EP4355762A1 - Improved bacillus host cell - Google Patents

Improved bacillus host cell

Info

Publication number
EP4355762A1
EP4355762A1 EP22733942.1A EP22733942A EP4355762A1 EP 4355762 A1 EP4355762 A1 EP 4355762A1 EP 22733942 A EP22733942 A EP 22733942A EP 4355762 A1 EP4355762 A1 EP 4355762A1
Authority
EP
European Patent Office
Prior art keywords
host cell
polypeptide
interest
gene
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22733942.1A
Other languages
German (de)
French (fr)
Inventor
Leendert HAMOEN
Biwen Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Publication of EP4355762A1 publication Critical patent/EP4355762A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group

Definitions

  • the present invention relates to a recombinant Bacillus host cell, and a process for producing a polypeptide or a compound of interest wherein the Bacillus host cell is used.
  • Bacillus strains produce and secrete a large number of useful proteins and metabolites. Because of their GRAS (General Recognised As Safe) status, Bacillus hosts, e.g. belonging to the species Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, are largely used in the production of important proteins for the food, feed and pharmaceutical industry.
  • GRAS General Recognised As Safe
  • US7,585,674 discloses that the productivity of a protein or polypeptide of interest in host cells, e.g. Bacillus host cells can be improved by deleting from the genome of said host cells specific genes participating in sporulation stage II, III, IV or V.
  • WO2015/118126 discloses that when Bacillus strains have been made deficient in the production of a neutral protease and / or an alkaline protease, this results in an increased yield of of a protein or polypeptide of interest during fermentation.
  • CtsR transcription repressor is a regulator of stress and heat shock response and controls clp and molecular chaperone gene expression in Gram-positive bacteria.
  • transcriptional fusions were constructed between the clpP and clpC promotor regions and the bgaB gene of Bacillus stearothermophilus, which encodes a thermostable b-galactosidase.
  • the present invention relates to a recombinant Bacillus host cell comprising a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest, wherein the host cell is deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes.
  • CtsR transcription repressor
  • the inventions further relates to a method for preparing a Bacillus host cell as disclosed herein, comprising deleting, disrupting or mutating a polynucleotide encoding a transcription repressor of class III heat shock genes.
  • the invention relates to a process for producing a polypeptide of interest, or a compound of interest, comprising cultivating the Bacillus host cell according to the present invention under conditions conducive for the production of the polypeptide or compound of interest.
  • derived from also includes the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” and “created from,” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.
  • expresses/produces is meant to include any steps involved in the expression and production of a polypeptide of interest in a host cell as described herein including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • gene refers to a polynucleotide that codes for a particular sequence of amino acids, which comprises all, or part of a protein coding sequence, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
  • the transcribed region of the gene may include untranslated regions (UTRs), including introns, 5 '-untranslated regions (UTRs), and 3'-UTRs, as well as the coding sequence.
  • Gene deletion techniques enable the partial or complete removal of the gene(s), thereby eliminating their expression, or expressing a non-functional (or reduced activity) polypeptide product.
  • the deletion of the gene(s) may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene.
  • gene refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all, or part of a protein coding sequence, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
  • the transcribed region of the gene may include untranslated regions (UTRs), including introns, 5 '-untranslated regions (UTRs), and 3'- UTRs, as well as the coding sequence.
  • gene disruption refers broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product.
  • Exemplary methods of gene disruptions include complete or partial deletion of any portion of a gene, including a polypeptide encoding sequence, a promoter, an enhancer, or another regulatory element, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof which disrupt/inactivate the target gene(s) and substantially reduce or prevent the production of the functional gene product (i.e. , a protein).
  • mutation refers to any change or alteration in a nucleic acid sequence.
  • heterologous refers to nucleic acid or amino acid sequences, such as a polynucleotide or a polypeptide, not naturally occurring in a host cell. In other words, the nucleic acid or amino acid sequence is not identical to that naturally found in the host cell.
  • homologous can be understood as referring to nucleic acid or amino acid sequences, such as a polynucleotide or a polypeptide naturally occurring in a host cell. Naturally occurring means, from the same or different but usually related species, which correspond in function to each other or are very similar to each other.
  • the term encompasses ortholog genes or proteins and paralog genes or proteins.
  • recombinant host cell refers to modified cell that comprises at least one genetic modification which is not present in a "parental" host cell from which the recombinant host cell is derived.
  • a "parental" host cell is altered (e.g. via one or more genetic modifications introduced into the parental host cell) to generate a modified (daughter) cell thereof.
  • a parental host cell may be referred to as a "control cell”, particularly when being compared with, or relative to, a modified or recombinant (daughter) cell.
  • a recombinant host cell as described herein may be constructed by a process of gene conversion.
  • a nucleic acid sequence corresponding to the gene(s) is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into the parental host cell to produce a defective gene.
  • the defective nucleic acid sequence replaces the endogenous gene.
  • the defective gene or gene fragment also encodes a marker which may be used for selection of transformants containing the defective gene.
  • the defective gene may be introduced on a non-replicating or temperature- sensitive plasmid in association with a selectable marker.
  • Selection for integration of the plasmid is effected by selection for the marker under conditions not permitting plasmid replication.
  • Selection for a second recombination event leading to gene replacement is effected by examination of colonies for loss of the selectable marker and acquisition of the mutated gene.
  • the defective nucleic acid sequence may contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.
  • a recombinant host cell as described herein may also be constructed by established anti- sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a host cell may be reduced (down-regulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • miRNA microRNA
  • antisense oligonucleotides and the like, all of which are well known to the skilled artisan.
  • a recombinant host cell may also be produced/constructed via CRISPR-Cas9 editing.
  • a gene encoding the transcription regulator ctsR can be disrupted (or deleted or down- regulated) by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g. Cas9) and Cpfl or a guide DNA (e.g. NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA.
  • This targeted DNA break becomes a substrate for DNA repair, and can recombine with a provided editing template to disrupt or delete the gene.
  • the gene encoding the nucleic acid guided endonuclease or a codon-optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the host cell and a terminator active in the host cell, thereby creating a host cell Cas9 expression cassette.
  • a promoter active in the host cell and a terminator active in the host cell thereby creating a host cell Cas9 expression cassette.
  • one or more target sites unique to the gene of interest are readily identified by a person skilled in the art.
  • the DNA break induced by the endonuclease is repaired/replaced with an incoming sequence.
  • Expression cassettes can be co-delivered to the host cell using many different methods. A transformed cell is screened by PCR amplifying the target gene locus, by amplifying the locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus. These fragments are then sequenced using a sequencing primer to identify edited
  • a recombinant host cell may also be constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and transposition. Modification of the gene may be performed by subjecting the parental host cell to mutagenesis and screening for mutant cells in which expression of the gene has been reduced or eliminated.
  • the mutagenesis which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'- nitrosoguanidine (NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
  • UV ultraviolet
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • NTG N-methyl-N'- nitrosoguanidine
  • EMS ethyl methane sulphonate
  • sodium bisulphite formic acid
  • nucleotide analogues examples include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'- nitroso
  • telomeres are directly transformed (i.e. an intermediate cell is not used to amplify, or otherwise process, the DNA construct prior to introduction into the host cell).
  • Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art to introduce DNA into a host cell, without insertion into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposomes and the like.
  • DNA constructs are co-transformed with a plasmid without being inserted into the plasmid.
  • a selective marker is deleted or substantially excised from the recombinant host cell by methods known in the art.
  • resolution of the vector from a host chromosome leaves the flanking regions in the chromosome, while removing the indigenous chromosomal region.
  • nucleic acid refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin, which may be double- stranded or single-stranded, whether representing the sense or antisense strand. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may encode a given polypeptide.
  • nucleic acid construct is herein referred to as a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
  • nucleic acid construct is synonymous with the term “expression cassette” or “expression vector” when the nucleic acid construct contains all the control sequences required for expression of a coding sequence, wherein said control sequences are operably linked to said coding sequence.
  • polynucleotides or nucleic acid molecules
  • vectors or vectors
  • plasmids and nucleic acid constructs
  • coding sequence refers to a nucleotide sequence, which directly specifies the amino acid sequence of its (encoded) polypeptide product.
  • the boundaries of the coding sequence are generally determined by an open reading frame (ORF), which usually begins with an ATG start codon.
  • the coding sequence typically includes DNA, cDNA, and recombinant nucleotide sequences.
  • promoter refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' (downstream) to a promoter sequence.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as constitutive promoters. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence (e.g. an ORF) when it is capable of affecting the expression of that coding sequence (i.e. that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • polypeptide refers to a polymer of any length comprising amino acid residues linked by peptide bonds.
  • the conventional one (1) letter or three (3) letter codes for amino acid residues are used herein.
  • the polypeptide may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the term polypeptide also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage.
  • codon optimisation are known in the art.
  • a preferred method to optimise codon usage of the nucleotide sequences to that of the chosen host cell is a codon pair optimization technology as disclosed in W02006/077258 and/or W02008/000632.
  • W02008/000632 addresses codon-pair optimization.
  • Codon-pair optimisation is a method wherein the nucleotide sequences encoding a polypeptide are modified with respect to their codon-usage, in particularthe codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.
  • Sequence identity Sequence identity, or sequence homology are used interchangeable herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences overthe reported aligned region.
  • the percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm.
  • the Needleman- Wunsch algorithm has been implemented in the computer program NEEDLE.
  • the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. Longden.l. and Bleasby.A.
  • the percentage of sequence identity between a query sequence and a sequence as disclosed herein is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.
  • nucleic acid and protein sequences of as disclosed herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • substitution as used herein in the context of a polypeptide or a sequence thereof means the replacement (i.e. substitution) of one amino acid with another amino acid.
  • the present invention relates to a recombinant Bacillus host cell comprising a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest, wherein the host cell is deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes.
  • CtsR transcription repressor which is the product of the first gene of the clpC operon, controls the expression of clpP and clpC heat shock genes in Bacillus subtilis, Derre et al., Mol. Microbiology (1999) 31 (1), 117-131.
  • a recombinant Bacillus host cell as described herein produces at least 0.5% more, at least 1% more, at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40% more, at least 45% more, at least 50% more, at least 55% more, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, at least 100% more, or even more of a polypeptide of interest and / or a compound of interest, relative to an unmodified (parental) cell.
  • a recombinant host cell as described herein exhibits an increased specific productivity of a polypeptide of interest and / or a compound of interest relative the (unmodified) parental cell.
  • a recombinant host cell as described herein comprises a specific productivity increase of at least 0.5%, at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 100%, or even more relative to an unmodified (parental) cell.
  • the Bacillus host cell according to the present invention is deficient in the expression of the transcription repressor comprising a polypeptide sequence of SEQ ID NO: 10, or a polypeptide sequence which has at least 70% identity to SEQ ID NO: 10.
  • the Bacillus host cell according to the present invention is deficient in the expression of a transcription repressor which comprises a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 10.
  • the Bacillus host cell according to the present invention may be made deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes by any suitable means known by a person skilled in the art.
  • a host cell as described herein comprises a genetic modification which deletes, disrupts, inactivates or interferes with the c tsR gene thereby rendering the recombinant host cell deficient in the expression of a functional CtsR transcription repressor.
  • a Bacillus host cell as described herein comprises a deletion, disruption or mutation of a polynucleotide encoding the transcription repressor of class III heat shock genes, wherein the polynucleotide sequence encoding the transcription repressor has at least 70% identity to SEQ ID NO: 9.
  • the polynucleotide sequence encoding the transcription repressor has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 9.
  • Class III heat shock genes in a Bacillus host cell as disclosed herein comprise heat shock genes clpC, clpE, clpX and clpP.
  • heat shock genes clpC, clpE, clpX and/or clpP are controlled by the transcription repressor CtsR as defined herein.
  • the heat shock gene clpC has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO: 29.
  • the heat shock gene clpE has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO: 31 .
  • the heat shock gene clpX has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO: 33.
  • the heat shock gene clpP has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO:35.
  • the heat shock gene clpC comprises polynucleotide sequence according to SEQ ID NO: 29
  • the heat shock clpE comprises polynucleotide sequence according to SEQ ID NO: 31
  • the heat shock gene clpX comprises polynucleotide sequence according to SEQ ID NO: 33
  • / or the heat shock gene clpP comprises polynucleotide sequence according to SEQ ID NO: 35.
  • the heat shock gene clpC as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 30 or comprises the clpC protein according to SEQ ID NO: 30.
  • the heat shock gene clpE as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 32 or encodes the polypeptide according to SEQ ID NO: 32.
  • the heat shock gene clpX as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 34, or encodes the polypeptide according to SEQ ID NO: 34.
  • the heat shock gene clpP as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 36 , or encodes the polypeptide according to SEQ ID NO: 36.
  • the Bacillus host cell does not comprise a transcriptional fusion between a class III heat shock gene and the polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest.
  • the Bacillus host cell does not comprise a transcriptional fusion between a clpC or clpP gene and the polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest.
  • a Bacillus host cell as disclosed herein does not comprise or contain a transcriptional fusion between the heat shock genes wherein the c tsR coding sequence has been deleted, and a polynucleotide encoding a polypeptide of interest or a polypeptide involved in the in the production of a compound of interest.
  • the polynucleotide encoding a polypeptide of interest or a polypeptide involved in the production of a compound of interest in the Bacillus host cell as disclosed herein, is not located in a transcriptional fusion with a heat shock gene, or a heat shock operon, such as the clpP operon.
  • the Bacillus host cell comprises a promoter sequence allowing for expression of the polynucleotide encoding a polypeptide of interest or a polypeptide involved in the synthesis of a compound of interest in the host cell.
  • the promoter may for instance be a bacteriophage promoter sequence, more preferably a bacteriophage SPQ1 promoter sequence.
  • a bacteriophage promoter sequence is herewith defined as a promoter sequence derived from a bacteriophage, i.e. derived from a virus that infects and replicates in bacteria.
  • a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the synthesis of a compound of interest may be produced recombinantly, for example using PCR (polymerase chain reaction) cloning techniques, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • a polynucleotide is typically provided in isolated and/or purified form.
  • the polypeptide of interest or the polypeptide involved in the synthesis of a compound of interest may be homologous or heterologous to the Bacillus host cell.
  • the polypeptide of interest or the polypeptide involved in the production of a compound of interest is usually overexpressed in the Bacillus host cell by known techniques in the art, for instance by inserting multiple copies of a polynucleotide encoding the polypeptide of interest or the polypeptide involved in the synthesis of a compound of interest .
  • the recombinant Bacillus host cell as disclosed herein preferably comprises a nucleic acid construct wherein the nucleic acid construct comprises a polynucleotide encoding a polypeptide of interest or a polypeptide involved in the synthesis of a compound of interest operably linked to a promoter sequence allowing for expression of the polynucleotide in the host cell.
  • the host cell according to the present disclosure is further deficient in a sporulation-related gene, preferably deficient in a gene selected from the group consisting of spoOA, spollSA, spollAC, sigE, sigF, spollSB, spollE, sigG, spoIVCB, spolllC, spollGA, spollAA, spolVFB, spollR, and spoilt, preferably deficient in a spollE or deficient in a spollAC gene.
  • the host cell according to the present disclosure is further deficient in the production of a neutral protease and / or an alkaline protease, such as disclosed in WO2015/118126, Example 2.
  • the host cell according to the present invention comprises a deletion, disruption or mutation of the nprE and / orthe aprE gene.
  • a Bacillus host cell as disclosed herein which further comprises a deficiency in a neutral protease gene and / or an alkaline protease gene, results in a further increased yield or productivity of a polypeptide of interest or a compound of interest, such as at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 100% as compared to a parental Bacillus host cell not containing a deficiency in a neutral protease and / or an alkaline protease.
  • a recombinant host cell as described herein comprises a genetic modification which deletes, disrupts, inactivates or interferes with a Lon protease gene, thereby rendering the recombinant host cell deficient in the expression (i.e. production) of a functional Lon protease.
  • the term “Lon protease” as used herein means an ATP-dependent serine peptidase belonging to the MEROPS peptidase family S16 (Lon protease family, clan SJ). On the basis of sequence homology and structure, Lon proteases can be divided into two subfamilies: LonA and LonB.
  • the Lon protease is a LonA protease such as a bacterial LonA protease, for example a Bacillus LonA protease.
  • the Lon protease is selected from the group consisting of (a) a Lon protease having at least 60% sequence identity to the polypeptide of SEQ ID NO:38 (b) a Lon protease encoded by a polynucleotide having at least 60% sequence identity to the polypeptide coding sequence of SEQ ID NO:37, and (c) a fragment of the Lon protease of (a) or (b) that has Lon protease activity.
  • the Lon protease has at least 70%, at least 75%, at least 80%, 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% sequence identity to the polypeptide of SEQ ID NO:38.
  • the Lon protease comprises the amino acid sequence of the polypeptide of SEQ ID NO:38.
  • the amino acid sequence of the Lon protease consists of the polypeptide of SEQ ID NO:38.
  • the Lon protease is encoded by a polynucleotide having at least 70%, at least 75%, at least 80%, 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% sequence identity to the polypeptide coding sequence of SEQ ID NO:37.
  • the Lon protease is encoded by a polynucleotide comprising the polypeptide coding sequence of SEQ ID NO: 37.
  • the Lon protease is encoded by a polynucleotide consisting of SEQ ID NO:37.
  • the Bacillus host cell belongs to a species selected from the group consisting of: B. agaradherens, B. alkalophilus, B. amyloliquefaciens, B. anthracis, B. atrophaeus, B. brevis, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. halodurans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. mojavensis, B. pumilus, B. puntis, B. sphaericus, B. stearothermophilus, B. subtilis, B. thuringiensis, and B.
  • Bacillus host cell belongs to B. subtilis, B. amyloliquefaciens or B. licheniformis, most preferably the Bacillus host cell is a B. subtilis host cell.
  • the host cell may also be an Alicyclobacillus sp. , for instance Alicyclobacillus pohliae.
  • the present invention also relates to a method for preparing a Bacillus host cell according to the present invention comprising deleting, disrupting or mutating a polynucleotide encoding a transcription repressor of class III heat shock genes.
  • a person skilled in the art knows how to delete, disrupt or mutate a polynucleotide in a Bacillus host cell as described further herein above.
  • the present invention also relates to a process for producing a polypeptide of interest, or a compound of interest, comprising cultivating the Bacillus host cell according to the present invention under conditions conducive for the production of the polypeptide or compound of interest.
  • Cultivating the Bacillus host cell comprises producing a polypeptide of interest or a compound of interest, wherein the Bacillus host cell is fermented in a suitable medium.
  • a suitable medium usually comprises a carbon source, a nitrogen source and inorganic salts known to a person skilled in the art.
  • Cultivating a Bacillus host cell may be performed in any suitable culture such as a batch, fed batch or a continuous culture known to a person skilled in the art. Cultivating a Bacillus host cell is performed under aerobic conditions.
  • the process for producing a polypeptide of interest or a compound of interest further comprises a step of recovering the polypeptide of interest or a compound of interest from the culture medium, by known procedures in the art, for instance by filtration, spray drying evaporation, or centrifugation.
  • the protein of interest may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.
  • any suitable polypeptide of interest may be produced in a process as disclosed herein and is encoded by the polynucleotide in the Bacillus host cell of the present invention.
  • the polypeptide of interest preferably is an enzyme.
  • the polypeptide of interest is a xylanase, hemicellulase, cellulase, glucanase, alpha-galactosidase, phytase, protease, pectinase, lipase, phospholipase or an amylase, such as an alpha-amylase or a maltogenic amylase.
  • polypeptide of interest or the polypeptide involved in the synthesis of a compound of interest may be homologous (endogenous) or heterologous to the Bacillus host cell.
  • the polynucleotide coding for a polypeptide involved in the production of the compound of interest may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, alcohols, lipids, carotenoids, and vitamins.
  • a primary or secondary metabolite such as organic acids, alcohols, lipids, carotenoids, and vitamins.
  • Such enzyme may be a lyase, esterase, protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase, phospholipase etc.
  • Metabolites encompasses both primary and secondary metabolites. Metabolites comprise citric acid, gluconic acid, adipic acid, fumaric acid, itaconic acid and succinic acid.
  • the primary metabolite may be, but is not limited to, an amino acid, fatty acid, triacylglycerol, nucleoside, nucleotide, sugar, triglyceride, or vitamin.
  • an amino acid fatty acid, triacylglycerol, nucleoside, nucleotide, sugar, triglyceride, or vitamin.
  • vitamin A vitamin B2, pantothenic acid (vitamin B5), vitamin B6, vitamin C, vitamin D or vitamin E.
  • the secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, orterpene. Examples
  • BSB1 is a tryptophan-prototrophic (frp + ) derivative of Bacillus subtilis 168 (ATCC 23857) (trpC2) described by Anagnostopoulos C. and Spizizen J. (J. Bacteriol. (1961) 81(5): 741-746).
  • BSB1 was obtained by transforming Bacillus subtilis 168 with chromosomal DNA from the trp + strain HVS495 and selecting for colonies growing on minimal medium without tryptophan.
  • Trp + colony was named BSB1 , (Nicolas P. et al., Science. 2012 Mar 2;335(6072):1103-1106).
  • the xynA gene was deleted using a marker free gene deletion clean knockout (Morimoto T. Genes Genet Syst. 2009 Aug;84(4):315-8).
  • Three DNA fragments upstream and downstream of xynA were amplified from the BSB1 genomic DNA using primer pairs BW45 (SEQ ID NO: 4) and BW46 (SEQ ID NO: 5), BW41 (SEQ ID NO: 1) and BW42(SEQ ID NO: 2), BW41 (SEQ ID NO: 1) and BW44 (SEQ ID NO: 3).
  • a toxin DNA fragment (SEQ ID NO: 6) present in strain TMO310 (Morimoto et al (2009) Genes Genet Syst. 84, p. 315-318), with lac operator, inducible P spac -masF and a spectinomycin resistance cassette was amplified using primers BW05 (SEQ ID NO: 7) and BW06 (SEQ ID NO: 8).
  • the four PCR fragments were purified and fused using Gibson Assembly and directly transformed to competent BSB1 cells. Transformants were selected on LB agar with 150 pg/ml spectinomycin. Cells were spread on LB agar with 1 mM IPTG to induce the MasF toxin and select for colonies that excised the xynA and the toxin fragment from the chromosome via homologous recombination. This resulted in the xynA deletion strain BWB06 with the following genotype trpC+, AxynA.
  • amyE gene was deleted using the same clean knockout method.
  • Three DNA fragments upstream and downstream of amyE were amplified from the BSB1 genomic DNA using primer pairs BW49 (SEQ ID NO: 23) and BW50 (SEQ ID NO: 24), BW51 (SEQ ID NO: 25) and BW52 (SEQ ID NO: 26), and BW53 (SEQ ID NO: 27) and BW54 (SEQ ID NO: 28).
  • the three a yE fragments and together with the toxin DNA fragment were purified and fused using Gibson Assembly and directly transformed to competent BWB06 cells. Following a similar selection process, this resulted in the xynA and amyE double deletion strain BWB09 with the following genotype trpC + , AxynA, AamyE.
  • the ctsR gene (SEQ ID NO: 9 ) encoding the transcription repressor of class III heat shock protein CtsR (SEQ ID NO: 10) was deleted from strain BWB09.
  • Strain BWB09 was transformed with a ctsR::ery deletion construct ( Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291- 305) and an erythromycin resistant colony containing the ctsR deletion was named strain SGB03, with the following phenotype trpC + , AxynA, AamyE ctsRwery.
  • Strain BWB09 and ctsR deletion strain SGB03 were transformed with the xylanase expression plasmid pDBC4XAS-1 as described above, which resulted in strains BWB09/ pDBC4XAS-1 and SGB03/ pDBC4XAS-1 , respectively.
  • Strains BWB09/ pDBC4XAS-1 , SGB03/ pDBC4XAS-1- were grown in 10 ml LB medium with a start O ⁇ boo of 0.05 in 100 ml flasks under continuous shaking with 210 rpm at 37 °C, 50 pg/ml kanamycin was added to maintain the plasmids.
  • Supernatants of cell cultures were sampled at 2, 3, 4, 6, 8, 10 and 15 hours. The cell density (O ⁇ boo) was measured. Supernatants were collected by flash freezing in liquid N2, and stored at -80 °C.
  • the xylanase activity was determined using the fluorescence based assay EnzChek® Ultra Xylanase Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions.
  • the commercial Xylanase (Sigma, X2753) was used for construction of standard curve for the detection of xylanase and amylase enzyme activity. Growth and xylanase production of strains BWB09/pDBC4XAS-1 , and SGB03/pDBC4XAS-1 was followed in time and depicted in Table 1.
  • Strain SGB03/pDBC4XAS-1 which contains the ctsR deletion produces more xylanase than strain BWB09/pDBC4XAS-1 with intact ctsR gene. The difference in xylanase yield increases in time.
  • Strain BWB143 was constructed by three-step deletion. Firstly, strain BSB1 was transformed with a spollE::ery deletion construct ( Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291- 305) and an erythromycin resistant colony was further transformed with pDR244 (temperature- sensitive plasmid with constitutively expressed Cre recombinase) and selected for spectinomycin resistant colony at 30 °C. The plasmid pDR244 was then removed by serial cultivation in LB liquid at 42°C, resulting in marker-less spollE deletion mutant named BWAB01 , with the following phenotype trpC + , AspollE.
  • BWAB02 was constructed similarly by transforming BWAB01 with a nprE::ery deletion construct (Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291-305) followed by pDR244, and resulting in marker-less spollE and nprE double deletion, with the following phenotype trpC + , AspollE, AnprE.
  • BWB143 was constructed similarly by transforming BWAB02 with an aprE::ery deletion construct (Koo et al., Cell Syst.
  • BWB144 was constructed by transforming strain BWB143 with a ctsR::ery deletion construct ( Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291-305) and an erythromycin resistant colony containing the ctsR deletion was named strain BWB144, with the following phenotype trpC + , AspollE, AnprE, AaprE, ctsRv.ery.
  • Strain BWB143 and BWB144 containing a ctsR deletion which were constructed as described in Example 3, were transformed with the xylanase expression plasmid pDBC4XAS-1 as described above, which resulted in strains BWB143/pDBC4XAS-1 and BWB144/pDBC4XAS-1 .
  • Strains BWB143/pDBC4XAS-1 and BWB144/pDBC4XAS-1 were grown in 10 ml LB medium with a start OD600 of 0.05 in 100 ml flasks under continuous shaking with 210 rpm at 37 °C. 50 pg/ml kanamycin was added to maintain the plasmids.
  • Strain BWB144/pDBC4XAS-1 which contains the ctsR deletion produces more xylanase than strain BWB143/pDBC4XAS-1 with intact ctsR gene.
  • the difference in xylanase yield increases in time.
  • the relative xylanase activity in the medium of BWB144/pDBC4XAS-1 is 1.8-fold higher than the xylanase activity in the medium of BWB143/pDBC4XAS-1 .
  • Examples 2 and 4 show that a deletion of ctsR in a Bacillus strain increases the yield of a polypeptide of interest, such as a xylanase, in the Bacillus strain.
  • Example 4 shows that a further deletion of the sporulation gene ( AspollE) and a deletion of neutral and alkaline protease ( AnprE , AaprE) in a Bacillus host strain further increases the yield of the polypeptide of interest by the Bacillus strain.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a recombinant Bacillus host cell comprising a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest, wherein the host cell is deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes. The invention further relates to a process for producing a polypeptide of interest, or a compound of interest, comprising cultivating the Bacillus host cell of the invention under conditions conducive for the production of the polypeptide or compound of interest.

Description

IMPROVED BACILLUS HOST CELL
Field
The present invention relates to a recombinant Bacillus host cell, and a process for producing a polypeptide or a compound of interest wherein the Bacillus host cell is used.
Background
Bacillus strains produce and secrete a large number of useful proteins and metabolites. Because of their GRAS (General Recognised As Safe) status, Bacillus hosts, e.g. belonging to the species Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, are largely used in the production of important proteins for the food, feed and pharmaceutical industry.
To increase the production yields of proteins of interest and metabolites, several different genetic strategies have been used in Bacillus strains.
US7,585,674 discloses that the productivity of a protein or polypeptide of interest in host cells, e.g. Bacillus host cells can be improved by deleting from the genome of said host cells specific genes participating in sporulation stage II, III, IV or V.
WO2015/118126 discloses that when Bacillus strains have been made deficient in the production of a neutral protease and / or an alkaline protease, this results in an increased yield of of a protein or polypeptide of interest during fermentation.
Derre et a!., Mol. Microbiology (1999) 31 (1), 117-131 discloses that the CtsR transcription repressor is a regulator of stress and heat shock response and controls clp and molecular chaperone gene expression in Gram-positive bacteria. In order to follow heat shock induction, transcriptional fusions were constructed between the clpP and clpC promotor regions and the bgaB gene of Bacillus stearothermophilus, which encodes a thermostable b-galactosidase.
Despite the advances that have been made, there is still a need for methods to increase expression of proteins or polypeptides in Bacillus hosts.
Summary
The present invention relates to a recombinant Bacillus host cell comprising a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest, wherein the host cell is deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes.
The inventions further relates to a method for preparing a Bacillus host cell as disclosed herein, comprising deleting, disrupting or mutating a polynucleotide encoding a transcription repressor of class III heat shock genes. In another aspect, the invention relates to a process for producing a polypeptide of interest, or a compound of interest, comprising cultivating the Bacillus host cell according to the present invention under conditions conducive for the production of the polypeptide or compound of interest.
Definitions
The term "derived from" also includes the terms "originated from," "obtained from," "obtainable from," "isolated from," and "created from," and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.
The term “expresses/produces” is meant to include any steps involved in the expression and production of a polypeptide of interest in a host cell as described herein including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term “gene” as used herein refers to a polynucleotide that codes for a particular sequence of amino acids, which comprises all, or part of a protein coding sequence, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions (UTRs), including introns, 5 '-untranslated regions (UTRs), and 3'-UTRs, as well as the coding sequence.
Gene deletion techniques enable the partial or complete removal of the gene(s), thereby eliminating their expression, or expressing a non-functional (or reduced activity) polypeptide product. In such methods, the deletion of the gene(s) may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene.
The term “gene” as used herein refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all, or part of a protein coding sequence, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions (UTRs), including introns, 5 '-untranslated regions (UTRs), and 3'- UTRs, as well as the coding sequence.
As used herein, “gene disruption” refers broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product. Exemplary methods of gene disruptions include complete or partial deletion of any portion of a gene, including a polypeptide encoding sequence, a promoter, an enhancer, or another regulatory element, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof which disrupt/inactivate the target gene(s) and substantially reduce or prevent the production of the functional gene product (i.e. , a protein).
The term “mutation” as used herein refers to any change or alteration in a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frame shift mutations, splicing mutations and the like. Mutations may be performed specifically (e.g. via site directed mutagenesis) or randomly (e.g. via chemical agents, passage through repair minus bacterial strains).
The term "heterologous" as used herein refers to nucleic acid or amino acid sequences, such as a polynucleotide or a polypeptide, not naturally occurring in a host cell. In other words, the nucleic acid or amino acid sequence is not identical to that naturally found in the host cell.
The term “homologous” can be understood as referring to nucleic acid or amino acid sequences, such as a polynucleotide or a polypeptide naturally occurring in a host cell. Naturally occurring means, from the same or different but usually related species, which correspond in function to each other or are very similar to each other. The term encompasses ortholog genes or proteins and paralog genes or proteins.
The term "recombinant host cell" refers to modified cell that comprises at least one genetic modification which is not present in a "parental" host cell from which the recombinant host cell is derived. A "parental" host cell is altered (e.g. via one or more genetic modifications introduced into the parental host cell) to generate a modified (daughter) cell thereof. In an embodiment a parental host cell may be referred to as a "control cell", particularly when being compared with, or relative to, a modified or recombinant (daughter) cell. It is to be understood that when the expression and/or production of a polypeptide of interest or compound of interest in an "unmodified" (parental) host cell is being compared to the expression and/or production of the same polypeptide of interest or a compound of interest in a modified or recombinant (daughter) cell, the modified and unmodified host cells are grown/cultivated/fermented under the same conditions (e.g. the same conditions such as media, temperature, pH and the like).
A recombinant host cell as described herein may be constructed by a process of gene conversion. For example, in the gene conversion method, a nucleic acid sequence corresponding to the gene(s) is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into the parental host cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants containing the defective gene. For example, the defective gene may be introduced on a non-replicating or temperature- sensitive plasmid in association with a selectable marker. Selection for integration of the plasmid is effected by selection for the marker under conditions not permitting plasmid replication. Selection for a second recombination event leading to gene replacement is effected by examination of colonies for loss of the selectable marker and acquisition of the mutated gene. Alternatively, the defective nucleic acid sequence may contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.
A recombinant host cell as described herein may also be constructed by established anti- sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a host cell may be reduced (down-regulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated. Such anti- sense methods include, but are not limited to RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, and the like, all of which are well known to the skilled artisan.
A recombinant host cell may also be produced/constructed via CRISPR-Cas9 editing. For example, a gene encoding the transcription regulator ctsR can be disrupted (or deleted or down- regulated) by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g. Cas9) and Cpfl or a guide DNA (e.g. NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair, and can recombine with a provided editing template to disrupt or delete the gene. For example, the gene encoding the nucleic acid guided endonuclease or a codon-optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the host cell and a terminator active in the host cell, thereby creating a host cell Cas9 expression cassette. Likewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art. In certain embodiments, the DNA break induced by the endonuclease is repaired/replaced with an incoming sequence. Expression cassettes can be co-delivered to the host cell using many different methods. A transformed cell is screened by PCR amplifying the target gene locus, by amplifying the locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus. These fragments are then sequenced using a sequencing primer to identify edited colonies.
A recombinant host cell may also be constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and transposition. Modification of the gene may be performed by subjecting the parental host cell to mutagenesis and screening for mutant cells in which expression of the gene has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods. Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'- nitrosoguanidine (NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parental cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells exhibiting reduced or no expression of the gene.
Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells. Indeed, such methods as transformation including protoplast transformation and congression, transduction, and protoplast fusion are known and suited for use in the present invention. In addition to commonly used methods, host cells are directly transformed (i.e. an intermediate cell is not used to amplify, or otherwise process, the DNA construct prior to introduction into the host cell). Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art to introduce DNA into a host cell, without insertion into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposomes and the like. In additional embodiments, DNA constructs are co-transformed with a plasmid without being inserted into the plasmid. In further embodiments, a selective marker is deleted or substantially excised from the recombinant host cell by methods known in the art. In some embodiments, resolution of the vector from a host chromosome leaves the flanking regions in the chromosome, while removing the indigenous chromosomal region.
The term “nucleic acid” as used herein refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin, which may be double- stranded or single-stranded, whether representing the sense or antisense strand. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may encode a given polypeptide.
The term "nucleic acid construct" is herein referred to as a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term “expression cassette” or “expression vector” when the nucleic acid construct contains all the control sequences required for expression of a coding sequence, wherein said control sequences are operably linked to said coding sequence.
It is understood that the polynucleotides (or nucleic acid molecules) described herein include “genes”, “vectors”, “plasmids” and “nucleic acid constructs”.
As used herein, the term “coding sequence” refers to a nucleotide sequence, which directly specifies the amino acid sequence of its (encoded) polypeptide product. The boundaries of the coding sequence are generally determined by an open reading frame (ORF), which usually begins with an ATG start codon. The coding sequence typically includes DNA, cDNA, and recombinant nucleotide sequences. The term “promoter” as used herein refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' (downstream) to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as constitutive promoters. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” as used herein refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence (e.g. an ORF) when it is capable of affecting the expression of that coding sequence (i.e. that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “polypeptide” as used herein refers to a polymer of any length comprising amino acid residues linked by peptide bonds. The conventional one (1) letter or three (3) letter codes for amino acid residues are used herein. The polypeptide may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term polypeptide also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
To increase the likelihood that a polypeptide is expressed in active form by a cell of the invention, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage. Several methods for codon optimisation are known in the art. A preferred method to optimise codon usage of the nucleotide sequences to that of the chosen host cell is a codon pair optimization technology as disclosed in W02006/077258 and/or W02008/000632. W02008/000632 addresses codon-pair optimization. Codon-pair optimisation is a method wherein the nucleotide sequences encoding a polypeptide are modified with respect to their codon-usage, in particularthe codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.
“Sequence identity”. Sequence identity, or sequence homology are used interchangeable herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences overthe reported aligned region. The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman- Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. Longden.l. and Bleasby.A. Trends in Genetics 16, (6) pp276 — 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence as disclosed herein is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.
The nucleic acid and protein sequences of as disclosed herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.
The term “substitution” as used herein in the context of a polypeptide or a sequence thereof means the replacement (i.e. substitution) of one amino acid with another amino acid.
Detailed description
The present invention relates to a recombinant Bacillus host cell comprising a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest, wherein the host cell is deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes. The CtsR transcription repressor, which is the product of the first gene of the clpC operon, controls the expression of clpP and clpC heat shock genes in Bacillus subtilis, Derre et al., Mol. Microbiology (1999) 31 (1), 117-131.
Surprisingly, it was found that the yield of a polypeptide of interest or a compound of interest is increased when a Bacillus host cell has been made deficient in the expression of a transcription repressor as compared to a parental cell.
A recombinant Bacillus host cell as described herein produces at least 0.5% more, at least 1% more, at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40% more, at least 45% more, at least 50% more, at least 55% more, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, at least 100% more, or even more of a polypeptide of interest and / or a compound of interest, relative to an unmodified (parental) cell.
A recombinant host cell as described herein exhibits an increased specific productivity of a polypeptide of interest and / or a compound of interest relative the (unmodified) parental cell. In an embodiment a recombinant host cell as described herein comprises a specific productivity increase of at least 0.5%, at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 100%, or even more relative to an unmodified (parental) cell.
In one embodiment, the Bacillus host cell according to the present invention is deficient in the expression of the transcription repressor comprising a polypeptide sequence of SEQ ID NO: 10, or a polypeptide sequence which has at least 70% identity to SEQ ID NO: 10. Preferably, the Bacillus host cell according to the present invention is deficient in the expression of a transcription repressor which comprises a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 10.
The Bacillus host cell according to the present invention may be made deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes by any suitable means known by a person skilled in the art. A host cell as described herein comprises a genetic modification which deletes, disrupts, inactivates or interferes with the c tsR gene thereby rendering the recombinant host cell deficient in the expression of a functional CtsR transcription repressor. In one embodiment a Bacillus host cell as described herein comprises a deletion, disruption or mutation of a polynucleotide encoding the transcription repressor of class III heat shock genes, wherein the polynucleotide sequence encoding the transcription repressor has at least 70% identity to SEQ ID NO: 9. Preferably, the polynucleotide sequence encoding the transcription repressor has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 9.
Class III heat shock genes in a Bacillus host cell as disclosed herein comprise heat shock genes clpC, clpE, clpX and clpP. Preferably, heat shock genes clpC, clpE, clpX and/or clpP are controlled by the transcription repressor CtsR as defined herein. Preferably, the heat shock gene clpC has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO: 29. Preferably, the heat shock gene clpE has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO: 31 . Preferably, the heat shock gene clpX has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO: 33. Preferably, the heat shock gene clpP has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the polynucleotide according to SEQ ID NO:35. Preferably, the heat shock gene clpC comprises polynucleotide sequence according to SEQ ID NO: 29, the heat shock clpE comprises polynucleotide sequence according to SEQ ID NO: 31 , the heat shock gene clpX comprises polynucleotide sequence according to SEQ ID NO: 33, and / or the heat shock gene clpP comprises polynucleotide sequence according to SEQ ID NO: 35.
The heat shock gene clpC as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 30 or comprises the clpC protein according to SEQ ID NO: 30. The heat shock gene clpE as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 32 or encodes the polypeptide according to SEQ ID NO: 32. The heat shock gene clpX as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 34, or encodes the polypeptide according to SEQ ID NO: 34. The heat shock gene clpP as disclosed herein encodes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 36 , or encodes the polypeptide according to SEQ ID NO: 36.
In one embodiment the Bacillus host cell does not comprise a transcriptional fusion between a class III heat shock gene and the polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest. For instance, the Bacillus host cell does not comprise a transcriptional fusion between a clpC or clpP gene and the polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest.
A Bacillus host cell as disclosed herein does not comprise or contain a transcriptional fusion between the heat shock genes wherein the c tsR coding sequence has been deleted, and a polynucleotide encoding a polypeptide of interest or a polypeptide involved in the in the production of a compound of interest.
The polynucleotide encoding a polypeptide of interest or a polypeptide involved in the production of a compound of interest in the Bacillus host cell as disclosed herein, is not located in a transcriptional fusion with a heat shock gene, or a heat shock operon, such as the clpP operon.
The Bacillus host cell according to the invention comprises a promoter sequence allowing for expression of the polynucleotide encoding a polypeptide of interest or a polypeptide involved in the synthesis of a compound of interest in the host cell. The promoter may for instance be a bacteriophage promoter sequence, more preferably a bacteriophage SPQ1 promoter sequence. A bacteriophage promoter sequence is herewith defined as a promoter sequence derived from a bacteriophage, i.e. derived from a virus that infects and replicates in bacteria.
A polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the synthesis of a compound of interest, may be produced recombinantly, for example using PCR (polymerase chain reaction) cloning techniques, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. A polynucleotide is typically provided in isolated and/or purified form.
The polypeptide of interest or the polypeptide involved in the synthesis of a compound of interest may be homologous or heterologous to the Bacillus host cell. The polypeptide of interest or the polypeptide involved in the production of a compound of interest is usually overexpressed in the Bacillus host cell by known techniques in the art, for instance by inserting multiple copies of a polynucleotide encoding the polypeptide of interest or the polypeptide involved in the synthesis of a compound of interest .
The recombinant Bacillus host cell as disclosed herein preferably comprises a nucleic acid construct wherein the nucleic acid construct comprises a polynucleotide encoding a polypeptide of interest or a polypeptide involved in the synthesis of a compound of interest operably linked to a promoter sequence allowing for expression of the polynucleotide in the host cell.
In one embodiument the host cell according to the present disclosure is further deficient in a sporulation-related gene, preferably deficient in a gene selected from the group consisting of spoOA, spollSA, spollAC, sigE, sigF, spollSB, spollE, sigG, spoIVCB, spolllC, spollGA, spollAA, spolVFB, spollR, and spoilt, preferably deficient in a spollE or deficient in a spollAC gene. A Bacillus host cell as disclosed herein which further comprises a deficiency in any of the sporulation genes, results in a further increased yield or productivity of a polypeptide of interest or a compound of interest, such as at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 100% as compared to a parental Bacillus host cell not containing a deficiency a sporulation gene.
In another embodiment the host cell according to the present disclosure is further deficient in the production of a neutral protease and / or an alkaline protease, such as disclosed in WO2015/118126, Example 2. Preferably, the host cell according to the present invention comprises a deletion, disruption or mutation of the nprE and / orthe aprE gene. A Bacillus host cell as disclosed herein which further comprises a deficiency in a neutral protease gene and / or an alkaline protease gene, results in a further increased yield or productivity of a polypeptide of interest or a compound of interest, such as at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 100% as compared to a parental Bacillus host cell not containing a deficiency in a neutral protease and / or an alkaline protease. In an embodiment a recombinant host cell as described herein comprises a genetic modification which deletes, disrupts, inactivates or interferes with a Lon protease gene, thereby rendering the recombinant host cell deficient in the expression (i.e. production) of a functional Lon protease. The term “Lon protease” as used herein means an ATP-dependent serine peptidase belonging to the MEROPS peptidase family S16 (Lon protease family, clan SJ). On the basis of sequence homology and structure, Lon proteases can be divided into two subfamilies: LonA and LonB. In a preferred embodiment the Lon protease is a LonA protease such as a bacterial LonA protease, for example a Bacillus LonA protease.
In an embodiment the Lon protease is selected from the group consisting of (a) a Lon protease having at least 60% sequence identity to the polypeptide of SEQ ID NO:38 (b) a Lon protease encoded by a polynucleotide having at least 60% sequence identity to the polypeptide coding sequence of SEQ ID NO:37, and (c) a fragment of the Lon protease of (a) or (b) that has Lon protease activity. In an embodiment the Lon protease has at least 70%, at least 75%, at least 80%, 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% sequence identity to the polypeptide of SEQ ID NO:38. In an embodiment the Lon protease comprises the amino acid sequence of the polypeptide of SEQ ID NO:38. In an embodiment the amino acid sequence of the Lon protease consists of the polypeptide of SEQ ID NO:38.
In an embodiment the Lon protease is encoded by a polynucleotide having at least 70%, at least 75%, at least 80%, 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% sequence identity to the polypeptide coding sequence of SEQ ID NO:37. In an embodiment the Lon protease is encoded by a polynucleotide comprising the polypeptide coding sequence of SEQ ID NO: 37. In an embodiment the Lon protease is encoded by a polynucleotide consisting of SEQ ID NO:37.
The Bacillus host cell according to the present invention belongs to a species selected from the group consisting of: B. agaradherens, B. alkalophilus, B. amyloliquefaciens, B. anthracis, B. atrophaeus, B. brevis, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. halodurans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. mojavensis, B. pumilus, B. puntis, B. sphaericus, B. stearothermophilus, B. subtilis, B. thuringiensis, and B. vallismortis. Preferably the Bacillus host cell belongs to B. subtilis, B. amyloliquefaciens or B. licheniformis, most preferably the Bacillus host cell is a B. subtilis host cell. The host cell may also be an Alicyclobacillus sp. , for instance Alicyclobacillus pohliae.
The present invention also relates to a method for preparing a Bacillus host cell according to the present invention comprising deleting, disrupting or mutating a polynucleotide encoding a transcription repressor of class III heat shock genes. A person skilled in the art knows how to delete, disrupt or mutate a polynucleotide in a Bacillus host cell as described further herein above. The present invention also relates to a process for producing a polypeptide of interest, or a compound of interest, comprising cultivating the Bacillus host cell according to the present invention under conditions conducive for the production of the polypeptide or compound of interest. Cultivating the Bacillus host cell comprises producing a polypeptide of interest or a compound of interest, wherein the Bacillus host cell is fermented in a suitable medium. A suitable medium usually comprises a carbon source, a nitrogen source and inorganic salts known to a person skilled in the art.
Cultivating a Bacillus host cell may be performed in any suitable culture such as a batch, fed batch or a continuous culture known to a person skilled in the art. Cultivating a Bacillus host cell is performed under aerobic conditions.
In one embodiment, the process for producing a polypeptide of interest or a compound of interest further comprises a step of recovering the polypeptide of interest or a compound of interest from the culture medium, by known procedures in the art, for instance by filtration, spray drying evaporation, or centrifugation. The protein of interest may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.
Any suitable polypeptide of interest may be produced in a process as disclosed herein and is encoded by the polynucleotide in the Bacillus host cell of the present invention. The polypeptide of interest preferably is an enzyme. Preferably, the polypeptide of interest is a xylanase, hemicellulase, cellulase, glucanase, alpha-galactosidase, phytase, protease, pectinase, lipase, phospholipase or an amylase, such as an alpha-amylase or a maltogenic amylase.
The polypeptide of interest or the polypeptide involved in the synthesis of a compound of interest may be homologous (endogenous) or heterologous to the Bacillus host cell.
The polynucleotide coding for a polypeptide involved in the production of the compound of interest may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, alcohols, lipids, carotenoids, and vitamins. Such enzyme may be a lyase, esterase, protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase, phospholipase etc.
The term "metabolite" encompasses both primary and secondary metabolites. Metabolites comprise citric acid, gluconic acid, adipic acid, fumaric acid, itaconic acid and succinic acid.
The primary metabolite may be, but is not limited to, an amino acid, fatty acid, triacylglycerol, nucleoside, nucleotide, sugar, triglyceride, or vitamin. For example, vitamin A, vitamin B2, pantothenic acid (vitamin B5), vitamin B6, vitamin C, vitamin D or vitamin E.
The secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, orterpene. Examples
Materials and Methods Molecular biology techniques
Molecular biology techniques used are known to the skilled person and described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001). B. subtilis transformations were performed as described by Anagnostopolous, C., and J. Spizizen (1961 , Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81 : 741- 746). Polymerase chain reaction (PCR) was performed on a thermocycler with Phusion High- Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland) according to the instructions of the manufacturer.
Example 1. Strain construction BWB09 and SGB03 1.1 Construction of BWB09
BSB1 is a tryptophan-prototrophic (frp+) derivative of Bacillus subtilis 168 (ATCC 23857) (trpC2) described by Anagnostopoulos C. and Spizizen J. (J. Bacteriol. (1961) 81(5): 741-746). BSB1 was obtained by transforming Bacillus subtilis 168 with chromosomal DNA from the trp+ strain HVS495 and selecting for colonies growing on minimal medium without tryptophan. One Trp+ colony was named BSB1 , (Nicolas P. et al., Science. 2012 Mar 2;335(6072):1103-1106).
The xynA gene was deleted using a marker free gene deletion clean knockout (Morimoto T. Genes Genet Syst. 2009 Aug;84(4):315-8). Three DNA fragments upstream and downstream of xynA were amplified from the BSB1 genomic DNA using primer pairs BW45 (SEQ ID NO: 4) and BW46 (SEQ ID NO: 5), BW41 (SEQ ID NO: 1) and BW42(SEQ ID NO: 2), BW41 (SEQ ID NO: 1) and BW44 (SEQ ID NO: 3).
A toxin DNA fragment (SEQ ID NO: 6) present in strain TMO310 (Morimoto et al (2009) Genes Genet Syst. 84, p. 315-318), with lac operator, inducible Pspac-masF and a spectinomycin resistance cassette was amplified using primers BW05 (SEQ ID NO: 7) and BW06 (SEQ ID NO: 8).
The four PCR fragments were purified and fused using Gibson Assembly and directly transformed to competent BSB1 cells. Transformants were selected on LB agar with 150 pg/ml spectinomycin. Cells were spread on LB agar with 1 mM IPTG to induce the MasF toxin and select for colonies that excised the xynA and the toxin fragment from the chromosome via homologous recombination. This resulted in the xynA deletion strain BWB06 with the following genotype trpC+, AxynA.
Subsequently, the amyE gene was deleted using the same clean knockout method. Three DNA fragments upstream and downstream of amyE were amplified from the BSB1 genomic DNA using primer pairs BW49 (SEQ ID NO: 23) and BW50 (SEQ ID NO: 24), BW51 (SEQ ID NO: 25) and BW52 (SEQ ID NO: 26), and BW53 (SEQ ID NO: 27) and BW54 (SEQ ID NO: 28).The three a yE fragments and together with the toxin DNA fragment were purified and fused using Gibson Assembly and directly transformed to competent BWB06 cells. Following a similar selection process, this resulted in the xynA and amyE double deletion strain BWB09 with the following genotype trpC+, AxynA, AamyE.
1.2. Construction of SGB03.
The ctsR gene (SEQ ID NO: 9 ) encoding the transcription repressor of class III heat shock protein CtsR (SEQ ID NO: 10) was deleted from strain BWB09. Strain BWB09 was transformed with a ctsR::ery deletion construct ( Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291- 305) and an erythromycin resistant colony containing the ctsR deletion was named strain SGB03, with the following phenotype trpC+, AxynA, AamyE ctsRwery.
1.3. Construction of xylanase expression plasmid pDBC4XAS-1
Construction of the xylanase expression plasmid pDBC4XAS-1 was previously described in WO2015118126, in the materials and method section and Figure 1.
Example 2. Expression endo-1 ,4-beta-xylanase in B. subtilis
Strain BWB09 and ctsR deletion strain SGB03 were transformed with the xylanase expression plasmid pDBC4XAS-1 as described above, which resulted in strains BWB09/ pDBC4XAS-1 and SGB03/ pDBC4XAS-1 , respectively. Strains BWB09/ pDBC4XAS-1 , SGB03/ pDBC4XAS-1- were grown in 10 ml LB medium with a start Oϋboo of 0.05 in 100 ml flasks under continuous shaking with 210 rpm at 37 °C, 50 pg/ml kanamycin was added to maintain the plasmids. Supernatants of cell cultures were sampled at 2, 3, 4, 6, 8, 10 and 15 hours. The cell density (Oϋboo) was measured. Supernatants were collected by flash freezing in liquid N2, and stored at -80 °C.
The xylanase activity was determined using the fluorescence based assay EnzChek® Ultra Xylanase Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The commercial Xylanase (Sigma, X2753) was used for construction of standard curve for the detection of xylanase and amylase enzyme activity. Growth and xylanase production of strains BWB09/pDBC4XAS-1 , and SGB03/pDBC4XAS-1 was followed in time and depicted in Table 1.
Strain SGB03/pDBC4XAS-1 which contains the ctsR deletion produces more xylanase than strain BWB09/pDBC4XAS-1 with intact ctsR gene. The difference in xylanase yield increases in time.
Table 1. Growth (Oϋboo) and relative xylanase activity (X) of strains BWB09/pDBC4XAS-1 (wf) and SGB03/pDBC4XAS-1 (ActsR) followed in time. Data represent average value of three independent biological replicates.
Example 3. Construction of B subtilis sporulation- and protease-deficient strain BWB143 and corresponding ctsR mutant BWB144 3.1. Construction of BWB143
Strain BWB143 was constructed by three-step deletion. Firstly, strain BSB1 was transformed with a spollE::ery deletion construct ( Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291- 305) and an erythromycin resistant colony was further transformed with pDR244 (temperature- sensitive plasmid with constitutively expressed Cre recombinase) and selected for spectinomycin resistant colony at 30 °C. The plasmid pDR244 was then removed by serial cultivation in LB liquid at 42°C, resulting in marker-less spollE deletion mutant named BWAB01 , with the following phenotype trpC+, AspollE. Secondly, BWAB02 was constructed similarly by transforming BWAB01 with a nprE::ery deletion construct (Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291-305) followed by pDR244, and resulting in marker-less spollE and nprE double deletion, with the following phenotype trpC+, AspollE, AnprE. Thirdly, BWB143 was constructed similarly by transforming BWAB02 with an aprE::ery deletion construct (Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291-305) followed by pDR244, and resulting in marker-less spollE, nprE and aprE triple deletion mutant named BWB143, with the following phenotype trpC+, AspollE, AnprE, AaprE. The marker-less deletion of spollE, nprE and aprE was verified but both internal and external primer pairs, TS22 (SEQ ID NO: 11) & TS23 (SEQ ID NO: 12) (spollE- Intern a i), ZT153 (SEQ ID NO: 13) & ZT155 (SEQ ID NO: 14) (spollE- External), BW278 (SEQ ID NO: 15) & BW279 (SEQ ID NO: 16) (nprE- Intern a I), BW274 (SEQ ID NO: 17) & BW275 (SEQ ID NO: 18) (nprE -External), BW280 (SEQ ID NO: 19) & BW281 (SEQ ID NO: 20) (aprE -Internal), BW276 (SEQ ID NO: 21) & BW277 (SEQ ID NO: 22) (aprE -External) respectively.
3.2. Construction of BWB144 Strain BWB144 was constructed by transforming strain BWB143 with a ctsR::ery deletion construct ( Koo et al., Cell Syst. 2017 Mar 22; 4(3): 291-305) and an erythromycin resistant colony containing the ctsR deletion was named strain BWB144, with the following phenotype trpC+, AspollE, AnprE, AaprE, ctsRv.ery.
Example 4. Expression endo-1,4-beta-xylanase in B. subtilis protease and sporulation-deficient background
Strain BWB143 and BWB144 containing a ctsR deletion, which were constructed as described in Example 3, were transformed with the xylanase expression plasmid pDBC4XAS-1 as described above, which resulted in strains BWB143/pDBC4XAS-1 and BWB144/pDBC4XAS-1 . Strains BWB143/pDBC4XAS-1 and BWB144/pDBC4XAS-1 were grown in 10 ml LB medium with a start OD600 of 0.05 in 100 ml flasks under continuous shaking with 210 rpm at 37 °C. 50 pg/ml kanamycin was added to maintain the plasmids. Supernatants of cell cultures were sampled at 0, 2, 3, 4, 6 and 8 hours. The cell density (OD600) was measured. Supernatants were collected by flash freezing in liquid N2, and stored at -80 °C. The xylanase activity was determined using method as described above. Growth and xylanase production of strains BWB143/pDBC4XAS-1 and BWB144/pDBC4XAS-1 was followed in time and depicted in Table 2.
Strain BWB144/pDBC4XAS-1 which contains the ctsR deletion produces more xylanase than strain BWB143/pDBC4XAS-1 with intact ctsR gene. The difference in xylanase yield increases in time. At 8 h, the relative xylanase activity in the medium of BWB144/pDBC4XAS-1 is 1.8-fold higher than the xylanase activity in the medium of BWB143/pDBC4XAS-1 .
Table 2. Growth (Oϋboo) and relative xylanase activity (X) of strains BWB143/pDBC4XAS-1 and BWB144/pD BC4XAS-1 ( ActsR ) followed in time. Data represent average value of two independent biological replicates.
The results in Table 3 show that the additional deletions AspollE, AnprE, AaprE in the Bacillus host strain (BWB143 / pDBC4XAS-1) and (BWB144 / pDBC4XAS-1) further increases the xylanase activity in the fermentation broth as compared to a Bacillus strain not containing these deletions. Table 3. Xylanase activity (U / ml) in the fermentation broth after 8 h of cultivation of strains BWB09/ pDBC4XAS-1 and SGB03/ pDBC4XAS-1 from Examples 1 and 2, and strains BWB143 / PDBC4XAS-1 and BWB144 / pDBC4XAS-1 of Examples 3 and 4.
Conclusion
Examples 2 and 4 show that a deletion of ctsR in a Bacillus strain increases the yield of a polypeptide of interest, such as a xylanase, in the Bacillus strain. Example 4 shows that a further deletion of the sporulation gene ( AspollE) and a deletion of neutral and alkaline protease ( AnprE , AaprE) in a Bacillus host strain further increases the yield of the polypeptide of interest by the Bacillus strain.

Claims

1. A recombinant Bacillus host cell comprising a polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the production of a compound of interest, wherein the host cell is deficient in the expression of a transcription repressor (CtsR) of class III heat shock genes.
2. The Bacillus host cell according to claim 1 , wherein the Bacillus host cell does not comprise a transcriptional fusion between a class III heat shock gene and the polynucleotide encoding a polypeptide of interest or encoding a polypeptide involved in the in the production of a compound of interest.
3. The host cell according to claim 1 or 2, wherein the transcription repressor comprises a polypeptide sequence of SEQ ID NO: 10, or a polypeptide sequence which has at least 70% identity to SEQ ID NO: 10.
4. The host cell according to any one of the claims 1 to 3, wherein the host cell comprises a deletion, disruption or mutation of a polynucleotide encoding the transcription repressor ( ctsR ) of class III heat shock genes, wherein the polynucleotide sequence encoding the transcription repressor has at least 70% identity to SEQ ID NO: 9.
5. The host cell according to any one of the claims 1 to 4, wherein the class III heat shock genes comprise clpC, clpE, clpX and / or clpP.
6. The host cell according to any one of the claims 1 to 5, wherein the host cell is further deficient in a sporulation-related gene, preferably deficient in a gene selected from the group consisting of spoOA, spollSA, spollAC, sigE, sigF, spollSB, spollE, sigG, spoIVCB, spolllC, spollGA, spollAA, spolVFB, spollR, and spolll.
7. The host cell according to any one of the claims 1 to 6, wherein the host cell is further deficient in the production of a neutral protease and / or an alkaline protease.
8. The host cell according to claim 7, wherein the host cell has a disruption, deletion or mutation in the gene nprE and / or aprE.
9. The host cell according to any one of the claims 1 to 8, wherein the Bacillus host cell belongs to a species selected from the group consisting of: B. agaradherens, B. alkalophilus, B. amyloliquefaciens, B. anthracis, B. atrophaeus, B. brevis, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. halodurans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. mojavensis, B. pumilus, B. puntis, B. sphaericus, B. stearothermophilus, B. subtilis, B. thuringiensis, and B. vallismortis.
10. A method for preparing a Bacillus host cell according to any one of the claims 1 to 9, comprising deleting, disrupting or mutating a polynucleotide encoding a transcription repressor of class III heat shock genes.
11 . A process for producing a polypeptide of interest, or a compound of interest, comprising cultivating the Bacillus host cell according to anyone of the claims 1 to 9 under conditions conducive for the production of the polypeptide or compound of interest.
12. The process according to claim 11 , further comprising recovering the polypeptide of interested or the compound of interest.
13. The host cell according to any one of the claims 1 to 9, or the method according to claim 10, or the process according to 11 or 12, wherein the polypeptide of interest comprises a xylanase, hemicellulase, cellulase, glucanase, alpha-galactoside, protease, pectinase, lipase, phospholipase or an amylase, such as an alpha amylase or maltogenic amylase, or any combination thereof.
14. The host cell according to any one of the claims 1 to 9 and 13, or the method according to claim 10 and 13, or the process according to claim 11 to 13, wherein the compound of interest is a metabolite, such as vitamin A, vitamin B2, pantothenic acid (vitamin B5), vitamin B6, vitamin C, vitamin D or vitamin E. .
EP22733942.1A 2021-06-17 2022-06-15 Improved bacillus host cell Pending EP4355762A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP21180004 2021-06-17
PCT/EP2022/066294 WO2022263504A1 (en) 2021-06-17 2022-06-15 Improved bacillus host cell

Publications (1)

Publication Number Publication Date
EP4355762A1 true EP4355762A1 (en) 2024-04-24

Family

ID=76807484

Family Applications (1)

Application Number Title Priority Date Filing Date
EP22733942.1A Pending EP4355762A1 (en) 2021-06-17 2022-06-15 Improved bacillus host cell

Country Status (2)

Country Link
EP (1) EP4355762A1 (en)
WO (1) WO2022263504A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118599879B (en) * 2024-07-03 2025-03-04 长江大学 Homologous recombinant Bacillus subtilis expressing cecropin B gene and construction method thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7585674B2 (en) 2001-05-29 2009-09-08 Kao Corporation Host microorganisms
AU2003218833A1 (en) * 2002-03-11 2003-09-22 Wageningen Centre For Food Sciences Listeria monocytogenes ctsr mutants presenting low virulence and stress-resistance
US7888489B2 (en) 2005-01-24 2011-02-15 Dsm Ip Assets B.V. Method for producing a compound of interest in a filamentous fungal cell
WO2008000632A1 (en) 2006-06-29 2008-01-03 Dsm Ip Assets B.V. A method for achieving improved polypeptide expression
WO2015118126A1 (en) 2014-02-07 2015-08-13 Dsm Ip Assets B.V. Improved bacillus host
EP3481959A1 (en) * 2016-07-06 2019-05-15 Novozymes A/S Improving a microorganism by crispr-inhibition
CN112225785B (en) * 2020-10-23 2022-05-10 中国科学院天津工业生物技术研究所 GntR family transcription inhibitor mutant, mutant gene and application thereof in preparation of vitamin B2In (1)

Also Published As

Publication number Publication date
WO2022263504A1 (en) 2022-12-22

Similar Documents

Publication Publication Date Title
JP7231228B2 (en) Compositions and methods for increased protein production in Bacillus licheniformis
CN105992817B (en) Improved bacillus host
CN101652468B (en) Recombinant microorganism
JP2009225804A (en) Enhanced protein expression in bacillus
EP2133416B1 (en) Recombinant microorganism
JP2019511218A (en) Engineered ribosomal promoter for protein production in microorganisms
JP2021510070A (en) Mutations and genetically modified Bacillus cells for increased protein production, and methods thereof
JP2025072450A (en) Compositions and methods for increased protein production in Bacillus licheniformis
JP5294666B2 (en) Recombinant microorganism
EP4355762A1 (en) Improved bacillus host cell
JP5513759B2 (en) Method for producing protein or polypeptide
JP7656603B2 (en) Compositions and methods for enhanced protein production in Bacillus cells
JP4832153B2 (en) Recombinant microorganism
JP4850011B2 (en) Recombinant microorganism
JP4676848B2 (en) Recombinant microorganism
JP2007130012A (en) Recombinant microorganism
JP7706628B2 (en) Modified 5' untranslated region and method for producing target substance using same
JP4842749B2 (en) Recombinant microorganism
JP6791623B2 (en) Recombinant microorganisms and their use
JP4643999B2 (en) Recombinant microorganism
JP7702230B2 (en) Method for producing monoacylglycerol lipase
JP4842750B2 (en) Recombinant microorganism
JP2025102402A (en) Method for modifying the 5' untranslated region
JP4685521B2 (en) Recombinant microorganism
CN117693587A (en) Modified Bacillus host cells with altered RemA/RemB proteins

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20231128

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: DSM IP ASSETS B.V.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20251113