass of the parent cell, preferably decreased at least 18% relative to the biomass of the parent cell.

89. The cell according to any one of the proceeding paragraphs, wherein the biomass is decreased relative to the biomass of the parent cell, after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

90. The cell according to any one of the proceeding paragraphs, wherein the cultivation is a fed-batch, batch or continuous cultivation process, preferably a fed-batch cultivation process.

91. A method for producing one or more polypeptides of interest, the method comprising, a) providing a cell according to any one of the previous paragraphs, b) cultivating said cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally recovering the one or more polypeptide of interest.

92. A nucleic acid construct comprising a second heterologous promoter and/or a mutated Shine- Dalgarno sequence operably linked to the second polynucleotide according to any preceding embodiments.

93. The nucleic acid construct of any of paragraph 92, which is isolated.

94. The nucleic acid construct of any of paragraphs 920-93, which is purified.

95. An expression vector comprising the nucleic acid construct according to paragraphs 92-94.

Claims

Claims:

1 . A mutant cell comprising in its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptide, wherein expression of the one or more Rpo subunit polypeptide is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell.

2. The mutant cell according to claim 1 , wherein: a) the second polynucleotide is operably linked to a second heterologous promoter, b) the second polynucleotide is operably linked to a mutated Shine-Dalgarno sequence derived from a parent Shine-Dalgarno sequence, c) the second polynucleotide comprises one or more nucleic acid insertion, deletion, or substitution, d) expression of the second polynucleotide is decreased by a CRISPR inhibition construct, and/or e) expression of the second polynucleotide is decreased by RNA interference.

3. The mutant cell according to any one of claims 1 -2, wherein the parent Shine-Dalgarno sequence has a sequence identity of 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% to the polynucleotide sequence of AAGGAGG or of SEQ ID NO: 58.

4. The mutant cell according to any one of claims 1-3, wherein the second polynucleotide is native to the cell.

5. The mutant cell according to any one of claims 1 -4, wherein the second polynucleotide encodes a Rpo subunit polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO:40.

6. The mutant cell according to any one of the proceeding claims, wherein the cell comprises at least two copies, e.g., three, four, or five, or six, or more copies, of the first polynucleotide in its genome.

7. The mutant cell according to any one of claims 1 to 6, wherein the cell is a prokaryotic cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus , Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

8. The mutant cell according to any one of the proceeding claims, wherein the mutated Shine- Dalgarno sequence comprises a nucleic acid substitution at a position corresponding to position 3 of the parent Shine Dalgarno sequence with the nucleic acid sequence “AAGGAGG” with Adenine (A), G3A, with Cytosine (C), G3C, or with Thymine (T), G3T, or at a position corresponding to position 5 of SEQ ID NO: 58 with Adenine (A), G5A, with Cytosine (C), G5C, or with Thymine (T), G5T.

9. The mutant cell according to any one of the preceding claims, wherein expression of the one or more Rpo subunit polypeptide is decreased by at least 10%, e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to expression of the one or more Rpo subunit polypeptide of the parent cell when cultivated under identical conditions.

10. The mutant cell according to any one of the preceding claims, wherein yield of the polypeptide of interest is increased compared to yield of the parent cell when cultivated under identical conditions.

11. The mutant cell according to any one of the preceding claims, wherein biomass formation is decreased during cultivation of the mutant cell relative to the biomass formation during cultivation of the parent cell when cultivated under identical conditions.

12. The mutant cell according to any one of the preceding claims, wherein the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alphagalactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase; even more preferably the one or more polypeptide of interest comprises an amylase or a protease. The mutant cell according to any one of the preceding claims, wherein the polypeptide of interest is an amylase, such as an amylase which comprises, or consists of the mature polypeptide having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 7. The mutant cell according to any one of claims 1- 12, wherein the polypeptide of interest is a protease, such as a protease which comprises, or consists of the mature polypeptide having a sequence identity of 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% to the amino acid sequence of SEQ ID NO: 9. A method for producing one or more polypeptides of interest, the method comprising, a) providing a mutant cell according to any of claims 1 to 14, b) cultivating said cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally recovering the one or more polypeptide of interest.