WO2021224152A1 - Amélioration de l'expression dans les processus de fermentation - Google Patents

Amélioration de l'expression dans les processus de fermentation Download PDF

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WO2021224152A1
WO2021224152A1 PCT/EP2021/061506 EP2021061506W WO2021224152A1 WO 2021224152 A1 WO2021224152 A1 WO 2021224152A1 EP 2021061506 W EP2021061506 W EP 2021061506W WO 2021224152 A1 WO2021224152 A1 WO 2021224152A1
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promoter
gene
bacillus
region
sequence
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PCT/EP2021/061506
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Max Fabian FELLE
Christopher Sauer
Mathis APPELBAUM
Maximilian HILKMANN
Thomas Schweder
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Basf Se
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

Definitions

  • the present invention is concerned with materials and methods for industrial fermentation prolapses.
  • the invention is concerned with expression cassettes to facilitate the ex pression of a target gene under the control of a heterologous promoter.
  • the invention further pertains the construction of such promoters, vectors and host cells comprising such expression cassettes and fermentation methods using such host cells.
  • the invention provides materials obtained by such fermentation.
  • Promoters for expression of target genes in industrial fermentation processes have been exten sively studied. Of particular interest are promoters which are inducer independent. Such pro moters are allowed to express the target gene in a fermentation process without having to con stantly supply and inducer to the fermentation medium to achieve expression of the target gene.
  • promoters are the aprE promoter, amyL promoter, veg promoter, bacteriophage SP01 promoter, and the cry3A promoter
  • aprE promoter The native promoter from the gene encoding the Bacillus subtilisin Carlsberg protease, also referred to as aprE promoter, is well described in the art.
  • the aprE gene is transcribed by sigma factor A (sigA) and its expression is highly controlled by several regulators - DegU acting as activator of aprE expression, whereas AbrB, ScoC (hpr) and SinR are repressors of aprE ex pression (Ferrari, E., D.J.Henner, M.Perego, and J.A.Hoch. 1988. Transcription of Bacillus sub- tilis subtilisin and expression of subtilisin in sporulation mutants.
  • sigA sigma factor A
  • hpr ScoC
  • SinR SinR
  • J Bacteri ol 171 2657-2665; Gaur.N.K., J.Oppenheim, and I. Smith. 1991.
  • the Bacillus subtilis sin gene a regulator of alternate developmental processes, codes for a DNA-binding protein. J Bacteriol 173: 678-686; Kallio, P.T. , J.E.Fagelson, J.A.Hoch, and M.A.Strauch. 1991.
  • the transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein. Journal of Biological Chemistry 266: 13411-13417).
  • the core promoter region comprising the sigma factor A binding sites -35 and -10 have been mapped to the region nt -1 - nt -45 relative to the transcriptional start site (Park.S.S., S.L.Wong, L.F.Wang, and R.H.Doi. 1989. Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo. J Bacteriol 171: 2657-2665).
  • WO0151643 describes the increase of expression by mutating the -35 site of the wild type aprE promoter from TACTAA to the canonical TTGACA -35 site motif (Helmann.J.D. 1995. Compila tion and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for ex tended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23: 2351-2360).
  • the transcriptional start site is located at nt -58 relative to the start GTG of the aprE gene.
  • the 5’UTR comprises the ribosome binding site (Shine Dalgarno) and a sequence within nt -58 - nt -33 relative to the start GTG forming a very stable stem-loop structure of the 5’-end of the mRNA being responsible for high mRNA transcript stability of up to 25 min (Hambraeus, et al. , 2000, Microbiology. 146 Pt 12:3051-3059; Hambraeus et al. , 2002, Microbi ology.148(Pt 6): 1795- 1803).
  • nt -141 - nt -161 The region of nt -141 - nt -161 relative to the transcriptional start site has be shown to be re sponsible for full induction in a DegU (SacU) and DegQ (SacQ) dependent manner, whereas regions 5’ of nt -200 up to nt -600 are negatively regulated by ScoC (Hpr) (Henner.D.J.,
  • the ScoC (hpr) binding sites within the Bacillus subtilis aprE promoter region have been more precisely mapped revealing additional binding sites within the above 48 mentioned core pro moter region (Kallio, P.T., J.E.Fagelson, J.A.Hoch, and M.A.Strauch. 1991.
  • the transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein. Journal of Biological Chemistry 266: 13411-13417).
  • the binding site of the repressing transition state regulator ArbB has been mapped to nt -58 to + nt 15 relative to the transcriptional start site (Strauch.M.A., G.B.Spiegelman, M.Perego,
  • the transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J 8: 1615-1621).
  • the binding sites of the repressor SinR have been mapped to nt -233 to nt -268 relative to the transcriptional start site (Gaur.N.K., J.Oppenheim, and I. Smith. 1991.
  • the Bacillus subtilis sin gene a regulator of alternate developmental processes, codes for a DNA-binding protein. J Bacteriol 173: 678-686).
  • Jacobs et al Jacobs M, Eliasson M, Uhlen M, Flock Jl. 1985. Cloning, sequencing and expres sion of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res 13: 8913-8926; Ja cobs, M.F. 1995. Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis. Gene 152: 69-74) discloses the sequence of the aprE (subC) gene and its 5’ region of the Bacillus licheniformis NCIB6816 strain (GenBank accession No. X03341).
  • the transcriptional start site (TSS) is located at nt -73 and ac cordingly the 5’ UTR comprising nt -73 to nt -1 relative to the start ATG.
  • the ribosome binding site (Shine Dalgarno) is located at position nt -16 to nt -9.
  • the recognition sequence -10-site (TATAAT-box) of the sigma factor A is highly conserved and located at nt -84 to nt -79 whereas the -35 site (TACCAT) located 17 nt upstream of the -10 site is less conserved compared to standard sigma factor A dependent promoters in Bacillus (Helmann et al., 1995, Nucleic Acids Res. 23: 2351-2360).
  • Promoter truncations from the 5’ end comprising nt -122 to nt -1 and nt - 181 to nt -1 show 20-40 fold reduced subtilisin Carlsberg protease expression activities compared to ex pression with promoter fragment nt -225 to nt -1 (mutant 769, as described in Jacobs et al., 1995) in Bacillus subtilis strains with elevated regulators DegU (degU32H) or DegQ (degQ36H). Therefore, the binding sites of the regulator degU stimulating subtilisin Carlsberg expression lie within the region comprising nt -225 to nt -182.
  • W09102792 discloses the functionality of the promoter of the alkaline protease gene for the large-scale production of subtilisin Carlsberg-type protease in Bacillus licheniformis.
  • the subtil- isin Carlsberg is produced in a fermentation process using complex media components as ni trogen and carbon sources.
  • WO9102792 describes the 5’ region of the subtilisin Carlsberg protease encoding aprE gene of Bacillus licheniformis ( Figure 27) comprising the functional aprE gene promoter and the 5’UTR comprising the ribosome binding site (Shine Dalgarno sequence).
  • the truncated fragment thereof starting with the Aval restriction endonuclease site comprises the functional aprE gene promoter and the 5’UTR comprising the ribosome binding site (Shine Dal garno sequence) as exemplified by expression of subtilisin Carlsberg fusion protein consisting of the signal peptide of the aprE gene from Bacillus licheniformis and the propeptide sequence and mature sequence of the Bacillus lentus alkaline protease gene.
  • subtilisin Carlsberg fusion protein consisting of the signal peptide of the aprE gene from Bacillus licheniformis and the propeptide sequence and mature sequence of the Bacillus lentus alkaline protease gene.
  • the invention thus aspires to provide materials and methods to improve industrial fermentation processes, in particular by providing an expression cassette to achieve increased expression of a target gene under the control of a promoter.
  • the invention provides an expression cassette comprising a target gene under the control of a heterologous promoter of aprE-type, the promoter comprising a sigma factor A site comprising a -10 and a -35 motif, and a first enhancer element having a motif score of at least 100 and, preferably, a second enhancer element having a motif score of at least 100.
  • the invention also provides an expression cassette, obtainable or obtained by a method com prising or consisting of the steps: i) obtaining a promoter sequence having an HMM score above 50, ii) if required altering the promoter sequence to conform to the promoter as defined in any of the preceding claims, iii) bringing a heterologous target gene under the control of the promoter.
  • the invention provides a vector comprising an expression cassette according to the present invention.
  • the invention further provides a host cell comprising a vector of the present invention or, inte grated into its genome, an expression cassette according to the present invention.
  • the invention also provides a fermentation method for the production of a target protein, com prising cultivating a host cell according to the present invention in a suitable medium to produce the protein, preferably further comprising the step of isolating or purifying the target protein.
  • the invention provides a fermentation broth obtained by the method according to the present invention.
  • the invention provides the use of an expression cassette according to the present inven tion, a vector according to the present invention or a host cell according to the present invention for the production of a target protein coded by the target gene of the expression cassette.
  • Figure 1 shows an annotated sequence (SEQ ID NO. 6) of the 5' region to the aprE gene com prising a minimal promoter aprE (SEQ ID NO. 45) for use in an expression cassette according to the present invention.
  • Figure 2 shows an alignment of the aprE type promoters of SEQ ID NO. 2, 3, 4, 5 and 6.
  • Figure 3 shows the relative promoter activity in percent plotted against the indicated B. licheni- formis strains for the 24h and 48h timepoints of cultivation.
  • B. licheniformis strains M609.1A and M609.1 B carry a GFP reporter gene under control of the truncated and full-length aprE promot er of B. licheniformis DSM641, respectively.
  • B. licheniformis strains M609.2A and M609.2B car ry the GFP reporter gene under control of the truncated and full-length aprE promoter of B. li cheniformis DSM13, respectively.
  • the figure shows that the promoter activity of the aprE type promoter of the present invention is significantly increased over similar promoters both within 24h and 48h of fermentation.
  • nucleic acid optionally includes, as a practical matter, many copies of that nucleic acid mole cule; similarly, the term “probe” optionally (and typically) encompasses many similar or identical probe molecules.
  • probe optionally (and typically) encompasses many similar or identical probe molecules.
  • word “comprising” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
  • composition when used in reference to a measurable value, for example an amount of mass, dose, time, temperature, sequence identity and the like, refers to a variation of ⁇ 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or even 20% of the specified value as well as the specified value.
  • com prising about 50% X
  • the composition com prises 50% X whilst in other embodiments it may comprise anywhere from 40% to 60% X (i.e., 50% ⁇ 10%).
  • the term "gene” refers to a biochemical information which, when materialised in a nucleic acid, can be transcribed into a gene product, i.e. a further nucleic acid, preferably an RNA, and preferably also can be translated into a peptide or polypeptide.
  • the term is thus also used to indicate the section of a nucleic acid resembling said information and to the sequence of such nucleic acid (herein also termed "gene sequence").
  • alleles or nucleotide sequence variants of the invention have at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%,
  • nucleotide sequence identity to the nucleotide sequence of the wild type gene.
  • nucleic acid sequence of the allele has at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%,
  • amino acid identity to the respective wild type peptide or polypeptide.
  • Mutations or alterations of amino or nucleic acid sequences can be any of substitutions, dele tions or insertions; the terms “mutations” or “alterations” also encompass any combination of these.
  • Protein or nucleic acid variants may be defined by their sequence identity when compared to a parent protein or nucleic acid. Sequence identity usually is provided as “% sequence identity” or “% identity”. To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e. , a pairwise global alignment). The align ment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p.
  • the preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.
  • Seq A AAGATACTG length: 9 bases
  • Seq B GATCTGA length: 7 bases
  • sequence B is sequence B.
  • Seq B The ⁇ ” symbol in the alignment indicates identical residues (which means bases for DNA or amino acids for proteins). The number of identical residues is 6.
  • the symbol in the alignment indicates gaps.
  • the number of gaps introduced by alignment within the sequence B is 1.
  • the number of gaps introduced by alignment at borders of se quence B is 2, and at borders of sequence A is 1.
  • the alignment length showing the aligned sequences over their complete length is 10.
  • the alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence).
  • sequence A is the sequence of the invention
  • alignment length showing se quence B over its complete length would be 8 (meaning sequence B is the sequence of the in vention).
  • %-identity (identical residues / length of the alignment region which is showing the respective sequence of this invention over its complete length) *100.
  • sequence identity in relation to comparison of two amino acid sequences according to the invention is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respec tive sequence of this invention over its complete length. This value is multiplied with 100 to give “%-identity”.
  • hybridisation is a process wherein substantially complementary nucleotide sequences anneal to each other.
  • the hybridisation process can occur entirely in so lution, i.e. both complementary nucleic acids are in solution.
  • the hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • the hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a sili ceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • the nucleic acid molecules are generally thermal ly or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • stringency refers to the conditions under which a hybridisation takes place.
  • the stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below Tm, and high stringency conditions are when the temperature is 10°C below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the de generacy of the genetic code. Therefore, medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.
  • the “Tm” is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe.
  • the Tm is dependent upon the solution con ditions and the base composition and length of the probe. For example, longer sequences hy bridise specifically at higher temperatures.
  • the maximum rate of hybridisation is obtained from about 16°C up to 32°C below Tm.
  • the presence of monovalent cations in the hybridisation solu tion reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher con centrations, this effect may be ignored).
  • Formamide reduces the melting temperature of DNA- DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisa tion will be lowered.
  • Base pair mismatches reduce the hybridisation rate and the thermal stabil ity of the duplexes.
  • the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids: DNA-DNA hybrids (Meinkoth and Wahl, Anal.
  • Tm 79.8 + 18.5 (log10[Na+] ⁇ a ⁇ ) + 0.58 (%G/C ⁇ b ⁇ ) + 11.8 (%G/C ⁇ b ⁇ )2 - 820/L ⁇ c ⁇
  • ⁇ In ⁇ effective length of primer 2* (no. of G/C)+(no. of A/T)
  • Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterolo gous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase.
  • a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively lower ing the formamide concentration (for example from 50% to 0%).
  • annealing temperature for example from 68°C to 42°C
  • progressively lower ing the formamide concentration for example from 50% to 0%.
  • hybridisation typically also depends on the function of post-hybridisation washes.
  • samples are washed with dilute salt solutions.
  • Critical factors of such washes in clude the ionic strength and temperature of the final wash solution: the lower the salt concentra tion and the higher the wash temperature, the higher the stringency of the wash. Wash condi tions are typically performed at or below hybridisation stringency.
  • a positive hybridisation gives a signal that is at least twice of that of the background.
  • suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected.
  • typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% forma- mide, followed by washing at 65°C in 0.3x SSC.
  • Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC.
  • the length of the hybrid is the anticipated length for the hybridising nucleic acid.
  • the hybrid length may be determined by aligning the se quences and identifying the conserved regions described herein.
  • 1 xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1.0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
  • high stringency conditions is hybridisation at 65°C in 0.1x SSC comprising 0.1 SDS and optionally 5x Denhardt's reagent, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by the washing at 65°C in 0.3x SSC.
  • nucleic acid construct refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or is synthetic.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a polynucleo tide.
  • control sequence is defined herein to include all sequences affecting the expression of a polynucleotide, including but not limited thereto, the expression of a polynucleotide encod ing a polypeptide.
  • Each control sequence may be native or foreign to the polynucleotide or na tive or foreign to each other.
  • control sequences include, but are not limited to, promoter sequence, 5’-UTR (also called leader sequence), ribosomal binding site (RBS, Shine Dalgarno sequence), 3’-UTR, and transcription start and stop sites.
  • a regulatory element including but not limited thereto a promoter
  • further regulatory elements including but not limited thereto a terminator
  • a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide se quence such that the control sequence directs the expression of the coding sequence of a poly peptide.
  • a “promoter” or “promoter sequence” is a nucleotide sequence located upstream of a gene on the same strand as the gene that enables that gene's transcription. Promoter is followed by the transcription start site of the gene. A promoter is recognized by RNA polymerase (together with any required transcription factors), which initiates transcription. A functional fragment or func- tional variant of a promoter is a nucleotide sequence which is recognizable by RNA polymerase, and capable of initiating transcription.
  • active promoter fragment describes a fragment or variant of the nucleotide sequences of a promoter, which still has promoter activity.
  • an “inducer dependent promoter” is understood herein as a promoter that is increased in its activity to enable transcription of the gene to which the promoter is operably linked upon addi tion of an "inducer molecule" to the fermentation medium.
  • an inducer-dependent pro- moter the presence of the inducer molecule triggers via signal transduction an increase in ex pression of the gene operably linked to the promoter.
  • the gene expression prior activation by the presence of the inducer molecule does not need to be absent, but can also be present at a low level of basal gene expression that is increased after addition of the inducer molecule.
  • the "inducer molecule” is a molecule which presence in the fermentation medium is capable of af- fecting an increase in expression of a gene by increasing the activity of an inducer-dependent promoter operably linked to the gene.
  • the inducer molecule is a carbohydrate or an analogue thereof.
  • the inducer molecule is a secondary carbon source of the Bacillus cell. In the presence of a mixture of carbohydrates cells selectively take up the car bon source that provide them with the most energy and growth advantage (primary carbon source). Simultaneously, they repress the various functions involved in the catabolism and up take of the less preferred carbon sources (secondary carbon source).
  • a primary car bon source for Bacillus is glucose and various other sugars and sugar derivates being used by Bacillus as secondary carbon sources.
  • Secondary carbon sources include e.g. mannose or lac tose without being restricted to these.
  • the activity of promoters that do not depend on the presence of an inducer molecule are either constitutively active or can be increased regardless of the presence of an induc er molecule that is added to the fermentation medium.
  • the inducer- independent promoter is an aprE promoter.
  • aprE promoter is the nucleotide se quence (or parts or variants thereof) located upstream of an aprE gene, i.e. , a gene coding for a Bacillus subtilisin Carlsberg protease, on the same strand as the aprE gene that enables that aprE gene’s transcription.
  • transcription start site or “transcriptional start site” shall be understood as the location where the transcription starts at the 5’ end of a gene sequence.
  • +1 is in general an adenosine (A) or guanosine (G) nucleotide.
  • A adenosine
  • G guanosine
  • expression means the transcription of a specific gene or specif- ic genes or specific nucleic acid construct.
  • expression in par ticular means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
  • vector is defined herein as a linear or circular DNA molecule that comprises a poly nucleotide that is operably linked to one or more control sequences that provides for the ex pression of the polynucleotide.
  • isolated DNA molecule refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state.
  • isolated preferably refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state.
  • DNA molecules fused to regulatory or coding sequences with which they are not normally asso ciated, for example as the result of recombinant techniques, are considered isolated herein.
  • Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, in that they are not in their native state.
  • PCR polymerase chain reaction
  • Polynucleotide molecules, or fragment thereof can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthe sizer.
  • a polynucleotide can be single-stranded (ss) or double- stranded (ds).
  • Double-stranded refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions.
  • the polynucleotide is at least one selected from the group consisting of sense single- stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), a double-stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; a mixture of pol ynucleotides of any of these types can be used.
  • heterologous or exogenous or foreign or recombinant or non-native polypeptide is defined herein as a polypeptide that is not native to the host cell, a polypeptide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to alter the native polypeptide, or a polypeptide native to the host cell whose expression is quantitatively altered or whose expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter.
  • heterologous polynucleotide refers to a polynucleotide that is not native to the host cell, a polynucleotide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to alter the native polynucleotide, or a polynucleotide native to the host cell whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter, or a polynucleotide native to the host cell, but integrated not within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques.
  • heterologous is used to characterized that the two or more polynucleotide sequences or two or more amino acid sequences are naturally not occurring in the specific combination with each other.
  • heterologous when referring to a promoter-gene combination means that the specific combination of promoter and gene is not found in nature.
  • a promotor is heterologous to a gene and vice versa in particular when (a) a promoter, which in a wild type cell is operably linked to a gene A, is now operably linked instead to another gene B, or (b) where a promotor not found in nature is operably linked to a gene, or (c) where a promotor is operably linked to a gene of a sequence not found in nature.
  • host cell includes any cell type that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector.
  • host cell includes cells that have the capacity to act as a host or ex pression vehicle for a newly introduced DNA sequence, in particular for expression of a target gene comprised in said newly introduced DNA sequence.
  • recombinant when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation.
  • a gene sequence open reading frame is recombinant if (a) that nu cleotide sequence is present in a context other than its natural one, for example by virtue of being (i) cloned into any type of artificial nucleic acid vector or (ii) moved or copied to another location of the original genome, or if (b) the nucleotide sequence is mutagenized such that it differs from the wild type sequence.
  • the term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid is a recombinant plant.
  • transgenic refers to an organism, preferably a plant or part thereof, or a nucleic acid that comprises a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to refer to any cell or cell line the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell.
  • a "recombinant" organism preferably is a "transgenic” organism.
  • mutagenized refers to an organism or nucleic acid thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wildtype organism or nucleic acid, wherein the alteration(s) in genetic material were induced and/or selected by human action.
  • Methods of inducing muta tions can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e. , can be directed mutagenesis techniques), such as by use of a genoplasty technique.
  • a nucleic acid can also be mutagenized by using mutagenesis means with a preference or even specificity for a particular site, thereby creating an artificially induced heritable allele ac cording to the present invention.
  • Such means for example site specific nucleases, including for example zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nu- celases (TALENS) (Mal leopard et al., Cell Biosci, 2017, 7:21) and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA (for example as a single-guide RNA, or as modified crRNA and tracrRNA mol ecules which form a dual molecule guide), and methods of using this nucleases to target known genomic locations, are well-known in the art (see reviews by Bortesi and Fischer, 2015, Bio technology Advances 33: 41-52; and by Chen and Gao, 2014, Plant Cell Rep 33: 575-583, and references within).
  • ZFNs zinc finger nucleases
  • TALENS transcription activator-like effector nu- celases
  • GMO genetically modified organism
  • the source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant).
  • nucleotide or polypeptide refers to the cell or organism as found in nature and to the polynucleotide or polypeptide in question as found in a cell in its natural form and genetic environment, respectively (i.e. , without there being any human intervention).
  • wildtype or "corresponding wildtype plant” means the typical form of an organ ism or its genetic material, as it normally occurs, as distinguished from e.g. mutagenized and/or recombinant forms.
  • control cell or “wildtype host cell” is intended a cell that lacks the particular polynucleotide of the invention that are disclosed herein.
  • wildtype is not, therefore, intended to imply that a host cell lacks recombinant DNA in its ge nome, and/or does not possess fungal resistance characteristics that are different from those disclosed herein.
  • the invention provides an expression cassette comprising a target gene under the control of a heterologous promoter.
  • the promoter is of aprE-type. Preferred promoters are described herein and are available to the skilled person.
  • the promoter comprises a sigma factor A site comprising a -10 and a -35 motif, and a first enhancer element having a motif score of at least 100 and preferably a second enhancer element having a motif score of at least 100.
  • a promoter with these features leads to an increased expression of a target gene operably linked thereto. This was particularly unexpected as promoters of aprE-type had been well studied before.
  • the motif score is calculated as follows: The putative promoter sequence is aligned to the se quence according to SEQ ID NO. 44. Let n be the length of the motif to be assessed. Then, starting at the first nucleotide of the putative promoter, the nucleotides up to position n are com pared to their respective scores in the corresponding motif score table and the respective scores are totalled.
  • the score value for the first nucleotide according to table 1 is added to the score of the second nucleotide according to table 1 and so on until the score of the seventh nucleotide according to table 1 is added. Then, if the sum is at least 100, the presence of a first enhancer element at sequence position 1-7 is confirmed. If the respective element cannot be confirmed at position 1 to n, then the window of analysis is shifted to the next consecutive nucleotides 2 to n+1 of the putative promoter sequence and the process is repeated. The process is repeated until the presence of the respective enhancer has been confirmed. Of course, when the number of remaining nucleo tides is smaller than n then the presence of the enhancer element cannot be confirmed for the remaining nucleotides; the respective enhancer element is thus not found at those positions.
  • the invention is not limited to any particular biological enhancer or transcription factor recogniz ing any of the first, second or third enhancer elements or of the repressor element.
  • the term “enhancer” or “repressor” is attached to each element only to denote that, for an enhancer ele ment, the presence of this element is preferred according to the invention, or discouraged for a repressor element.
  • the first enhancer element is preferably located in the region of -202 to -102, more preferably in the region of -191 to -169, even more preferably in the region of -186 to -174 and most prefera bly in the region of -183 to -177.
  • a binding site is defined to be position number -12, all nucleo- tides more upstream of this are then numbered by decreasing integers (-13, -14 and so on).
  • nucleotide in 3' direction next to position -1 is numbered +1 and all further nucleotides in 3' direction are successively numbered by increasing integers (2, 3 and so on). This numbering is depicted in figure 1. As shown in the examples absence of such first enhancer element leads to a significant decrease in expression of a target gene.
  • the second enhancer element is preferably located in the region of -189 to -100, more prefera bly in the region of -179 to -154, even more preferably in the region of -174 to -159 and most preferably in the region of -171 to -162.
  • the promoter further preferably comprises, immediately downstream of the second enhancer element, a region of 10 nucleotides length enriched in strong or weak type nucleotides such that the ratio of strong:weak nucleotides is either at least 6:4 or at least 4:6, more preferably at least 7:3 or at least 3:7, respectively.
  • the nucleotides adenine (A) and thymidine (T) are collectively called “weak” type nucleotides and the remaining cytidine and guanosine nucleotides are called "strong" type nucleotides.
  • the promoter comprises a third enhancer element having a motif score of at least 100.
  • the third enhancer element if present, is preferably located in the region of -152 to - 100, more preferably in the region of -142 to -115, even more preferably in the region of -137 to -120 and most preferably in the region of -134 to -123. As shown in the examples the presence of this enhancer element is not sufficient to achieve the significant increase in expression strength of the promoter of the present invention. However, the third enhancer element is found in preferred promoters of the present invention, for example according to SEQ ID NO. 6, 44 or 45, and it is thus preferred that the element is present.
  • the promoter according to the present invention preferably does not comprise a repressor ele ment which has the sequence of SEQ ID NO. 47 or differs from SEQ ID NO. 47 by up to 2 nu cleotides and wherein the repressor element covers position -131.
  • a repressor ele ment which has the sequence of SEQ ID NO. 47 or differs from SEQ ID NO. 47 by up to 2 nu cleotides and wherein the repressor element covers position -131.
  • Such repressor element is found in some aprE-type promoters. However, such repressor element would collide with the presence of the preferred third enhancer element.
  • the promoter according to the present invention preferably does not comprise such repressor element, or if it does, then preferably position -131 of the promoter does not fall within the repressor element.
  • the promoter preferably further comprises a degU binding motif.
  • DegU is a transcription factor known to increase expression of aprE-type promoters.
  • the promoter further comprises one more more binding motifs of regulatory factors selected from ScoC, hpr, SinR and AbrB. These regulatory factors are primarily negative regula tors. However, presence of such binding sites is preferred to improve correct timing of expres sion of the target gene during an industrial fermentation process.
  • the promoter comprises a 5'UTR.
  • This is a transcribed but not translated re gion downstream of the -1 promoter position.
  • Such untranslated region for example should con tain a ribosome binding site to facilitate translation in those cases where the target gene codes for a peptide or polypeptide.
  • the invention in particular teaches to combine the promoter of the present invention with a 5'UTR comprising one or more stabilising elements.
  • the mRNAs synthesized from the promoter region may be processed to generate mRNA transcript with a stabilizer sequence at the 5' end of the transcript.
  • a stabilizer sequence at the 5'end of the mRNA transcripts increases their half-life as described by Hue et al, 1995, Journal of Bacteriology 177: 3465-3471.
  • Suitable mRNA stabilizing elements are those de scribed in
  • WO0814857 preferably SEQ ID NO. 1 to 5 of W008140615, or fragments of these se quences which maintain the mRNA stabilizing function, and in
  • W008140615 preferably Bacillus thuringiensis CrylllA mRNA stabilising sequence or bac teriophage SP82 mRNA stabilising sequence, more preferably a mRNA stabilising sequence according to SEQ ID NO. 4 or 5 of W008140615, more preferably a modified mRNA stabilising sequence according to SEQ ID NO. 6 of W008140615, or fragments of these sequences which maintain the mRNA stabilizing function.
  • Preferred mRNA stabilizing elements are selected from the group consisting of aprE, grpE, cotG, SP82, RSBgsiB, CrylllA mRNA stabilizing elements, preferably mRNA stabilising ele ments according to SEQ ID NO. 48 to 52 respectively (corresponding to SEQ ID NO. 1 to 5, respectively, of WO08148575), and according to SEQ ID NO. 53 or 54 (corresponding to SEQ ID NO. 4 and 6, respectively of W008140615), or according to fragments of these sequences which maintain the mRNA stabilizing function.
  • a preferred mRNA stabilizing element is the grpE mRNA stabilizing element, preferably according to SEQ ID NO. 49 (corresponding to SEQ ID NO. 2 of WO08148575).
  • the 5'UTR also preferably comprises a modified rib leader sequence located downstream of the promoter and upstream of an ribosome binding site (RBS).
  • a rib leader is herewith defined as the leader sequence upstream of the riboflavin biosyn thetic genes (rib operon) in a Bacillus cell, more preferably in a Bacillus subtilis cell.
  • the rib operon comprising the genes involved in riboflavin biosynthesis, include ribG (ribD), ribB (ribE), ribA, and ribH genes. Transcription of the riboflavin operon from the rib pro moter (Prib) in B.
  • subtilis is controlled by a riboswitch involving an untranslated regulatory lead er region (the rib leader) of almost 300 nucleotides located in the 5'-region of the rib operon be tween the transcription start and the translation start codon of the first gene in the operon, ribG.
  • rib leader an untranslated regulatory lead er region
  • Suitable rib leader sequences are described in WO2015/1181296, in particular pages 23-25, incorporated herein by reference.
  • the invention also provides an expression cassette obtainable or obtained by a method com prising or consisting of the steps: i) obtaining a promoter sequence sequence having an HMM score above 50, ii) if required altering the promoter sequence to conform to the promoter as defined in any of the preceding claims, iii) bringing a heterologous target gene under the control of the promoter.
  • the "HMM score” is the score value obtained by the method used in Example 2.
  • suitable further promoters are found - for example the aprE-type promoters of sequences SEQ ID NO. 49 to 196 as described in example 2 - , converted into promoters according to the present invention and made use of by coupling them with a target gene to be expressed.
  • suitable promot ers can be reliably identified in highly varying sources, and still can be converted into strong promoters for the expression of a target gene, preferably for a microbial host as described here in.
  • the present invention advantageously not only provides, for use in an expression cas sette of the present invention, the promoters according to SEQ ID NO.
  • promot ers having at least 80%, more preferably at least 85%, more preferably at least 89%, more pref erably at least 90%, more preferably at least 91%, more preferably at least 92%, more prefera bly at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to any of sequences SEQ ID NO. 6, 44 or 45, provided that the promot ers that differ from SEQ ID NO. 44 comprise the first and preferably also one or more of the further elements (other than the putative repressor element at position -131) as described here in, further preferably at the respective locations as described with respect to the elements.
  • the target gene preferably codes for an enzyme, and more preferably the enzyme is selected from the group consisting of amylase, catalase, cellulase, chitinase, cutinase, galactosidase, beta-galactosidase, glucoamylase, glucosidase, hemicellu- lase, invertase, laccase, lipase, mannanase, mannosidase, nuclease, oxidase, pectinase, phosphatase, phytase, protease, ribonuclease, transferase and xylanase, more preferably a protease, amylase or lipase, most preferably a protease.
  • the promoter of the expression cassette is capable of driving expression of a variety of target genes common in industrial fermentation processes.
  • the invention also provides a vector comprising an expression cassette as described herein.
  • the vector facilitates the transformation of host cells to have the target gene expressed, prefer ably in an industrial fermentation process.
  • the invention also provides a host cell comprising a vector according to the present invention, and the invention provides a host cell having integrated into its genome an expression cassette according to the present invention.
  • the host cell according to the invention preferably belongs to the taxonomic genus Bacillus, more preferably to any of the species Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus subtilis or Bacillus velezensis, and most preferably belongs to the species Bacillus li cheniformis.
  • Bacillus microorganisms are commonly used in fermentation processes.
  • the expression cassette provided herein is adapted to facilitate or cause a strong expression of the target gene in such Bacillus microorganisms, thereby helping to increase the yield of a target protein in an industrial fermentation process.
  • Bacillus licheniformis is selected from the group consisting of Bacillus licheni formis ATCC 14580, ATCC 31972, ATCC 53926, ATCC 53757, ATCC 55768, DSM 13, DSM 394, DSM 641, DSM 1913, DSM 11259, and DSM 26543.
  • the host cell accord ing to the invention belongs to a Bacillus licheniformis species encoding a restriction modifica tion system having a recognition sequence GCNGC.
  • a bacterial host cell may additionally contain modifications, e.g., deletions or disrup tions, of other genes that may be detrimental to the production, recovery or application of a pol ypeptide of interest.
  • a bacterial host cell is a protease-deficient cell.
  • the bacterial host cell e.g., Bacillus cell, preferably comprises a disruption or deletion of extracellu lar protease genes including but not limited to aprE, mpr, vpr, bpr, and / or epr. Further prefera bly the bacterial host cell does not produce spores.
  • the bacterial host cell comprises a disruption or deletion of spollAC, sigE, and / or sigG.
  • the bacterial host cell e.g., Bacillus cell
  • the bacterial host cell comprises a disruption or deletion of one of the genes involved in the biosynthesis of polyglutamic acid.
  • Oth er genes including but not limited to the amyE gene, which are detrimental to the production, recovery or application of a polypeptide of interest may also be disrupted or deleted.
  • the invention also provides a fermentation method for the production of a target protein, comprising cultivating a host cell according to the invention in a suitable medium to pro prise the protein, preferably further comprising the step of isolating or purifying the target pro tein.
  • the host comprises an expression cassette according to the invention which, in turn, comprises a target gene coding for the target protein.
  • the invention also provides a fermentation broth obtained by the fermentation method accord ing to the present invention.
  • Such fermentation broth comprises high concentrations of the tar get protein when the protein is secreted or the host cells are ruptured during downstream pro cessing; if the protein is not secreted, then the fermentation broth comprises host cells which, in turn, feature a high concentration of the target protein.
  • the fermentation broth advantageously lends itself to the production of a product comprising or made using the target protein.
  • the invention teaches the use of an expression cassette ac cording to the present invention, a vector according to the present invention or a host cell ac cording to the present invention for the production of a target protein coded by the target gene of the expression cassette.
  • Such use realises the advantages conferred by the promoter-gene combination provided by the present invention.
  • the invention is further described by way of examples. These examples are for illustrative pruposes and are not intended to limit the scope of the invention or of the claims.
  • Example 1 Comparison of a promoter of the present invention to similar promoters
  • Transformation of DNA into Bacillus licheniformis strain DSM641 is performed via electro poration. Preparation of electrocompetent Bacillus licheniformis cells and transformation of DNA is performed as essentially described by Brigidi et al (Brigidi.P., Mateuzzi.D. (1991). Biotechnol. Techniques 5, 5) with the following modification: Upon transformation of DNA, cells are recov ered in 1 ml LBSPG buffer and incubated for 60 min at 37°C (Vehmaanpera J., 1989, FEMS Microbio. Lett., 61 : 165-170) following plating on selective LB-agar plates.
  • plasmid DNA is isolated from Ec#098 cells as described below.
  • plasmid DNA is isolated from E. coli INV110 cells (Life technologies).
  • Transformation of DNA into Bacillus subtilis ATCC6051a is performed via electroporation as described for Bacillus licheniformis. Plasmid DNA isolated from E.coli DH10B cells can be readi ly used for transfer into Bacillus subtilis.
  • Plasmid DNA was isolated from Bacillus and E. coli cells by standard molecular biology meth ods described in (Sambrook, J. and Russell, D.W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) or the alkaline lysis method (Birnboim, H. C., Doly, J. (1979). Nucleic Acids Res 7(6): 1513-1523). Bacillus cells were in comparison to E. coli treated with 10 mg/ml lysozyme for 30 min at 37°C prior to cell lysis.
  • oligonucleotides 1.0.4 Annealing of oligonucleotides to form oligonucleotide-duplexes. Oligonucleotides were adjusted to a concentration of 100pM in water. 5pl of the forward and 5pl of the corresponding reverse oligonucleotide were added to 90pl 30mM Hepes-buffer (pH 7.8). The reaction mixture was heated to 95°C for 5min following annealing by ramping from 95°C to 4°C with decreasing the temperature by 0.1°C/sec (Cobb, R. E., Wang, Y., & Zhao, H. (2015). High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synthetic Biology, 4(6), 723-728).
  • E. coli strain Ec#098 is an E. coli INV110 strain (Life technologies) carrying the DNA- methyltransferase encoding expression plasmid pMDS003 WO2019016051.
  • Plasmid DNA was isolated from indi vidual clones and used for subsequent transfer into Bacillus licheniformis strains.
  • the isolated plasmid DNA carries the DNA methylation pattern of Bacillus licheniformis strains DSM641 re spectively and is protected from degradation upon transfer into B. licheniformis.
  • Electrocompetent Bacillus licheniformis DSM641 cells (US5352604) were prepared as de scribed above and transformed with 1 pg of pDel006 restrictase gene deletion plasmid isolated from E. coli Ec#098 following plating on LB-agar plates containing 5 pg/ml erythromycin at 30°C.
  • the gene deletion procedure was performed as follows:
  • Plasmid carrying Bacillus licheniformis cells were grown on LB-agar plates with 5 pg/ml eryth romycin at 45°C driving integration of the deletion plasmid via Campbell recombination into the chromosome with one of the homology regions of pDel006 homologous to the sequences 5’ or 3’ of the restrictase gene.
  • Clones were picked and cultivated in LB-media without selection pressure at 45°C for 6 hours, following plating on LB-agar plates with 5 pg/ml erythromycin and incubation overnight at 30°C. Individual clones were picked and screened by colony-PCR anal- ysis with oligonucleotides SEQ ID NO.
  • Electrocompetent B. licheniformis P304 cells were prepared as described above and trans formed with 1 pg of pDel005 sigF gene deletion plasmid isolated from E. coli INV110 cells (Life technologies) following plating on LB-agar plates containing 5 pg/ml erythromycin and incuba tion overnight at 30°C.
  • the gene deletion procedure was performed as described for the restrictase gene.
  • the deletion of the sigF gene was analyzed by PCR with oligonucleotides SEQ ID NO. 25 and SEQ ID NO. 26.
  • the resulting B. licheniformis strain with a deleted sigF gene is designated B. licheniformis P305 and is no longer able to sporulate as described (WO9703185).
  • Electrocompetent Bacillus licheniformis P305 cells were prepared as described above and transformed with 1 pg of pDel003 aprE gene deletion plasmid isolated from E. coli INV110 cells following plating on LB-agar plates containing 5 pg/ml erythromycin and incubation overnight at 30°C.
  • the gene deletion procedure was performed as described for the deletion of the restrictase gene.
  • the deletion of the aprE gene was analyzed by PCR with oligonucleotides SEQ ID NO. 22 and SEQ ID NO. 23
  • the resulting Bacillus licheniformis strain with deleted aprE gene was named B. licheniformis P307.
  • Bacillus licheniformis M309 deleted poly-gamma glutamate synthesis genes
  • Electrocompetent Bacillus licheniformis P307 cells were prepared as described above and transformed with 1 pg of pDel007 pga gene deletion plasmid isolated from E. coli INV110 cells (Life technologies) following plating on LB-agar plates containing 5 pg/ml erythromycin and in cubation overnight at 30°C.
  • the gene deletion procedure was performed as described for the deletion of the restrictase gene.
  • Electrocompetent Bacillus licheniformis M309 cells were prepared as described above and transformed with 1 pg of pCC043 plasmid isolated from E. coli INV110 cells following plating on LB-agar plates containing 20 pg/ml kanamycin and incubation overnight at 37°C.
  • B. licheniformis strain M609.1B was constructed as described for B. licheniformis strain M609.1A, however plasmid pCC047 with expression construct PaprE fl. DSM641-GFPmut2 was used.
  • B. licheniformis strain M609.2A was constructed as described for B. licheniformis strain M609.1A, however plasmid pCC048 with expression construct PaprE trunc. DSM13-GFPmut2 was used.
  • B. licheniformis strain M609.2B was constructed as described for B. licheniformis strain M609.1A, however plasmid pCC049 with expression construct PaprE fl. DSM13-GFPmut2 was used.
  • the plasmid pE194 is PCR-amplified with oligonucleotides SEQ ID NO. 9 and SEQ ID NO. 10 with flanking Pvull sites, digested with restriction endonuclease Pvull and ligated into vector pCE1 digested with restriction enzyme Smal.
  • pCE1 is a pUC18 derivative, where the Bsal site within the ampicillin resistance gene has been removed by a silent mutation.
  • the ligation mix ture was transformed into E. coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37°C on LB-agar plates containing 100pg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting plasmid is named pEC194S.
  • the type-ll-assembly mRFP cassette is PCR-amplified from plasmid pBSd141R (accession number: KY995200) (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Ba- cillus subtilis. Sci. Rep. 7: 14134) with oligonucleotides SEQ ID NO. 11 and SEQ ID NO. 12, comprising additional nucleotides for the restriction site BamHI.
  • the PCR fragment and pEC194S were restricted with restriction enzyme BamHI following ligation and transformation into E. coli DH10B cells (Life technologies).
  • Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest.
  • the resulting plasmid pEC194RS carries the mRFP cassette with the open reading frame opposite to the reading frame of the erythromycin resistance gene.
  • the gene deletion plasmid for the aprE gene of Bacillus licheniformis was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID NO. 21 comprising the genomic regions 5’ and 3’ of the aprE gene flanked by Bsal sites compatible to pEC194RS.
  • the type-ll- assembly with restriction endonuclease Bsal was performed as described (Radeck et al. , 2017; Sci. Rep. 7: 14134) and the reaction mixture subsequently transformed into E. coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37°C on LB-agar plates containing 100pg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting aprE deletion plasmid is named pDel003.
  • the gene deletion plasmid for the sigF gene (spollAC gene) of Bacillus licheniformis was con structed as described for pDel003, however the gene synthesis construct SEQ ID NO. 24 com prising the genomic regions 5’ and 3’ of the sigF gene flanked by Bsal sites compatible to pEC194RS was used.
  • the resulting sigF deletion plasmid is named pDel005.
  • the gene deletion plasmid for the restrictase gene (SEQ ID NO. 14) of the restriction modifica tion system of Bacillus licheniformis DSM641(SEQ ID NO. 13) was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID NO. 15 comprising the genomic regions 5’ and 3’ of the restrictase gene flanked by Bsal sites compatible to pEC194RS.
  • the type-ll- assembly with restriction endonuclease Bsal was performed as described above and the reac tion mixture subsequently transformed into E. coli DH10B cells (Life technologies). Trans formants were spread and incubated overnight at 37°C on LB-agar plates containing 100pg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting restrictase deletion plasmid is named pDel006.
  • the deletion plasmid for deletion of the genes involved in poly-gamma-glutamate (pga) produc tion namely ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), ywtC (pgsE) of Bacillus licheniformis was constructed as described for pDel006, however the gene synthesis construct SEQ ID NO. 18 comprising the genomic regions 5’ and 3’ flanking the ywsC(pgsB), ywtA (pgsC), ywtB (pgsA), ywtC (pgsE) genes flanked by Bsal sites compatible to pEC194RS was used.
  • the resulting pga deletion plasmid is named pDel007. 1.0.16.6 Plasmid p689-T2A-lac
  • the E. coli plasmid p689-T2A-lac comprises the lacZ-alpha gene flanked by Bpil restriction sites, again flanked 5’ by the T 1 terminator of the E. coli rrnB gene and 3’ by the TO lambda terminator and was ordered as gene synthesis construct (SEQ ID NO. 27).
  • the truncated promoter of the aprE gene from Bacillus licheniformis DSM641 (SEQ ID NO. 2) of plasmid pCB56C (US5352604) was PCR-amplified with oligonucleotides SEQ ID NO. 28 and SEQ ID NO. 29.
  • the GFPmut2 gene variant (accession number AF302837) with flanking Bpil restriction sites (SEQ ID NO. 30) was ordered as gene synthesis fragment (Geneart Regens burg).
  • the gene expression construct comprising the truncated (trunc.) PaprE promoter from Bacillus licheniformis DSM641 fused to the GFPmut2 variant was cloned into plasmid p689- T2A-lac by type-ll-assembly with restriction endonuclease Bpil as described (Radeck et al. , 2017; Sci. Rep. 7: 14134) and the reaction mixture subsequently transformed into electrocom- petent E. coli DH10B cells. Transformants were spread and incubated overnight at 37°C on LB- agar plates containing 100pg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting plasmid is named p890 PaprE trunc. DSM641-GFPmut2.
  • the plasmid p891 PaprE fl. DSM641-GFPmut2 was constructed as described for p890 PaprE trunc. DSM641-GFPmut2, however the full-length (fl.) promoter of the aprE gene from Bacillus licheniformis DSM641 (SEQ ID NO. 3) was PCR-amplified with oligonucleotides SEQ ID NO. 31 and SEQ ID NO. 32 using genomic DNA as a template.
  • the plasmid p892 PaprE trunc. DSM13-GFPmut2 was constructed as described for p891 PaprE fl. DSM641-GFPmut2, however the truncated (trunc.) promoter of the aprE gene from Bacillus licheniformis DSM13 (SEQ ID NO. 5) was PCR-amplified with oligonucleotides SEQ ID NO. 33 and SEQ ID NO. 32 using genomic DNA as a template.
  • the plasmid p893 PaprE fl. DSM13-GFPmut2 was constructed as described for p892 PaprE trunc. DSM13-GFPmut2, however the full-length (fl.) promoter of the aprE gene from Bacillus licheniformis DSM13 (SEQ ID NO. 6) was PCR-amplified with oligonucleotides SEQ ID NO. 31 and SEQ ID NO. 32 using genomic DNA as a template
  • Plasmid pJOE8999.1 was produced as described in Altenbuchner J., 2016, Editing of the Bacil lus subtilis genome by the CRISPR-Cas9 system, Appl Environ Microbiol 82:5421-5. 1.0.16.12 Plasmid pJOE-T2A
  • the CRISPR/Cas9 plasmid pJOE8889.1 was modified as follows.
  • the type-ll-assembly mRFP cassette from plasmid pBSd141R (accession number: KY995200) (Radeck et al., J 2017; Sci. Rep. 7: 14134) was modified such to remove multiple restriction sites and the Bpil restriction sites and ordered as gene synthesis fragment with flanking Sfil re striction sites (SEC ID NO. 34).
  • the plasmid is named p#732.
  • Plasmid p#732 and plasmid pJOE8999.1 were digested with Sfil (New England Biolabs, NEB) and the mRFP cassette of p#732 ligated into Sfil-digested pJOE8999.1 following transformation into competent E. coli DH10B cells. Positive clones were screened on IPTG/X-Gal and kanamycin (20 pg/ml) contain ing LB agar plates for purple colonies (blue-white screening and mRFP1 expression). The re sulting sequence-verified plasmid was named pJOE-T2A.
  • Plasmid pCC027 is derivative of the plasmid pJOE-T2A, where the promoter PmanP was re placed by a promoter fragment (SEQ ID NO. 35) comprising in the 5’ to 3’ orientation the termi nator region of pMutin2 (accession number AF072806) followed by a Pveg promoter variant derived from Guiziou et al (Guiziou.S., V.Sauveplane, H.J. Chang, C.CIerte, N.Declerck,
  • the 20 bp target sequence of the amyB gene for the sgRNA were ordered as oligonucleotides SEC ID NO. 36 and Seq ID NO. 37 with 5' phosphorylation following annealing to form an oligo nucleotide duplex.
  • the 5’ and 3’ regions of the amyB gene of Bacillus licheniformis were PCR- amplified with oligonucleotides SEC ID NO. 38 and SEQ ID NO. 39 and SEQ ID NO. 40 and SEQ ID NO.41, respectively.
  • the CRISPR/Cas9 based gene integration plasmid replacing the amyB gene of Bacillus licheni formis was constructed by type-ll-assembly with restriction endonuclease Bsal with the follow ing components: pCC027, the oligonucleotide duplex (SEQ ID NO. 36, SEQ ID NO. 37), the PCR-fragment of the 5’ homology region of the amyB gene, p890-PaprE trunc. DSM641- GFPmut2 and the PCR-fragment of the 3’ homology regions of the amyB gene.
  • the reaction mixture was transformed into E. coli INV110 cells (Life technologies).
  • the plasmid pCC047 was constructed as for plasmid pCC043, however the plasmid p891 was used.
  • the plasmid pCC048 was constructed as for plasmid pCC043, however the plasmid p892 was used.
  • the plasmid pCC049 was constructed as for plasmid pCC043, however the plasmid p893 was used.
  • Bacillus licheniformis strains M609.1A, M609.1B, M609.2A and M609.2B (as listed in Table 4) were cultivated in a fed-batch based process and promoter strength recorded for individual cells by measurement of the fluorescence signal produced by GFP.
  • Table 4 Description of strains note: Bacillus licheniformis strains DSM13 and ATCC1450 are isogenic.
  • Bacillus licheniformis strains were cultivated in a microtiter plate-based fed-batch process (Ha- bicher et al. , 2019 Biotechnol J.;15(2)). All cultivations were conducted in an orbital shaker with a diameter of 25 mm (Innova 42, New Brunswick Scientific, Eppendorf AG; Hamburg, Germany) at 30 °C and 400 rpm. Strains were cultivated in two subsequent precultures in FlowerPlates (MTP-48-OFF, m2p-labs GmbH) for synchronization of growth. The first preculture was carried out in 800 pi TB medium inoculated with a fresh single colony from the strain streaked onto LB agar plates.
  • the second preculture containing 800 mI V3 minimal medium (Meissner et al., 2015, Journal of industrial microbiology & biotechnology 42 (9): 1203-1215) as inoculated with 8 mI of the first preculture and cultivated for 24 h.
  • Microtiter plate-based fed-batch main cul tivations were conducted using 48-well round- and deep-well-microtiter plates with glucose- containing polymer on the bottom of each well (FeedPlate, article number: SMFP08004, Kuhner Shaker GmbH; Herzogenrath, Germany). 7 mI of the second preculture were used to inoculate 760 mI V3 minimal medium without glucose. Main cultures were incubated for 48 h.
  • Precultures were covered with a sterile gas-permeable sealing foil (AeraSeal film, Sigma-Aldrich) to avoid contamination.
  • FeedPlates were sealed with a sterile gas-permeable, evaporation reducing foil (F-GPR48-10, m2p-labs GmbH) to reduce evaporation and to avoid contamination.
  • F-GPR48-10 sterile gas-permeable, evaporation reducing foil
  • Microscope slides were prepared using 1.5 % agarose in phosphate-buffered saline, which was molded into a 125 pi GeneFrame (Thermo Fisher Scientific; Waltham, USA) to immobilize cells and ensure an even focus plane.
  • 2 pg/ml DAPI 4’,6- diamidino-2-phenylindole
  • 0.5 mI cell-suspension were applied on an agarose gel slide and excess moisture was allowed to evaporate before applying the co- verslip. Images were captured using a Zeiss Axio Imager.
  • the measurement of the mean GFP intensity of single cells was carried out using the ImageJ software with the embedded ObjectJ plugin NucT racer (Syvertsson et al. , 2016, PloS one 11 (3): e0151267).
  • NucT racer uses the DAPI stained nucleoid as identifier for individual cells and measures GFP fluorescence in the corresponding image obtained in the GFP channel.
  • Micro scopic images of phase-contrast, DAPI and GFP channels were imported into ImageJ software and stored in a TIFF-stack format containing the images arranged by channels. The analysis was performed with 16-bit tiff files.
  • Figure 3 summarizes the relative promoter strength at the timepoints of 24 h and 48 h of cultiva tion for the four different promoter constructs.
  • the promoter activity of the full-length PaprE promoter from B. licheniformis DSM641 (M609.1 B) is lower compared to the truncated version of the PaprE promoter (Bacillus licheniformis strain M609.1A).
  • the promoter activities of both the truncated and full-length PaprE promoters from Bacillus licheniformis DSM13 are higher compared to the trun cated version of the PaprE promoter from Bacillus licheniformis DSM641 (Bacillus licheniformis strain M609.1A) with the full-length promoter PaprE fl.
  • DSM13 showing a promoter activity over 150% compared to the reference PaprE trunc.-DSM641. At the 48h timepoint the PaprE pro moters of B.
  • licheniformis DSM641 both truncated and full-length
  • the truncated Pap rE promoter of B. licheniformis DSM13 show comparable promoter activities, however lower compared to the 24h timepoint PaprE trunc. DSM641 reference (B. licheniformis M609.1A).
  • the full-length PaprE promoter of B. licheniformis DSM13 (B. licheniformis strain M609.2B) shows highest promoter activity of 150% relative to the reference.
  • Example 2 Calculation of HMM score
  • upstream sequences of aprE-coding genes were extracted, subject to the following conditions: a) Upstream extraction size was 200 nucleotides. If there was an upstream gene/CDS anno tation closer than 200 nucleotides, then a shorter fragment was extracted. If fragment length was less than 50 nucleotides, such a fragment was not extracted. b) Extracted upstream sequences were grouped by BLAST hit bitscore, and sorted in de scending order by the same bitscore. To avoid bias, identical upstream sequences from the same bitscore group were deduplicated.
  • an hmm was build using HMMER 3.1 b1 (Wheeler, Travis J, and Sean R Eddy. (2013) “nhmmer: DNA homology search with profile HMMs.” Bioinformatics (Oxford, England) vol. 29,19 (2013): 2487-9), by running the command: hmmbuild -n PaprE PaprE.hmm ⁇ aligned. mfa ⁇ . This hmm was then pressed using: hmmpress PaprE.hmm, resulting in a model that can be run over any sequence.
  • the HMMER software can be run using the command: nhmmscan PaprE.hmm ⁇ sequence ⁇ , where ⁇ sequence ⁇ represents a fasta for matted file containing any DNA sequence.
  • ⁇ sequence ⁇ represents a fasta for matted file containing any DNA sequence.
  • This will output a list of sequences matching the model (given by start and end of the match), together with an e-value and a score.
  • Calibration of the hmm indicated that any score above a cutoff of 50 is indicative of a match. Using this cutoff to extract matching sequences from a database of over 8000 non-Bacilli genomes, a false dis covery rate of zero was confirmed.
  • Figure 1 shows the 5' region (SEQ ID NO. 6) of the B. licheniformis aprE gene coding for the AprE protease (SEQ ID NO. 48).
  • the sigma factor A -10 and -35 motifs are each indicated in bold face.
  • Underlined is the promoter region, wherein the 5'UTR between the -1 promotor posi tion and the start codon (SEQ ID NO. 46) is indicated by a curly line.
  • the first, second and third enhancer elements are each enclosed in a box.
  • Optional promoter regions are indicated by oblique typeface.
  • Figure 2 shows an alignment of the promoters according to SEQ ID NO. 2, 3, 4, 5 and 6. Only the sequence of SEQ ID NO. 3 is spelled out. For all other sequences only the nucleotides that differ at a given position from the corresponding nucleotide of SEQ ID NO. 3 are indicated; “.” indicates that the nucleotide is identical to that of SEQ ID NO. 3 at the respective position; "-" indicates that the respective nucleotide is missing.

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Abstract

La présente invention concerne des matériaux et des procédés pour des processus de fermentation industriels. En particulier, l'invention concerne des cassettes d'expression pour faciliter l'expression d'un gène cible sous le contrôle d'un promoteur hétérologue. L'invention concerne en outre la construction de tels promoteurs, vecteurs et cellules hôtes comprenant de telles cassettes d'expression et des procédés de fermentation utilisant de telles cellules hôtes. En outre, l'invention concerne des matériaux obtenus par une telle fermentation.
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CN112662599A (zh) * 2021-01-27 2021-04-16 吉林省农业科学院 一株禽源贝莱斯芽孢杆菌cl-4及其应用
CN115851541A (zh) * 2022-11-09 2023-03-28 安徽师范大学 一种益生菌微生态制剂及其制备方法和应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662599A (zh) * 2021-01-27 2021-04-16 吉林省农业科学院 一株禽源贝莱斯芽孢杆菌cl-4及其应用
CN112662599B (zh) * 2021-01-27 2022-06-14 吉林省农业科学院 一株禽源贝莱斯芽孢杆菌cl-4及其应用
CN115851541A (zh) * 2022-11-09 2023-03-28 安徽师范大学 一种益生菌微生态制剂及其制备方法和应用

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