Altered RNA polymerase activity

Abstract

The present invention relates to mutant host cells in which RNA polymerase subunit expression is reduced, polynucleotides and expression vectors for reducing the expression of RNA polymerase subunits in host cells, and host cells and methods for producing polypeptides of interest.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. [Background technology]

[0002] The present invention relates to mutant host cells with reduced RNA polymerase subunit expression, polynucleotides and expression vectors for the reduced expression of RNA polymerase subunits in host cells, and host cells and methods for producing polypeptides of interest.

[0003] 2. Description of Related Art Recombinant gene expression in recombinant host cells, such as bacterial or fungal host cells, is a common method for recombinant protein production. The recombinant proteins produced in these systems are enzymes and other valuable proteins. For industrial and commercial purposes, the productivity of the applied cell system, i.e., the total protein produced per unit of fermentation, is an important factor in production costs. Traditionally, yield enhancement has been achieved by mutagenesis, signal peptide optimization, and screening of numerous mutants for increased production of the protein of interest. However, this approach is primarily useful only for overproduction of endogenous proteins in isolates containing the enzyme of interest. Therefore, extensive strain and process development programs are required to achieve productivity improvements for each new protein or enzyme product.

[0004] For the overexpression of heterologous proteins in recombinant host cell systems, the production process is recognized as a complex, multi-step 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.

[0005] Various approaches to improve transcription have been used in bacteria. For the expression of heterologous genes, codon-optimized synthetic genes can improve transcription rates (WO 9923211, Novozymes A / S). To obtain high-level expression of a specific gene, a well-established procedure is to target multiple copies of a recombinant gene construct to the locus of a highly expressed endogenous gene.

[0006] However, multi-copy strains often reach the expression limit of the host cell, after which integration of additional copies of the recombinant gene does not further improve recombinant yields.

[0007] At the molecular level, expression of a protein of interest can be divided into the following steps: i) transcription of the gene of interest from DNA to RNA, ii) translation of the RNA into a polypeptide, and iii) maturation and secretion of the polypeptide of interest.

[0008] During step i), RNA in all cellular organisms is synthesized by a complex molecular machine: DNA-dependent RNA polymerase (RNAP, or Rpo). In its simplest bacterial form, this enzyme contains at least four subunits with a total molecular weight of approximately 400 kDa. The eukaryotic enzyme contains more than 12 subunits with a total molecular weight of approximately 500 kDa. The catalytically competent bacterial core Rpo (subunit composition: 2 × RpoA (α), 1 × RpoB (β), 1 × RpoB' (β'), and 1 × RpoZ (ω)) is evolutionarily conserved in sequence, structure, and function from bacteria to eukaryotes, including fungal and mammalian cells (Borukhov & Nudler, Trends in Microbiology, 16(3), 2008, 126-134).

[0009] Despite these proposed approaches, there is a continuing interest in further improving recombinant protein production in genetically modified host cells. It is an object of the present invention to provide modified host cells and methods of protein production with increased recombinant protein productivity and / or yield. Summary of the Invention [Means for solving the problem]

[0010] As disclosed herein, the inventors of the present invention have demonstrated that one bottleneck in increasing yield during recombinant protein production can be explained by the suboptimal transcription capacity of the host cell. In other words, transcript (RNA) formation for a polypeptide of interest must compete with all other transcription processes occurring in the host cell, i.e., all other transcribed genes in the host cell.

[0011] Surprisingly, the present inventors have shown that reducing the expression of native RNA polymerase subunits in recombinant cells producing a recombinant protein of interest improves the secretion and / or yield of the recombinant protein of interest. After reducing the expression of RNA polymerase subunit alpha (RpoA), recombinant protein yield was significantly improved, i.e., by 12%, compared to protein yield from host cells in which RpoA expression was not modified, and biomass formation was reduced by 18% compared to biomass formation from host cells in which RpoA expression was not modified. As described in the Examples, the present inventors have surprisingly shown that reducing the expression of RNAP subunits results in increased yields of different classes of proteins of interest (amylases and proteases). Therefore, the present inventors expect that these findings will also apply to other proteins of interest, such as other enzymes, and in particular other heterologous proteins. In addition, the reduction in RNA polymerase subunit expression resulted in a completely unexpected decrease in biomass formation (18% reduction), which is beneficial for fermentation (e.g., more efficient downstream processing) and product formulation, since less biomass must be removed per polypeptide product.

[0012] Without wishing to be bound by any theory, particularly 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, reducing the expression of the RNA polymerase subunit can resolve the transcription and / or translation bottleneck of the polypeptide of interest, thus providing increased yield of the polypeptide of interest. Fine-tuning the expression of the RNA polymerase subunit can ensure that more RNA transcripts of the gene of interest can be produced relative to the mRNA formation of other host cell genes, thereby resulting in an overall increased yield of the polypeptide of interest. Furthermore, surprisingly, reducing the expression of the RNA polymerase subunit can be used as a means to reduce biomass formation without compromising product yield or cell viability, in fact, while simultaneously increasing product yield.

[0013] Thus, in a first aspect, the present invention relates to a mutant cell comprising within its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotides encoding one or more RNA polymerase (Rpo) subunit polypeptides, wherein expression of the one or more Rpo subunit polypeptides is reduced or eliminated compared to a non-mutated but otherwise isogenic or parental cell.

[0014] In a second aspect, the present invention provides a method for producing one or more polypeptides of interest, comprising the steps of: a) providing a mutant cell according to the first aspect; b) culturing the cells under conditions conducive to expression of one or more polypeptides of interest; c) optionally recovering one or more polypeptides of interest; The present invention relates to a method comprising:

[0015] In a third aspect, the present invention relates to a nucleic acid construct comprising a heterologous promoter and / or a mutant Shine-Dalgarno sequence operably linked to a second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptides.

[0016] In a fourth aspect, the present invention relates to an expression vector comprising a nucleic acid construct according to the third aspect.

[0017] definition In accordance with the detailed description, the following definitions apply: It should be noted that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0018] Unless otherwise defined 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.

[0019] Amylase: The term "amylase" refers to a polypeptide having amylase activity, such as α-amylase (EC 3.2.1.1), which catalyzes the hydrolysis of 1,4-α-glucosidic bonds in amylose and amylopectin. A non-limiting example of an amylase is the α-amylase set forth in SEQ ID NO:7.

[0020] Amylase activity: Amylase activity can be determined in a variety of ways known to those skilled in the art. For example, amylase activity can be measured by the assays described in the "Enzyme Assays" section in the Examples.

[0021] Bacterial RNA polymerase subunit: The term "bacterial RNA polymerase subunit" refers to any bacterial RNA polymerase subunit polypeptide selected from the list of subunit beta (β), subunit beta' (β'), subunit alpha (α), and subunit omega (ω).

[0022] Biomass: In the context of the present invention, the term "biomass" refers to the accumulation of cells in a culture. For bacterial cell fermentation, the term "biomass" also includes spores and other cellular structures. For fungal cell fermentation, the term "biomass" also includes hyphae and other cellular structures. Biomass is typically measured as the dry or wet weight of a plurality of fungal cells. Additionally or alternatively, biomass can be measured for bacterial cell cultures by determining the optical density of the culture broth at a specific wavelength, for example, 650 nm wavelength.

[0023] cDNA: The term "cDNA" refers to 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, which is processed through a series of steps, including splicing, before emerging as a mature, spliced ​​mRNA.

[0024] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which generally 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 can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0025] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, and can also be 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 transcriptional or translational initiator and terminator sequences. At a minimum, control sequences include a promoter and transcriptional and translational termination signals. A non-limiting example of a promoter is the P3 promoter, having SEQ ID NO: 38. Control sequences can be provided with linkers intended to introduce specific restriction sites facilitating ligation of the control sequence with the coding region of a polynucleotide encoding a polypeptide.

[0026] Eukaryotic RNA polymerase: The term "eukaryotic RNA polymerase" refers to any eukaryotic polymerase, including RNA polymerase I, RNA polymerase II, or RNA polymerase III, including any subunit derived from a eukaryotic RNA polymerase.

[0027] Expression: The term "expression" refers to all steps involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0028] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, where the coding sequence is operably linked to a suitable control sequence capable of effecting the 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, an enhancer, and sequences that control the termination of transcription and translation.

[0029] Elongation: The term "elongation" refers to the addition of one or more amino acids to the amino and / or carboxyl terminus of an RNA polymerase subunit polypeptide, wherein the "elongated" subunit polypeptide alters RNA polymerase activity, e.g., reduces RNA polymerase activity.

[0030] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids that lacks the amino and / or carboxyl terminus of a mature RNA polymer as a subunit polypeptide, wherein the fragment alters RNA polymerase activity, e.g., reduces RNA polymerase activity.

[0031] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which a polypeptide is fused to the N-terminus and / or C-terminus of a polypeptide of the present invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present disclosure, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and involve linking coding sequences encoding the polypeptides so that the coding sequences are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, which creates a fusion polypeptide after translation (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). Fusion polypeptides can further include a cleavage site between the two polypeptides. Upon secretion of the fusion protein, this site is cleaved to release the two polypeptides. Examples of cleavage sites include, but are not limited to, those described 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. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0032] Gene: The term "gene," such as "rpoA gene," refers to a polynucleotide sequence that includes a polynucleotide sequence encoding a polypeptide product / POI (protein of interest), a promoter sequence, and a Shine-Dalgarno sequence (ribosome binding site, RBS) upstream of the polynucleotide sequence encoding the POI. Transcription of the gene and / or translation of the gene product can be altered, for example, by using a different promoter and / or a modified Shine-Dalgarno sequence, respectively. As a non-limiting example, transcription of the rpoA gene can be altered by replacing the native promoter with a heterologous promoter. As a further non-limiting example, translation of the RpoA polypeptide can be altered by providing in the rpoA gene a mutant Shine-Dalgarno sequence that includes one or more nucleic acid substitutions in the native Shine-Dalgarno sequence of "AAGGAGG."

[0033] Heterologous: The term "heterologous," with respect to a host cell, means that a polypeptide or nucleic acid does not naturally occur in that host cell. The term "heterologous," with respect to a polypeptide or nucleic acid, means that a regulatory sequence of the polypeptide or nucleic acid, e.g., a promoter, is not naturally associated with that polypeptide or nucleic acid, i.e., the regulatory sequence is derived from a gene other than the gene encoding the mature polypeptide.

[0034] 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 containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0035] 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.

[0036] Isogenic cell: The term "isogenic," with respect to a host cell, refers to a parental or clonal host cell that has essentially the same genotype, e.g., a parental host cell that has certain differences due to additional mutations or polynucleotides that are subsequently introduced into the daughter cells, resulting in daughter cells that have the additional mutations and / or polynucleotides, but that are otherwise isogenic to the parental cell, but have essentially the same background mutations as the daughter cells.

[0037] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form that does not occur in nature. Isolated polypeptides include, but are not limited to, culture broth containing the polypeptide expressed and secreted in a host cell.

[0038] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal and / or C-terminal processing (e.g., removal of a signal peptide). In one aspect, the mature polypeptide is SEQ ID NO:3.

[0039] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0040] Nucleic Acid: The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. 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 the 5' to 3' direction.

[0041] Nucleic acid construct: The term "nucleic acid construct" means a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner that would not occur in nature, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0042] Operably linked: The term "operably linked" means that the specified components are in a relationship, including but not limited to, a juxtaposition, permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence. In another example, an SD sequence is operably linked to a coding sequence such that both sequences are transcribed into an mRNA.

[0043] Protease: The term "protease" refers to a polypeptide having protease activity that catalyzes the hydrolysis of proteins or polypeptides into smaller amino acid polymers (EC 3.4.21.-). A non-limiting example of a protease is the polypeptide set forth in SEQ ID NO:9.

[0044] Protease activity: Protease activity can be determined in a variety of ways known to those skilled in the art. For example, protease activity can be measured by the assays described in the "Enzyme Assays" section in the Examples.

[0045] Purified: The term "purified" refers to a nucleic acid, polypeptide, or cell that is substantially free of 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, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more pure (e.g., by weight or molar percentage). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule after application of a purification or concentration 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 higher relative or absolute concentration than in the starting composition.

[0046] In one aspect, the term "purified," as used herein, refers to a polypeptide or cell that is essentially free from components, particularly insoluble components, from the producing organism. In other aspects, the term "purified" refers to a polypeptide that is essentially free from insoluble components, particularly insoluble components, from the natural organism from which the polypeptide is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which the polypeptide is recovered. The polypeptide can be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0047] Thus, a polypeptide can be purified so that only trace amounts of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" can refer to the removal of other components, particularly other proteins, most particularly other enzymes, present in the cell from which the polypeptide originates. A polypeptide can be "substantially pure," i.e., free from other components from the organism in which the polypeptide is produced (e.g., the host organism for recombinantly produced polypeptides). In one aspect, the polypeptide is at least 40% pure, by weight, of the total polypeptide material present in a 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 a preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains up to 10%, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and even most preferably up to 0.5% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0048] Thus, a substantially pure polypeptide is preferably 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, and most preferably at least 99.5% pure, by weight of total polypeptide material present in the preparation. The polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which the polypeptide is naturally or recombinantly associated). This can be achieved, for example, by preparing the polypeptide by well-known recombinant or classical purification methods.

[0049] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form configurations that differ from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been altered from its natural state. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions that differ from those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0050] Recover: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, e.g., by collecting polypeptide crystals, or by filtration, e.g., depth filtration (using filter aids or packed filter media, fabric filtration in a chamber filter, rotary drum filtration, drum filtration, rotary vacuum drum filter, candle filter, horizontal leaf filter, or similar, using seed or pad filtration in a frame or modular setup), 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 a decanter centrifuge, disc centrifuge, hydrocyclone, or similar), or by precipitating the polypeptide and collecting it from the broth medium by using particle size fractionation using a suitable solid-liquid separation method. Recovery encompasses isolation and / or purification of the polypeptide.

[0051] RNA polymerase activity: The term "RNAP activity" or "RNA polymerase activity" refers to the ability to synthesize an RNA molecule from a DNA template through the process of transcription.

[0052] As a non-limiting example, RNAP activity can be determined using a rifampicin dilution assay, in which RNAP activity is assessed by determining rifampicin resistance, i.e., cell survival in the presence of rifampicin is associated with increased RNAP activity, whereas cell death in the presence of rifampicin is associated with decreased RNAP activity. Such an assay is shown in Example 6.

[0053] RNA polymerase subunit polypeptide: The term "RNA polymerase subunit polypeptide" or "RNAP subunit" or "RPO subunit" refers to any subunit of RNA polymerase throughout the animal kingdom. RNA polymerase, which contains several subunits, is responsible for catalyzing the transcription of DNA into RNA using 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, this enzyme contains at least four subunits with a total molecular weight of approximately 400 kDa. The eukaryotic enzyme contains more than 12 subunits with a total molecular weight of approximately 500 kDa. The catalytically competent bacterial core Rpo (subunit composition: 2x RpoA(α), 1x RpoB(β), 1x RpoB'(β'), and 1x RpoZ(ω)) is evolutionarily conserved in sequence, structure, and function from bacteria to eukaryotes, including fungal and mammalian cells (see Table 1) (Borukhov & Nudler, Trends in Microbiology, 16(3), 2008, 126-134).

[0054] [Table 1]

[0055] RpoA Polypeptide: The term "RpoA polypeptide" or "RNAP subunit alpha" or "RpoA subunit" refers to the DNA-dependent RNA polymerase subunit alpha, which is involved in catalyzing the transcription of DNA to RNA using the four ribonucleoside triphosphates as substrates. This reaction is also known as EC:2.7.7.6. A non-limiting example of an RpoA polypeptide is the Bacillus licheniformis RpoA polypeptide set forth in SEQ ID NO:2.

[0056] rpoA gene: The term "rpoA gene" refers to a polynucleotide sequence encoding an RNA polymerase subunit alpha polypeptide, RpoA, such as DNA-dependent RNA polymerase subunit alpha (EC 2.7.7.6), also known as RNAP subunit alpha. RpoA / RNAP subunit alpha is a DNA-dependent RNA polymerase that catalyzes the transcription of DNA to RNA using four ribonucleoside triphosphates as substrates. A non-limiting example of an rpoA gene is the rpoA gene from Bacillus licheniformis set forth in SEQ ID NO:3, which contains the 7-nucleotide Shine-Dalgarno sequence "AAGGAGG" at positions 1 to 7 of its 5' end. A non-limiting example of an RpoA polypeptide is the RpoA polypeptide from Bacillus licheniformis set forth in SEQ ID NO:2.

[0057] The term "rpoA gene" includes a polynucleotide sequence upstream of the start codon "ATG," including the Shine-Dalgarno (SD) sequence "AAGGAGG," as shown in SEQ ID NO: 3. During transcription of the rpoA gene, the SD sequence, along with the polynucleotide sequence encoding rpoA, is transcribed into mRNA. The SD sequence is a ribosome binding site, generally located 5 to 9 bases upstream of the start codon AUG. The SD RNA sequence serves to recruit ribosomes to mRNA and 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 polypeptide levels, respectively (Velaquez et al., Journal of Bacteriology, May 1991, pp. 3261-3264). Therefore, modification of the SD sequence in the rpoA gene can reduce or increase RpoA polypeptide levels. This change results from a decrease or increase in the efficiency of rpoA mRNA pairing with ribosomes. A non-limiting example of a mutated SD sequence is the mutated SD sequence in the B. licheniformis rpoA gene represented by the nucleotide sequence of SEQ ID NO: 4, in which G is replaced by A at position corresponding to position 3 "G3A" of SEQ ID NO: 3 (native SD+rpoA sequence). During translation, such a mutation affects mRNA-ribosome pairing efficiency, but the mutation does not disrupt the sequence of the mature RpoA polypeptide.

[0058] Eukaryotic homologs of RpoA include the RNA polymerase I subunits RPAC40 and RPAC19, the RNA polymerase II subunits RPB3 and RPB11, and the RNA polymerase III subunits RPAC40 and RPAC19 (see Table 1).

[0059] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0060] For purposes of the present invention, sequence identity between two amino acid sequences is determined as a "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably 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), version 6.6.0 or later. The parameters used are a gap opening 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 on the command line. The "longest identity" output displayed by Needle is calculated as follows: (Identical residues × 100) / (length of alignment − total number of gaps in the alignment)

[0061] For purposes of the present invention, sequence identity between two polynucleotide sequences is determined as a "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the 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 on the command line. The "longest identity" output displayed by Needle is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment − total number of gaps in alignment)

[0062] Shine-Dalgarno sequence: The term "Shine-Dalgarno sequence" or "SD sequence" refers to a ribosome-binding site on an RNA sequence, generally located 5 to 9 bases upstream of the AUG initiation codon in bacterial and archaeal cells. The SD RNA sequence serves to recruit ribosomes to mRNA and initiate protein synthesis by aligning the ribosome with the initiation codon. Mutations in the SD sequence can reduce or increase translation in host cells, resulting in reduced or increased polypeptide levels, respectively (Velaquez et al., Journal of Bacteriology, May 1991, pp. 3261-3264). Therefore, modification of the SD sequence in the rpo subunit gene can reduce or increase RPO subunit polypeptide levels. This change in RPO subunit levels results from a decrease or increase in the efficiency of rpo subunit mRNA pairing with ribosomes.

[0063] Mutant Shine-Dalgarno sequence: The term "mutant Shine-Dalgarno sequence" or "mutated SD sequence" refers to an SD sequence that contains one or more nucleic acid modifications, such as a nucleic acid substitution, deletion, or insertion. As described above, modifications of the SD sequence can decrease or increase translation of a gene located downstream of the modified or mutated SD sequence, depending on the mutation.

[0064] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates the secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0065] Subsequence: The term "subsequence" refers to a polynucleotide having one or more nucleotides lacking the 5' and / or 3' end of a mature RNA polymerase subunit polypeptide coding sequence; herein, a subsequence encodes a fragment of an RNA polymerase subunit that does not reduce RNA polymerase activity.

[0066] Therapeutic Polypeptide: The term "therapeutic polypeptide" refers to any polypeptide or protein, or variant thereof, suitable for use in the treatment of a disease or condition in humans or for use in veterinary medicine. Non-limiting examples of therapeutic polypeptides are antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons (e.g., interferon alpha-2b), interleukins, lactoferrin, alpha-lactalbumin, beta-lactalbumin, ovomucoid, ovostatin, cytokines, obestatin, human galactosidases (e.g., human alpha-galactosidase A), and thrombolytic drugs.

[0067] Variant: The term "variant" refers to an RNA polymerase subunit polypeptide having RNA polymerase activity that contains an artificial mutation, i.e., a substitution, an insertion (including an extension), and / or a deletion (e.g., a truncation), at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0068] Wild-type: The term "wild-type" in reference to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to anything found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to anything not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or variants of a wild-type sequence). DETAILED DESCRIPTION OF THE INVENTION

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] Decreased expression of RNA polymerase subunits Decreased RNA polymerase activity in host cells In one aspect, the present invention relates to reducing the transcription and / or translation of one or more RNA polymerase subunits, which is effected in reducing overall RNA polymerase (RNAP) activity. Reducing transcription and / or translation has been shown to increase the yield of a polypeptide of interest in a recombinant host cell while also reducing biomass formation.

[0073] The reduction in RNAP activity and / or the reduction in transcription and / or the reduction in translation of one or more RNA polymerase subunits can be achieved, for example, by: a) operably linking a second polynucleotide encoding an RNAP subunit to a second heterologous promoter that is weaker than the native promoter of the gene encoding the RNAP subunit, e.g., by replacing the native promoter with the weaker second heterologous promoter; b) operably linking said second polynucleotide to a mutant Shine-Dalgarno sequence that comprises one or more nucleic acid modifications compared to the native SD sequence of the gene encoding the RNAP subunit, wherein the mutated SD sequence results in weaker ribosome binding of the RNA during translation of the RNAP subunit; c) targeting the coding sequence of an RNAP subunit gene or its transcript during transcription or translation, respectively, using CRISPRi, RNAi, or other interference techniques known to those skilled in the art; d) deleting one or more genes encoding RNAP subunits, and / or e) introducing one or more mutations into one or more of the RNAP subunit genes to provide mutated RNAP subunits that reduce RNAP activity; This can be achieved by:

[0074] In certain embodiments, the second polynucleotide is operable with a second heterologous promoter, wherein the second heterologous promoter results in reduced transcription compared to transcription of the native promoter of the gene encoding the RNAP subunit, and wherein the SD sequence upstream of the gene encoding the RNAP subunit is not mutated.

[0075] The present invention relates to reducing the expression of one or more RNA polymerase (RNAP) subunit polypeptides. In one aspect, the present invention provides a method for reducing the expression of one or more RNA polymerase (RNAP) subunit polypeptides, comprising: (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 their cDNA sequence; (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-terminus is extended by the addition of one or more amino acids; and (e) a fragment of the polypeptide of (a), (b), (c), or (d). The present invention relates to a reduction in the expression of one or more RNAP subunit polypeptides selected from the group consisting of:

[0076] In one aspect, the one or more RNAP subunit polypeptides have 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% 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.

[0077] The one or more RNAP subunit polypeptides preferably comprise, consist essentially of, or consist 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 the mature polypeptide thereof.

[0078] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, 1 to 5 amino acids.

[0079] In some embodiments, the second polynucleotide encoding one or more RNA subunit polypeptides has 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% 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 their cDNA sequence.

[0080] 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.

[0081] In another aspect, the RNAP subunit polypeptide is 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. In another aspect, the polypeptide is derived from the mature polypeptide 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 by 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 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 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino- or carboxy-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding module.

[0082] Essential amino acids in a polypeptide can be identified using 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 resulting molecules are tested for RNA polymerase activity to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures, such as those determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., 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 alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or with polypeptides / proteins (typically with similar three-dimensional structure, function, and significant sequence similarity) derived from a common ancestor within a polypeptide or protein family. Additionally or alternatively, protein structure prediction tools for protein structure modeling can be used to identify essential amino acids and / or the active site of a polypeptide. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold," Nature 596:583-589.

[0083] 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 appropriate screening procedures, 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; U.S. Pat. No. 5,223,409; WO 92 / 06204), and site-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0084] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0085] The RNAP subunit polypeptide can be a fusion polypeptide.

[0086] host cell The present invention also relates to recombinant host cells containing 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.

[0087] A construct or vector containing a polynucleotide is introduced into a host cell, as described above, so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. The choice of host cell will largely depend on 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 regulatory sequences can be heterologous to the first polynucleotide encoding the polypeptide of interest. Additionally or alternatively, the one or more regulatory sequences can be operably linked to a second polynucleotide, preferably heterologous to the second polynucleotide. The recombinant host cell can contain 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 can contain 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 an RNAP subunit polypeptide.

[0088] The host cell can be any microbial cell, such as a prokaryotic or fungal cell, useful for the recombinant production of a polypeptide of interest.

[0089] According to a first aspect, the present invention relates to a mutant cell comprising within its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotides encoding one or more RNA polymerase (Rpo) subunit polypeptides, wherein expression of the one or more Rpo subunit polypeptides is reduced or eliminated compared to a non-mutated but otherwise isogenic or parental cell.

[0090] In one embodiment, the second polynucleotide is operably linked to a second heterologous promoter.

[0091] In one embodiment, the second polynucleotide is operably linked to a variant Shine-Dalgarno sequence derived from the parent Shine-Dalgarno sequence.

[0092] In another embodiment, the second polynucleotide comprises one or more nucleic acid insertions, deletions, or substitutions.

[0093] In one embodiment, the expression of the second polynucleotide is reduced by a CRISPR inhibitor construct.

[0094] In another embodiment, the expression of the second polynucleotide is reduced by RNA interference.

[0095] In one embodiment, the parent Shine-Dalgarno sequence has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polynucleotide sequence of AAGGAGG or to SEQ ID NO: 58.

[0096] In one embodiment, the second polynucleotide is native to the cell.

[0097] 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 an RNA polymerase subunit polypeptide.

[0098] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more bacterial RNA polymerase subunit polypeptides selected from the list of subunit beta (β), subunit alpha (α), and subunit omega (ω).

[0099] In one embodiment, the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α) RpoA.

[0100] In one embodiment, the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β) RpoB.

[0101] In one embodiment, the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits beta' (β') RpoB'.

[0102] In one embodiment, the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω)RpoZ.

[0103] In one embodiment, the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α)RpoA, and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β)RpoB and / or (β')RpoB'.

[0104] In one embodiment, the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α) RpoA, and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω) RpoZ.

[0105] In one embodiment, the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β) RpoB, and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω) RpoZ.

[0106] In one embodiment, the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits beta' (β')RpoB' and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω)RpoZ.

[0107] In one embodiment, the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α)RpoA, the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β)RpoB and / or (β')RpoB', and the one or more third second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω)RpoZ.

[0108] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoA polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0109] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoA polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1.

[0110] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoB polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:11.

[0111] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoB polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0112] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoB' polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:40.

[0113] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoB' polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 39.

[0114] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoZ polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0115] In one embodiment, the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoZ polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0116] In one embodiment, the second polynucleotide is heterologous to the cell.

[0117] In one embodiment, the first polynucleotide is operably linked to one or more promoters that direct the production of the polypeptide of interest, preferably the promoters are heterologous to the first polynucleotide.

[0118] In one embodiment, the heterologous promoter comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 38.

[0119] In one embodiment, the cell comprises at least two copies of the first polynucleotide in its genome, for example, at least three, at least four, or at least five, or at least six, or more copies.

[0120] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more archaeal RNA polymerase subunit polypeptides selected from the list of Rpo1, Rpo2, Rpo3, Rpo11, Rpo4, Rpo5, Rpo6, Rpo8, Rpo10, Rpo12, Rpo7, or Rpo13.

[0121] In one embodiment, the one or more RNA polymerase subunit polypeptides are eukaryotic RNA polymerase I, RNA polymerase II, and / or RNA polymerase III subunit polypeptides.

[0122] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase I subunit polypeptides selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

[0123] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase subunit polypeptides selected from the list of RPAC40 (AC40), RPAC19 (AC19), RPO3, RPO11, RPB3, and RPB11.

[0124] In one embodiment, the second polynucleotide encodes a RPAC40 (AC40) polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0125] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase I subunit polypeptides selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

[0126] In one embodiment, the second polynucleotide encodes a RPAC40 (AC40) polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19.

[0127] In one embodiment, the second polynucleotide encodes a RPAC19 (AC19) polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

[0128] In one embodiment, the second polynucleotide encodes a RPAC19 (AC19) polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.

[0129] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase II subunit polypeptides selected from the list of RPB1, RPB2, RPB3, RPB11, RPB6, RPB5, RPB8, RPB10, RPB12, RPB4, RPB7, RPB9, TFIIFα, and TFIIFβ.

[0130] In one embodiment, the second polynucleotide encodes an RPB3 polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.

[0131] In one embodiment, the second polynucleotide encodes an RPB3 polypeptide comprising or consisting of an amino acid sequence having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31.

[0132] In one embodiment, the second polynucleotide encodes an RPB11 polypeptide and comprises or consists of a nucleic acid sequence having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.

[0133] In one embodiment, the second polynucleotide encodes an RPB11 polypeptide comprising or consisting of an amino acid sequence having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.

[0134] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase III subunit polypeptides 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.

[0135] In one embodiment, the one or more RNA polymerase subunit polypeptides are one or more yeast RNA polymerase subunit polypeptides selected from the list of Rpb5 (ABC27), Rpb6 (ABC23, or Rpo26), Rpb8 (ABC14.5), Rpb10 (ABC10β), and Rpb12 (ABC10α).

[0136] In one embodiment, one or more RNA polymerase subunit polypeptides comprise an N-terminal and / or C-terminal extension of 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably an extension of 1 to 6 amino acid residues at the N-terminus and / or 1 to 6 amino acids, e.g., 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 amino acids, at the C-terminus, wherein the extended polypeptide has RNA polymerase activity.

[0137] In one embodiment, the cell is a eukaryotic cell.

[0138] In one embodiment, the cell is a mammalian cell.

[0139] In one embodiment, the cell is a prokaryotic cell.

[0140] In one embodiment, the cell is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, e.g., Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cells.

[0141] In one embodiment, the cell is a filamentous fungal recombinant host cell, such as a cell of the genus Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nelumbo nucifera, or the like. Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cells, in particular Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis kallegeacaregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola 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 heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseumroseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma koningii, Trichoderma longibrachiatum longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0142] In one embodiment, the cell is an Aspergillus cell.

[0143] In one embodiment, the cell is an Aspergillus niger cell.

[0144] In one embodiment, the cell is an Aspergillus oryzae cell.

[0145] In one embodiment, the cell is a Trichoderma cell.

[0146] In one embodiment, the cell is Trichoderma reesei.

[0147] In one embodiment, the cell is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cell, or a Campylobacter, E. coli, or a Gram-positive cell selected from the group consisting of a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cell.Gram-negative bacteria selected from the group consisting of Bacillus coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, 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.

[0148] In one embodiment, the cell is a Bacillus cell.

[0149] In one embodiment, the cell is a Bacillus licheniformis cell.

[0150] In one embodiment, the cell is a Bacillus subtilis cell.

[0151] In one embodiment, the cells are isolated.

[0152] In one embodiment, the cells are purified.

[0153] In one embodiment, the second heterologous promoter operably linked to the second polynucleotide results in reduced transcription of the second polynucleotide compared to transcription of the second polynucleotide when operably linked to its native or endogenous promoter.

[0154] In one embodiment, the variant Shine-Dalgarno sequence operably linked to the second polynucleotide results in reduced transcription of the second polynucleotide compared to transcription of the second polynucleotide when operably linked to its native or endogenous Shine-Dalgarno sequence.

[0155] In one embodiment, the polypeptide of interest comprises an enzyme; preferably, the enzyme is a 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, or alpha-galactosidase. Preferably, the one or more polypeptides of interest are selected from the group consisting of: glutamate, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase; even more preferably, the one or more polypeptides of interest comprise an amylase or a protease.

[0156] 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, an 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 interferon alpha-2b), an interleukin, lactoferrin, alpha-lactalbumin, beta-lactalbumin, ovomucoid, ovostatin, a cytokine, obestatin, a human galactosidase (such as human alpha-galactosidase A), a vaccine, a protein vaccine, and a thrombolytic drug.

[0157] In one embodiment, the polypeptide of interest comprises a Nanobody (Nb), preferably a Nanobody consisting of a single variable light chain (VL).

[0158] In one embodiment, the first polynucleotide encodes a polypeptide having amylase activity and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0159] In one embodiment, the polypeptide of interest is an amylase, such as an amylase comprising or consisting of a mature polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0160] In one embodiment, the first polynucleotide encodes a polypeptide having protease activity and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0161] In one embodiment, the polypeptide of interest is a protease, such as a protease comprising or consisting of a mature polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0162] In one embodiment, the expression of one or more Rpo subunit polypeptides is reduced by at least 10%, e.g., 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 the expression of one or more Rpo subunit polypeptides in a parent cell when cultured under the same conditions.

[0163] In one embodiment, the variant Shine-Dalgarno sequence comprises a nucleic acid substitution at the position corresponding to position 5 of a parent Shine-Dalgarno sequence having the nucleic acid sequence of SEQ ID NO: 58 with adenine (A) (G5A), with cytosine (C) (G5C), or with thymine (T) (G5T).

[0164] In one embodiment, the variant Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and / or deletion at one or more positions of nucleotides corresponding to positions 1 to 7 of the nucleic acid sequence "AAGGAGG," or of the nucleic acid sequence at positions 1 to 7 of SEQ ID NO: 3.

[0165] In one embodiment, the mutant Shine-Dalgarno sequence comprises at least one nucleic acid substitution, insertion, and / or deletion at one or more nucleotide positions of the nucleic acid sequence "GAGGGGTG," "AAGGGAG," or "GGAGGTTG."

[0166] In one embodiment, the mutant 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 in the nucleic acid sequence at positions 1 to 7 of SEQ ID NO:3.

[0167] In one embodiment, the mutant Shine-Dalgarno sequence comprises a nucleic acid substitution with adenine (A) (G3A), with cytosine (C) (G3C), or with thymine (T) (G3T) at the position corresponding to position 3 of the nucleic acid sequence "AAGGAGG."

[0168] In one embodiment, the mutant Shine-Dalgarno sequence comprises a nucleic acid substitution at position 3 of the nucleic acid sequence "AAGGAGG" with an adenine (A) (G3A).

[0169] In one embodiment, the variant Shine-Dalgarno sequence comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence "AAAGAGG" or to the nucleic acid sequence at positions 1 to 7 of SEQ ID NO:4.

[0170] In one embodiment, the variant Shine-Dalgarno sequence comprises or consists of the nucleic acid sequence "AAAGAGG" or the nucleic acid sequence of positions 1 to 7 of SEQ ID NO:4.

[0171] In one embodiment, the second polynucleotide is operably linked to a variant Shine-Dalgarno sequence to form a coding nucleic acid sequence that comprises or consists of a coding nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence "AAGGAGG" or "AAAGAGG", or to the nucleic acid sequence at positions 1 to 7 of SEQ ID NO: 3 or 4, or to the nucleic acid sequence "GAGGGGTG", "AAGGGAG", or "GGAGGTTG".

[0172] In one embodiment, transcription and / or translation of the second polynucleotide is reduced compared to the parent cell when cultured under the same conditions.

[0173] In one embodiment, the transcription and / or translation of the second polynucleotide (RNAP subunit) is 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%, or at least 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%.

[0174] In one embodiment, transcription and / or translation of the second polynucleotide (RNAP subunit) is reduced relative to transcription and / or translation in the parent cell after at least 24 hours of culture, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of culture.

[0175] In one embodiment, the yield of the polypeptide of interest is increased compared to the parent cell when cultured under the same conditions.

[0176] In one embodiment, the yield of the polypeptide of interest is increased by 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 parental cells.

[0177] In one embodiment, the yield of the polypeptide of interest is increased by at least 12% relative to the yield of the parental cell.

[0178] In one embodiment, the yield of the polypeptide of interest is increased relative to the yield of the parent cells after at least 24 hours of culture, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of culture.

[0179] In one embodiment, during the culture of the cells, the biomass is reduced relative to the biomass during the culture of the parent cells when cultured under the same conditions.

[0180] In one embodiment, the biomass is reduced by 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 cells.

[0181] In one embodiment, biomass formation is reduced by at least 18% relative to the biomass formation of the parent cell.

[0182] In one embodiment, the biomass is reduced relative to the biomass of the parent cells after at least 24 hours of culturing, such as at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of culturing.

[0183] In one embodiment, the culture is a fed-batch, batch, or continuous culture process, preferably a fed-batch process.

[0184] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, including, but 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, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0185] Bacterial host cells include, but are 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 spp. The Bacillus cell can be any Bacillus cell, including Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0186] For purposes of the present invention, the class / genus / species of Bacillus shall be defined as set forth in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0187] The bacterial host cell can also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0188] The bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0189] 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, and transduction, in which DNA is introduced as a linear or circular polynucleotide. Those skilled in the art will be able to easily identify a suitable method for introducing DNA into a given prokaryotic cell, for example, depending on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, 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.

[0190] The host cell may be a fungal cell. As used herein, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming 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).

[0191] Fungal cells may be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic bombardment, and shock wave-mediated transformation, as outlined by the procedures set forth in Li et al., 2017, Microbial Cell Factories 16:168 and EP 238023, as well as procedures described in Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, Christensen et al., 1988, Bio / Technology 6:1419-1422, 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 a linearized or circular polynucleotide.

[0192] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the fungi imperfecti (Blastomycetes). For purposes of the present invention, yeast is defined as set forth in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0193] Yeast host cells can be Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, and the like. The yeast host cell may be a Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0194] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and the subdivision Oomycota (as defined by Hawksworth et al., 1995, supra). 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 can be fermentative.

[0195] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nelumbo nucifera, and others. The cell may be a Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In preferred embodiments, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell. In further preferred embodiments, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0196] For example, filamentous fungal host cells may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girvescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum (Fusari, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium 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, TrametesThe cell may be a Trichoderma villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0197] In one embodiment, the host cell is isolated.

[0198] In another embodiment, the host cells are purified.

[0199] Method of production In a second aspect, the present invention provides a method for producing one or more polypeptides of interest, comprising the steps of: a) providing a mutant cell according to the first aspect; b) culturing the cells under conditions conducive to expression of one or more polypeptides of interest; c) optionally recovering one or more polypeptides of interest; The present invention relates to a method comprising:

[0200] The host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells can be cultured in laboratory or industrial fermentors, in a suitable medium, and under conditions that allow the polypeptide to be expressed and / or isolated, by shake flask culture or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation). Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in catalogs 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, the polypeptide can be recovered from cell lysates.

[0201] A polypeptide can be detected using methods known in the art that are specific for that polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or assays that determine the relative or specific activity of the polypeptide.

[0202] The polypeptide can be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, a fermentation whole broth containing the polypeptide is recovered. In another aspect, a cell-free fermentation broth containing the polypeptide is recovered.

[0203] Polypeptides can be purified by various 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).

[0204] In an alternative embodiment, the polypeptide is not recovered.

[0205] Polynucleotides The present invention also relates to one or more second polynucleotides that encode the RNAP subunit polypeptides of the invention, as described herein.

[0206] The second polynucleotide may be operably linked to a second heterologous promoter.

[0207] Additionally or alternatively, a second polynucleotide may be operably linked to the mutated SD sequence.

[0208] The mutated SD sequence and / or the second heterologous promoter is located upstream of the second polynucleotide.

[0209] The second polynucleotide, second heterologous promoter, and / or mutated SD sequence can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof. The second polynucleotide and / or mutated SD sequence can be cloned from a strain of Bacillus, Trichoderma, Aspergillus, or a related organism.

[0210] In one embodiment, the second polynucleotide and / or mutated SD sequence of the present invention is isolated from a Bacillus licheniformis cell.

[0211] In one embodiment, the second polynucleotide and / or mutated SD sequence of the present invention is isolated from a Bacillus subtilis cell.

[0212] In one embodiment, the second polynucleotide and / or mutated SD sequence of the invention is isolated from an Aspergillus niger cell.

[0213] In one embodiment, the second polynucleotide and / or mutated SD sequence of the present invention is isolated from an Aspergillus oryzae cell.

[0214] In one embodiment, the second polynucleotide and / or mutated SD sequence of the present invention is isolated from a Trichoderma reesei cell.

[0215] The mutated SD sequence is mutated by introducing a nucleotide substitution, insertion, or deletion that does not result in a change in the amino acid sequence of the RNAP subunit polypeptide.

[0216] Additionally or alternatively, the second polynucleotide sequence is mutated by introducing nucleotide substitutions, insertions, or deletions that do not result in a change in the amino acid sequence of the RNAP subunit polypeptide, but that are consistent with the codon usage of the host organism in which the polypeptide of interest is intended to be produced, or by introducing nucleotide substitutions that may result in a different amino acid sequence (see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107, for a general description of nucleotide substitutions).

[0217] In one aspect, the polynucleotide is isolated.

[0218] In another embodiment, the polynucleotide is purified.

[0219] nucleic acid construct The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention linked to one or more control sequences that direct expression of an RNAP subunit coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0220] Polynucleotides can be manipulated in a variety of ways to provide for expression of RNAP subunit polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0221] promoter The control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell for expression of a first polynucleotide encoding a polypeptide of interest. The control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell for expression of a second polynucleotide encoding an RNAP subunit. A promoter contains transcriptional control sequences that mediate expression of a polypeptide. A promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0222] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells 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.

[0223] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as those described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications" and by Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0224] For expression in yeast hosts, 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 Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0225] Terminator The control sequence may also be a transcription terminator, which is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used in the present invention.

[0226] Preferred terminators for bacterial host cells can be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0227] Preferred terminators for filamentous fungal host cells can be obtained from Aspergillus or Trichoderma species, such as from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I (e.g., terminators described by Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology).

[0228] Preferred terminators for yeast host cells can 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.

[0229] mRNA stabilizers A regulatory sequence can also be an mRNA stabilizing region downstream of the promoter and upstream of the coding sequence of the gene, which increases expression of the gene encoding a polypeptide of interest.

[0230] Examples of suitable mRNA stabilization regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0231] Examples of mRNA stabilization regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0232] Leader sequence The control sequence may also be a leader, i.e., a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of a first polynucleotide encoding a polypeptide of interest and / or to a second polynucleotide encoding an RNAP subunit. Any leader that is functional in the host cell can be used.

[0233] Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12):3051-3059, and by Kaberdin and Blaesi, 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0234] Preferred leaders for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0235] Suitable leaders for yeast host cells can 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).

[0236] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, i.e., a sequence operably linked to the 3' end of the first polynucleotide and / or second polynucleotide that, when transcribed, is recognized by the host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0237] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0238] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0239] signal peptide The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide of interest and directs the polypeptide of interest into the secretory pathway of the cell. The 5' end of the coding sequence of the first polynucleotide may naturally contain a signal peptide coding sequence linked in translation reading frame with the segment of the coding sequence encoding the polypeptide of interest. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence can simply replace the native 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 the host cell may be used.

[0240] Signal peptide coding sequences useful in bacterial host cells include those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories 17:52.

[0241] Signal peptide coding sequences that are effective in filamentous fungal host cells include those 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, e.g., the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0242] 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.

[0243] Propeptide The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or, in some cases, a zymogen). Propolypeptides are generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizobium mucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0244] When both a signal peptide and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence. Additionally or alternatively, when both a signal peptide and a propeptide sequence are present, the polypeptide can comprise only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide can comprise a mixture of mature polypeptides and polypeptides comprising partial or full-length propeptide and / or signal peptide sequences.

[0245] Regulatory sequences It may be desirable to add regulatory sequences to regulate expression of the polypeptide of interest during growth of the host cell. It may also be desirable to add regulatory sequences to regulate expression of the RNAP subunit polypeptide during growth of the host cell. Examples of regulatory sequences are those that turn gene expression on or off in response to chemical or physical stimuli, 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 can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase, which is amplified in the presence of methotrexate, and metallothionein genes, which are amplified using heavy metals.

[0246] Shine-Dalgarno sequence The regulatory sequence may also be a Shine-Dalgarno (SD) sequence. The SD sequence is a ribosome-binding site on an RNA sequence, generally located 5–9 bases upstream of the AUG initiation codon. At the DNA level, the SD sequence is located upstream of the ATG initiation codon. The SD RNA sequence serves to recruit ribosomes to mRNA and initiate protein synthesis by aligning the ribosome with the initiation codon. Mutations in the SD sequence can reduce or increase translation in host cells, resulting in reduced or increased polypeptide levels, respectively (Velaquez et al., Journal of Bacteriology, May 1991, pp. 3261–3264). Therefore, modification of the SD sequence in the rpo / rnap subunit gene can reduce or increase RNAP subunit polypeptide levels. This change in RNAP subunit levels is due to a decrease or increase in the efficiency of ribosome pairing of the rnap subunit mRNA.

[0247] A mutant Shine-Dalgarno sequence can be obtained by one or more nucleic acid modifications, such as nucleic acid substitutions, deletions, or insertions. As described above, modifications of the SD sequence can decrease or increase translation of a gene located downstream of the modified or mutated SD sequence, depending on the mutation.

[0248] In a third aspect, the present invention relates to a nucleic acid construct comprising a second heterologous promoter and / or a mutant Shine-Dalgarno sequence operably linked to a second polynucleotide encoding one or more RNA polymerase (Rpo) subunit polypeptides.

[0249] In one embodiment, the nucleic acid construct is isolated.

[0250] In one embodiment, the nucleic acid construct is purified.

[0251] Expression vector In a fourth aspect, the present invention relates to an expression vector comprising a nucleic acid construct according to the third aspect.

[0252] The present invention also relates to recombinant expression vectors comprising the polynucleotides of the present invention, promoters, and transcription and translation termination signals. Various nucleotides and control sequences can be ligated together to produce recombinant expression vectors, which may contain one or more convenient restriction sites that allow for the insertion or substitution of a polynucleotide encoding a polypeptide at such a site. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide, or a nucleic acid construct containing the polynucleotide, into a vector suitable for expression. When creating an expression vector, a coding sequence is placed in the vector so that the coding sequence is operably linked to a control sequence suitable for expression.

[0253] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a polynucleotide. The choice of 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.

[0254] The vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it is integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids may be used that together contain the entire DNA to be introduced into the genome of the host cell, or a transposon.

[0255] Vectors preferably contain one or more selectable markers that permit easy selection of cells, such as transformed, transfected, transduced, or the like. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0256] Preferably, the vector contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0257] For integration into the host cell genome, the vector can rely on the sequence of the polynucleotide 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).

[0258] For autonomous replication, the vector can further comprise an origin of replication that enables the vector to replicate autonomously in the host cell of interest. The origin of replication can be any plasmid origin of replication that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid origin of replication" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0259] Two or more copies of a polynucleotide of the present invention can be inserted into a host cell to increase polypeptide production. For example, two, three, four, five, or more copies can be inserted into a host cell. Increasing the copy number of a polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, whereby cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selectable agent.

[0260] Fermentation Broth Composition or Cell Composition The present invention also relates to fermentation broth formulations or cell compositions comprising live or dead cells of the present invention. The fermentation broth formulations or cell compositions further comprise additional components used in the fermentation process, such as, for example, a polypeptide of interest, cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a killed whole broth containing organic acids, dead cells and / or cell debris, and culture medium.

[0261] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes no or minimal recovery and / or purification. For example, a fermentation broth is produced when a culture of microorganisms is grown to saturation and incubated under carbon-limited conditions to allow protein synthesis (e.g., expression of enzymes by the host cells) and secretion into the cell culture medium. A fermentation broth can contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, a fermentation broth is unfractionated and includes spent culture medium and cellular debris present after microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, a fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0262] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and / or salt thereof, and a second organic acid component comprising at least one 6 or more carbon organic acid and / or 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.

[0263] In one aspect, the composition contains an organic acid and, optionally, further contains dead cells and / or cell debris, hi some embodiments, the dead cells and / or cell debris are removed from the cell-killed whole broth to provide a composition free of these components.

[0264] The fermentation broth formulation or cell composition may further comprise preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0265] The killed whole broth or cell composition can contain the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the killed whole broth or cell composition contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the killed whole broth or cell composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0266] A whole broth or cell composition as described herein is typically liquid but may contain insoluble components, such as dead cells, cell debris, culture medium components, and / or insoluble enzymes. In some embodiments, the insoluble components can be removed to provide a clarified liquid composition.

[0267] The complete broth formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0268] Elimination or reduction of RNAP activity The present invention also relates to a method for producing a mutant of a parent cell, comprising disrupting, modifying, substituting, or deleting a promoter or a portion thereof, and regulating the transcription of one or more RNAP subunit polypeptides, resulting in a mutant cell that contains less RNAP activity than the parent cell when cultured under the same conditions.

[0269] The present invention also relates to a method for producing a mutant of a parent cell, comprising disrupting, modifying, substituting, or deleting the SD sequence or a portion thereof upstream of a gene encoding an RNAP subunit, thereby reducing the ribosome binding strength of the RNAP subunit mRNA during translation. Mutation of the SD sequence thus results in a mutant cell that contains lower RNAP activity than the parent cell when cultured under the same conditions. For expression of a polypeptide of interest (product) that is toxic to the cell, reduced RNAP activity can be advantageous, resulting in more balanced cell growth and product expression, thereby increasing overall product yield.

[0270] The present invention also relates to methods for producing a mutant of a parent cell, comprising disrupting, modifying, substituting, or deleting a second polynucleotide encoding one or more RNAP subunit polypeptides, or a portion thereof, thereby resulting in a mutant cell that contains less RNAP activity than the parent cell when cultured under the same conditions.

[0271] Reducing the transcription and / or translation of one or more RNA polymerase subunits, resulting in a reduction in total RNA polymerase (RNAP) activity, has been shown to increase the yield of a polypeptide of interest in recombinant host cells while also reducing biomass formation.

[0272] The reduction in RNAP activity and / or the reduction in transcription and / or the reduction in translation of one or more RNA polymerase subunits can be achieved, for example, by: a) operably linking a second polynucleotide encoding an RNAP subunit to a second heterologous promoter that is weaker than the native promoter of the gene encoding the RNAP subunit, e.g., by replacing the native promoter with a weaker heterologous promoter; b) operably linking said second polynucleotide to a mutant Shine-Dalgarno sequence that comprises one or more nucleic acid modifications compared to the native SD sequence of the gene encoding the RNAP subunit, wherein the mutated SD sequence results in weaker ribosome binding of the RNA during translation of the RNAP subunit; c) targeting the coding sequence of an RNAP subunit gene or its transcript during transcription or translation, respectively, using CRISPRi, RNAi, or other interference techniques known to those skilled in the art; and / or d) Deleting or mutating one or more RNAP subunit genes This can be achieved by:

[0273] Mutant cells can be constructed by reducing or eliminating expression of the second polynucleotide using methods well known in the art, such as one or more nucleotide insertions, one or more gene disruptions, one or more nucleotide replacements, or one or more nucleotide deletions.

[0274] The second polynucleotide to be modified or inactivated can be, for example, a coding region or portion thereof essential for activity, or a regulatory or control element required for expression of the coding region, e.g., a functional portion of a promoter sequence and / or a regulatory or control element required for transcription or translation of the polynucleotide. Other control sequences for possible modification include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, signal peptide sequences, transcription terminators, and transcriptional activators.

[0275] The modification or inactivation of the second polynucleotide can be carried out by subjecting parent cells to mutagenesis and selecting for mutant cells in which expression of the second polynucleotide is reduced or eliminated. Mutagenesis can be directed or random, for example, by using suitable physical or chemical mutagens, by using suitable oligonucleotides, or by subjecting the DNA sequence to PCR-generated mutagenesis. Furthermore, mutagenesis can be carried out by using any combination of these mutagenizing agents.

[0276] Examples of physical or chemical mutagens include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs (see J.L.Bose, Springer Protocols 2016, Methods in Molecular Biology, The Genetic Manipulation of Staphylococci).

[0277] Additionally or alternatively, nucleotides can be inserted or removed to introduce a stop codon, remove the start codon, or change the open reading frame. Such modification or inactivation can be achieved by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art, or by targeted gene editing using one or more nucleases, such as zinc finger nucleases or CRISPR-associated nucleases. Additionally or alternatively, modification or inactivation can be achieved by gene silencing, gene suppression, gene activation, and / or post-translational mutagenesis, for example, by methods using 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), for example, as disclosed in WO 18009520. The modification may be transient and / or reversible, irreversible and / or stable, and the modification may depend on a chemical inducer or on culture conditions such as culture temperature.

[0278] The modification can be performed in vivo, ie, directly in the cell that expresses the second polynucleotide, or the modification can be performed in vitro.

[0279] An example of a convenient way to modify expression of a second polynucleotide is provided in Example 1.

[0280] Another convenient method for reducing or eliminating RNAP activity in cells is the use of RNA polymerase-directed antimycobacterial agents (antibiotics), such as rifampicin. This is particularly useful when the target polypeptide is translated using a T7 RNA polymerase-dependent expression system. The use of an antibiotic reduces or eliminates the native RNAP activity of the host cell without interfering with the expression of the target polypeptide. Suitable antibiotic concentrations will be known to those skilled in the art. Non-limiting examples of antibiotic concentrations in culture are 0.01 to 5.0 ng / μl, e.g., about 0.2 ng / μl. The concentration is selected so that i) it effectively inactivates and eliminates native RNAP activity and ii) it does not significantly interfere with cell viability. A too high concentration will significantly interfere with cell viability, while a too low concentration will not efficiently reduce native RNAP activity.

[0281] Further examples of convenient methods for eliminating or reducing the expression of a polynucleotide are based on gene replacement, gene deletion, or gene disruption techniques. For example, in gene disruption methods, a nucleic acid sequence corresponding to an endogenous polynucleotide is mutagenized in vitro to generate a deleted nucleic acid sequence, which is then transformed into a parent cell to generate a deleted gene. The deleted nucleic acid sequence replaces the endogenous polynucleotide by homologous recombination. It may be desirable for the deleted polynucleotide to also encode a marker that can be used to select transformants in which the polynucleotide has been modified or destroyed. In one aspect, the polynucleotide is disrupted with a selectable marker, such as those described herein.

[0282] The present invention further relates to a mutant cell of a parent cell that includes a disruption or deletion of a second polynucleotide encoding an RNAP subunit polypeptide or its regulatory sequence or silenced gene, resulting in the mutant cell producing less RNAP subunit polypeptide or no RNAP subunit polypeptide at all compared to the parent cell.

[0283] Mutant cells with reduced RNAP subunit polypeptide levels are useful as host cells for the expression of native and heterologous polypeptides of interest, as the mutants have at least two advantages: i) increased yield of the polypeptide of interest, and ii) reduced biomass formation.

[0284] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0285] KK AEB1517: This is a B. subtilis donor strain for B. licheniformis conjugation as previously described (see U.S. Pat. Nos. 5,695,976, 5,733,753, 5,843,720, 5,882,888, and WO 2006042548). This strain contains pLS20 and the methylase gene M. bli1 904II (U.S. Pat. No. 20130177942) expressed from a triple promoter at the amyE locus, and orf beta from pBC16 and the B. subtilis comS gene (and kanamycin resistance gene) expressed from a triple promoter at the alr locus (creating a strain requiring D-alanine).

[0286] PP3724: An AEB1517 derivative (see US 20190276855) in which a second gene cassette consisting of the comS gene expressed from a tripartite promoter was inserted into the pel locus (pectate lyase).

[0287] SJ1904: A derivative of B. licheniformis Ca63, described in WO 2008 / 066931.

[0288] AN865: SJ1904 derivative carrying an amylase gene under a tripartite promoter.

[0289] JA4468: SJ1904 derivative carrying a protease gene under a tripartite promoter.

[0290] BN01: SJ1904 derivative carrying the protease gene under a tripartite promoter.

[0291] BN02: SJ1904 derivative carrying the protease gene under a tripartite promoter.

[0292] Halo-9: an SJ1904 derivative carrying a protease gene under a tripartite promoter.

[0293] Plasmid pMDT411: a derivative of pMDT454, see US Patent No. 2021021670

[0294] pMDT417: a derivative of pMDT452, see US Patent No. 2021021670

[0295] pEB-prsA: a derivative of pMDT411 carrying a prsA expression cassette driven by a tripartite promoter.

[0296] pTNA634: a derivative of pEB-prsA carrying a GFP expression cassette driven by the constitutive amyL promoter (PamyL4199) and the amyL RBS (ribosome binding sequence).

[0297] pTNA635: A derivative of pTNA634 with the wild-type rpoA RBS instead of the amyL RBS.

[0298] pTNA636: A derivative of pTNA634 with the rpoA RBS instead of the amyL RBS.

[0299] pTNA637: A derivative of pTNA634 that does not have an RBS in place of the amyL RBS.

[0300] Media and Solutions LB: 10 g / l tryptone, 5 g / l yeast extract, 5 g / l sodium chloride, adjusted to pH 7.0.

[0301] LB agar: LB containing 15g / l Bacto agar

[0302] TY: 20g / L tryptone, 5g / L yeast extract, 7mg / L FeCI2, 1mg / L MnCI2, 15mg / L MgCI2

[0303] TY agar: TY containing 15g / l Bacto agar

[0304] M-9 buffer solution: disodium hydrogen phosphate, 2H2O 8.8 g / L; potassium dihydrogen phosphate 3 g / L; sodium chloride 4 g / L; magnesium sulfate, 7H2O 0.2 g / L

[0305] PRK-50: 110 g / l soybean grits; disodium hydrogen phosphate, 2H2O 5 g / l; antifoaming agent (Struktol SB2121; Schill & Seilacher, Hamburg, Germany) 1 ml / l, pH adjusted to 8.0 with NaOH / H2PO4 before sterilization.

[0306] Constituent medium: Tryptone (casein hydrolysate from Difco), Bacto™ (tryptone, pancreatic digest of casein 211699) 30 g / l; magnesium sulfate, 7H2O 4 g / l; dipotassium hydrogen phosphate 7 g / l; disodium hydrogen phosphate, 2H2O 7 g / l; diammonium sulfate 4 g / l; citric acid 0.78 g / l; vitamins (thiamine dichloride 34.2 mg / l; riboflavin 2.9 mg / l; nicotinic acid 23 mg / l; D-calcium pantothenate 28.5 mg / l; pyridoxal-HCl 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, CuSO4, 5H2O 15.6 mg / l; ZnCl2 15.6 mg / l); antifoaming agent (Struktol SB2121; Schill & Seilacher, Hamburg, Germany) 1.25 ml / l; pH adjusted to 6.0 with NaOH / H2PO4 before sterilization.

[0307] Feed medium: glucose, 1H2O 820g / l

[0308] culture Bacillus strains were grown on LB or TY agar plates or in liquid medium. To select for erythromycin resistance, 5 μg / ml erythromycin was added to the agar and liquid medium. To select for tetracycline resistance, 15 μg / ml tetracycline was added to the agar and liquid medium. D-alanine was added to a final concentration of 0.1 mg / ml to the growth medium for strains carrying the alr gene disruption. Bacillus transformations were in Spizizen I medium, consisting of 1× 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 pH 7.0), 0.5% glucose, 0.1% yeast extract, and 0.02% casein hydrolysate.

[0309] Enzyme assay Amylase assay: Amylase activity was measured in the culture broth using a Pureauto S AMY-G7 (Sekisui Medical). The culture broth was first diluted in lysis buffer (0.03 M CaCl2; 0.0025% Brij L23; 6.67 M urea), and then dilution buffer (0.03 M CaCl2; 0.0025% Brij L23) was used for subsequent sample dilution. Enzyme activity measurements were performed using a Gallery Plus automated photometric analyzer. 16 μL of the diluted sample was mixed with 200 μL of Reagent 1 from the Pureauto kit, followed by the addition of 20 μL of Reagent 2. After 3 minutes of incubation at 37°C, absorbance was measured at 405 nm over 2 minutes. The final activity value was calculated by subtracting the amylase standard from the KNU(T) / g.

[0310] Protease assay: Serine endopeptidase hydrolyzes the substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide. The reaction was carried out at 37°C and pH 9.0. Release of pNA results in an increase in absorbance at 405 nm, which is proportional to the enzyme activity measured against a standard.

[0311] In vivo GFP assay: In vivo GFP expression levels were measured as follows. First, Bacillus strains carrying the GFP expression plasmid were grown overnight in LB liquid medium supplemented with 100 μg / ml D-Ala and 5 μg / ml erythromycin. 25 μL of the broth was transferred to a 96-well black plate and mixed with 75 μL of fresh LB medium for dilution. The diluted broth was subjected to GFP intensity measurement using a Synergy2 spectrophotometer (BioTek Instruments, Inc.) with Ex 485 nm / Em 528 nm.

[0312] molecular biological methods DNA manipulations and transformations were performed by standard molecular biology methods as described below: - 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.

[0313] Competent cells and transformation of B. subtilis were obtained as described by Yasbin et al. (1975, Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of propagation in competent cells. J. Bacteriol. 121, 296-304). Conjugation of B. licheniformis was carried out essentially as described in WO 1996 / 029418.

[0314] Genomic DNA was prepared using a commercially available QIAamp DNA Blood Kit (Qiagen). Each DNA fragment was amplified by PCR using the PrimeStar GXL DNA Polymerase System (TaKaRa). The PCR amplification reaction mixture contained 1 μL of template DNA, 2 μL of sense primer (20 pmol / μL), 2 μL of antisense primer (20 pmol / μL), 10 μL of 5× PCR buffer, 4 μL of dNTP mix, 30 μL of water, and 1 μL of DNA polymerase. The fragments were amplified using a thermocycler. The PCR products were purified from a 1.0% agarose gel containing 1× TAE buffer using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions.

[0315] The POE-PCR conditions were as follows: the purified PCR product was used in a subsequent PCR reaction to generate a single fragment using splice overlap extension PCR (SOE) with the PrimeStar GXL DNA polymerase system (TaKaRa) as follows: the extreme ends of the 5'-end fragment and the extreme 3'-end fragment have complementary ends, which allow the SOE to be concatenated into the POE PCR product. The PCR amplification reaction mixture contained 50 ng of each gel-purified PCR product. POE PCR was performed as described in You, C et al. (2017) Methods Mol. Biol. 116, 183-92.

[0316] Fed-batch fermentation procedure using laboratory tank fermenters 1. Inoculation Step a) Grow strains on LB agar plates overnight at 37°C. b) Wash the agar with M-9 buffer and collect the cell suspension. Measure the OD650 by photometer. c) Inoculate a PRK-50 shake flask (OD650 x cell suspension (ml) = 1). d) Incubate the shake flasks at 220 rpm at 37° C. overnight. e) The main fermentation is initiated by adding the growing shake flask culture (10% of the constituent medium, i.e., 80 ml to 800 ml).

[0317] 2. Fermentation equipment A standard laboratory fermentor equipped with a temperature control system, pH control using aqueous ammonia and phosphoric acid, and a dissolved oxygen electrode to measure >20% oxygen saturation throughout the fermentation.

[0318] 3.Fermentation parameters: Temperature: 37℃: Aqueous ammonia and phosphoric acid are used to maintain a pH of 6.8 to 7.2. Aeration: 1.5L / min / kg (broth weight) Agitation: 1500 rpm.

[0319] Example 1. Construction of plasmid DNA for introduction of mutations in the rpoA gene. The purpose of this experiment was to prepare plasmid DNA for introducing a single nucleotide mutation into the native rpoA gene of a B. licheniformis strain to alter its ribosome binding sequence (SD, Shine-Dalgarno sequence). From now on, this mutation will be referred to as the rpoASD mutation.

[0320] The plasmid DNA pMDT411 contains an expression cassette for a single guide RNA (sgRNA) to recruit Mad7 nuclease to the sgRNA-complementary region on the genome. A protospacer was designed and cloned into pMDT411 to edit the ribosome-binding region of the rpoA gene (see Table 2).

[0321] [Table 5]

[0322] The oligo DNAs used for cloning are listed in Table 2. The protospacer and homologous regions carrying the desired rpoASD mutation were inserted into pMDT411 by PoE PCR. First, each PCR fragment was amplified and purified by gel extraction using a QIAquick Gel Extraction Kit (Qiagen). Table 3 shows the primer pairs used. The purified fragments were then combined by PoE PCR as described in the Methods section. The PoE PCR products were then directly used to transform the D-alanine auxotrophic B. subtilis host PP3724 (hereafter referred to as PP3724-pMDT411-rpoA). Transformants were plated on TY+erythromycin and D-alanine agar plates and incubated at 34°C 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 containing the expression cassette for Mad7 nuclease was transformed into the B. subtilis host PP3724 (hereafter referred to as PP3724-pMDT417). Transformants were plated onto TY+tetracycline and D-alanine agar plates and incubated at 34°C for 1-2 days.

[0323] [Table 6]

[0324] Example 2. Transformation of B. licheniformis strains AN865 and JA4468 for integration of rpoASD mutations The goal of this experiment was to generate the desired rpoA mutant in a B. licheniformis strain. First, to introduce the plasmid DNA, the B. subtilis donor strain, PP3724-pMDT411-rpoA, was conjugated with the B. licheniformis recipient strain, AN865 or JA4468. The conjugates were plated on TY+erythromycin agar plates and incubated at 34°C for 1–2 days. Correct conjugates in B. licheniformis were selected by erythromycin resistance and the absence of D-alanine auxotrophs. Next, the erythromycin-resistant B. licheniformis strain was conjugated with PP3724-pMDT417. The conjugates were plated on TY + erythromycin and tetracycline agar plates and incubated for 2-3 days at 34°C. Correct conjugates in B. licheniformis were selected by double resistance to erythromycin and tetracycline.

[0325] Colonies on the double-selection plates were then transferred to LB liquid medium containing tetracycline and erythromycin for overnight incubation at 34°C. To isolate single colonies from the liquid culture, the culture broth was serially diluted and plated onto TY + erythromycin and tetracycline agar plates and incubated at 34°C for 2–3 days. Single colonies were then screened for the presence of the desired rpoA mutation in the genome by genomic PCR and Sanger sequencing. We identified the desired rpoA mutants from strains AN865 and JA4468 (hereafter referred to as AN865-rpoA-5 and JA4468-rpoA-3-3, respectively). The plasmid DNA used for genome editing was removed by culturing these strains in LB liquid medium overnight at 50°C. Finally, erythromycin- and tetracycline-dual-sensitive clones were selected and stored in glycerol.

[0326] Example 3. Increased amylase productivity after introduction of the rpoASD mutation The purpose of this experiment was to test whether the introduced rpoA mutation affected the amylase productivity of B. licheniformis strain AN865 in shake flasks (SF). First, 100 μl of frozen stocks of AN865 (wild-type rpoA) and AN865-rpoA-5 (mutant rpoA) were added to 100 ml of PRK-50 medium in 500 ml SF. The SF was incubated overnight at 37°C using an SF shaker at 220 rpm. Next, 10 ml of the culture broth was inoculated into 100 ml of 10R-av-30CG medium in 500 ml SF. The SF was incubated at 220 rpm at 37°C for 3 days. Finally, amylase activity in the culture broth was measured as described in the Enzyme Assay section. The relative amylase activity is shown in Table 4.

[0327] [Table 7]

[0328] The above data showed that the rpoASD mutation significantly improved the productivity of amylase expression in B. licheniformis strain AN865 by 12%.

[0329] Example 4. Increased protease expression after introduction of rpoASD mutations The purpose of this experiment was to test whether the introduced rpoA mutations affected the protease productivity of B. licheniformis strain JA4468 in laboratory tank fermenters using the process described in the Methods section. Finally, protease activity in the culture broth was measured as described in the Enzyme Assays section. The relative protease activities are shown in Table 5.

[0330] [Table 8]

[0331] The above data showed that the rpoA SD mutation significantly improved the productivity of protease expression in B. licheniformis strain JA4468 by 12%. Together with Example 3, this demonstrates that the rpoASD mutation has a positive impact on the recombinant production of various protein products in B. licheniformis strains.

[0332] Example 5. Reduced biomass formation after introduction of rpoASD mutations The purpose of this experiment was to confirm that the rpoA SD mutation affected not only enzyme productivity but also cell growth during fermentation. The amylase strains AN865 and AN865-rpoA-5 and the protease strains JA4468 and JA4468-rpoA-3-3 were cultured in 100 ml of LB liquid medium in a 500 ml SF tank. The cultures were grown overnight at 37°C at 220 rpm. After 24 hours of fermentation, 5 μl of the culture broth was mixed with 195 μl of deionized water for dilution, and the OD650 values ​​were then measured using a photometer. The results are summarized in Table 6.

[0333] [Table 9]

[0334] The data above showed that the rpoASD mutation significantly reduced cell density at the end of fermentation (18% reduction). A similar effect was observed in the fermentations performed in Examples 3 and 4. Reducing biomass after fermentation is a highly beneficial feature for bioproduction, as it reduces product purification and formulation costs.

[0335] Example 6: rpoASD mutations result in increased rifampicin susceptibility. The purpose of this experiment was to investigate the effect of the rpoASD mutation at the molecular level. Because the Shine-Dalgarno (SD) region functions as a ribosome-binding sequence, the rpoASD mutation can alter the translation level of the RpoA protein within the cell. Bacterial RNA polymerase is composed of two alpha (encoded by the rpoA gene), beta, beta prime, and omega subunits. The RNA polymerase complex is inhibited by the antibiotic rifampicin (see E.A. Campbell et al., "Structural mechanism for rifampicin inhibition of bacterial RNA polymerase," Cell, 2001). We hypothesized that the rpoA SD mutant would be more sensitive to rifampicin if the cellular RpoA protein levels in the rpoA SD mutant were lower than those in the wild-type strain.

[0336] To test this hypothesis, the protease strains listed in Table 7 were plated on agar plates containing rifampicin (Rif) to confirm the susceptibility of each strain to Rif. Strain JA4468 showed a decrease in viability when increasing the amount of Rif applied in TY-based agar plates. However, strain BN02, which has a known rpoB (encoding the β subunit) mutation (A478D) that confers Rif resistance, showed no decrease in viability, demonstrating the validity of this Rif susceptibility assay. Next, protease strains with and without the rpoA SD mutation (halo-9 and BN01, respectively) were analyzed. As shown in Table 7, halo-9, which has the rpoA SD mutation, showed lower viability than BN01, indicating that halo-9 is more susceptible to Rif. Therefore, we conclude that the identified rpoASD mutations reduce RNAP activity in host cells, for example, by reducing rpoA expression.

[0337] [Table 10]

[0338] Example 7: Construction of plasmid DNA for GFP expression linked to various SD sequences The purpose of this experiment was to confirm that the SD mutations cause a reduction in RpoA expression and, therefore, a reduction in RNAP activity. To do this, we prepared plasmid DNA to examine the effect of various SD sequences on GFP expression. As shown in Example 6, the rpoA SD mutations should reduce rpoA protein expression levels. To further examine this in a quantitative manner, we generated a series of GFP-expressing plasmid DNAs.

[0339] Plasmid DNA pEB-prsA, a derivative of pMDT411, contains an overexpression cassette of prsA driven by a tripartite promoter with multiple RBSs in the form of SD sequences. To create a simple expression cassette to compare the effects of SD sequences, a single amyL promoter (PamyL4199) was chosen instead of the tripartite promoter. PamyL4199 and various RBSs with GFP CD (including amyL SD, wild-type rpoA with wild-type SD, wild-type rpoA with mutated SD, or no SD) were then cloned into the pEB-prsA backbone by PoE PCR, yielding pTNA634–637, respectively. These constructs were transformed into the B. subtilis host PP3724 as described in Example 1. Oligonucleotides used for cloning are listed in Table 8. The SD sequences used in this study are summarized in Table 9 and annotated as ribosome binding sites (RBSs).

[0340] [Table 11]

[0341] [Table 12]

[0342] Example 8: rpoASD mutations result in reduced GFP expression. The purpose of this experiment was to compare in vivo GFP expression levels between wild-type and mutant rpoA SD sequences. Single colonies of PP3724 transformants of pTNA634-637 were cultured in LB liquid medium supplemented with 100 μg / ml d-Ala and 5 μg / ml erythromycin. After overnight incubation at 32°C, the culture broth was harvested and the OD650 and GFP intensity were measured as described in the assay section. The results are summarized in Table 10.

[0343] [Table 13]

[0344] As shown in Table 10, the transformants of pTNA634-637 showed various levels of GFP expression, while the negative control transformant (pEB-prsA) showed no GFP expression. (a) Because GFP intensity should be affected by the amount of cells in the broth, GFP intensity was normalized by OD650 (GFP / OD650 (b) , hereafter referred to as normalized GFP expression). The relative values ​​of normalized GFP expression were calculated using pTNA635 as a reference (see the rightmost column in Table 10). pTNA635 encodes a GFP cassette with a wild-type rpoA SD. As can be seen from Table 10, pTNA636 (mutant rpoA SD) resulted in an 85% reduction in GFP expression compared to that of pTNA635. This is in good agreement with the Rif sensitivity experiments seen in Example 6. In conclusion, these Examples show that the rpoA SD mutation resulted in an approximately 80-90% reduction in rpoA expression. Furthermore, we demonstrated that the rpoA SD mutation resulted in increased recombinant protein yields (Examples 3-4) and reduced biomass formation (Example 5).

[0345] Therefore, reduced rpoA expression directly correlates with the observed reduction in biomass and increased product yield.

[0346] The present invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to be illustrative of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of the 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.

[0347] The invention is further defined by the following numbered paragraphs:

[0348] 1. A mutant cell comprising within its genome a first heterologous promoter operably linked to a first polynucleotide encoding a polypeptide of interest, and one or more second polynucleotides encoding one or more RNA polymerase (Rpo) subunit polypeptides, wherein expression of the one or more Rpo subunit polypeptides is reduced or eliminated compared to a non-mutated but otherwise isogenic or parent cell.

[0349] 2. a) a second polynucleotide is operably linked to a second heterologous promoter; b) a second polynucleotide is operably linked to a variant Shine-Dalgarno sequence derived from the parent Shine-Dalgarno sequence; c) the second polynucleotide comprises one or more nucleic acid insertions, deletions, or substitutions; d) expression of the second polynucleotide is reduced by a CRISPR inhibitor construct; and / or e) the expression of the second polynucleotide is reduced by RNA interference; 2. The mutant cell of paragraph 1.

[0350] 3. The cell of any one of paragraphs 1-2, wherein the expression of one or more Rpo subunit polypeptides is reduced by at least 10%, e.g., 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 the expression of one or more Rpo subunit polypeptides in the parent cell when cultured under the same conditions.

[0351] 4. The cell of any one of paragraphs 1 to 3, wherein the second polynucleotide is native to the cell.

[0352] 5. The cell of any of paragraphs 1 to 4, wherein the second heterologous promoter is heterologous to the second polynucleotide.

[0353] 6. The cell of any of paragraphs 1-5, wherein the second heterologous promoter results in reduced 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 cultured under identical conditions.

[0354] 7. The cell of any of paragraphs 1 to 6, wherein the parent Shine-Dalgarno sequence has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polynucleotide sequence of AAGGAGG or with SEQ ID NO: 58.

[0355] 8. The cell of any one of paragraphs 1 to 7, wherein the cell comprises at least two second polynucleotides, e.g., at least three, or at least four second polynucleotides, each second polynucleotide encoding an RNA polymerase subunit polypeptide.

[0356] 9. The cell of any one of paragraphs 1 to 8, wherein the one or more RNA polymerase subunit polypeptides are one or more bacterial RNA polymerase subunit polypeptides selected from the list of subunit beta (β), subunit alpha (α), and subunit omega (ω).

[0357] 10. The cell of any one of paragraphs 1 to 9, wherein the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α) RpoA.

[0358] 11. The cell of any one of paragraphs 1 to 10, wherein the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β) RpoB.

[0359] 12. The cell of any one of paragraphs 1 to 11, wherein the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits beta' (β') RpoB'.

[0360] 13. The cell of any one of paragraphs 1 to 12, wherein the one or more second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω)RpoZ.

[0361] 14. The cell of any one of paragraphs 1 to 13, wherein the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α)RpoA, and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β)RpoB and / or (β')RpoB'.

[0362] 15. The cell of any one of paragraphs 1 to 14, wherein the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α) RpoA and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω) RpoZ.

[0363] 16. The cell of any one of paragraphs 1 to 15, wherein the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β) RpoB, and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω) RpoZ.

[0364] 17. The cell of any one of paragraphs 1 to 16, wherein the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits beta' (β') RpoB', and the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω) RpoZ.

[0365] 18. The cell of any one of paragraphs 1 to 17, wherein the one or more first second polynucleotides encode one or more bacterial RNA polymerase subunits alpha (α)RpoA, the one or more second second polynucleotides encode one or more bacterial RNA polymerase subunits beta (β)RpoB and / or (β')RpoB', and the one or more third second polynucleotides encode one or more bacterial RNA polymerase subunits omega (ω)RpoZ.

[0366] 19. The cell of any one of paragraphs 1 to 18, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoA polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0367] 20. The cell of any one of paragraphs 1 to 19, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, comprises or consists of a nucleic acid sequence that encodes an RpoA polypeptide and has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1.

[0368] 21. The cell of any one of paragraphs 1 to 20, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoB polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0369] 22. The cell of any one of paragraphs 1 to 21, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, comprises or consists of a nucleic acid sequence that encodes an RpoB polypeptide and has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 10.

[0370] 23. The cell of any one of paragraphs 1 to 22, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoB' polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0371] 24. The cell of any one of paragraphs 1 to 23, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, comprises or consists of a nucleic acid sequence encoding an RpoB' polypeptide and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 39.

[0372] 25. The cell of any one of paragraphs 1 to 24, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, encodes an RpoZ polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0373] 26. The cell of any one of paragraphs 1 to 25, wherein the second polynucleotide, e.g., the first, second, or third second polynucleotide, comprises or consists of a nucleic acid sequence that encodes an RpoZ polypeptide and has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0374] 27. The cell of any one of paragraphs 1 to 26, wherein the second polynucleotide is heterologous to the cell.

[0375] 28. The cell of any one of paragraphs 1 to 27, wherein the first polynucleotide is operably linked to one or more first promoters that direct the production of the polypeptide of interest, preferably the first promoters being heterologous to the first polynucleotide.

[0376] 29. The cell of any one of paragraphs 1 to 28, wherein the first heterologous promoter comprises or consists of a nucleic acid sequence having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 38.

[0377] 30. The cell of any one of paragraphs 1 to 29, wherein the cell comprises at least two copies of the first polynucleotide in its genome, such as at least three, at least four, or at least five, or at least six, or more copies.

[0378] 31. The cell of any one of paragraphs 1 to 30, wherein the one or more RNA polymerase subunit polypeptides are one or more archaeal RNA polymerase subunit polypeptides selected from the list of Rpo1, Rpo2, Rpo3, Rpo11, Rpo4, Rpo5, Rpo6, Rpo8, Rpo10, Rpo12, Rpo7, or Rpo13.

[0379] 32. The cell of any one of paragraphs 1 to 31, wherein the one or more RNA polymerase subunit polypeptides are eukaryotic RNA polymerase I, RNA polymerase II, and / or RNA polymerase III subunit polypeptides.

[0380] 33. The cell of any one of paragraphs 1 to 32, wherein the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase I subunit polypeptides selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

[0381] 34. The cell of any one of paragraphs 1 to 33, wherein the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase subunit polypeptides selected from the list of RPAC40 (AC40), RPAC19 (AC19), RPO3, RPO11, RPB3, and RPB11.

[0382] 35. The cell of any one of paragraphs 1 to 34, wherein the second polynucleotide comprises or consists of a nucleic acid sequence encoding a RPAC40 (AC40) polypeptide and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0383] 36. The cell of any one of paragraphs 1 to 35, wherein the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase I subunit polypeptides selected from the list of RPA190, RPBA135, RPAC40 (AC40), RPAC19 (AC19), RPB6, RPB5, RPB8, RPB10, RPB12, RPA14, RPA43, RPA12, RPA49, and RPA34.5.

[0384] 37. The cell of any one of paragraphs 1 to 36, wherein the second polynucleotide encodes a RPAC40 (AC40) polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0385] 38. The cell of any one of paragraphs 1 to 37, wherein the second polynucleotide comprises or consists of a nucleic acid sequence encoding a RPAC19 (AC19) polypeptide and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

[0386] 39. The cell of any one of paragraphs 1 to 38, wherein the second polynucleotide encodes a RPAC19 (AC19) polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.

[0387] 40. The cell of any one of paragraphs 1 to 39, wherein the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase II subunit polypeptides selected from the list of RPB1, RPB2, RPB3, RPB11, RPB6, RPB5, RPB8, RPB10, RPB12, RPB4, RPB7, RPB9, TFIIFα, and TFIIFβ.

[0388] 41. The cell of any one of paragraphs 1 to 40, wherein the second polynucleotide comprises or consists of a nucleic acid sequence encoding an RPB3 polypeptide and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.

[0389] 42. The cell of any one of paragraphs 1 to 41, wherein the second polynucleotide encodes an RPB3 polypeptide comprising or consisting of an amino acid sequence having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31.

[0390] 43. The cell described in any one of paragraphs 1 to 42, wherein the second polynucleotide comprises or consists of a nucleic acid sequence encoding an RPB11 polypeptide and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.

[0391] 44. The cell of any one of paragraphs 1 to 43, wherein the second polynucleotide encodes an RPB11 polypeptide comprising or consisting of an amino acid sequence having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.

[0392] 45. The cell of any one of paragraphs 1 to 44, wherein the one or more RNA polymerase subunit polypeptides are one or more eukaryotic RNA polymerase III subunit polypeptides 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.

[0393] 46. ​​The cell of any one of paragraphs 1 to 45, wherein the one or more RNA polymerase subunit polypeptides are one or more yeast RNA polymerase subunit polypeptides selected from the list of Rpb5 (ABC27), Rpb6 (ABC23, or Rpo26), Rpb8 (ABC14.5), Rpb10 (ABC10β), and Rpb12 (ABC10α).

[0394] 47. The cell of any one of paragraphs 1 to 46, wherein one or more RNA polymerase subunit polypeptides comprise an N-terminal and / or C-terminal extension of 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably an extension of 1 to 6 amino acid residues at the N-terminus and / or 1 to 6 amino acids, e.g., 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 amino acids, at the C-terminus, and wherein the extended polypeptides have RNA polymerase activity.

[0395] 48. The cell of any one of paragraphs 1 to 47, wherein the cell is a eukaryotic cell.

[0396] 49. The cell of any one of paragraphs 1 to 48, wherein the cell is a mammalian cell.

[0397] 50. The cell of any one of paragraphs 1 to 49, wherein the cell is a prokaryotic cell.

[0398] 51. Yeast recombinant host cells, for example, Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, for example, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, 51. The cell of any one of paragraphs 1 to 50, which is a Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0399] 52. Filamentous fungal recombinant host cells, such as Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neochalimus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma cells, in particular Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis kallegeacaregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola 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 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 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 52. The cell of any one of paragraphs 1 to 51, which is a Trichoderma reesei or Trichoderma viride cell.

[0400] 53. The cell of any one of paragraphs 1 to 52, which is an Aspergillus cell.

[0401] 54. The cell of any one of paragraphs 1 to 53, which is an Aspergillus niger cell.

[0402] 55. The cell of any one of paragraphs 1 to 54, which is an Aspergillus oryzae cell.

[0403] 56. The cell of any one of paragraphs 1 to 55, which is a Trichoderma cell.

[0404] 57. The cell of any one of paragraphs 1 to 56, which is a Trichoderma reesei cell.

[0405] 58. Prokaryotic recombinant host cells, for example, Gram-positive cells selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or Campylobacter, Escherichia coli (E.Gram-negative bacteria selected from the group consisting of Bacillus coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, 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.58. The cell of any one of paragraphs 1 to 57, which is a Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cell.

[0406] 59. The cell of any one of paragraphs 1 to 58, which is a Bacillus cell.

[0407] 60. The cell of any one of paragraphs 1 to 59, which is a Bacillus licheniformis cell.

[0408] 61. The cell of any one of paragraphs 1 to 60, which is a Bacillus subtilis cell.

[0409] 62. The cell of any one of paragraphs 1 to 61, which is isolated.

[0410] 63. The cell of any one of paragraphs 1 to 62, which is purified.

[0411] 64. The cell of any one of paragraphs 1 to 63, wherein the second heterologous promoter operably linked to the second polynucleotide results in reduced transcription of the second polynucleotide compared to transcription of the second polynucleotide when operably linked to its native or endogenous promoter.

[0412] 65. The cell of any one of paragraphs 1 to 64, wherein the mutant Shine-Dalgarno sequence operably linked to the second polynucleotide results in reduced transcription of the second polynucleotide compared to transcription of the second polynucleotide when operably linked to its native or endogenous Shine-Dalgarno sequence.

[0413] 66. The polypeptide of interest comprises an enzyme; preferably, the enzyme is a 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-gluc ... 66. The cell of any one of paragraphs 1 to 65, wherein the one or more polypeptides of interest are selected from the group consisting of: glucosidases, beta-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, nucleases, oxidases, pectinolytic enzymes, peroxidases, phosphodiesterases, phytases, polyphenol oxidases, proteases, ribonucleases, transglutaminases, xylanases, and beta-xylosidases; even more preferably, the one or more polypeptides of interest comprise an amylase or a protease.

[0414] 67. The cell of any one of paragraphs 1 to 66, wherein the polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of an antibody, an antibody fragment, an antibody-based drug, an 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 interferon alpha-2b), an interleukin, lactoferrin, alpha-lactalbumin, beta-lactalbumin, ovomucoid, ovostatin, a cytokine, obestatin, a human galactosidase (such as human alpha-galactosidase A), a vaccine, a protein vaccine, and a thrombolytic drug.

[0415] 68. The cell according to any one of paragraphs 1 to 67, wherein the polypeptide of interest comprises a nanobody (Nb), preferably wherein the nanobody consists of a single variable light chain (VL).

[0416] 69. The cell of any one of paragraphs 1 to 68, wherein the first polynucleotide encodes a polypeptide having amylase activity and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0417] 70. The cell of any one of paragraphs 1 to 69, wherein the polypeptide of interest is an amylase, such as an amylase comprising or consisting of a mature polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0418] 71. The cell of any one of paragraphs 1 to 70, wherein the first polynucleotide encodes a polypeptide having protease activity and comprises or consists of a nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0419] 72. The cell of any one of paragraphs 1 to 71, wherein the polypeptide of interest is a protease, such as a protease comprising or consisting of a mature polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0420] 73. The variant Shine-Dalgarno sequence is at one or more of the following nucleotide positions: - corresponding to positions 1 to 7 of the nucleic acid sequence "AAGGAGG" or positions 1 to 7 of the nucleic acid sequence of SEQ ID NO: 3, -corresponding to positions 1 to 8 of the nucleic acid sequence "GAGGGGTG", corresponding to positions 1 to 7 of the nucleic acid sequence "AAGGGAG", or -corresponding to positions 1 to 8 of the nucleic acid sequence "GGAGGTTG" 73. The cell of any one of paragraphs 1 to 72, comprising at least one nucleic acid substitution, insertion, and / or deletion.

[0421] 74. The cell of any one of paragraphs 1 to 73, wherein the mutant 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 having the nucleic acid sequence "AAGGAGG" or in the nucleic acid sequence at positions 1 to 7 of SEQ ID NO: 3.

[0422] 75. The cell of any one of paragraphs 1 to 74, wherein the mutant Shine-Dalgarno sequence comprises a nucleic acid substitution with adenine (A) (G3A), with cytosine (C) (G3C), or with thymine (T) (G3T) at the position corresponding to position 3 of the nucleic acid sequence "AAGGAGG."

[0423] 76. The cell of any one of paragraphs 1 to 75, wherein the mutant Shine-Dalgarno sequence comprises a nucleic acid substitution at the position corresponding to position 3 of the parent SD sequence having the nucleic acid sequence "AAGGAGG" with an adenine (A) (G3A).

[0424] 77. A cell according to any one of paragraphs 1 to 76, wherein the variant Shine-Dalgarno sequence comprises or consists of a nucleic acid sequence having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence "AAAGAGG" or to the nucleic acid sequence at positions 1 to 7 of SEQ ID NO: 4.

[0425] 78. A cell according to any one of paragraphs 1 to 77, wherein the mutant Shine-Dalgarno sequence comprises or consists of the nucleic acid sequence "AAAGAGG" or the nucleic acid sequence at positions 1 to 7 of SEQ ID NO: 4.

[0426] 79. The cell of any one of paragraphs 1 to 78, wherein the second polynucleotide is operably linked to a variant Shine-Dalgarno sequence forming a coding nucleic acid sequence comprising or consisting of a coding nucleic acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence "AAGGAGG" or "AAAGAGG", or to the nucleic acid sequence at positions 1 to 7 of SEQ ID NO: 3 or 4, or to the nucleic acid sequence "GAGGGGTG", "AAGGGAG", or "GGAGGTTG".

[0427] 80. The cell of any one of paragraphs 1 to 79, wherein the mutant Shine-Dalgarno sequence comprises a nucleic acid substitution at the position corresponding to position 5 of SEQ ID NO: 58 with adenine (A) (G5A), with cytosine (C) (G5C), or with thymine (T) (G5T).

[0428] 81. A cell described in any one of paragraphs 1 to 80, wherein transcription and / or translation of the second polynucleotide is reduced compared to a parent cell that is otherwise isogenic to the mutant cell when cultured under identical conditions, the parent cell not comprising either a) a heterologous promoter operably linked to the second polynucleotide or b) a mutant Shine-Dalgarno sequence operably linked to the second polynucleotide.

[0429] 82. The transcription and / or translation of the second polynucleotide (RNAP subunit) is 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%, or 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 1 8%, 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%, 82. The cell of any one of paragraphs 1-81, wherein the cell is reduced by 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%.

[0430] 83. The cell of any one of paragraphs 1 to 82, wherein transcription and / or translation of the second polynucleotide (RNAP subunit) is reduced relative to transcription and / or translation in the parent cell after at least 24 hours of culture, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of culture.

[0431] 84. The cell of any one of paragraphs 1 to 83, wherein the yield of the polypeptide of interest is increased compared to the parent cell when cultured under the same conditions.

[0432] 85. The cell of any one of paragraphs 1 to 84, wherein the yield of the polypeptide of interest is increased by at least 1%, such as 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 by at least 12% relative to the yield of the parent cell.

[0433] 86. The cell of any one of paragraphs 1 to 85, wherein the yield of the polypeptide of interest is increased relative to the yield of the parent cell after at least 24 hours of culture, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of culture.

[0434] 87. The cell of any one of paragraphs 1 to 86, wherein during culturing the cell the biomass is reduced relative to the biomass during culturing of the parent cell when cultured under the same conditions.

[0435] 88. The cell of any one of paragraphs 1 to 87, wherein the biomass is reduced by at least 1%, such as 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 by at least 18% relative to the biomass of the parent cell.

[0436] 89. The cell of any one of paragraphs 1 to 88, wherein the biomass is reduced relative to the biomass of the parent cell after at least 24 hours of culture, for example, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of culture.

[0437] 90. The cell according to any one of paragraphs 1 to 89, wherein the culture is a fed-batch, batch, or continuous culture process, preferably a fed-batch culture process.

[0438] 91. A method for producing one or more polypeptides of interest, comprising: a) providing a cell according to any one of paragraphs 1 to 90; b) culturing the cells under conditions conducive to expression of one or more polypeptides of interest; c) optionally recovering one or more polypeptides of interest; A method comprising:

[0439] 92. A nucleic acid construct comprising a second heterologous promoter and / or a mutant Shine-Dalgarno sequence operably linked to a second polynucleotide of any preceding embodiment.

[0440] 93. The nucleic acid construct of any of paragraph 92, which is isolated.

[0441] 94. The nucleic acid construct of any of paragraphs 920-93, which is purified.

[0442] 95. An expression vector comprising a nucleic acid construct according to paragraphs 92 to 94.

Claims

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

2. a) the second polynucleotide is operably linked to a second heterologous promoter; b) the second polynucleotide is operably linked to a variant Shine-Dalgarno sequence derived from a parent Shine-Dalgarno sequence; c) the second polynucleotide comprises one or more nucleic acid insertions, deletions, or substitutions; d) expression of said second polynucleotide is reduced by a CRISPR inhibition construct, and / or e) the expression of said second polynucleotide is reduced by RNA interference; The mutant cell of claim 1.

3. 3. The mutant cell of any one of claims 1 to 2, wherein the parent Shine-Dalgarno sequence has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polynucleotide sequence of AAGGAGG or of SEQ ID NO:

58.

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

5. 5. The mutant cell of any one of claims 1 to 4, wherein the second polynucleotide encodes an Rpo subunit polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ 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. 6. The mutant cell of any one of claims 1 to 5, wherein the cell comprises at least two copies, such as three, four, or five, or six or more copies of the first polynucleotide in its genome.

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

8. 8. The mutant cell of any one of claims 1 to 7, wherein the mutant Shine-Dalgarno sequence comprises a nucleic acid substitution with adenine (A) (G3A), with cytosine (C) (G3C), or with thymine (T) (G3T) at a position corresponding to position 3 of the parent Shine-Dalgarno sequence having the nucleic acid sequence "AAGGAGG", or with adenine (A) (G5A), with cytosine (C) (G5C), or with thymine (T) (G5T) at a position corresponding to position 5 of SEQ ID NO:

58.

9. The mutant cell of any one of claims 1 to 8, wherein the expression of the one or more Rpo subunit polypeptides is reduced by at least 10%, such as 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 the expression of the one or more Rpo subunit polypeptides in a parent cell when cultured under the same conditions.

10. The mutant cell according to any one of claims 1 to 9, wherein the yield of the target polypeptide is increased compared to the parent cell when cultured under the same conditions.

11. 11. The mutant cell of any one of claims 1 to 10, wherein biomass formation is reduced during culture of the mutant cell relative to biomass formation during culture of the parent cell when cultured under identical conditions.

12. The polypeptide of interest comprises an enzyme; preferably, the enzyme is a 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, or alpha-glucosidase.

12. The mutant cell of claim 1, wherein the one or more polypeptides of interest are selected from the group consisting of: sidases, beta-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, nucleases, oxidases, pectinolytic enzymes, peroxidases, phosphodiesterases, phytases, polyphenol oxidases, proteases, ribonucleases, transglutaminases, xylanases, and beta-xylosidases; even more preferably, the one or more polypeptides of interest comprise amylases or proteases.

13. 13. The mutant cell of any one of claims 1 to 12, wherein the polypeptide of interest is an amylase, such as an amylase comprising or consisting of a mature polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

7.

14. 13. The mutant cell of any one of claims 1 to 12, wherein the polypeptide of interest is a protease, such as a protease comprising or consisting of a mature polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

9.

15. 1. A method for producing one or more polypeptides of interest, comprising: a) providing a mutant cell according to any one of claims 1 to 14; b) culturing the cells under conditions conducive to expression of the one or more polypeptides of interest; c) optionally recovering said one or more polypeptides of interest; A method comprising:

Images