WO2023156585A1

WO2023156585A1 - Systems, tools, and methods for engineering bacteria

Info

Publication number: WO2023156585A1
Application number: PCT/EP2023/054015
Authority: WO
Inventors: Carlos PIÑERO LAMBEA; María LLUCH SENAR
Original assignee: Pulmobiotics, S.L.
Priority date: 2022-02-18
Filing date: 2023-02-17
Publication date: 2023-08-24

Abstract

The present invention concerns systems, tool, and methods for engineering bacteria. The invention is in particular directed to specific relative configurations of different elements that allows for robust genomic modification of bacterial genomes and is convenient to use. In particular embodiments of the systems, tools, and methods genetically modified bacterium can be obtained that does not contain any scar sequences. Further aspects of the invention are directed to innovative inducible expression systems suitable for obtaining regulated gene expression in bacteria.

Description

SYSTEMS, TOOLS, AND METHODS FOR ENGINEERING BACTERIA

FIELD OF THE INVENTION

The invention is situated in the technical field of molecular biology, more specifically in the technical field of genome engineering. Aspects of the invention relate to tools, systems, and methods for engineering bacteria. The invention is further related to innovative inducible expression systems particularly suitable for use in bacteria such as Mycoplasma bacteria.

BACKGROUND OF THE INVENTION

In 1998, new logic to engineer bacterial genomes called recombineering was developed (Murphy et al., J Bacteriol, 1998; Zhang et al., Nat Genet, 1998). This technology relies on phage-derived proteins, such as those encoded by the Red operon from X phage or RecET from the Rac prophage. Both systems code for at least two different protein activities: (i) a 5' to 3' dsDNA exonuclease (Exo and RecE, respectively described in Little et al., J Biol Chem, 1967; and Clark et al., Cold Spring Harb Symp Quant Biol, 1984) and (ii) a single-stranded (ss)DNA annealing protein (SSAP) (Beta and RecT, respectively described in Li et al., J Mol Biol, 1998; and Hall et al., J Bacteriol, 1993). Thanks to the coordinated action of these two activities, recombineering protocols uses linear dsDNA fragments as a substrate for recombination, even with regions of homology as short as 40 nucleotides (nt) (Datsenko, Proc Natl Acad Sci US A, 2000).

However, recombineering with dsDNA substrates was mostly circumscribed to E. coll genome engineering, and this technology only really flourished when it was found that synthetic ssDNA molecules (i.e. commercially available oligonucleotides) could be used as recombinogenic substrates (Ellis et al.. Proc Natl Acad Sci USA, 2001). This process, known as oligonucleotide recombineering (hereafter, oligo-recombineering), only requires the activity of the phage-derived SSAP that mediates the homology-driven hybridization of the editing oligonucleotide with the lagging strand of the replication fork. Oligo-recombineering was initially set up in E. coll with the beta protein of X phage as SSAP, but attempts to directly transfer this technology to other bacteria gave limited results (Ricaurte et al., Microb Biotechnol, 2018; Sun et al., Appl Microbiol Biotechnol, 2015). In fact, the performance of phage-derived recombinases is not maintained across different bacterial genera and depends on the phylogenetic distance between the native host of the phage and the bacteria being engineered (Filsinger et al., Nat Chem Biol, 2021). These findings prompted a survey of phage genomes to identify efficient SSAPs for a wide variety of microbes. This has allowed oligo-recombineering protocols to be used in at least 29 different bacterial species (Wannier et al., Nar Rev Methods Primers, 2021), and the recent development of a method termed “Serial Enrichment for Efficient Recombineering” (SEER, Wannier et al.. Proc Natl Acad Sci USA, 2020) promises to further expand the range of editable microorganisms. However, oligo recombineering is also characterized by a major drawback stemming from the small size of the recombinogenic substrate employed in this technique, which precludes the introduction of large inserts of DNA in a precise location. This limits the technology to gene deletions, nucleotide substitutions or the introduction of very small sequences. Importantly, this same limitation precludes the inclusion of a selective marker, although this would facilitate selection of edited cells. In some species, this is not a major issue, as small editing can be obtained at ultra-high frequencies (for instance, 5.1 x 10 ¹ in E. coll to change a single nucleotide), yet oligo-recombineering protocols adopted for most strains show only modest editing rates (10 ² to 10 ⁵), even for small changes (Wannier et al., Proc Natl Acad Sci USA, 2020). Moreover, in all species, the oligo incorporation rate tends to drop in a power trend that correlates with the size of the attempted editing (Wang et al.. Nature, 2009; Pinero Lambea et al., ACS Synth Biol, 2020). This makes the selection of cells carrying large genetic changes a cumbersome process even for species in which there are highly efficient oligo-recombineering protocols available. To solve this, oligo-recombineering protocols have been merged with a counterselection method based on CRISPR/Cas9, in which sgRNAs are designed to create a double-strand break (DSB) in only unmodified cells (Aparicio et al., Biotechnol J, 2018). Nonetheless, the genetic parts encoding the CRISPR/Cas9 system can accumulate mutations with a frequency (10 ³ to 10^) (Aparicio et al., Biotechnol J, 2018; Oh and Van Pijkeren, Nucleic Acids Res, 2014) that might exceed that of oligo- recombineering for large (i.e. sizeable) genetic changes, hampering selection of edited cells.

There is thus an unmet need for genome engineering tools and methods that combine the ease of use and high editing efficiency of oligo recombineering with the great selective capacity of antibiotic resistance inclusion. Ideally, such tools would allow for the generation of i) several seamless mutants (i.e. mutants lacking any remnant scar sequence) carrying gene deletions in different loci (comprising edited genomic regions of up to 30 kb), and ii) deletions of large DNA segments containing non- essential and/or essential genes. Such a marker-free, scarless editing method would greatly facilitate the engineering of different bacterial species and ultimately foster further developments in the field of synthetic biology.

SUMMARY OF THE INVENTION

As evidenced in detail by the examples enclosed herewith, the present invention relates to tools, systems, and methods to modify the genomic sequence of bacteria. The inventors have defined a set of components, preferably nucleotide arrangements, that allow for modification of the genomic sequence of a bacterial cell in a robust manner by using tools, systems, and methods that are convenient to use. The genome engineering workflow relies on specific positioning of recombinase sites and the use of their corresponding recombinases to allow modification of the genomic sequence of virtually any bacterium, including bacterial cells that are notoriously difficult to genetically modify under controlled conditions such as Mycoplasma bacterium. Embodiments of the invention enable the obtainment of genetically modified bacteria that only differ in their genomic sequence from their naturally occurring (i.e. not genetically modified) counterpart by the inserted nucleotide sequence and are void of any redundant and remnant sequences which were used in the genome engineering process. Additionally, the inventors have developed an innovative inducible expression system which allows highly regulated expression of gene products of interest.

The invention therefore provides the following aspects:

Aspect 1. An oligonucleotide modification system for genetic modification of a bacterium, wherein said bacterium comprises an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein, and wherein the oligonucleotide modification system comprises: a first nucleotide arrangement comprising:

- two nucleotide sequences (HA1 and HA2) each comprising a stretch of at least 5 contiguous nucleotides (located) respectively 5’ and 3’ of a target genomic sequence that is to be genetically modified, and

- a recognition sequence for a first site-specific recombinase (RSA), arranged such that said first nucleotide arrangement comprises one of said two nucleotide sequences (HA1) comprising a stretch of at least 5 contiguous nucleotides (located) 5 ’of said RSA recognition sequence and the other nucleotide sequence (HA2) comprising a stretch of at least 5 contiguous nucleotides (located) 3’ of said RSA recognition sequence; and a second nucleotide arrangement comprising:

- the recognition sequence for said first site-specific recombinase (RSA), and

- at least two recognition sequences for a second site-specific recombinase (RSB), arranged such that said second nucleotide arrangement comprises at least one RSB recognition sequence (located) 5’ of the RSA recognition sequence and at least one RSB recognition sequence (located) 3’ of the RSA sequence.

Aspect 2. The oligonucleotide modification system according to aspect 1, wherein the two nucleotide sequences (HA1 and HA2) each comprise a stretch of at least 8 contiguous nucleotides, preferably each comprise a stretch of at least 10 contiguous nucleotides, more preferably each comprise a stretch of at least 15 contiguous nucleotides. Aspect 3. The oligonucleotide modification system according to aspect 1 or 2, wherein the ssDNA annealing protein is a third recombinase, preferably a GP35 recombinase, more preferably a GP35 recombinase having a nucleotide sequence encoding a protein which is at least 65% identical, preferably at least 75% identical, more preferably at least 85% identical, most preferably at least 95% identical to the amino acid sequence of GP35 from Bacillus subtilis bacteriophage SPP1 as defined by SEQ ID NO: 1 based on the total length of the amino acid sequence of the recombinase.

Aspect 4. The oligonucleotide modification system according to any one of aspects 1 to 3, wherein the first recombinase, second recombinase, and/or third recombinase lack cross-reactivity.

Aspect 5. The oligonucleotide modification system according to any one of the preceding aspects, wherein the second nucleotide arrangement further comprises a nucleotide sequence that encodes at least one selection marker that allows for phenotypic selection of bacteria that comprise the second nucleotide arrangement.

Aspect 6. The oligonucleotide modification system according to aspect 5, wherein the selection marker is a luminescent protein, an antibiotic resistance protein, a protein mitigating auxotrophy, a component of a toxin-antitoxin system, or any combination thereof.

Aspect 7. The oligonucleotide modification system according to any one of the preceding aspects, wherein the second nucleotide arrangement is a circular double-stranded DNA construct, preferably wherein said second nucleotide arrangement is a plasmid.

Aspect 8. The oligonucleotide modification system according to any one of the preceding aspects, wherein the second nucleotide arrangement further comprises a nucleotide sequence encoding the first recombinase (RSA) and/or a nucleotide sequence encoding the second recombinase (RSB), preferably wherein each recombinase is operably linked to a promoter sequence.

Aspect 9. The oligonucleotide modification system according to aspect 8, wherein the first recombinase (RSA) and the second recombinase (RSB) are each operably and independently linked to promoter sequences.

Aspect 10. The oligonucleotide modification system according to aspect 8 or 9, wherein the first recombinase (RSA) and the second recombinase (RSB) are each operably linked to a distinct promoter sequence.

Aspect 11. The oligonucleotide modification system according to any one of aspects 8 to 10, wherein at least the first recombinase (RSA) or the second recombinase (RSB) is operably linked to an inducible operator sequence, preferably wherein the second recombinase (RSB) is operably linked to an inducible operator sequence. Aspect 12. The oligonucleotide modification system according to any one of aspects 8 to 11, wherein at least one of the recombinases is operably linked to a cumate promoter and operator sequence (CuO), preferably a cumate promoter and operator sequence (CuO) having a sequence identity- of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10, most preferably a cumate promoter and operator sequence (CuO) comprising SEQ ID NO: 6 or SEQ ID NO: 10.

Aspect 13. The oligonucleotide modification system according to any one of the preceding aspects, wherein the first nucleotide arrangement further comprises an intron-encoded endonuclease restriction site sequence located

- between the RSA recognition sequence and the stretch of at least 5 contiguous nucleotides of a genomic sequence (HA1) of the bacterium to be modified (located) 5’ of the RSA recognition sequence, or

- between the RSA recognition sequence and the stretch of at least 5 contiguous nucleotides of genomic sequence (HA2) of the bacterium to be modified (located) 3 ’of the RSA recognition sequence, preferably wherein the intron-encoded endonuclease restriction site is an I-Scel endonuclease restriction site.

Aspect 14. The oligonucleotide modification system according to any one of aspects 1 to 12, wherein the first arrangement consists essentially of, or consists of two nucleotide sequences of at least 5 nucleotides of the genomic sequence of a bacterium and a site-specific recognition site sequence of a first recombinase (RSA).

Aspect 15. The oligonucleotide modification system according to any one of the preceding aspects, wherein the first recombinase and/or the second recombinase is selected from the group consisting of ere recombinase, Sere recombinase, Vika recombinase, and Bxbl recombinase.

Aspect 16. The oligonucleotide modification system according to any one of the preceding aspects, wherein the bacterium is a bacterium selected from the group consisting of: Mycoplasma bacteria, Pseudomonas bacteria, and Lactobacillus bacteria, preferably wherein the bacterium is a Mycoplasma bacterium, more preferably wherein the bacterium is a Mycoplasma pneumoniae bacterium.

Aspect 17. Use of an oligonucleotide modification system as defined in any one of the preceding aspects for genetic modification of a bacterium.

Aspect 18. The use according to aspect 17, wherein the oligonucleotide modification system is used to introduce nucleotide sequences encoding heterologous gene products into the genomic sequence of the bacterium. Aspect 19. The use according to aspect 17 or 18, wherein the oligonucleotide modification system is used to replace and/or delete nucleotide sequences encoding one or more virulence factors.

Aspect 20. The use according to aspect 19, wherein use of the oligonucleotide modification system results in a genetically modified bacterium that has reduced pathogenicity upon introduction into a host organism when compared to the naturally occurring and non-modified bacterium.

Aspect 21. A method to genetically modify a bacterium, the method comprising the steps of:

- transforming a culture of said bacterium comprising an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein with (i) an oligonucleotide modification system as defined in any one of aspects 1 to 16 and (ii) a first recombinase (RS A),

- selecting bacteria from the culture comprising a genomic sequence wherein the second nucleotide arrangement is integrated,

- expressing a second recombinase (RSB) in the bacterium, thereby removing the second nucleotide arrangement from the genomic sequence of the bacterium.

Aspect 22. The method according to aspect 21, wherein the method comprises a further step of selecting bacteria from the culture wherein the second nucleotide arrangement is deleted from the genomic sequence of the bacterium.

Aspect 23. Tire method according to aspect 21 or 22, wherein the first recombinase (RSA) and/or the second recombinase (RSB) is encoded in a nucleotide sequence of a third nucleotide arrangement.

Aspect 24. The method according to aspect 21 or 22, wherein the second recombinase (RSB) is encoded in a nucleotide sequence of the second nucleotide arrangement.

Aspect 25. The method according to any one of aspects 21 to 24, wherein the first and second recombinase are encoded in a nucleotide sequence of the second nucleotide arrangement.

Aspect 26. The method according to any one of aspects 21 to 25, wherein the second nucleotide arrangement is a circular double-stranded DNA construct, preferably wherein said second nucleotide arrangement is a plasmid.

Aspect 27. The method according to any one of aspects 21 to 26, wherein the method comprises individual regulation of the expression of the first and/or the second recombinase, preferably wherein the method comprises sequential regulation of expression of the second recombinase.

Aspect 28. The method according to any one of aspects 21 to 26, wherein the method comprises a further step of contacting said bacterium with a nucleotide arrangement consisting of HA1 and HA2, and contacting said bacterium with an intron-encoded endonuclease, preferably wherein the intron- encoded endonuclease is a I-Scel endonuclease. Aspect 29. The method according to aspect 28, which is a method wherein the resulting bacterium does not contain any site-specific recombinase scar sequence in its genomic sequence.

Aspect 30. A genetically modified bacterium obtained by tire method defined in any one of aspects 21 to 29.

Aspect 31. The genetically modified bacterium according to aspect 30, wherein said bacterium does not comprise any site-specific recombinase scar sequence in its genomic sequence.

Aspect 32. The genetically modified bacterium according to aspects 30 or 31, wherein the bacterium is a bacterium selected from the group consisting of: Mycoplasma bacteria, Pseudomonas bacteria, and Lactobacillus bacteria, preferably wherein the bacterium is a Mycoplasma bacterium, more preferably wherein the bacterium is a Mycoplasma pneumoniae bacterium.

Aspect 33. A cumate promoter and operator sequence (CuO) comprising a nucleotide sequence having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 and wherein said cumate promoter and operator sequence (CuO) does not comprise or consists of SEQ ID NO: 9 or SEQ ID NO: 11, most preferably a cumate promoter and operator sequence (CuO) comprising SEQ ID NO: 6 or SEQ ID NO: 10.

Aspect 34. The cumate promoter and operator sequence (CuO) according to aspect 33, consisting essentially of, or consisting of a nucleotide sequence having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 and wherein said cumate promoter and operator sequence (CuO) does not comprise or consists of SEQ 9 or SEQ ID NO: 11, most preferably a cumate promoter and operator sequence (CuO) consisting of SEQ ID NO: 6 or SEQ ID NO: 10.

Aspect 35. A cumate-inducible expression system comprising:

- a repressor protein CymR or a nucleotide sequence encoding CymR, and

- a nucleotide sequence comprising

1) a cumate promoter and operator sequence (CuO) according to aspect 33 or 34, and

3) a nucleotide sequence encoding a gene product or forming an insertion site suitable for insertion of a nucleotide-encoded gene product, wherein said cumate promoter and operator sequence is positioned 5’ of the nucleotide sequence encoding a gene product or forming an insertion site suitable for insertion of a nucleotide-encoded gene product.

Aspect 36. The cumate-inducible expression system according to aspect 35, wherein the promoter sequence is a promoter sequence suitable for achieving expression of a gene product in a prokaryotic cell, preferably a promoter sequence suitable for achieving expression of a gene product in a bacterial cell.

Aspect 37. A kit of parts comprising the cumate-inducible expression system defined in aspects 35 or 36, and cumate.

Aspect 38. A cell comprising the cumate-inducible expression system of aspect 35 or 36.

Aspect 39. The cell according to aspect 38, wherein said cell is a prokaryotic cell, preferably a bacterial cell.

Aspect 40. A method for obtaining inducible gene expression in a cell, the method comprising the steps of:

- introducing the cumate-inducible expression system of aspects 35 or 36 into a cell, operably linked to a gene of interest,

- contacting the cell with cumate, thereby achieving inducible expression of said gene.

The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Rationale of the genome engineering method/system of the invention. Cells carrying a wild-type (WT) genome are co-transformed with an editing oligo and a selector plasmid. The editing oligo is composed of two homology arms (HA1 and HA2) that hybridize at the regions flanking the target gene and a site-specific recombinase A recognition site (RSA). The selector plasmid carries one copy of RSA and two copies of site-specific recombinase B recognition site (RSB), as well as an antibiotic resistance gene (not depicted). After an oligo-recombineering event catalysed by a phage- derived SSAP, edited genomes with an “unselectable” phenotype are generated. Site-specific recombinase A can then mediate tire recombination between the RSA introduced in the edited locus and the RSA present in the selector plasmid, thereby integrating the vector and generating a selectable phenotype for edited cells. Finally, site-specific recombinase B can drive the excision of the vector through recombination between the two RSBs.

Figure 2. Validation of the genome engineering system at four different loci. (A) Scheme depicting the chromosomal locations of the four loci selected to test the functionality of the system. Inserts: detailed schem e of the target area indicating the coding sequences present in either the plus strand (top) or the minus strand (bottom), as well as the molecules transformed into the cells. The shadowed triangles indicate the places in which the sequences used as HA are present in the chromosome, which are at a 1-kb distance to each other. (B) Scheme showing as an example the chromosomal conformation of mpn088 locus before editing (non-edited genome) and after editing with the plasmid inserted (edited genome) or excised (edited + resolved genome). Black arrows above and below the genome represent the primers used for the PCR screening; the expected size for each is indicated above the dashed line connecting them. (C) Top: electrophoretic analysis of PCRs conducted at the indicated loci in five different puromycin-resistant colonies (analysed colonies); a negative control and a WT sample are included as references. The ratio of edited clones at each locus is based on the observed size of the PCR products. Bottom: electrophoretic analysis of PCR products from the indicated loci in five different colonies (analysed colonies) obtained after transforming one clone carry ing the plasmid inserted at the edited locus with a suicide plasmid coding for Vcre. A negative control and a WT sample are included as references.

Figure 3. Engineering efficiency of the genome engineering method at different loci. Histogram showing the number of puromycin-resistant colonies found in all four transformations performed to deplete a Ikb fragment in mpn088. mpn256. mpn440 and mpn583 genes. Only 40 colonies were found in the negative controls, corresponding to cells transformed with selector plasmid without editing oligos. Thus, the insertion of the plasmid depended on previous placement of a landing pad by the editing oligo.

Figure 4. Performance of the methodology in the largest removeable region of M. pneumoniae genome. (A) Chromosome section representing the largest removeable region in M. pneumoniae genome and showing the battery-’ of designed editing oligos. The coding sequences present in either the plus strand (top) or the minus strand (bottom) are represented by arrows and the gene identifiers associated to them for essential genes (mpn489 and mpn515), and for non-essential genes (remainder of genes annotated). The shadow triangles between the chromosome and the oligos indicate the places in which the sequences used as HA are present in the genome (“A” or “delta” indicates a deletion hereafter throughout the present disclosure). All editing oligos share a common 5’ HA, whereas the 3’ HA binds to chromosomal regions placed at different distances from the 5 ’ HA, as indicated above each editing oligo. (B) Scheme showing as an example the expected chromosomal rearrangements upon the A90 bp (left) and A30 kb (right) genome editing. Non-edited genomes for both cases are also shown. Black arrows above and below the genome represent the primers used for the PCR screening; the expected size for each situation is indicated above the dashed line that connects them. For all editing, modified genomes should produce a PCR product slightly bigger than that of the selector plasmid (3.4 kb), whereas WT genomes should generate a PCR product slightly bigger than that of the attempted deletion. Note that for deletions of 10 kb and larger, the processivity of the polymerase is not efficient enough to amplify the product. (C) Electrophoretic analysis of the PCRs conducted for the indicated edits in five different puromycin-resistant colonies (e.g., the analysed colonies); a negative control and a WT sample are included as references. The ratio of edited clones at each locus is indicated at the bottom of each picture.

Figure 5. Further exploration of the genome engineering methodology. Histogram showing the number of puromycin-resistant colonies found in different transformations performing depletions ranging from 90 bp to 30 kb of a genomic region for up to 5.48% of the non-essential (NE) genome (Lluch-Senar et al., Mol Syst Biol, 2015), in which 25 NE coding genes are located (from mpn490 to mpn514) (Fig 3A). There is a trend in which the number of puromycin-resistant colonies decreased with the size of the attempted deletion, albeit all transformations showed a higher number of colonies compared to the control without primer.

Figure 6. Scarless genome engineering method. (A) Cells carrying a WT genome were cotransformed with the SSR-A-mediated plasmid integration and an editing oligo that contained a restriction site for the Seel enzyme and an RSA site between the two homology arms (HA1 and HA2). (B) Upon integration of the vector, edited cells had the resistance marker included in the genome. After picking colonies and isolating genomic DNA, cells were analysed by PCR. Four out of the five analysed clones showed a positive band of 3681 bp, consistent with the edited genome. (C) Positive clones were transformed with the pSSR-B vector, which mediated the excision of the integrated vector, thereby eliminating the resistance marker and leaving a scar of a 326 bp sequence that contained the Seel site and the lox sequences from the recombination event. Four out of five analysed clones were positive by PCR. (D, E) The cells were transformed with an oligo that eliminated the 326 bp of the genome and a selector vector that allowed the expression of Seel enzyme. (D) If the cells were edited and they had eliminated the scar, the Seel enzyme did not cut. NT, cells that were non-transformed (e.g., the DNA was from the cells in the previous step) and carried the editing scar (cells that were not co-transformed with oligo and See coding plasmid). (E) If the cells were not edited by the oligo, the genome was cleaved and the cells died. All five clones of the analysed clones had Seel site excised out, confirming that the scar had been eliminated.

Figure 7. Result of the scarless genome editing method. Histogram showing the number of colony forming units resistant to puromycin from the co-transformation of the editing oligo and the selector vector to deplete a 90-bp region of the mpn490 gene and include simultaneously the I-Scel restriction site. The number of colonies obtained was significantly higher in the co-transformation of the editing oligo and the selector vector, than in the control transformation with only pLoxPuro plasmid.

Figure 8. Compatibility of the genome engineering method with different SSRs. (A) The structure of the different constructed selector plasmids, together with the editing primers used to test their activity. All primers shared the same homology arms (HA1 and HA2) that flank the indicated RS. (B) Scheme depicting the chromosomal location of the locus selected to compare the efficiency of the different selector plasmids. A more detailed view of the target area is shown within the square, indicating the coding sequences present in either the plus strand (top) or the minus strand (bottom), as well as the molecules transformed into the cells. Has of the editing primer have a sequence of the chromosome plus strand, whereas the RS present in the editing oligo and the selector plasmid are depicted in multicolour, to reflect that they change their sequence depending on the particular RS/SSR pair used. The shadowed triangles indicate the places in which the sequences used as HA are present in the chromosome, which are at a 0.84-kb distance to each other. (C) Scheme showing the chromosomal conformation of mpn088 locus before editing (non-edited genome) and after editing with the plasmid inserted there (edited genome). Black arrows above and below the genome represent the primers used for the PCR screening and the expected size for each situation is indicated on top of the dashed line that connects them. Note that while the size shown corresponds to that expected using pLoxPuroCre as selector plasmid, all selector plasmids will generate a similar-sized PCR product. (D) Electrophoretic analysis of the PCRs conducted for each selector plasmid in five different puromycin-resistant colonies (analysed colonies); a negative control and a WT sample are included as a reference. The ratio of edited clones at each locus is indicated at the bottom of each image.

Figure 9. Expanding the genome engineering method logic to different site-specific recombinases. Histogram representing the number of puromycin-resistant cells obtained after co-transforming the editing oligo with each of these selector plasmids in a strain that solely expresses GP35 as a SSAP (M129-GP35 strain). We observed variable numbers of puromycin-resistant colonies, with pLoxPuroCre resulting in the most and pSloxPuroScre in the least productive vectors for the number of colonies obtained. Control transformations with the selector plasmid showed limited number of colonies in most cases; however, control transformation with pVoxPuroVika gave approximatively a third of the number of the colonies obtained in the co-transformation of this vector with the editing oligo. This suggests the existence of a vox-like sequence in the genome ofM. pneumoniae that led to an unspecific integration of the vector, independent of any previous placement at the target area of a vox landing by the editing oligo.

Figure 10. Gene platforms by the genome engineering method. Histogram showing the number of CFUs resistant to puromycin obtained from the co-transformation performed with the oligo designed to delete the whole area of 5.5 kb (from mpn633 to mpn638), and a selector plasmid termed pLoxPuroCreCOMP636-637. Co-transformation of both molecules in the M129-GP35 strain produced substantially more colonies than a control transformation with only selector plasmid.

Figure 11. Insertion of gene platforms at a desired location. (A) Scheme depicting the chromosomal location of the region in which the targeted insertion of gene platforms was tested. A more detailed view of the target area is shown within the square. Genes encoded in this area are only present in the plus strand (top strand), whereas the gene identifiers associated with them are essential genes (mpn636 and mpn637) or non-essential genes (mpn633, mpn634, mpn635, mpn638). Above the chromosome, the editing oligo and the selector plasmid transformed into the cells are depicted. HAs of the editing oligo have the sequence of the plus strand of the chromosome; the shadowed triangles indicate the genomic locations of the HA sequences, which are at a 5.5kb distance to each other (B) Illustration depicting the main features included in the selector plasmid used for the targeted insertion of gene platforms (in this case, the mpn636 and mpn637 genes). Note that the features important for the engineering process are indicated for the sake of clarity. (C) Scheme showing the chromosomal conformation of tire modified area before the editing (non-edited genome) and after the editing with the plasmid inserted there (edited genome) or with the plasmid excised (edited + resolved genome). Note that after excision, the two essential genes of the vector wherein the cloning occurred are maintained in the chromosome. Black arrows above and below the genome represent the primers used for the PCR screening; the expected size for each situation is indicated above the dashed line connecting them. (D) Top: electrophoretic analysis of the PCR conducted in five different puromycin-resistant colonies (analysed colonies); a negative control and a WT sample are included as references. The ratio of edited clones is indicated based on the observed size of the PCR products. Bottom: electrophoretic analysis of the PCRs of five different colonies (analysed colonies) obtained after transforming one clone carrying the plasmid inserted at the edited locus with a suicide plasmid coding for Vcre. A negative control and a WT sample are included as references.

Figure 12. Development of a novel inducible system for M. pneumoniae based on the Pcum2.1 design. (A) Schematic representation of the regulatory region of the cumate inducible system. The DNA sequences of the three tested promoters derived from Pveg (Pcuml; PCum2 and Pcum2.1) are shown in the dashed box. The Pcuml design was based on the WT sequence of PVeg, which has two different binding sites for RNA polymerase (RNApol)78. In PCum2, the RNApol binding site more distant to the CuO was removed. Pcum2.1 is a derivative of PCum2 in which seven nucleotides (lowercase letters in the sequence) were changed to increase the affinity of RNApol complex towards the sequence. Genes are represented by arrows. (B) Histogram showing the fluorescence signal emitted by different strains expressing the venus protein under different promoter sequences and inducers doses. The cumate inducible system is compared side by side with the inducible system based on the tetracycline repressor (for each condition from left to right: M129 WT; M129 PCumlVenus; M129 PCum2Venus; M129 PCum2.1Venus; M129 PTetVenus). The WT strain not containing the venus coding gene was included as a negative control for all the induction conditions. (C) Histogram depicting the leakiness (i.e. the ratio between the fluorescent signal in the absence of inducer, and that observed in the WT strain) for all systems. It was negligible for all the strains except for the PCuml system, which produced a fluorescent signal 8-times higher than that observed in the control strain. (D) Histogram depicting the inducibility (i.e. the fold-change in venus expression between the optimal induction condition and the uninduced state), which was almost three-times higher in Pcum2. 1 than in Ptet.

Figure 13. Selection of small- and large-scale genome editing with an all-in-one selector plasmid. (A) Illustration depicting the main features included in the all-in-one selector plasmid capable to mediate its own integration and excision on demand. (B) Left side, scheme indicating the expected chromosomal conformations upon the indicated genome edits selected with pLoxPuroCreVcre plasmid. Black arrows above and below the genome represent the primers used for the PCR screening and the expected size for each situation is indicated on top of the dashed line that connects them. Right side: Electrophoretic analysis of the PCRs conducted for the indicated editing in 5 different puromycin- resistant colonies (analysed colonies), a negative control and a WT sample are included as a reference. The ratio of edited clones at each locus is indicated at the bottom of each picture. (C) Picture of the 12- well plate in which 3 clones carrying the A 30 kb edit selected with pLoxPuroCreVcre were inoculated and grown in the presence (+) or absence (-) of puromycin and cumate as indicated. Growth is monitored by measuring M. pneumoniae-associated acidification, corresponding to a colour change of the growth medium which contains phenol red. Note: the extreme right column of the photograph is characterised by a strong red colour (dark grey), whereas the three left columns are characterised by a yellow colour (light grey) (D) Left, scheme indicating the expected chromosomal conformations of the area modified in the A 30 kb editing, after excision of the vector. Black arrows above and below the genome represent the primers used for the PCR screening, and the expected size is indicated above the dashed line. Right, electrophoretic analysis of the PCRs conducted in the three clones carrying the A 30 kb edit selected with pLoxPuroCreVcre, after being grown in the presence of cumate (+) to induce Vcre expression. A non-induced clone (-) is shown as a reference.

Figure 14. AH in one vector. (A) Histogram showing the number of puromycin-resistant colonies obtained in the different co-transformations (left: A 90 bp; right: A 30 kb). Regardless of the attempted modification, co-transformations of the editing oligo and pLoxPuroCre selector plasmid resulted in the highest number of puromycin-resistant colonies. All co-transformations resulted in an amount of puromycin-resistant colonies clearly higher than that obtained in their respective control transformations in which only the selector plasmid was included. (B) pLoxPuro selector vector. (C) pLoxPuroCre selector vector. (D) PCR analysis of five puromycin-resistant colonies performed to confirm the correct identity of the obtained clones with the transformation using pLoxPuro selector vector. (E) PCR analysis clones obtained with the pLoxPuroCre as selector vector. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from ... to ... ” or the expression “between . . . and ... ” or another expression.

The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/- 1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment”, or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

Amino acids are referred to herein with their full name, their three-letter abbreviation or their one letter abbreviation. Unless explicitly stated otherwise, reference herein to any peptide, polypeptide, protein, or nucleic acid, or fragment thereof may generally also encompass modified forms of said peptide, polypeptide, protein, or nucleic acid, or fragment thereof, such as bearing post-expression modifications including the following non-limiting examples: phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, combinations thereof.

The terms “plasmid”, “plasmid vector”, “expression vector” or “vector” as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined cell or vehicle organism such that the cloned sequence is reproducible. A vector may also preferably contain a selection marker, such as, e.g., an antibiotic resistance gene, to allow selection of recipient cells that contain the vector.

“Gene product” as used herein is indicative for any molecule directly derived from a gene or functional fragment of a gene. A skilled person is aware that the term “gene product” may also be indicative for the product derived from a non-naturally occurring operon comprised in a Mycoplasma bacterium, as indicated by the term "heterologous gene product" or “exogenous gene product”. The term may therefore cover any protein of biotechnological interest.

The term “oligonucleotide modification system” as used in the context of the invention refers to a collection, i.e . a multitude of distinct molecular elements, capable of specific targeting and subsequently modifying an nucleotide sequence. Oligonucleotide modification systems have been described in the art, and can be introduced in a target cell under various forms such as described further herein, including DNA, RNA, proteins, or any combination hereof. A skilled person understands that in the context used herein, the term modification system is indicative for a genome engineering system, thus a collection of distinct molecular elements (i.e. components) that attribute to a change in sequence of a targeted nucleotide sequence. The term “oligonucleotide modification system” does by no means impose any limitation on the physical entities comprised in said oligonucleotide modification system, and only reflects the intended entity to be modified. Hence, the oligonucleotide system may comprise one or more components that do not fall under the term “oligonucleotide”, but for example would be appreciated by a skilled person as e.g. proteins. Additionally, a skilled person readily appreciates that the absolute and/or relative amounts of the distinct components of the oligonucleotide modification system may need to be optimised on a case-by-case basis. Non-limiting illustrative parameters that may influence the absolute and/or relative amounts of the oligonucleotide modification system include the bacterium (i.e. bacterial strain) that is to be modified, the permissibility of said bacterium, and/or the genomic sequence that is to be modified to the introduction of foreign (i.e. exogenous) components by e.g. transformation, the genomic region or position that is to be modified, and/or the purity of the components of the oligonucleotide modification system.

“Nucleotide arrangements” as used herein, or synonymously “nucleotide sequences”, “polynucleotide arrangements”, “polynucleotide sequences”, refers to a sequence of a multitude of nucleotides physically connected to form a nucleotide sequence. Unless the contrary is explicitly mentioned, the nucleotide arrangements are not presented as part of, or embedded in their naturally occurring genome. Means and methods to obtain, generate and modify isolated polynucleotide sequences are well known to a person skilled in the art (Alberts et al., Molecular Biology of the Cell. 4th edition, 2002).

By means of guidance and not limitation, any nucleotide arrangement can be part of an expression vector such as a plasmid optionally a non-replicative plasmid, a phagemid, a bacteriophage, a bacteriophage-derived vector, an artificial chromosome, a minicircle, a lentiviral vector, a retroviral vector, an adenoviral or adeno-associated viral vector, a piggyback vector, or a tol2 vector. A skilled person is aware of these different types of constructs and their generation and manipulation has been detailed at numerous instances (Sambrook et al., Molecular cloning: a laboratory manual, ISBN 0879693096, 1989 and the corresponding updated 4^th Edition, Cold Spring Harbor Laboratory Press, 2012). Furthermore, it is evident to a skilled person that plasmid DNA, or (circular) recombinant DNA is commonly referred to in the art as copy DNA, complement DNA, or even referred to by the abbreviation “cDNA”, which may each be used interchangeably. In certain embodiments, a nucleotide arrangement comprised in the oligonucleotide modification system is part of a bicistronic expression construct. In further embodiments, the nucleotide arrangement is incorporated, i.e. inserted, in a cellular genome, preferably a genomic sequence of a bacterium, more preferably the genomic sequence of the bacterium whose genomic sequence is to be modified. In yet further embodiments, the nucleotide arrangement is part of a cellular genome, e.g. a de novo designed cellular genome or a mutagenized or synthetic bacterium. In further embodiments, the nucleotide arrangement is comprised in a bacterial artificial chromosome or a yeast artificial chromosome.. In certain embodiments, the 5’ and/or 3’ end of the polynucleotide arrangement is modified to improve the stability of the sequence in order to actively avoid degradation. Suitable modifications in this context include but are not limited to biotinylated nucleotides and phosphorothioate nucleotides. In certain embodiments, the polynucleotide sequence, multiple nucleotide arrangements, or the complete oligonucleotide modification system as disclosed herein is comprised in a bacteriophage. In further embodiments, the polynucleotide sequence, multiple nucleotide arrangements, or the complete oligonucleotide modification system as disclosed herein is comprised in a bacteriophage in the form of a gene drive. A skilled person is aware of the term “gene drive” as it is further described in the present disclosure. The term “bacteriophage” as described herein is indicative for a virus that infects and optionally is able to replicate within bacteria and archaea, which may be modified for therapeutic purposes as has been described in the art (e.g. Principi et al., Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections, Frontiers in Pharmacology, 2019).

“Genetic modification” as referenced thereto in the present specification refers to any change in nucleotide sequence of an organism, in the present context a bacterium. The type of modification is not particularly limited and therefore includes both insertions, rearrangements, deletions, point mutations, inversions, exchanges, or combinations thereof of one or more nucleotides in the target genomic sequence of the bacterium. The effect that the resulting genetic modification imparts on the transcriptome and/or proteome of said bacterium is not particularly limiting. Similarly “genetically modified” as used herein is indicative for an organism or a cell that comprises a genomic sequence aberrant from the genomic sequence of that organism or cell occurring in natural conditions. Hence, “genetically modified” refers to a non-naturally occurring organism or cell that is obtained by targeted mutagenesis, i.e. by artificial intervention. “Genetically modified” as used herein thus excludes organisms that are newly found in nature and are the product of natural selection and/or natural evolutionary mutagenesis.

Suitable bacteria that can be modified using the tools, systems, and methods described herein are not particularly limited. Therefore, the bacterium may be a naturally occurring and/or commercially available bacterium. A skilled person is aware of repositories and vendors where bacteria may be acquired, non-limiting examples thereof being the Leibniz Institute DSMZ -German Collection of Microorganisms and Cell Cultures, the American Type Culture Collection, or the National Collection of Type Cultures. Given the notion that the collection of suitable bacteria that can be modified using the oligonucleotide modification system described herein is not particularly limited, the bacterium may be a bacterium having a naturally occurring genomic sequence or a bacterium having a non-naturally occurring genomic sequence (e.g. a bacterium that has been the subject of at least one earlier artificially introduced genomic modification).

Throughout the present disclosure, relative positions of certain nucleotide sequences are indicated by the terms “5’ (5 prime)” and “3’ (3 prime)”. A skilled person readily appreciates the meaning of these indications and is aware that these refer to a certain location of a certain nucleotide sequence relative to a second location of a certain nucleotide sequence. The term 5’ may be used interchangeably with the term “upstream” which is equally common in the art. The term 3 ’ may be used interchangeably with the term “downstream” which is equally common in the art. It is evident that these terms indicate the relative position of a nucleotide sequence or portion thereof vis-a-vis another nucleotide sequence or portion thereof in a larger nucleotide sequence. Throughout the art and in the present disclosure, typically the 5’ to 3’ strand of a double stranded nucleotide sequence is visualized as the top strand. In such representations, a 5’ location is typically visually depicted on a more left position of a 5’ to 3’ strand, while a 3’ location is typically visually depicted on a more right position of a 5’ to 3’ strand. Such an illustration does not impose limitations to the actual relative position of elements or portions of the nucleotide sequence and is merely a conventional method of displaying nucleotide sequence.

Throughout the disclosure, certain components of the oligonucleotide modification system may be exogenous components. “Exogenous” in this context indicates a component that is comprised in a cell which is not the cell type and/or organism wherein said component is expressed in a naturally occurring situation (i.e. a wild-type, non-genetically modified, or non-manipulated cell or organism). “Exogenous” may be interchangeably used with the term “heterologous” in accordance with the accepted nomenclature in the art and technical field of molecular biology. For example, a person skilled in the art appreciates that a protein exclusively expressed in wild-type E. coli bacteria, when expressed in a Mycoplasma bacterium by manipulation of said Mycoplasma bacterium would be considered an exogenous (i.e. heterologous) protein in said Mycoplasma bacterium. Alternatively, a protein exclusively expressed in e.g. non-manipulated liver cells would be considered an exogenous protein when expressed in a neuronal cell. Evidently, the terms “exogenous” and “heterologous” are not restricted to be used in a context of proteins, and may equally be used to indicate foreign nucleotide sequences such as DNA sequences or RNA sequences (i.e. transcripts). Finally, said terms do not impose any restriction whatsoever on where the exogenous component is located in the manipulated cell and thus includes components that are introduced as such in the cell (e.g. recombinant proteins), components that are introduced in the cell which lead to expression of the exogenous component(s) (e.g. expression constructs, expression vectors, RNA sequences, etc.), and components that have been stably inserted in the genomic sequence of the manipulated cell by e.g. genome engineering methodologies. Means and methods to express proteins such as the construction of suitable expression vectors, and methods contributing to the establishment of any biological framework comprising cells that mediate expression of one or more genes encoded in such expression vectors are known to a skilled person and have been described in the art on numerous occasions (e.g. Srinivasan et al., Fundamentals of Molecular Biology, Current Developments in Biotechnology and Bioengineering, 2017).

A first aspect of the invention is directed to an oligonucleotide modification system that is suitable for genetic modification of a bacterium. More particularly, the aspect is directed to an oligonucleotide modification system that is suitable for genetic modification of a bacterium, said bacterium comprising an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein and wherein the oligonucleotide modification system comprises a first nucleotide arrangement and a second nucleotide arrangement. The first nucleotide arrangement comprises two nucleotide sequences (HA1 and HA2) each comprising a stretch of at least 5 contiguous nucleotides of a target genomic sequence of the bacterium (i.e. the bacterium that is to be genetically modified), and a recognition sequence for a first site-specific recombinase (RS A). The two nucleotide sequences (HA1 and HA2) and the recognition sequence for the first site-specific recombinase (RSA) are arranged such that said first nucleotide arrangement comprises one of said two nucleotide sequences (HA1) comprising a stretch of at least 5 contiguous nucleotides (located) 5 ’of said RSA recognition sequence and the other nucleotide sequence (HA2) comprising a stretch of at least 5 contiguous nucleotides (located) 3’ of said RSA recognition sequence. The second nucleotide arrangement comprises the recognition sequence for said first site-specific recombinase (RSA), and at least two recognition sequences for a second site-specific recombinase (RSB). The recognition sequence for said first site-specific recombinase (RSA) and the at least two recognition sequences for a second site-specific recombinase (RSB) are arranged such that said second nucleotide arrangement comprises at least one RSB recognition sequence (located) 5 ’ of the RSA recognition sequence and at least one RSB recognition sequence (located) 3’ of the RSA sequence. Such an oligonucleotide modification system provides a particularly interesting and convenient framework for the efficient introduction of genomic modifications into a host bacterium.

“A stretch of x contiguous nucleotides” in the context of the present disclosure is indicative for “a linear nucleotide sequence of x nucleotides” and “an unbranched nucleotide sequence of x nucleotides”. “Target genomic sequence” as used herein with the term “target sequence” refers to any genomic sequence that is to be modified (by e.g. insertion of one or more nucleotides, deletion of one or more nucleotides, replacement of one or more nucleotides, or any combination thereof) by the oligonucleotide modification system. The term is therefore not particularly limiting for the encoded “information” in said sequence, and therefore includes both target (genomic) sequences that encode one or more gene products and target (genomic) sequences that do not encode for a gene product (such as but not limited to regulatory elements such as a promoter sequence). Neither limiting is the length of a genomic sequence of interest, and a skilled person readily appreciates that a target genomic sequence is smaller when attempting to introduce a single nucleotide mutation when compared to a target genomic sequence when attempting to delete a nucleotide sequence encoding a complete gene product such as a protein.

A skilled person appreciates that the abbreviations “HA1” and “HA2” refer to homology arms (HAs). Use of the term “homology arms” is widespread in the technical field of genome engineering and in the context of the invention the term is therefore to be interpreted accordingly. The terms “homology arm” or “homology region” therefore indicates a stretch (i.e. sequence) of contiguous nucleotides that are identical or essentially identical to a part of the target genomic sequence of the bacterium subjected to the genetic modification. A skilled person is further aware that an increasing number of mismatches of a HA to the corresponding bacterial target genomic sequence to be genetically modified in general leads to increased diminishment of engineering efficiency (Fels et al., Front Microbiol, 2020).

“ssDNA annealing protein” or the abbreviation “SSAP” as used herein refers to a protein capable of performing the process of single strand (ss) annealing. More particularly, in the present context reference to an ssDNA annealing protein refers to a protein capable to mediate (i.e. execute, perform, provoke, initiate, complete) homology-driven hybridization of an editing oligonucleotide with the lagging strand of the replication fork. In embodiments described herein, the ssDNA annealing protein is a recombinase. In preferred embodiments, the ssDNA annealing protein is an exogenous ssDNA annealing protein such as an exogenous recombinase. Notwithstanding, in certain embodiments the ssDNA annealing protein may be an endogenous ssDNA annealing protein, such as an endogenous recombinase. In certain embodiments wherein the ssDNA annealing protein is encoded by an oligonucleotide, tire oligonucleotide may be comprised in the cell as part of an expression vector, or the oligonucleotide may alternatively be integrated (i.e. comprised in, part of) the genomic sequence of the bacterium. In yet alternative embodiments, the ssDNA annealing protein may be introduced in the cell as a recombinant protein, or introduced in the cell as an RNA oligonucleotide transcript.

"Recombinase" as defined herein indicates an enzyme capable of effectuating genetic recombination. A skilled person is aware that genetic recombination is interchangeably annotated in the art by the term “genetic reshuffling”, which when used in a context of genetic engineering refers to an artificial and deliberate recombination of distinct pieces of DNA to create recombinant DNA. Depending on the number and orientation of the DNA sites, there can be an inversion, rearrangement, deletion or insertion of DNA. Recombinases catalyse DNA exchange reactions, which are directionally sensitive, between short target site sequences that are specific to each recombinase. In certain embodiments, the distinct pieces of DNA originate from different organisms. In certain embodiments, one distinct piece of DNA is integrated in the genome of a host cell, and a second distinct piece of DNA is recombinant DNA. In the art, the process describing the use of a recombinase for exchanging DNA sequences is alternatively and commonly referred to as "recombineering". Recombineering is an efficient homologous recombination-based method for genome engineering and allows precise insertion, deletion, or any kind of alteration of any DNA sequence (Sharan et al., Nature Protocols, 2009). It is evident to a person skilled in the art that the term "recombinase" as used herein is in its broadest interpretation indicative for any protein that may aid, assist, or contribute to any sort of recombination activity, or provide any sort of recombination activity.

It is well known to a person skilled in the art that different recombinases have been described in eukaryotes, bacteria, archaea, phages and viruses. Both recombinases that only rely on homology between the distinct pieces of DNA and recombinases that only function upon inclusion of (a) specific nucleotide sequence(s) in the distinct pieces of DNA have been described. Several systems comprising site specific recombinases and short target sequences are used in molecular biology. The group of site specific DNA recombinase systems popular for use in genome engineering and synthetic biology includes but is by no means limited to the Hin/hix system, the Cre/lox system, the Flp/FRT system, the XerCD/dif system, the FimBE/fims system, the KD/KDRT system, the B2/B2RT system, the B3/B3RT system, the R/RSRT system, the VCreNloxP system, the SCre/SloxP system, the Vika/vox system, the Dre/rox system, the A-lnt/attP-attB system, the HK022/attP-attB system, the cpC3 1/attP-attB system, the Bxb 1/attP-attB system, the Gin/gix system, the Nigri/nox system, the Panto/pox system and the Tn3 res sitel system. Different site specific recombinase systems have been reviewed extensively at several occasions (Grindly etal., Molecular Medical Microbiology (Second edition - chapter 15), 2015).

Accordingly, the term “recognition sequence for a site-specific recombinase” is indicative for a certain nucleotide sequence that is recognised by a site-specific recombinase and is essential to initiate and/or complete the recombination process. In general, recombination sites have a nucleotide length ranging of from about 30 to about 200 nucleotides and consist of at least two motifs with a partial inverted- repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. The pairs of sites between which the recombination occurs may be identical (e.g. Lox sites) or non-identical (e.g. attP and attB sites of lambda integrase). Various pairs of recognition sequences (interchangeably indicated in the art by the term “recognition site”) and sitespecific recombinases have been described in the art and are known to a skilled person (e.g. Olorunniji et al., Biochem J, 2016). Site-specific recombinases are commonly abbreviated in the art as “SSRs”. In certain embodiments, the recombinases referred to herein (e.g. RSA, RSB, and optionally the SSAP) are tyrosine recombinases. In alternative embodiments, the recombinases referred to herein (e.g. RSA, RSB, and optionally the SSAP) are tyrosine recombinases. In yet alternative embodiments, the recombinases referred to herein (e.g. RSA, RSB, and optionally the SSAP) are a combination of serine and tyrosine recombinases. The molecular mechanisms of both serine recombinases and tyrosine recombinases have been described in the art (respectively in e.g. Stark, Mirobiol Spectr, 2014; and Jayaram et al., Microbiol Spectr, 2015).

As indicated above, the recognition sequence for the first site-specific recombinase (RSA) is preferably identical in the first and second nucleotide arrangement.

Instead of indicating the relative position of the different features of the oligonucleotide modification system by terms such as 5’, 3’, upstream, and downstream, the relative position may additionally or alternatively be indicated by terms such as “flanking”. It is to be understood that “flanking” merely indicates that a certain nucleotide sequence has at both 5’ and 3’ of said nucleotide sequence two other sequences, which may or may not be identical sequences. Furthermore, “flanking” does therefore not necessarily equates expression such “immediately adjacent to” or “neighboring”, but may be attributed this interpretation in certain embodiments.

In certain embodiments, one or more alternative nucleotide arrangements reading on the first or second nucleotide arrangement of the oligonucleotide system described herein are additionally comprised in the oligonucleotide modification system. For example, a first nucleotide arrangement described herein and at least two distinct second nucleotide arrangements described herein may be comprised in the oligonucleotide modification system. Alternatively, at least two distinct first nucleotide arrangements described herein and a second nucleotide arrangement described herein may be comprised in the oligonucleotide modification system. Yet alternatively, at least two distinct first nucleotide arrangements described herein and at least two distinct second nucleotide arrangements described herein may be comprised in the oligonucleotide modification system. It is envisaged by the inventors that such compositions of the oligonucleotide modification system allow for multiplex genome engineering (i.e. simultaneous or near simultaneous modification of distinct genomic regions and/or positions).

In certain embodiments, the first nucleotide arrangement has a nucleotide length of from 20 to 2500 nucleotides. In preferred embodiments, the first nucleotide arrangement has a nucleotide length of from 40 to 1250 nucleotides, preferably of from 44 to 1000 nucleotides, preferably of from 45 to 750 nucleotides, preferably of from 50 to 500 nucleotides, preferably of from 55 to 300 nucleotides, preferably of from 58 to 250 nucleotides, preferably of from 100 to 150 nucleotides. In certain embodiments such as those described above, the recognition sequence for a first site-specific recombinase (RSA) is a sequence having a sequence length of at least 34 nucleotides, preferably at least 47 nucleotides, more preferably at least 48 nucleotides. In certain embodiments, the first nucleotide arrangement has a nucleotide length of at least 44 nucleotides, preferably at least 45 nucleotides, preferably at least 50 nucleotides, preferably at least 55 nucleotides, preferably at least 58 nucleotides, preferably at least 60 nucleotides, preferably at least 65 nucleotides, preferably at least 70 nucleotides, preferably at least 75 nucleotides, preferably at least 80 nucleotides, preferably at least 85 nucleotides, preferably at least 90 nucleotides, preferably at least 95 nucleotides, preferably at least 100 nucleotides.

In certain embodiments, the oligonucleotide modification system suitable for genetic modification of a bacterium, said bacterium comprising an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein, consisting of a first nucleotide arrangement and a second nucleotide arrangement wherein the first nucleotide arrangement consists of two nucleotide sequences (HA1 and HA2) each consisting of a stretch of at least 5 contiguous nucleotides of a target genomic sequence of the bacterium (i.e. the bacterium that is to be genetically modified), and a recognition sequence for a first site-specific recombinase (RSA) arranged such that said first nucleotide arrangement consists one of said two nucleotide sequences (HA1) consisting of a stretch of at least 5 contiguous nucleotides (located) 5 ’of said RSA recognition sequence and the other nucleotide sequence (HA2) consisting of a stretch of at least 5 contiguous nucleotides (located) 3’ of said RSA recognition sequence; and a second nucleotide arrangement comprising the RSA recognition sequence for said first site-specific recombinase, and at least two recognition sequences for a second site-specific recombinase (RSB), arranged such that said second nucleotide arrangement consists of at least one RSB recognition sequence (located) 5’ of the RSA recognition sequence and at least one RSB recognition sequence (located) 3’ of the RSA sequence.

In certain embodiments, the oligonucleotide modification system suitable for genetic modification of a bacterium, said bacterium comprising an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein, comprising a first nucleotide arrangement and a second nucleotide arrangement wherein the first nucleotide arrangement consists essentially of or consists of two nucleotide sequences (HA1 and HA2) each consisting of a stretch of at least 5 contiguous nucleotides of a target genomic sequence of the bacterium (i.e. the bacterium that is to be genetically modified), and a recognition sequence for a first site-specific recombinase (RSA) arranged such that said first nucleotide arrangement comprises one of said two nucleotide sequences (HA1) consisting of a stretch of at least 5 contiguous nucleotides (located) 5 ’of said RSA recognition sequence and the other nucleotide sequence (HA2) consisting of a stretch of at least 5 contiguous nucleotides (located) 3 ’ of said RSA recognition sequence; and a second nucleotide arrangement comprising the RSA recognition sequence for said first site-specific recombinase, and at least two recognition sequences for a second site-specific recombinase (RSB), arranged such that said second nucleotide arrangement comprises at least one RSB recognition sequence (located) 5 ’ of the RSA recognition sequence and at least one RSB recognition sequence (located) 3’ of the RSA sequence. In embodiments wherein the method described herein results in the generation of a genetically modified bacterium that is characterised by one or more inserted exogenous genes or functional gene fragments, the one or more exogenous sequences (i.e. the sequences that are to be permanently inserted into the genomic sequence of the bacterium) that are to be inserted are preferably located in the second nucleotide arrangement. The length of these sequences are not particularly limiting for the invention.

In certain embodiments, HA1 and HA2 each comprise a stretch of at least 6 contiguous nucleotides, preferably each comprise a stretch of at least 7 contiguous nucleotides, preferably each comprise a stretch of at least 8 contiguous nucleotides, preferably each comprise a stretch of at least 9 contiguous nucleotides, preferably each comprise a stretch of at least 10 contiguous nucleotides, preferably each comprise a stretch of at least 12 contiguous nucleotides, preferably each comprise a stretch of at least 15 contiguous nucleotides, preferably each comprise a stretch of at least 20 contiguous nucleotides. It is by no means essential that HA1 and HA2 are equal in terms of their nucleotide length. Thus, in certain embodiments, HA1 and HA2 each comprise a stretch of from about 5 to about 200 nucleotides, preferably each comprise a stretch of from about 6 to 175 contiguous nucleotides, preferably each comprise a stretch of from about 7 to 150 contiguous nucleotides, preferably each comprise a stretch of from 8 to 125 contiguous nucleotides, preferably each comprise a stretch of from 9 to 100 contiguous nucleotides, preferably each comprise a stretch of from 10 to 75 contiguous nucleotides, preferably each comprise a stretch of from 12 to 50 contiguous nucleotides, preferably each comprise a stretch of from 15 to 25 contiguous nucleotides, preferably each comprise a stretch of about 20 contiguous nucleotides.

In certain embodiments, the ssDNA annealing protein is a third recombinase.

In preferred embodiments, the ssDNA annealing protein is a GP35 recombinase. In a yet more preferred embodiment, the ssDNA annealing protein is a GP35 recombinase having an amino acid sequence which is at least 65% identical, preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 92.5% identical, more preferably at least 95% identical, most preferably at least 97.5% identical to the amino acid sequence of GP35 from Bacillus subtilis bacteriophage SPP1 as defined by SEQ ID NO: 1 based on the total length of the amino acid sequence of the recombinase. In certain embodiments, the ssDNA annealing protein is a homologue ororthologue of GP35 from Bacillus subtilis bacteriophage SPP1. In preferred embodiments wherein the ssDNA annealing protein is a GP35 recombinase having an amino acid sequence which is at least 95% identical to the amino acid sequence of GP35 from Bacillus subtilis bacteriophage SPP1 as defined by SEQ ID NO: 1, the bacterium is a. Mycoplasma bacterium, preferably ^.Mycoplasma pneumoniae bacterium.

The SPP1 GP35 amino acid sequence annotated under NCBI reference sequence NP 690727.1 is reproduced below (SEQ ID NO: 1): MATKKQEELKNALAQQNGAVPQTPVKPQDKVKGYLERMMPAIKDVLPKHLDADRLSRIAMNVIRTNPK LLECDTASLMGAVLESAKLGVEPGLLGQAYILPYTNYKKKTVEAQFILGYKGLLDLVRRSGHVSTISA QTVYKNDTFEYEYGLDDKLVHRPAPFGTDRGEPVGYYAVAKMKDGGYNFLVMSKQDVEKHRDAFSKSK NREGVVYGPWADHFDAMAKKTVLRQLINYLPISVEQLSGVAADERTGSELHNQFADDDNIINVDINTG EIIDHQEKLGGETNE

In alternative preferred embodiments, the ssDNA annealing protein is a recT recombinase. In a yet more preferred embodiment, the ssDNA annealing protein is a Lactobacillus reuteri recT recombinase or a Enterococcus faecalis recT recombinase. In certain embodiments, the ssDNA annealing protein is a homologue or orthologue of Lactobacillus reuteri recT recombinase or a Enterococcus faecalis recT recombinase. In yet further preferred embodiments, the recT recombinase is a recombinase having an amino acid sequence which is at least 65% identical, preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 92.5% identical, more preferably at least 95% identical, most preferably at least 97.5% identical to the amino acid sequence of Lactobacillus reuteri recT recombinase as defined by SEQ ID NO: 2 based on the total length of the amino acid sequence of the recombinase. In preferred embodiments wherein the ssDNA annealing protein is a Lactobacillus reuteri recT recombinase having an amino acid sequence which is at least 95% identical to the amino acid sequence of Lactobacillus reuteri recT recombinase as defined by SEQ ID NO: 2, the bacterium is a Lactobacillus bacterium.

The Lactobacillus reuteri recT recombinase amino acid sequence annotated under NCBI reference sequence WP 003668036.1 is reproduced below (SEQ ID NO: 2):

MTNQVAQQQKPTKLTDLVLDRVKQMQDTQDLSLPKNYNASNALNAAFLELQKVQDRNHRPALEVCSHD SIVKSLLDMTLQGLSPAKDQCYFIVYGNELQMQRSYFGTVAAVKRLDGVKKVRAEVVHEKDDFEIGAN EDMELVVKRFVPKFENQDNQI IGAFAMIKTDEGTDFTVMTKKEIDQSWAQTRQKNNKVQQNFSQEMAK RTVLNRAAKMFINTSDDSDLLTGAINDTTSNEYDDERRDVTPVEDEKQSTDKLLEGFQKSQEAKAKGV SNDGNSNEGKETSEEVADGQTELFSEGTIKPADEADS

Iii yet alternative further preferred embodiments, the recT recombinase is a recombinase having an amino acid sequence which is at least 65% identical, preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 92.5% identical, more preferably at least 95% identical, most preferably at least 97.5% identical to the amino acid sequence of Enterococcus faecalis recT recombinase as defined by SEQ ID NO: 3 based on the total length of the amino acid sequence of the recombinase. In preferred embodiments wherein the ssDNA annealing protein is a Enterococcus faecalis recT recombinase having an amino acid sequence which is at least 95% identical to the amino acid sequence of Enterococcus faecalis recT recombinase as defined by SEQ ID NO: 3, the bacterium is a Lactobacillus bacterium.

The Enterococcus faecalis recT recombinase amino acid sequence annotated under GenBank reference sequence AAO81865.1 is reproduced below (SEQ ID NO: 3):

MGNELIVSVQNRIQEMQHGEGLRLPTGYSVGNALNSAYLILSDNSKGKSLLEKCHPTSVSKALLNMAI QGLSPAKNQCYFVPYGDQCTLMRSYFGSVSILERLSNVKKVHAEVI FEGDEFEIGSEDGRTVVTNFKP SFLNRDNPI IGAFAWVEQTDGIKVYTIMTKKEIYKSWSKAKTKNVQNDYPQEMAKRTVLSRAAKMFIN SSSDNDLLVKAINETTEDEYDNNQPRKDITPNPPNIEKLEKSI FNQDENKKIAQDMIDSIDLNQADKD LQEELNIEFPDPSKNYLATGEVNGDVENEDGPYPF

In alternative preferred embodiments, the ssDNA annealing protein is a rec2 recombinase or Ssr recombinase. In a yet more preferred embodiment, the ssDNA annealing protein is a Pseudomonas putida rec2 recombinase or a Pseudomonas putida Ssr recombinase. In certain embodiments, the ssDNA annealing protein is a homologue or orthologue of Pseudomonas putida rec2 recombinase or a Pseudomonas putida Ssr recombinase. In yet further preferred embodiments, the rec2 recombinase is a recombinase having an amino acid sequence which is at least 65% identical, preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 92.5% identical, more preferably at least 95% identical, most preferably at least 97.5% identical to the amino acid sequence of Pseudomonas putida rec2 recombinase as defined by SEQ ID NO: 4 based on the total length of the amino acid sequence of the recombinase. In preferred embodiments wherein the ssDNA annealing protein is a Pseudomonas putida rec2 recombinase having an amino acid sequence which is at least 95% identical to the amino acid sequence of Pseudomonas putida rec2 recombinase as defined by SEQ ID NO: 4, the bacterium is a Pseudomonas bacterium.

The Pseudomonas putida rec2 recombinase amino acid sequence defined by NCBI identifier WP 009397165.1 is reproduced below (SEQ ID NO: 4):

MSNAALAERQNETQLLDAPAGAVQATESSMMIQMIQRAAADPAVDVDKMERLMQMHERFVDRQASAAF NAAMVRAQRRIKPVARRALNVQTNSTYARLEDIDREISPI FTEEGFSLSFGTGDSHLAGYIRVICDVM HDQGHTRQYKMDLPLDATGIGGKTNKTGVHAHGSTNSYARRYLTMNIFNVVMANEDTDGNAEPPEEPV ITSRQAAQLEALLKKCSPTMAGLFIEKYGCASNVYKSEFDEVLAKLTRSANRTQQEPVDANHH

In yet further preferred embodiments, the Ssr recombinase is a recombinase having an amino acid sequence which is at least 65% identical, preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 92.5% identical, more preferably at least 95% identical, most preferably at least 97.5% identical to the amino acid sequence of Pseudomonas putida Ssr recombinase as defined by SEQ ID NO: 5 based on the total length of the amino acid sequence of the recombinase. In preferred embodiments wherein the ssDNA annealing protein is a Pseudomonas putida Ssr recombinase having an amino acid sequence which is at least 95% identical to the amino acid sequence of Pseudomonas putida Ssr recombinase as defined by SEQ ID NO: 5, the bacterium is a Pseudomonas bacterium .

The Pseudomonas putida Ssr recombinase amino acid sequence defined by UniProtKB identifier I7AX46 is reproduced below (SEQ ID NO: 5):

MSQVARVETHSQPQAVAAESATILQI IQQVAMSPNADIDKMERLMAMHRQHQAQQAQQAFDAALAAMQ EELPVIRERGAIKDKYKNVQSTYALWEDINEELKPILAKHGFALTFRIPRTDKGIEVEGVLSHRDGHR ETTSILLPADATGSKNAVQAVASSVSYGKRYTAGALLNFTTTGEDDDGQGAVPMQVPDEPVITPRQAA QLDGLLKKCSQVLVDNFTAKYGCAANVYKSEFDVVLARLTKSASRPQE

Methods and tools to verify sequence homology or sequence identity between different sequences of amino acids or nucleic acids are well known to a person skilled in the art and include non-limiting tools such as Protein BLAST, ClustalW2, SIM alignment tool, TranslatorX and T-COFFEE. The percentage of identity between two sequences may show minor variability depending on the algorithm choice and parameters. The term “sequence identity” refers to the relationship between sequences at the nucleotide (or amino acid) level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. A reference window is chosen and the “% identity” is then calculated by determining the number of nucleotides (or amino acids) that are identical between the sequences in the window, dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence.

The terms “homologue” and “orthologue” are to be interpreted according to their commonly accepted meaning in the art. Hence, a skilled person appreciates that a “homologue” or “homolog” (interchangeably used with terms such as “homologous gene”) is a gene inherited in two species by a common ancestor. Similarly, a skilled person appreciates that an “orthologue” or “ortholog” (interchangeably used with terms such as “orthologous gene”) is a gene in a different species that evolved from a common ancestral gene by speciation and generally retains the same function during the course of evolution.

In certain embodiments, the first recombinase (RSA), the second recombinase (RSB), and/or optionally the third recombinase are recombinases that are characterised by less than 50% cross-reactivity, preferably less than 40% cross-reactivity, more preferably less than 30% cross-reactivity, more preferably less than 25% cross-reactivity, more preferably less than 20% cross-reactivity, more preferably less than 20% cross-reactivity, more preferably less than 15% cross-reactivity, more preferably less than 10% cross-reactivity. In further embodiments, the first recombinase (RSA), the second recombinase (RSB), and/or optionally the third recombinase are recombinases that not display detectable cross-reactivity. “Cross-reactivity” in the context of the present specification indicates that a recognition sequence intended to be exclusively engaged by a first recombinase also displays a certain degree of reactivity when engaged with a second recombinase. Method to evaluate cross-reactivity of recombinases and recognition sites have been described in the art (e.g. in Suzuki and Nakayama, Nucleic Acids Res, 2011) and are therefore known to a person skilled in the art.

In certain embodiments, the second nucleotide arrangement further comprises a nucleotide sequence that encodes at least one gene product that allows for phenotypic selection of bacteria that comprise the second nucleotide arrangement. A skilled person appreciates that a “gene product that allows for phenotypic selection” is interchangeably and commonly referred to as a “selection marker” in the art. These terms are therefore to be interpreted as any gene encoding a gene product that confers a trait suitable for artificial selection. In the context of the present disclosure, these selection markers are therefore highly suitable to separate bacteria that did not recombine with the second nucleotide arrangement from bacteria that did recombined with the second nucleotide arrangement. Traits that a selection marker introduces to the subject bacterial cells are not particularly limited and therefore also includes reporter genes. In certain embodiments, the at least one selection marker is a positive selection marker that confers a selective advantage to the bacterial cell. Hallmark non-limiting examples hereof are antibiotic resistance genes. In alternative embodiments, the at least one selection marker is a negative selection marker (i.e. a counter selectable marker) that reduces or arrests growth of the subject bacterial cell. A hallmark non-limiting example hereof is thymidine kinase, which is known to render a cell susceptible to ganciclovir selection. In yet alternative embodiments, the at least one selection marker is both a positive and negative selection marker. A hallmark yet non-limiting example hereof is a gene of which the corresponding gene product confers a selective advantage to the bacterial cell in a first condition, but reduces or arrests growth of the bacterial cell in a second condition. A hallmark nonlimiting example hereof is an enzyme capable of mitigating an auxotrophy but may also be capable of converting a certain molecule or substance to a molecule that is toxic for the bacterial cell. A skilled person appreciates that “auxotrophy” refers to the inability of a cell to synthesize a product which is essential for its growth and/or survival. It is further evident that a skilled person aware of the above equally envisages combinations of selection markers in certain situations, e.g. in situations where multiple second oligonucleotide arrangements are used or when at least two selection criteria are required to arrive at a desired genetically modified bacterial cell. Therefore, without limitation suitable selection markers include luminescent proteins, an antibiotic resistance protein, a protein conferring auxotrophy, a component of a toxin-antitoxin system, enzymes, or any combination thereof. In certain embodiments, the second nucleotide arrangement is (part of) a circular construct. In certain embodiments, the second nucleotide arrangement is (part of) a double stranded DNA construct. In further embodiments, the second nucleotide arrangement is (part of) a circular double-stranded DNA construct. In yet further embodiments, the second nucleotide arrangement is (part of) an expression construct. In even further embodiments, the second nucleotide arrangement is (part of) a plasmid, “plasmid” refers to a circular double stranded DNA construct wherein additional DNA fragments (i.e. DNA nucleotide sequences) can be inserted by molecular cloning methods.

In certain embodiments, the second nucleotide arrangement further comprises a nucleotide sequence encoding the first recombinase (RSA) and/or a nucleotide sequence encoding the second recombinase (RSB), preferably wherein each recombinase is operably linked to a promoter sequence. The wording “operably linked” refers to a multitude of genetic elements that are joined as part of the same nucleic acid molecule, suitably positioned and oriented fortranscription to be initiated from the promoter. Thus, “operably linked” indicates that a certain nucleotide sequence encoding a particular gene product of interest (in context of the present invention encoding the first recombinase (RSA) and/or the second recombinase (RSB)) is linked to a regulatory element DNA such as a promoter sequence in an arrangement that allows for expression of the gene product of interest. In certain embodiments, the first recombinase (RSA) and the second recombinase (RSB) are encoded to be expressed as fusion proteins and the expression of said fusion protein is regulated by a single promoter sequence. In alternative embodiments, the first recombinase (RSA) and the second recombinase (RSB) are encoded to be expressed as one transcript wherein the expression of said transcript is regulated by a single promoter sequence, and wherein the single transcript into two physically distinct recombinases upon translation. In such embodiments, the nucleotide sequences encoding the first recombinase (RSA) and the second recombinase (RSB) are flanking a third nucleotide sequence that causes separation during translation, for example a sequence encoding a 2A peptide. It has been established in the art that 2A peptides are short peptides that cause produce equimolar levels of multiple genes from the same mRNA. The ribosome skips the synthesis of a peptide bond at the C -terminus of a 2A peptide, leading to separation between the end of the 2A sequence and the next peptide downstream. This skipping occurs between the Glycine and Proline residues found on the C -terminus meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline that is part of the 2A sequence (Liu et al. , Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector, Scientific Reports, 2017). An alternative non-limiting example of a third nucleotide sequence that causes separation during translation the a so-called Internal Ribosome Entry' Sites (IRES) sequence (Bochkov and Palmenberg, Biotechniques, 2018).

In alternative embodiments, the nucleotide sequence encoding the first recombinase (RSA) and the nucleotide sequence encoding the second recombinase (RSB) are each operably linked to a distinct promoter sequence, wherein the distinct promoter sequence may or may not be identical to each other. Hence, in further embodiments, the nucleotide sequence encoding the first recombinase (RSA) and the nucleotide sequence encoding the second recombinase (RSB) are each operably linked to a dedicated promoter sequence. In such embodiments, one can appreciate that the nucleotide sequence encoding the first recombinase (RS A) and the nucleotide sequence encoding the second recombinase (RSB) are each operably linked to a promoter sequence that that is not operably linked to the other recombinase. The term “unique” in this context does not impose a limitation on envisaged embodiments that the promoter sequences should comprise different nucleotide sequences, although this may occur. Thus, in certain embodiments, the nucleotide sequence encoding the first recombinase (RSA) and the nucleotide sequence encoding the second recombinase (RSB) are each operably linked to a unique promoter sequence wherein the promoter sequences do not have 100% sequence identity to one another. In alternative embodiments, the nucleotide sequence encoding the first recombinase (RSA) and the nucleotide sequence encoding the second recombinase (RSB) are each operably linked to a unique promoter sequence wherein the promoter sequences do not have 100% sequence identity to one another. In certain of the above embodiments, the promoter sequences allow for expression of the first recombinase (RSA) and the second recombinase (RSB) independently from one another. In such embodiments, the oligonucleotide modification system comprises functionally independent promoter sequences.

The term “promoter” as defined herein is a region of DNA that initiates transcription of a particular gene and hence enables a gene to be transcribed. A promoter is thus an example of a regulatory element. A promoter is recognized by RNA polymerase, which then initiates transcription. Thus, a promoter contains a DNA sequence that is either bound directly by, or is involved in the recruitment, of RNA polymerase. A promoter sequence can also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the transacting factors) to enhance transcription levels of genes in a gene-cluster. The enhancer, while typically at the 5 ’ end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3’ to the coding region of the gene. Promoters may be located in close proximity of the start codon of genes, in preferred embodiments on the same strand and typically located upstream (5’) of the gene. Promoters may vary in size, and are preferably from about 100 to 1000 nucleotides long. In certain embodiments, the promoter may be a constitutive promoter. A constitutive promoter is understood by a skilled person to be a promoter whose expression is constant under the standard culturing conditions, i.e. a promoter which expresses a gene product at a constant expression level. Alternatively, it is also appreciated that inducible (conditional) promoters are promoters which are responsive at least one induction cue. Inducible promoters, and more specifically bacterial inducible promoter systems have been described in great detail in the art (inter alia in Brautaset et al. , Positively regulated bacterial expression systems, Microbial biotechnology, 2009). Thus, in certain embodiments at least the first recombinase (RS A) and/or the second recombinase (RSB) is operably linked to an inducible operator sequence. In certain embodiments, the inducible promoter is chemically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducing agent such as an alcohol, tetracycline, a steroid, a metal, or other small molecule) or physically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high or low temperatures). An inducible promoter can also be regulated by other transcription factors that are constitutive or are themselves directly regulated by chemical or physical cues. In preferred embodiments, the first recombinase (RSA) is operably linked to a constitutive promoter and the second recombinase (RSB) is operably linked to an inducible operator sequence.

By means of example and not limitation, the inducible promoter may be a TetR promoter part of a Tet- On or Tet-off system (Krueger et al., Tetracycline derivatives: alternative effectors for Tet transregulators, Biotechniques, 2004, and, Loew et al., Improved Tet-responsive promoters with minimized background expression, BioMedCentral Biotechnology, 2010).

By means of alternative example and not limitation, at least one of the recombinases may be operably linked to a cumate promoter and operator sequence (CuO). In certain preferred embodiments, the cumate promoter and operator sequence (CuO) has a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 and does not comprise or consists of SEQ ID NO: 9 or SEQ ID NO: 11. In further preferred embodiments, the cumate promoter and operator sequence comprises SEQ ID NO: 6 or SEQ ID NO: 10.

SEQ ID NO: 6 and SEQ ID NO: 10 are further objects of the invention as discussed further in the present specification and correspond to the following nucleotide sequences:

TTTATTTGACAAAAATGAGATTATTTGGTATAATTAATGTAGTAACAAACAGACAATCTGGTCTGTTT GTATTAATATAAACTAATAAGGAGGACAAAC (SEQ ID NO: 6)

ATTTTGTCAAAATTTTATTTGACAAAAATGAGATTATTTGGTATAATTAATGTAGTAACAAACAGACA ATCTGGTCTGTTTGTATTAATATAAACTAATAAGGAGGACAAAC (SEQ ID NO: 10)

In certain embodiments, the first nucleotide arrangement is a nucleotide sequence consisting essentially of or consisting of a recognition sequence for the first recombinase (RSA) flanked by two homology arms (HA1 and HA2), each of at least 5 contiguous nucleotides. Such embodiments do not exclude any embodiments wherein the first nucleotide arrangement is a nucleotide sequence consisting essentially of or consisting of a recognition sequence for the first recombinase (RSA) flanked by two homology arms (HA1 and HA2), each of at least 5 contiguous nucleotides and wherein at least one nucleotide is chemically modified. For example, in any of the embodiments described herein, the 5 ’ nucleotide and/or 3 ’ nucleotide may comprise a chemical modification which protects the first nucleotide arrangement from degradation upon introduction into the bacterium that is to be genetically modified. A non-limiting example of a suitable modification is a phosphorothioate modification.

In alternative embodiments, the first nucleotide arrangement further comprises an intron-encoded endonuclease restriction site sequence located between the RSA recognition sequence and the stretch of at least 5 contiguous nucleotides of genomic sequence (HA1) of the bacterium to be modified (located) 5’ of the RSA recognition sequence. In yet alternative embodiments, the first nucleotide arrangement further comprises an intron-encoded endonuclease restriction site sequence located between the RSA recognition sequence and the stretch of at least 5 contiguous nucleotides of genomic sequence (HA2) of the bacterium to be modified (located) 3 ’of the RSA recognition sequence. In preferred embodiments, the intron-encoded endonuclease restriction site is an I-Scel endonuclease restriction site. In further preferred embodiments, the intron-encoded endonuclease restriction site comprises the sequence 5’-TAGGGATAACAGGGTAAT-3’ (SEQ ID NO: 8). In certain embodiments, the first nucleotide arrangement consists essentially of the following components listed in a 5’ to 3’ orientation: a stretch of at least 5 contiguous nucleotides of genomic sequence of the bacterium to be modified (HA1), a I-Scel endonuclease restriction site, a RSA recognition sequence, and a second stretch of at least 5 contiguous nucleotides of genomic sequence of the bacterium to be modified (HA2). In certain embodiments, the first nucleotide arrangement consists essentially of the following components listed in a 5’ to 3’ orientation: a stretch of at least 5 contiguous nucleotides of genomic sequence of the bacterium to be modified (HA1), a RSA recognition sequence, a I-Scel endonuclease restriction site, and a second stretch of at least 5 contiguous nucleotides of genomic sequence of the bacterium to be modified (HA2). In preferred embodiments, the first nucleotide arrangement has a nucleotide length of from 62 to 1250 nucleotides, preferably of from 65 to 1000 nucleotides, preferably of from 70 to 750 nucleotides, preferably of from 75 to 500 nucleotides, preferably of from 76 to 300 nucleotides, preferably of from 80 to 250 nucleotides, preferably of from 85 to 150 nucleotides. In certain embodiments such as those described above, the recognition sequence for a first site-specific recombinase (RSA) is a sequence having a sequence length of at least 34 nucleotides, preferably at least 47 nucleotides, more preferably at least 48 nucleotides. In certain embodiments, the first nucleotide arrangement has a nucleotide length of at least 62 nucleotides, preferably at least 65 nucleotides, preferably at least 75 nucleotides, preferably at least 76 nucleotides, preferably at least 80 nucleotides, preferably at least 85 nucleotides, preferably at least 90 nucleotides, preferably at least 95 nucleotides, preferably at least 100 nucleotides, preferably at least 105 nucleotides, preferably at least 110 nucleotides, preferably at least 115 nucleotides, preferably at least 120 nucleotides, preferably at least 130 nucleotides. The choice of the first and second recombinases in the present invention is not particularly limiting. Illustrative non-limiting examples of suitable recombinases include ere recombinases. Sere recombinases, Vika recombinases, and Bxbl recombinases.

Neither particularly limiting are the bacteria that can be subjected to genetic modification by the oligonucleotide modification system as described herein. Non-limiting examples of bacteria include Mycoplasma bacteria, Pseudomonas bacteria, and Lactobacillus bacteria. Each of the bacteria used in conjunction with the herein described aspects of the invention may be a naturally occurring bacterial strain (i.e. a wild type strain), or a bacterial strain that was artificially genetically modified in a previous point in time. Additionally envisaged are so called synthetic bacterial strains, i.e. strains that have been synthesized de novo by human synthetic biology approaches.

Exemplar}’ Mycoplasma species include those of the following non-exhaustive list: M. adleri, M. agalactiae, M. agassizii, M. alkalescens, M. alligatoris, M. alvi, M. amphoriforme, M. anatis, M. anseris, M. arginine, M. arthritidis, M. auris, M. bovigenitalium, M. bovirhinis, M. bovis, M. bovoculi, M. buccale, M. buteonis, M. californicum, M. canadense, M. canis, M. capricolum, M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, M. caviae, M. cavipharyngis, M. ciconiae, M. citelli, M. cloacale, M. collis, M. columbinasale, M. columbinum, M. columborale, M. conjunctivae, M. corogypsi, M. cottewii, M. cricetuli, M. crocodyli, M. cynos, M. dispar, M. edwardii, M. elephantis, M. equigenitalium, M. equirhinis, M. falconis, M. fastidiosum, M. faucium, M. felifaucium, M. feliminutum, M. felis, M. feriruminatoris, M. fermentans, M. flocculare, M. gallinaceum, M. gallinarum, M. gallisepticum, M. gallopavonis, M. gateae, M. genitalium, M. glycophilum, M. gypis, M. haemocanis, M. haemofelis, M. haemomuris, M. hominis, M. hyopharyngis, M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. iguana, M. imitans, M. indiense, M. iners, M. iowae, M. lagogenitalium, M. leachii, M. leonicaptivi, M. leopharyngis, M. lipofaciens, M. lipophilum, M. maculosum, M. meleagridis, M. microti, M. moatsii, M. mobile, M. molare, M. mucosicanis, M. muris, M. mustelae, M. mycoides, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. neophronis, M. neurolyticum, M. opalescens, M. orale, M. ovipneumoniae, M. ovis, M. oxoniensis, M. penetrans, M. phocicerebrale, M. phocidae, M. phocirhinis, M. pirum, M. pneumoniae, M. primatum, M. pullorum, M. pulmonis, M. putrefaciens, M. salivarium, M. simbae, M. spermatophilum, M. spumans, M. sturni, M. sualvi, M. subdolum, M. suis, M. synoviae, M. testudineum, M. testudinis, M. tullyi, M. verecundum, M. wenyonii, M. yeatsii, M. coccoides. 'Mycoplasma" further includes the non-limiting list of candidate species Moeniiplasma glomeromycotorum, M. aoti, M. corallicola, M. erythrocervae, M. girerdii, M. haematoparvum, M. haemobos, M. haemocervae, M. haemodidelphidis, M. haemohominis, M. haemolamae, M. haemomacaque, M. haemomeles, M. haemominutum, M. haemomuris subsp. musculi, M. Haemomuris subsp. ratti, M. haemovis, M. haemozalophi, M. kahaneii, M. ravipulmonis, M. struthiolus, M. turicensis, M. haemotarandirangiferis, M. preputii and others such as M. insons, M. sphe nisei. M. vulturis, and M. zalophi. Similarly, it is evident to a skilled person that the term Mycoplasma additionally includes any Mycoplasma strain or species that is generated by genetic or chemical synthesis, or any sort of rational design and/or the reorganization of a naturally occurring Mycoplasma genomic sequence and that the term therefore also covers those Mycoplasma strains and species that are termed “synthetic Mycoplasma", alternatively “Mycoplasma laboraiorium". “Mycoplasma synthia”, or even short “Synthia” in the art (Gibson et al., Science, 2010). Hence, in certain embodiments described throughout this specification, the Mycoplasma species subject of the invention have as genomic sequence comprising 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 97% global sequence identity to a naturally occurring or commercially available Mycoplasma bacterium such as those described herein. In certain embodiments, the Mycoplasma bacterium is M. pneumoniae M129-B7 as available from the American Type Culture Collection accession number 29342.

Exemplary Pseudomonas species cover both P. aeruginosa species, P. chlororaphis species, P. fluorescens species, P. pertucinogena species, P. putida species, P. stutzeri species, P. syringae species, and incertae sedis and con sequently include those of the following non-exhaustive list: P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthi, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, and P. xanthomarina, It is evident to a skilled person that the term Pseudomonas additionally includes any Pseudomonas strain or species that is generated by genetic or chemical synthesis, or any sort of rational design and/or the reorganization of a naturally occurring Pseudomonas genomic sequence and that the term therefore also covers those Pseudomonas strains and species that are termed “synthetic Pseudomonas”, alternatively "Pseudomonas laboratorium” , or “Pseudomonas synthia. Hence, in certain embodiments described throughout this specification, the Pseudomonas species subject of the invention have as genomic sequence comprising 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 97% global sequence identity to a naturally occurring or commercially available Pseudomonas bacterium such as those described herein.

Exemplary Lactobacillus species include those of the following non-exhaustive list: L. acetotolerans, L. acidophilus, L. alvi, L. amylolyticus, L. amylovorus, L. apis, L. backi, L. bombicola, L. colini, L. crispatus, L. delbrueckii, L. equicursoris, L. fornicalis, L. gallinarum, L. gasseri, L. gigeriorum, L. ginsenosidimutans, L. hamster, L. helsingborgensis, L. helveticus, L. hominis, L. iners, L. intestinalis, L. Jensenii, L. jinshani, L. johnsonii, L. kalixensis, L. kefiranofaciens, L. kimbladii, L. kitasatonis, L. kullabergensis, L. melliventris, L. mulieris, L. nasalidis, L panisapium, L. paragasseri, L. pasteurii, L. porci, L. psittaci, L. raoultii, L. rodentium, L. rogosae, L. taiwanensis, L. thermophilus, L. timonensis, L. ultunensis, and L. xujianguonis . It is evident to a skilled person that the term Lactobacillus additionally includes any Lactobacillus strain or species that is generated by genetic or chemical synthesis, or any sort of rational design and/or the reorganization of a naturally occurring Lactobacillus genomic sequence and that the term therefore also covers those Lactobacillus strains and species that are termed “synthetic Lactobacillus” , alternatively “Lactobacillus laboratorium” , or “Lactobacillus synthia. Hence, in certain embodiments described throughout this specification, the Lactobacillus species subject of the invention have as genomic sequence comprising 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 97% global sequence identity to a naturally occurring or commercially available Lactobacillus bacterium such as those described herein.

A further aspect of the invention is related to the use of an oligonucleotide modification system as described herein for genetic modification (i.e. alteration of the genomic sequence) of a bacterium. Preferably, the use of an oligonucleotide modification as described herein for genetic modification of a bacterium is in vitro use thereof. In certain embodiments, the oligonucleotide modification system is used to introduce nucleotide sequences encoding one or more heterologous gene products into the genomic sequence of the bacterium . In alternative embodiments, the oligonucleotide modification system is used to introduce nucleotide sequences encoding additional copies of one or more endogenous gene products into the genomic sequence of the bacterium. In yet alternative embodiments, the oligonucleotide modification system is used to introduce one or more nucleotide sequences encoding one or more therapeutic gene products, such as but not limited to therapeutic proteins and therapeutic peptides. In yet alternative embodiments, the oligonucleotide modification system is used to introduce nucleotide sequences encoding a combination of at least one heterologous gene product and at least one additional copy of an endogenous gene product into the genomic sequence of the bacterium. In yet a further embodiment, the oligonucleotide modification system is used to rearrange certain portions of the genomic sequence of the bacterium. In yet an even further embodiment, the portion of the genomic sequence of the bacterium is a protein coding gene. In alternative embodiments, the oligonucleotide modification system is used to alter the sequence of a regulator}- genomic element of a bacterium. In further embodiments, the regulator}- genomic element is a nucleotide sequence which is capable of increasing or decreasing the expression of one or more specific genes. In certain embodiments, the oligonucleotide modification system is used to introduce nucleotide sequences encoding one or more heterologous gene products into the genomic sequence of the bacterium wherein said one or more heterologous gene products are capable of further inducing one or more modifications to the genomic sequence of the bacterium. Non-limiting illustrative examples hereof are “engineered” nucleases such as Zinc Zinger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) /CRISPR associated (Cas) systems (CRISPR/Cas systems). Exemplar}-’ Cas proteins include without limitation: Casl, Caslb, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9, CaslO, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, CaslO, Csm2, Cmr5, CaslO, Csxl l, CsxlO, Csfl, Cas9, Csn2, Cas4, Cpfl (i.e. Casl2), C2cl, C2c3, Casl3a, Casl 3b, Casl3c, and Casl3d. The terms ZFNs, TALENs, and CRISPR/Cas systems also encompass homologs or mutants of said ZFNs, TALENs, and CRISPR/Cas systems.

A skilled person appreciates that terms such as “regulator}- (genomic) element” and “regulatory (genomic) sequence” are interchangeably indicated in the art respectively by the terms “control element” and “control sequence”. By means of illustration and not limitation, the regulator}- element or regulator}- sequence may be an enhancer, a selection marker, an origin of replication, a linker sequence, a poly A sequence, a terminator sequence, or a degradation sequence.

The term “therapeutic protein” or “therapeutic peptide” is considered clear to a person skilled in the art and the skilled person understands that a wide range of therapeutic proteins have been described in the art. Therapeutic proteins can be stratified into five large groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Alternatively, therapeutic proteins may also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Therapeutic proteins and therapeutic peptides can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target such as monoclonal antibodies; (b) affecting covalent bonds such as enzymes; and (c) exerting activity without specific interactions, e.g. serum albumin. The above mentioned classifications and the contribution of each of the groups to the state of the art have been described in scientific literature (Dimitrov, Therapeutic proteins, Methods in Molecular Biology, 2012). Non limiting examples of classes of therapeutic proteins include cytokines, antibodies, nanobodies, (soluble) receptors, antibody-like protein scaffolds, and functional fragments hereof.

In further embodiments where the oligonucleotide modification system is used to insert a protein coding nucleotide sequence into the genomic sequence of the bacterium, the gene product may further comprise a peptide or protein tag sequence. Non-limiting examples of commonly used peptide tag sequences are the AviTag, C-tag, calmodulin-tag, polyglutamate tag, E-tag, Flag-tag, HA-tag, His-tag, Myc-tag, NE- tag, RholD4-tag, S-tag, SBP-tag, Softag 1, Softag 3, Spot-tag, Strep-tag, TC tag, Ty tag, V5 tag, VSV- tag, Xpress tag, isopeptag, SpyTag, SnoopTag, DogTag, and the SdyTag

In certain embodiments, the oligonucleotide modification system is used to replace, mutate and/or delete nucleotide sequences encoding one or more virulence factors. The term “virulence factor” is to be interpreted according to the common interpretation of said term in the art, i.e. any cellular structure, molecules, or regulatory system that enable pathogens to achieve colonization in the host (or a niche of the host), evasion of the host's immune response (either partially or completely), immunosuppression (either partially or completely), and obtain nutrition from the host. An immune response in the context of the current specification is a reaction which occurs within an organism for the purpose of counteracting a foreign invading organism by the host organism subject of exposure to said foreign organism. In certain prior art documents, an immune response is commonly described as repelling or inhibiting a foreign organism from proliferating or surviving in a host organism. Alternatively, the purpose of an immune response is known in the art to be the safeguarding of the host organism from the invading organism. A skilled person understands that in general, an immune response leads to an improved health state of the infected organism. However, in certain instances the improved health state of the infected organism is characteri zed by a prior temporary? decrease in health state of the infected organism. In certain embodiments, the virulence factor may be replaced by an exogenous gene product. Alternatively’ phrased, the genetically modified bacterium may be attenuated vis-a-vis the same bacterium prior to the start of the genomic modification process or introduction of the oligonucleotide modification system into the bacterium.

The term “attenuated” as described herein can be used interchangeably’ with terms such as "weakened" and "diminished". The wording "attenuated strain" is commonly used in the art and refers to weakened disease agents, i.e. attenuated pathogens. An attenuated bacterium is a weakened, less vigorous, less virulent bacterium when compared to the traditionally occurring counterpart. An attenuated bacterium according to embodiments of the invention is indicative for a genetically modified bacterium wherein expression of genes whereof the gene product is responsible for, or contributes to a certain degree of virulence or toxicity have been modified in order to diminish the adverse effect of said gene on an infected subject. In certain embodiments, expression of a gene product responsible for a degree of toxicity is completely impeded by use of the oligonucleotide modification system. In further embodiments, the promoter of the gene encoding the toxic gene product is inactivated by mutagenesis of the promoter sequence by use of the oligonucleotide modification system. In alternative further embodiments, a coding region, or exon, of a gene contributing to toxicity is mutagenized or removed by use of the oligonucleotide modification system. In yet a further embodiment, a frame shift in a gene contributing to toxicity is induced by use of the oligonucleotide modification system. In even further embodiments, a gene encoding atoxic or harmful gene product is replaced by a heterologous nucleotide- encoded gene product by use of the oligonucleotide modification system. In even further embodiments, the expression level of a toxic or harmfill gene product is diminished by by use of the oligonucleotide modification system. In further embodiments, one or more fragments of a toxic or harmful gene are removed by use of the oligonucleotide modification system, whereby optionally the one or more fragments are removed without altering the reading frame and hence the modified gene product is still expressed.

The tenn “subject” refers to animals, preferably warm-blooded animals, more preferably vertebrates, and even more preferably mammals specifically including humans and non-human mammals, that have been the object of treatment, observation or experiment. The term “mammals”, or “mammalian subjects” refers to any animal classified as such and include, but are not limited to, humans, domestic animals, commercial animals, farm animals, zoo animals, sport animals, pet and experimental animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows: primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Preferred subjects are human subjects including all genders and all age categories thereof.

In certain embodiments, the use of the oligonucleotide modification system results in a genetically modified bacterium that has reduced pathogenicity and/or immunogenicity upon introduction into a host organism when compared to the naturally occurring and non-modified bacterium. In further embodiments, the pathogenicity and/or immunogenicity of the modified bacterium is decreased by at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% when compared to a reference bacterium which may be the corresponding non-modified bacterium and/or the naturally occurring wild type bacterium with the proviso that said reference bacterium does not comprise said genomic modification introduced by the oligonucleotide modification system. In certain embodiments, the reduced pathogenicity and/or immunogenicity is reduced to such an extent that the genetically modified bacterium, in contrast to the naturally occurring corresponding bacterium, does not lead to a manifestation of clinical symptoms when introduced into a healthy human subject. In further embodiments, the reduced pathogenicity and/or immunogenicity is characterized by a reduction of toxicity by at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, when said bacterium is introduced to a host organism when compared to a suitable reference bacterium. A suitable reference bacterium in this context is a bacterium having an identical genomic sequence to the subject bacterium with the proviso that said reference bacterium does not comprise the genomic sequence. In yet further embodiments, the reduction of toxicity is assessed by measuring the inflammatory response by a host organism upon infection by the bacterium, preferably by measuring the expression levels of inflammatory cytokines in a host organism upon infection by the bacterium. In certain embodiments, the genetically modified bacterium is a replicative defective genetically modified bacterium.

A further aspect of the invention is directed to a method of genetically modifying a bacterium, the method comprising the steps of:

(i) transforming a culture of said bacterium comprising an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein with (i) an oligonucleotide modification system as defined in any one of the embodiments described herein, and (ii) a first recombinase (RSA),

(ii) selecting bacteria from the culture comprising a genomic sequence wherein the second nucleotide arrangement is integrated,

(iii) expressing a second recombinase (RSB) in the bacterium, thereby removing the second nucleotide arrangement from the genomic sequence of the bacterium.

Methods and protocols to introduce nucleotide arrangements into bacteria, i.e. methods of bacterial transformation, are known to a person skilled in the art (Johnston et al., Nature Rev Microbiol, 2014). The term “transformation” is indicative for a genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material. In this context specifically, “incorporation” does not indicate incorporation in the genomic sequence of the cell, but merely internalization of the exogenous genetic material into the cell. Transformation is a horizontal gene transfer process and is commonly used in context of introducing foreign DNA to a bacterial, yeast, plant, animal, or human cell. Cells capable of taking up foreign DNA are named competent cells. In other embodiments, transformation may be indicative for the insertion of new genetic material into animal and human cells, albeit the term “transfection” is more common for these cells. The term “transformation hence encompasses chemical, physical, and electrical process (or any combination thereof) that facilitates or forces the uptake of exogenous genetic material, such as DNA (Ren et al., Appl Microbiol Biotechnol, 2019). By means of illustration and not limitation, suitable transformation techniques thus include chemical treatment by cations, any method relying on the Yoshida effect (e.g. rubbing with nanoparticles), electroporation, ultrasound transformation, micro-shock waves, freeze-thawing, or any combination thereof.

For example, in heat shock transformation artificial competence is typically induced by making the cell permeable to DNA by subjecting them to non-physiological conditions. In such atypical transformation experiment, the cells are incubated in a solution containing divalent cations often in cold conditions, before the cells are exposed to a heat shock. It is theorized that exposure of the cells to divalent cations are responsible for a weakening of the cell surface structure, rendering it (more) permeable to DNA. The heat shock generates a thermal imbalance across the membrane, forcing entry of DNA through cell pores (i.e. adhesion zones or Bayer junctions) or through the damaged cell wall. Alternatively, electroporation is hypothesized to create pores in the cellular membrane. In electroporation the bacterial cells are briefly exposed to an electric field of 10-20kV/cm. After the shock, cellular membrane repair mechanisms remove the pores.

The method described herein starts from a bacterium comprising an ssDNA annealing protein. Different embodiments of the ssDNA annealing protein are described throughout the present specification and equally apply to the presently described method. The particulars of how and under which physical representation form the ssDNA annealing protein is introduced in the bacterium are not particularly limiting for the invention. Hence, in certain embodiments, the ssDNA annealing protein may be transiently expressed from an expression construct such as a plasmid in the bacterium. Alternatively, the ssDNA annealing protein may be introduced recombinantly into the bacterium. Yet alternatively, an RNA sequence encoding the ssDNA annealing protein may be introduced into the bacterium. Yet alternatively, a nucleotide sequence encoding the ssDNA annealing protein may be stably integrated into the target genomic sequence. In further embodiments, when stably integrated into the genomic sequence of the bacterium the nucleotide sequence encoding the ssDNA annealing protein may be operably linked to a constitutive promoter sequence. In alternative further embodiments, when stably integrated into the genomic sequence of the bacterium the nucleotide sequence encoding the ssDNA annealing protein may be operably linked to an inducible promoter sequence.

The oligonucleotide modification system as described herein and the first recombinase (RSA) may be introduced simultaneously or subsequently into the bacterium. In preferred embodiments, the oligonucleotide modification system is introduced in a first step into the bacterium and the first recombinase (RSA) is introduced into the bacterium in a second step, wherein the first step occurs at a first point in time and the second step occurs at a point in time which is later than the first point in time. The particulars of how and under which physical representation form the first recombinase (RSA) is introduced in the bacterium are not particularly limiting for the invention. Hence, in certain embodiments, the first recombinase (RS A) may be transiently expressed from an expression construct such as a plasmid in the bacterium. Alternatively, the first recombinase (RSA) may be introduced as a recombinant protein into the bacterium. Yet alternatively, an RNA sequence encoding the first recombinase (RSA) may be introduced into the bacterium.

In certain embodiments of the method, bacteria from the culture comprising a genomic sequence wherein the second nucleotide arrangement is integrated are selected from non-modified bacteria in said culture by addition of a molecule or substance to the culture medium. In further embodiments, the molecule or substance is an antibiotic or toxin. In alternative embodiments, bacteria from the culture comprising a genomic sequence wherein the second nucleotide arrangement is integrated are selected from non-modified bacteria in said culture by stratification of said bacteria based on a differentiating morphological feature. In further embodiments, the differentiating morphological feature is fluorescence-based or shape-based. In yet further embodiments, the selection is effectuated by a cell sorting step, which may optionally be a fluorescence activated cell sorting (FACS) step. In certain embodiments, the bacteria comprising a genomic sequence wherein the second nucleotide arrangement is integrated is selected from the culture by isolation of a number of bacteria from the culture (which comprises both modified and wild type bacteria), optionally followed by a clonal expansion, and analysing the genomic sequence of the different clonal populations. In further embodiments, analysis of the genomic sequence of the different clonal populations is achieved by means of polymerase chain reactions (PCR) on the genomic DNA of the bacteria, by sequencing of the genomic DNA of the bacteria or a portion thereof, by one or more restriction digests, or a combination of both PCR and sequencing. In certain embodiments wherein a portion of the genomic DNA is analysed by means of sequencing, the sequenced genomic region comprises at least the genetically modified region. In certain embodiments wherein PCR analysis of the genome is performed, the PCR analysis comprises performing junction PCR, overspanning PCR, or a combination thereof. A skilled person appreciates that a junction PCR is a PCR wherein a first primer is designed to engage with a DNA sequence part of the second nucleotide and a second primer is designed to engage with a genomic DNA sequence (located) 5 ’ or (located) 3 ’ of the envisaged insertion position of the second nucleotide arrangement, whereas an overspanning PCR relies on a first primer designed to engage with a genomic DNA sequence upstream of the insertion site of the second nucleotide arrangement and a second primer designed to engage with a genomic DNA sequence downstream of the insertion site of the second nucleotide arrangement. The term “restriction digest” and methods for conducting a restriction digest are known to a skilled person.

In certain embodiments of the method described herein, the method comprises a second step of selecting bacteria from the culture wherein the second nucleotide arrangement is deleted from the genomic sequence of the bacterium after expression of the second recombinase (RSB) in the bacterium . The particulars of how and under which physical representation fonn tire second recombinase (RSB) is introduced in the bacterium are not particularly limiting for the invention. Hence, in certain embodiments, the second recombinase (RSB) may be transiently expressed from an expression construct such as a plasmid in the bacterium. Alternatively, the second recombinase (RSB) may be introduced as a recombinant protein into the bacterium. Yet alternatively, an RNA sequence encoding the second recombinase (RSB) may be introduced into the bacterium.

Optionally, the first recombinase (RSA) may be encoded in a nucleotide sequence of a third nucleotide arrangement of the oligonucleotide modification system described herein, which forms an expression construct such as a plasmid. Alternatively, the second recombinase (RSB) may be encoded in a nucleotide sequence of a third nucleotide arrangement, which forms an expression construct such as a plasmid. Optionally, the first recombinase (RSA) and the second recombinase (RSB) are encoded in a nucleotide sequence of a distinct (i.e. different) nucleotide arrangement, each forming an independently functioning expression construct such as a plasmid.

In certain preferred embodiments, the second recombinase (RSB) is encoded in a nucleotide sequence comprised in the second nucleotide arrangement of the oligonucleotide modification system described herein. In further embodiments, the second recombinase (RSB) is optionally arranged to be encoded such that at least one RSB recognition sequence of the second nucleotide arrangement is (located) 5’ and at least one RSB recognition sequence of the second nucleotide arrangement is (located) 3 ’ of the nucleotide sequence encoding the second recombinase (RSB). In yet further embodiments, the second recombinase (RSB) is encoded 5’ of the RSA recognition sequence. In alternative further embodiments, the second recombinase (RSB) is encoded 3’ of the RSA recognition sequence.

In certain preferred embodiments, both the first recombinase (RSA) and the second recombinase (RSB) are encoded in a nucleotide sequence comprised in the second nucleotide arrangement of the oligonucleotide modification system described herein. In further embodiments, both the first recombinase (RSA) and the second recombinase (RSB) are optionally arranged to be encoded such that at least one RSB recognition sequence of the second nucleotide arrangement is (located) 5 ’ and at least one RSB recognition sequence of the second nucleotide arrangement is (located) 3 ’ of the nucleotide sequence encoding the first recombinase (RSA) and the nucleotide sequence encoding the second recombinase (RSB). In yet further embodiments, the first recombinase (RSA) and the second recombinase (RSB) are encoded 5’ of the RSA recognition sequence. In alternative further embodiments, the first recombinase (RS A) and the second recombinase (RSB) are encoded 3 ’ of the RSA recognition sequence. In preferred embodiments, transcription of the first recombinase (RSA) and the second recombinase (RSB) is independently controlled by distinct promoter sequences. In further preferred embodiments, transcription of the first recombinase (RSA) and the second recombinase (RSB) is independently controlled by distinct promoter sequences that allow for temporal control of expression of said recombinases. In yet further preferred embodiments, transcription of the first recombinase (RSA) or the second recombinase (RSB) is independently controlled by an inducible promoter sequence. In yet further preferred embodiments, transcription of both the first recombinase (RSA) and the second recombinase (RSB) is independently controlled by inducible promoter sequences, which are preferably distinct inducible promoter sequences.

It is a preferred embodiment that expression of the first recombinase (RSA) and/or expression of the second recombinase can be subjected to independent regulation, regardless of which embodiment is envisaged. It is an alternative preferred embodiment that at least expression of second recombinase (RSB) can be subjected to temporal regulation, regardless of which embodiment is considered. In a further preferred embodiment, expression of the second recombinase (RSB) can be regulation to such that said recombinase is expressed after expression of the first recombinase (RSA) is initiated and optionally terminated (i.e. sequential expression regulation).

Optionally, the method comprises a further step of contacting said genetically modified bacterium w ith a nucleotide arrangement consisting of HA1 and HA2, and contacting said bacterium with an intron- encoded endonuclease. In a preferred embodiment, the intron-encoded endonuclease is a I-Scel endonuclease. In such embodiments, the genetically modified bacterium is contacted with a nucleotide arrangement consisting of HA1 and HA2 after expression of the first recombinase (RS A) and after expression of the second recombinase (RSB). Additionally, in such embodiments it is preferred that the method as described herein comprised as first nucleotide arrangement an intron-encoded endonuclease restriction site sequence, preferably a I-Scel restriction site sequence located between the RSA recognition sequence and the stretch of at least 5 contiguous nucleotides of genomic sequence (HA1) of the bacterium to be modified (located) 5’ of the RSA recognition sequence, or located between the RS A recognition sequence and the stretch of at least 5 contiguous nucleotides of genomic sequence (HA2) of the bacterium to be modified (located) 3 ’of the RSA recognition sequence. In such embodiments, it is appreciated that a genetically modified bacterium is obtained that does not contain any scar sequence, for example a site-specific recombinase recognition site scar sequence, in its genomic sequence.

“Scar sequence” is a commonly used term in the field of genome engineering and synthetic biology. The term indicates any nucleotide sequence that remains excessively integrated in a genomic sequence of an organism after genome engineering. In the context of the present invention, the term encompasses any selection marker gene, any site-specific recombinase recognition sequence, or any recombinase that is not an essential sequence for obtaining the goal of the person using the oligonucleotide modification system. For example, a person aiming to genetically modify a genomic sequence of interest (i.e. a target genomic sequence) of a bacterium to express a therapeutic protein would, upon completion of the genome engineering protocol, consider any recombination site sequence or selection marker gene that is still embedded in the genomic sequence of the modified bacterium as a scar sequence, since these sequences to not contribute to or influence expressing said therapeutic protein . Alternatively w orded, a scarless genetically modified bacterium does not contain any sequences that may provide information or hint at how the genomic modification was introduced in said bacterium.

Also envisaged by the present disclosure is a genetically modified bacterium obtained by any of tire methods described herein. In certain embodiments, the genetically modified bacterium is inactivated and/or killed after performing said method. In alternative embodiments, the genetically modified bacterium is further cultivated after performing said method. In certain embodiments, the genetically modified bacterium obtained by any of the methods described herein expresses a heterologous ssDNA annealing protein, a first recombinase (RSA), a second recombinase (RSB), or any combination thereof.

In a preferred embodiment, the genetically modified bacterium does not comprise any site-specific recombinase scar sequence in its genomic sequence. In further preferred embodiments, the genetically modified bacterium does not comprise any scar sequence in its genomic sequence. In a preferred embodiment, the genetically modified bacterium is a bacterium selected from the group consisting of Mycoplasma bacteria, Pseudomonas bacteria, and Lactobacillus bacteria. In a further preferred embodiment, the genetically modified bacterium is a bacterium selected from the group consisting of a Mycoplasma bacterial species described herein, a Pseudomonas bacterial species described herein, and Lactobacillus bacteria described herein. In certain embodiments, the genetically modified bacterium is a component of a pharmaceutical composition which further comprises at least one excipient. The term “excipient”, commonly termed “carrier” in the art may be indicative for all solvents, including but by no means limited to: diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubilisers (e.g., Tween 80, Polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives (e.g., benzyl alcohol), antioxidants (such as, e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol) and the like. The use of such media and agents for formulating pharmaceutical compositions is well known in the art.

In certain embodiments, the genetically modified bacterium is a metabolic active bacterium. In alternative embodiments, the genetically modified bacterium is a metabolic inactive bacterium. In certain embodiments, the genetically modified bacterium obtained by any of the methods described herein is stored in a lyophilised form. The term “lyophilization” which may be used interchangeably with terms such as “freeze-drying” and “cryodesiccation” may be used interchangeably herein and refers to dehydration process which involves freezing the product (i.e. the genetically modified bacteria) without destroying the physical structure of the matter. Lyophilisation comprises at least a freezing step and a sublimation step. The sublimation step may comprise two stages of dry ing: a primary dry ing step and a secondary drying step. Advantages of lyophilisation may be but are not limited to improved aseptic handling, enhanced stability of a dry powder, the removal of water without excessive heating of the product, and enhanced product stability in a dry- state. In general, the quality of a rehydrated, lyophilized product is excellent and does not show an inferior quality to a non-lyophilized product. In context of the invention, quality of the bacterium may refer to any of the following non-limiting examples: growth rate, morphology, virulence, expression levels of heterologous nucleotide-encoded gene products, and metabolite production.

Cumate inducible expression systems have been reported in the art (e.g. SEQ ID NO: 9 and SEQ ID NO: 11, as described in Mullick et al., BMC Biotechnol, 2006; and Gaillet et al., Biotechnol. Bioeng. 2010). However the inventors have developed a particularly potent cumate promoter and operator sequence that is characterised by a high inducible expression level while maintaining low leakage expression levels in a ratio that is markedly improved over cumate promoter and operator sequences and cumate-inducible expression systems reported in the art suitable for use in Mycoplasma bacteria. Therefore another aspect of the invention is directed to a cum ate promoter and operator sequence (CuO) comprising a nucleotide sequence having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 that does not comprise or consists of SEQ ID NO: 9 or SEQ ID NO: 11. In a preferred embodiment, the cumate operator sequence (CuO) has a sequence comprising SEQ ID NO: 6 or SEQ ID NO: 10.

The term “cumate operator sequence” and its abbreviation “CuO” which is used in the present disclosure refers to a DNA element which is capable of being bound by a cumate repressor protein (CymR). The CuO is therefore a DNA recognition sequence for CymR. In the absence of the cumate small molecule, the cumate-repressor protein (CymR) binds to cumate operator sequence (CuO) within an associated cumate promoter sequence. If the operator sites is positioned proximal to the promoter sequence, the CymR protein blocks formation of the transcription initiation complex and thus prevents transcription by steric hindrance.

In certain embodiments, the cumate promoter and operator sequence (CuO) drives expression of an operably linked gene product in absence of cumate which is at least 2 fold, preferably at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 50 fold, at least 100 fold higher when compared to the expression of the operably linked gene product in presence of Cumate. In certain embodiments, the cumate promoter and operator sequence (CuO) described herein to upon presence of cumate drive expression of an operably linked gene product that is 1.5 fold, preferably 2 fold, preferably 5 fold, preferably 10 fold, preferably 15 fold, preferably 20 fold stronger than expression of a gene product operably linked to a cumate promoter and operator sequence (CuO) known in the art. In certain embodiments, the cumate promoter and operator sequence (CuO) described herein to upon presence of cumate drive expression of an operably linked gene product that is 1.5 fold, preferably 2 fold, preferably 5 fold, preferably 10 fold, preferably 15 fold, preferably 20 fold stronger than expression of a gene product operably linked to a cumate promoter and operator sequence (CuO) comprising SEQ ID NO: 9 or SEQ ID NO: 11 reproduced below:

AATTTTATTGACAACGTCTTATTAACGTTGATATAATTTAAATTTTATTTGACAAAAATGGGCTCGTG T TGT ACAAT AAAT GT AGT AACAAACAGACAAT CT GGT CT GT TT GT AT T AAT AT AAACT AAT AAGGAGG ACAAAC (SEQ ID NO: 9)

ATTTTGTCAAAATAATTTTATTGACAACGTCTTATTAACGTTGATATAATTTAAATTTTATTTGACAA AAATGGGCTCGTGTTGTACAATAAATGTAGTAACAAACAGACAATCTGGTCTGTTTGTATTAATATAA ACTAATAAGGAGGACAAAC (SEQ ID NO: 11)

In certain embodiments, the cumate promoter and operator sequence (CuO) described herein is characterised by a leakiness reduction of at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80% in absence of cumate when compared to a cumate promoter and operator sequence (CuO) comprising SEQ ID NO: 9 or SEQ ID NO: 11.

In an alternative preferred embodiment, the cumate promoter and operator sequence (CuO) consists essentially of, or consists of a nucleotide sequence having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 that does not comprise or consists of SEQ ID NO: 9 or SEQ ID NO: 11. In a further preferred embodiment, the cumate promoter and operator sequence (CuO) consists essentially of, or consists of SEQ ID NO: 6 or SEQ ID NO: 10.

In a further preferred embodiment, the cumate promoter and operator sequence (CuO) comprises an RNA polymerase binding domain corresponding to SEQ ID NO: 7 or SEQ ID NO: 12.

TTTATTTGACAAAAATGAGATTATTTGGTATAATTAATGTAGT (SEQ ID NO: 7)

ATTTTGTCAAAATTTTATTTGACAAAAATGAGATTATTTGGTATAATTAATGTAGT (SEQ ID NO: 12)

In yet a further preferred embodiment, the cumate promoter and operator sequence (CuO) comprises a nucleotide sequence having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 and comprises an RNA polymerase binding domain corresponding to SEQ ID NO: 7 or SEQ ID NO: 12 that does not comprise or consists of SEQ ID NO: 52 or SEQ ID NO: 53. In yet a further preferred embodiment, the cumate promoter and operator sequence (CuO) comprises, consists essentially of, or consists of a nucleotide sequence having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10 and consists essentially of, or consists of a single RNA polymerase binding domain corresponding to SEQ ID NO: 7 or SEQ ID NO: 12.

Similarly, an aspect of the invention is directed to a cumate-inducible expression system comprising a repressor protein CymR or a nucleotide sequence encoding CymR, and a nucleotide sequence comprising 1) a cumate promoter and operator sequence (CuO) as described herein, and 2) a nucleotide sequence encoding a gene product or forming (alternatively worded “representing” or “comprising”) an insertion site suitable for insertion of a nucleotide-encoded gene product. In such a cumate inducible expression system, the cumate promoter and operator sequence is operably linked and positioned 5 ’ of the nucleotide sequence encoding a gene product or forming an insertion site suitable for insertion of a nucleotide-encoded gene product. In certain embodiments, the cumate inducible expression system is encoded in an expression construct such as a vector or plasmid. In certain embodiments, the nucleotide sequence encoding CymR and the nucleotide sequence comprising a cumate promoter and operator sequence as described herein operably linked to a nucleotide sequence encoding a gene product are each comprised in a single nucleotide arrangement such as a vector or plasmid. In alternative embodiments, the nucleotide encoding CymR is arranged on a distinct nucleotide arrangement than the remaining components of the cumate inducible expression system. In preferred embodiments, the plasmid comprises as insertion site for insertion of a nucleotide encoded gene product a multiple cloning site. In alternative preferred embodiments, the plasmid comprises as nucleotide encoded gene product one or more recombinases, such as a first recombinase (RSA) and/or a second recombinase (RSB) a multiple cloning site.

The term “multiple cloning site”, interchangeably indicated by the term “polylinker”, enjoys widespread usage in the field of molecular biology and is therefore known to a skilled person as being a position in an expression construct which allows convenient integration of a certain DNA fragment into said position, and hence into said expression construct. A multiple cloning site is a relative short segment of DNA which contains distinct restriction sites. The number of restriction sites is not particularly limited, and a skilled person is capable of generating and using different multiple cloning sites depending on the envisaged functionality thereof.

In certain embodiments, the promoter sequence portion of the cumate promoter and operator sequence is a promoter sequence suitable for achieving expression of a gene product in a prokaryotic cell and/or a eukaryotic cell, preferably a promoter sequence suitable for achieving expression of a gene product in a bacterial cell.

In certain embodiments, the cumate-inducible expression system comprises a repressor protein CymR or a nucleotide sequence encoding CymR, and at least two, preferably at least three nucleotide sequences comprising 1) a cumate promoter and operator sequence (CuO) as described herein, and 2) a nucleotide sequence encoding a gene product or forming an insertion site suitable for insertion of a nucleotide-encoded gene product, which may optionally be a multiple cloning site.

Also envisaged by the present invention are kits of parts comprising the cumate-inducible expression system defined herein and cumate. In certain embodiments, the kit of parts further comprises a DNA ligase and optionally one or more restriction enzymes. In certain embodiments, the kit of parts further comprises a reagents and/or materials for purification of a nucleotide sequence comprising the cumate promoter and operator sequence, and an inserted and operably linked gene product-encoding nucleotide sequence. In further embodiments, the kit of parts further comprises a DNA ligase, and/or one or more restriction enzymes, and/or reagents and/or material for purification of a nucleotide sequence comprising the cumate promoter and operator sequence.

Evidently, any kit of parts may be accompanied by instructions on the use thereof, documents regarding safety, documents concerning quality assurance and any other information that is commonly provided in kit of parts.

Further envisaged are cells comprising the cumate inducible expression system described herein. In certain embodiments, the cell is a prokaryotic cell, preferably a bacterial cell. In alternative embodiments, the cell is a eukary otic cell. In further embodiments, the eukary otic cell is a, preferably a eukaryotic cell selected from the group of cells comprising: yeast cells, plant cells, insect cells, and mammalian cells. In a further preferred embodiment, the eukaryotic cell is a human cell.

In a final aspect of the invention, a method for obtaining inducible gene expression in a cell is envisaged, wherein the method comprises the steps of introducing the cumate-inducible expression system described herein (and operably linked to a gene of interest) into a cell, and contacting the cell with cumate, thereby achieving inducible expression of said gene. In certain embodiments, the cells are contacted with fresh culture medium whereto cumate is added prior to contacting said cells with said fresh medium. In alternative embodiments, the cells are contacted with cumate by addition of cumate to the culture medium of the cells.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects, statements, and embodiments of the invention are further supported by the following nonlimiting examples. EXAMPLES

Example 1. Rationale and validation of the system at four different loci

To overcome the main drawbacks associated with oligo-recombineering protocols (Fig. 1), we envisioned a system based on two components: an oligo and a non-replicative plasmid (hereinafter selector plasmid). First, the oligo performs the genetic modification through homology arms (HA) and introduces a recognition site (RS) for site-specific recombinases (SSR). The editing oligo would create the desired modification with a non-selectable phenotype but leaving in the edited locus a RS of SSR that can act as a landing pad. Second, the selector plasmid carries an antibiotic resistance gene and a RS compatible with that placed in the landing pad. Thus, co-transformation of the oligo with the selector plasmid results in plasmid integration and the generation of a selectable phenotype. Once the clones carrying the desired modification have been selected the plasmid can be removed from the edited area by inclusion of two RS incompatible with those used for the integration of the vector. The engineering rationale is illustrated in Figure 1.

We tested this hypothesis using M. pneumoniae, since this bacterium has been traditionally difficult to engineer (Halbedel et al., Int J Med Microbiol, 2007).

The basic enzymes required as components in the genome engineering protocol when applied to M. pneumoniae are: i) GP35, as a SSAP; ii) SSR-A, to catalyse plasmid integration; and iii) SSR-B, to perform vector deletion from edited clones. For a SSR-A, we selected Cre, a tyrosine recombinase derived from P 1 bacteriophage that is well-characterized and extensively used (Sternberg et al. , J Mol Biol, 1981; Nagy et al., Genesis, 2000). For SSR-B, we focused on Vcre, a lesser-known tyrosine recombinase encoded by a plasmid present in several Vibrio species (Suzuki et al. , Nucleic Acids Res, 2011). In line with these choices, we used lox motifs as RSA and vlox sequences as RSB. Of note, it has been previously demonstrated that Cre/lox and Vcre/vlox systems do not cross-react with each other (Suzuki et al. , Nucleic Acids Res, 2011). We selected these recombinase enzymes and SSR sites as they work in a broad variety of microorganisms.

First, we obtained by transposon delivery a strain expressing both GP35 (SSAP) and Cre (SSR-A) using a constitutive and inducible Tet promoter, respectively (M129-GP35-PtetCre strain). Then, we transformed this strain with oligos designed to delete 1 kb in four non-essential different locations of the chromosome (e.g., the mpn088, mpn256, mpn440 and mpn583 loci) and to insert a lox site at the edited locus (Fig 2A). Each oligo was then co-transformed with a selector plasmid (termed pLoxPuro) carrying a puromycin resistance cassette, one lox site as RSA and two vlox sites as RSBs (Fig. 2A). A high number of puromycin-resistant colonies were obtained in all four transformations, ranging from 890 to 1820 colony forming units (CFU) depending on the edited locus (Fig. 3). We found only 40 CFUs in the negative control (transformation with only selector plasmid but not oligo), indicating that the insertion of the plasmid is mainly dependent on the placement of a landing pad by the editing oligo. To confirm the intended deletions, five colonies from each transformation were analysed by PCR. A scheme showing the expected chromosomal conformations of mpn088 locus before and after the editing is shown as an example in Fig. 2B. The PCR screen showed that 60% of cells were edited in both at the mpn088 and mpn583 loci (3 out of 5 tested clones), 80% (4 out 5 clones) at the mpn256 locus and 100% (5 out 5 clones) at the mpn440 locus (Fig. 2C).

To conclude the protocol, we carried out a second transformation step with a suicide plasmid encoding SSR-B (i.e., Vcre), which led to vector excision in the 90% of the colonies analysed (i.e., 18 of 20; Fig. 2C). All edited loci were confirmed by sequencing. Therefore, the genome engineering protocol described herein allows clones carrying 1 kb, marker-free edits to be generated and selected with high efficiency in a rapid, cloning-free manner and irrespective of the position of the genomic region that is to be edited.

Example 2. Further exploration of the genome engineering method

We next evaluated whether the selective capacity of the genome engineering system enables clones that carry large genome modifications to be isolated. Based on the availability of a high coverage essentiality study for M. pneumoniae, we identified the largest chromosomal region that can be deleted without affecting any essential function for this bacterium. This region encompasses more than 30 kb and accounts for up to 5.48% of the non-essential (NE) genome, containing 25 NE coding genes (from mpn490 to mpn514) (Fig 4A). We designed a battery of editing primers in which the 5' HA is constantly located at the 3' end of mpn490 gene; in each primer, the 3' HA is displaced by different distances from the 5'-HA (ranging from 90 bp to 30 kb) (Fig 4A). In separate reactions, each of these oligos was transformed together with pLoxPuro (as the selector plasmid) into the M129-GP35-PtetCre strain. We observed a trend in which the number of puromycin-resistant colonies decreased with the increasing size of the attempted deletion; however, all transformations showed a higher number of colonies as compared to the control without primer (Fig. 5). A scheme depicting the expected chromosomal conformations before and after the smallest and largest edits is shown in Figure 4B as an example. For all the attempted deletions, more than 50% of all cells (i.e., 29 of the 35 colonies analysed by PCR) carried the expected modification (Fig 4C). Furthermore, for some conditions, this percentage increased up to 100% of the screened colonies (A90 bp, A10 kb and A30 kb edits). Sequencing of the PCR products confirmed the accuracy of the designed deletions and the consequent integration of the selector plasmid between the regions selected as HA in the editing primers. These results demonstrate that the genome engineering protocol described herein is able to execute small as well as large changes, and hence this method offers a simple way to carry out target editing, not only at gene level but also at a genome scale. Example 3. Scarless genome editing of bacteria by the presently described method

The inclusion of a lox66 site between the HAs of the editing oligo allows us to rescue ultra-rare events of oligo-recombineering, due to integration of a selector plasmid at the edited locus that carries a lox71 site compatible with that introduced by the oligo (Albert et al. , Plant J, 1995). When these two lox sites recombine to mediate the integration of the selector plasmid, a scar in the editing area containing a double mutant inactive lox72 site (in the chromosome) and a wild-type active loxP site (in the selector plasmid) is generated even after vector removal (Fig 2B). In this scenario, no further rounds of editing are possible; here, the likelihood is low that a lox-based selector plasmid would be preferentially inserted in this scar rather than in a novel lox landing created by a new event of oligo-recombineering.

To solve this limitation, we evaluated whether the presently described genome engineering rationale can be turned into a scarless genome editing method. To this end, we repeated the 90-bp deletion performed in Fig. 4 using a new oligo: besides introducing a lox site, it also mediates the insertion at the edited area of 18 bp that constitute the restriction site of I-Scel homing endonuclease (Fig. 6A) (Monteilhet et al., Nucleic Acids Res, 1990). The extended length of its restriction site, makes of this endonuclease a non-cutting enzyme in most bacterial genomes, which has favoured its use as a counter selective marker in some genome editing methods (Posfai et al. , Nucleic Acids Res, 1999). As expected, the number of colonies obtained was significantly higher in the co-transformation of the editing oligo and the selector vector when compared to the control transformation with only pLoxPuro plasmid (Fig. 7). Of the five puromycin-resistant colonies analysed, four showed PCR amplification products compatible with the insertion of the selector plasmid at the target site (Fig. 6B). One of these clones was expanded and transformed with a non-replicative plasmid coding for Vcre, leading to the excision of the vector in most of the analysed colonies (Fig. 6C). Of note, the editing scar retained after Vcre excision contained one vlox site, two lox site and the I-Sce-I restriction site (Fig. 6C). We took advantage of this to co-transform a suicide plasmid that expresses I-Sce-I endonuclease in an inducible manner along with a new editing oligo designed to delete the scar from the edited cells. Specifically, this oligo has the exact same sequence of the one used in the first editing step, but without the lox or the I-Sce-I restriction site. In this framework, upon the co-transformation and induction of I-Sce-I expression, only those cells that have incorporated the editing oligo and consequently removed the restriction site from the chromosome are expected to survive (Fig 6D and 6E). Accordingly, five survivor colonies analysed by PCR showed an amplification product compatible with deletion of the editing scar (Fig. 6D) and this deletion of the scar was confirmed by sequencing the PCR products. Altogether, these results demonstrated that the presently described method/system generated scarless edits in a simple and cloning-free manner, which will eventually allow iterative rounds of editing with a single selector vector (i.e. pLoxPuro). While inclusion of the I-Scel restriction site in this example is mediated by an editing oligo, it could be also included through the selector plasmid, which would reduce the length of the editing oligo and thereby decrease its cost and improve its editing capacity. Example 4. Expanding the presently described genome engineering rationale to different site-specific recombinases

We demonstrated that we could overcome the main limitation of the described genome engineering technology (i.e., the unavoidable presence a scar containing an active RS at the end of the editing protocol) by developing a "scarless" editing protocol. This approach will allow iterative rounds of genome editing to be carried out but involves an additional transformation step.

To accelerate eventual iterative rounds of editing and increase the modularity of the method/system, we created a collection of selector plasmids based on different SSRs. As a first step, using the pLoxPuro vector as a reference, we created four additional selector vectors (termed pSloxPuroScre, pRoxPuroDre, pVoxPuroVika and pAttBPuroBxbl) based on the Sere (Suzuki et al.. Nucleic Acids Res, 2011), Dre (Sauer et al., Nucleic Acids Res, 1999), Vika (Karimova et al., Nucleic Acids Res, 2013) and Bxbl (Kim et al., Mol Microbiol, 2003; Grindley et al., Annu Rev Biochem, 2006) recombinases, respectively; each ofthese carries the corresponding RS (e.g., slox, rox, vox and attB/attP, respectively). In contrast to our previous assays, these selector plasmids now contain the SSR-A coding gene under the control of the Tet inducible promoter, thus avoiding the requirement of obtaining in advance strains expressing the specific SSR-A. We then compared to the ability of these selector plasmids (e.g., this set and pLoxPuroCre) (Fig. 8A) to select an oligo-recombineering event that mediates a 0.84-kb deletion at the mpn507 locus (Fig. 8B). After co-transforming the editing oligo with each of these selector plasmids into a strain that solely expresses GP35 as SSAP (M129-GP35 strain), we observed variable numbers of puromycin-resistant colonies, with pLoxPuroCre being the most productive vector, and pSloxPuroScre, the least productive, for colonies obtained (Fig. 9). Control transformations with most of the selector plasmids showed only a low number of colonies. In contrast, the control transformation with pVoxPuroVika gave approximatively one-third as many colonies as obtained from the cotransformation of this vector with the editing oligo (Fig. 9). This suggests the existence of a vox-like sequence within the genome of M. pneumoniae that led to unspecific integration of the vector (e.g., independent of the editing oligo). A scheme showing the expected chromosomal conformations before and after the intended edit is shown in Figure 8C, using pLoxPuroCre selector vector as an example. PCR screening revealed that all plasmids mediated the selection of edited cells at high efficiencies (4 or 5 out of 5 screened cells; Fig. 8D), except from pVoxPuroVika, in which only 3 out of 5 of the screened colonies carried the intended modification (Fig 8D), probably as result of the relaxed specificity of the Vika/vox system in M. pneumoniae (Fig. 9). Sequencing the PCR products showed integration of the different selector plasmids and deletion of the target area in all cases. Altogether, these results demonstrated that the present logic is compatible with several SSRs regardless of the family to which they belong. This expanded modularity offers a faster approach to perform iterative rounds of genome editing as compared to the scarless approach. Example 5. Enabling of gene platforms to be introduced at the target locus

A second major drawback of oligo-recombineering protocols (besides the inability to directly select edited clones in the absence of clear phenotype) is the lack of a single-step process for introducing gene platforms. Given that the herein described technology involves the transient integration of a plasmid to facilitate the selection of edited clones, we envisioned that cloning the desired platform into a specific location of the vector might overcome this limitation of classical recombineering protocols (Y us et al. , Cell Syst, 2019; Breton et al., Microbiology (Reading), 2010); Mariscal et al., DNA Res, 2016; Krishnamurthy et al., BMC Microbiol, 2016; Gibson et al., Science, 2008).

We tested the ability of the present method/system to introduce gene platforms within a framework of genome streamlining. As a proof of concept, we focused our attention on a chromosome region close to the ori (origin of replication) that contains non-essential genes (mpn633, mpn634, mpn635 and mpn638) and flanks an E operon composed of two essential genes (mpn636 and mpn637) (Fig. 8A). For this, we designed i) an editing oligo to delete the entire area (5.5 kb; from mpn633 to mpn638) and ii) a selector plasmid (termed pLoxPuroCreCOMP636-637) in which we cloned the two E genes to complement the planned deletion (Fig. 8B). Co-transformation of both molecules into a M129-GP35 strain produced a substantially higher number of colonies than that observed in the control transformation with only selector plasmid (Fig. 10). We analysed five puromycin-resistant colonies from the co-transformation by PCR. For three of these, we obtained PCR products of a size compatible with the deletion of the whole area and the insertion of pLoxPuroCreCOMP636-637 vector (Fig. 8C and 8D). One of these clones was subsequently transformed with a suicide vector encoding Vcre, to excise the vector backbone but leaving the edited area in the E operon (Fig. 8C). PCR analyses (Fig. 8D) and sequencing of the amplification products confirmed that the vector had been successfully removed from the target area. These results demonstrate that the present technology enables gene platforms to be introduced into the target locus in a single step, a feature that might be of special interest in genome streamlining processes.

Example 6. Adaptation of a cumate inducible system for Mycoplasma, and its use for generating an all-in-one genome engineering vector

We have shown that the presently described genome engineering rationale is a highly versatile system that allows a wide variety of genome modifications to be carried out in a single step, with efficiencies always above 50% of the screened colonies regardless of the type of modification attempted or the SSR used. Nonetheless, to obtain marker-free modifications and to excise the inserted vector, an additional transformation step with a suicide vector coding for SSR-B (i.e., Vcre recombinase) needs to be done. As the inclusion of an SSR-A coding gene into the sequence of the selector plasmid still resulted in a functional system (Figs. 6-11) we reasoned that it should be also possible to clone the coding sequence of an SSR-B. In this framework, the selector plasmid would carry all the elements required for a functional genome engineering technology as presently described (except for a SSAP); in other words, producing an on-demand, self-integrative and self-excisable plasmid. For this purpose, the SSR-A and SSR-B should act in a strictly temporary order to mediate integration and excision of the vector at will. Thus, their expression needs to be driven by two different inducible systems. Unfortunately, only a tetracycline-inducible system was available for Mycoplasma,' this system was used to drive the expression of the SSR-A upon addition of anhydrotetracyline (aTc). This forced us to adapt a novel system based on cumate, in which the expression of SRR-B can be controlled in response of an external signal different from aTc. Based on this system we rationally designed different cumate responsive constructs for Mycoplasma (see Methods section) and tested their performance driving the expression of a reporter gene (i.e. the coding sequence of venus fluorescent protein). The levels of venus fluorescence obtained with the three designs (dubbed Pcuml, Pcum2 and Pcum 2.1) (Fig. 12A) were tested across different inducer doses, and compared side by side with those produced by the only inducible system that has been previously reported for Mycoplasma, based on the tetracycline repressor (Fig. 12B). The leakiness (i.e. the ratio between the fluorescent signal in the absence of inducer, and that observed in the WT strain) was neglectable for all systems except for PCuml system that produced a fluorescent signal 8 times higher than that observed in the control strain (Fig. 12C). In contrast the inducibility (i.e. the fold change in venus expression between the optimal induction condition and the uninduced state) was almost three times more pronounced in Pcum2.1 that in Ptet (Fig. 12D). Altogether, these results confirm the development of a novel inducible system for M. pneumoniae based on Pcum2.1 design that clearly outperforms the previously available system.

With this novel inducible system at hand, we constructed an all-in-one genome engineering selector plasmid that we termed pLoxPuroCreVcre (Fig. 13A). We aimed to compare the performance of this selector plasmid with that observed for the selector vector that only codes for the puromycin resistance gene (e.g., pLoxPuro) or the one that additionally carries an SSR-A coding gene (e.g., pLoxPuroCre). Hence, we assessed in parallel the ability of the three plasmids to select the clones carrying the smallest (A 90 bp) or the biggest (A 30 kb) edits tested in this work. To this end, equimolar amounts of the different selector plasmids were transformed together with the appropriate editing oligo into either M129-GP35-Cre (for pLoxPuro plasmid) or M129-GP35 (for pLoxPuroCre and pLoxPuroCreVcre plasmids). Regardless of the attempted modification, co-transformation of the editing oligo and pLoxPuroCre selector plasmid showed the highest number of puromycin-resistant colonies. In any case, all co-transformations resulted in an amount of puromycin-resistant colonies that was clearly higher that that obtained in their respective control transformations with only a selector plasmid (Fig. 14A). To confirm the correct identity of the obtained editing we analysed five puromycin-resistant colonies of each cotransformation by PCR with oligos flanking the edited area. According to this analysis, pLoxPuro selector vector mediated the intended modification in 4 or 5 of the 5 analysed colonies for the A 90 bp and A 30 kb edits, respectively (Fig. 14B and 14D), whereas these percentages were 100% and 60% when pLoxPuroCre was used as selector vector (Fig. 14C and 14E). Although the use of pLoxPuroCreVcre as selector plasmid resulted in diminished number of puromycin-resistant colonies compared to pLoxPuro or PloxPuroCre, the percentage of analysed colonies carrying the intended modifications was as high as that observed with pLoxPuro selector plasmid, being 100% for the A 90 bp editing and 80% for the A 30 kb editing (Fig. 13B). Next, three of the clones carrying the A30 kb edit selected with pLox66PuroCreVcre were grown in different combinations of puromycin and cumate to confirm the possibility of mediating vector excision without any additional round of transformation with a vector coding for Vcre. All clones were able to grow in all tested conditions except those involving the simultaneous presence of cumate and puromycin (Fig. 13C). This falls in line with the expected behaviour of the system, as cumate induction should lead to the expression of Vcre, which would excise the vector from the edited locus precluding the growth of the bacterium in puromycin. To further confirm this, we analysed by PCR the three clones grown in the presence of cumate, which gave PCR products with a size a compatible to the expected in case of vector excision (Fig. 13D). These products were sequenced confirming the removal of the selector plasmid from the edited area. Altogether, these results confirmed the functionality of the all-in-one genome engineering selector plasmid, producing marker-free, genome-scale (up to 30 kb) edits in a single step.

Alternative bacteria that may be of particular interest for genetic engineering may equally be subjected to the method(s) described above. Briefly, a 15mL culture of L. johnsonii strain expressing from either the chromosome or a replicative plasmid the RecT protein from Lactobacillus reuteri strain ATCC PTA 6475 (accession number WP_003668036.1) or alternatively RecT protein from Enterococcus faecalis (accession number AAO81865.1) was grown overnight at 37°C in static conditions in Man-Rogosa- Sharpe (MRS) medium. The next day, half of this preinocolum was added into a 50 mb tube containing 42 mb of a cell weakening version of MRS medium (1.1M Glycine, IM Sucrose) that also contains the inducer for RecT expression (nisin or cumate). The resulting cell suspension was incubated at 37°C at static conditions for 90 minutes. Cells were harvested at 5000 g 10 minutes and 4°C and washed 2 times with ice-chilled MilliQ water. Next, the cell pellet was resuspended in 1 mb of 50 mM EDTA and stand on ice for 5 minutes before a new round of two washing steps this time in 0.3 M sucrose. Finally, cell pellet was resuspended in 400 pL of 0.3M sucrose and 100 pL of this cell suspension were mixed with 5 pL of the desired editing oligo and 1 pg of a selector plasmid carrying either chloramphenicol or erythromycin resistance and the SSR coding gene under control of nisin or cumate responsive promoters. The resulting mix of cells and DNA was transferred into a 0.2 cm gap electroporation cuvette. After the transformation pulse (1.5 kV, 200 Q and 25 pF) cells were collected from the cuvette with 900 pL of MRS medium containing either nisin or cumate as inducers for SSAP and SSR expression and allowed to recover for at least 4 hours at 37°C, before plating into selective (chloramphenicol or erythromycin) MRS agar plates. Example 7: Materials and Methods

Bacterial strains and culture conditions

All strains are summarized in Table 1. The Mycoplasma pneumoniae wild-type (WT) strain M129-B7 (ATTC 29342, subtype 1, broth passage no. 35) and all its derivatives were grown at 37°C under 5% CO2 in tissue culture flasks or multi-well plates with Hayflick modified medium, as described elsewhere69. Hayflick broth was supplemented with puromycin (3 pg/ml), gentamicin (100 pg/ml) or chloramphenicol (20 pg/ml) for cell selection, as needed. To induce the Ptet or Pcum systems, anhydrotetracycline (5 ng/ml) or p-isopropyl benzoate (cumate) (100 pM) were used unless otherwise indicated. When growth on a plate was required, Hayflick broth was supplemented with 0.8% bacto agar.

Table 1. Used strains and description thereof.

For cloning purposes, E. coli NEB® 5-alpha High Efficiency strain was grown at 37°C in LB broth or on LB agar plates supplemented with ampicillin (100 pg /ml).

Plasmids and oligonucleotides

All plasmids generated in this work were assembled following the Gibson method70 and are listed in Table 2. Gene amplifications were carried out with Phusion DNA polymerase. When required, IDT performed gene synthesis. Oligonucleotides used as substrate for recombineering (editing oligos) as well as those used to screen edited clones were synthesized by IDT (Table 3). Editing oligonucleotides were designed by taking into consideration the number of occurrences of said sequence in the Ml 29 strain genome when allowing a maximum of three mismatches to select the sequences that were included in the 40 nucleotide homology arms (HAs) of the editing oligonucleotides, to ensure specificity and to minimize the number of off-target recombineering events. The correct identity of assembled plasmids and edited genomes was verified by Sanger sequencing (Eurofins genomics).

Table 2. Used plasmids and description thereof.

Table 3. Editing and screening oligonucleotides used.

Editing transformation

Electrocompetent cells from M129-GP35-PtetCre or M129-GP35 strains (depending on the presence or not of SSR-A coding gene in the used selector plasmid) were prepared as previously described23. Resulting cell suspensions (70 pl) were mixed with 0.5 nmol of the editing primer selected (i.e., 5 pl of a 100 pM oligo solution) and 2 pg of the desired selector vector, except for those used to compare performance of the three different systems (Fig. 14A), in which the amount of plasmid was adjusted to transform equimolar quantities (i.e. 1.23 pg, 2 pg or 2.7 pg for pLoxPuro, pLoxPuroCre or pLoxPuroCreVcre, respectively). In all cases, a control transformation only with the selector vector was carried out to estimate the frequency of spontaneous plasmid insertion in the absence of editing oligo. After the electroporation pulse, cells were harvested from the cuvette in Hayflick medium already supplemented with aTc and allowed to recover at 37°C for 2 h. The entire transformation volume was then inoculated in a 75 -cm2 flask containing 25 -ml Hayflick supplemented with puromycin and aTc, to induce expression of SSR-A, which mediates plasmid integration. After 24 h, cells were scraped from the flasks and seeded onto puromycin-selective Hayflick-agar plates. For all editing, a third-part of the transformation was seeded, except for A 90 bp editing, for which only 1% of the transformation volume was seeded. The total number of puromycin-resistant colonies in each transformation was calculated according to the seeded volumes for each editing.

Screening of edited clones

Colonies were picked from puromycin-selective Hayflick-agar plates and inoculated in 96 well plates filled with 200 pl of puromycin-supplemented Hayflick medium per well. When the cells were grown and reached confluency, genomic DNA was extracted using MasterPure DNA purification kit (Lucigen) following manufacturer’s instructions. For the screening around 30 ng the gDNA prep were used as template for the PCR reaction. PCR products were analysed by electrophoresis to estimate the size of the amplified products. To further confirm that the correct modifications were present, products were cleaned-up using QIAquick PCR purification kit and sequenced by Sanger method.

Vector backbone excision mediated by suicide plasmid coding for Vcre

Clones carrying any selector vector inserted at the edited area were grown in tissue culture flasks to prepare electrocompetent cells as previously described. The resulting cell suspensions (70 pl of each) were mixed with 2 pg of a suicide vector termed pGentaVcre. A control transformation with no plasmid was always performed in parallel. After the electroporation pulse, cells were allowed to recover at 37°C for 2 h before inoculating one-fifth of the transformation in a 75 -cm2 flask containing 25 -ml Hayflick supplemented with gentamicin. Flasks were incubated at 37°C for 5 days, a timeframe long enough to kill non-transformed cells and to excise the selector plasmid from the edited area in cells that received the pGentaVcre suicide vector. After this incubation, survivor cells were scraped from the flask and seeded onto non-selective Hayflick-agar plates. Colonies grown on 96-well plates containing 200 pl of non-selective Hayflick were picked; once expanded, genomic DNA was extracted as described above, with PCR confirming the excision of the selector plasmid from the edited area.

Scarless editing

Electrocompetent cells from edited + resolved clones (i.e. strains carrying the intended modification with the vector backbone excised from the edited area) were prepared as previously described23. The resulting cell suspensions (70 pl) were mixed with 0.5 nmol of the editing primer intended to delete the scar at the modified locus (i.e., 5 pl of a 100 pM oligo solution) and 2 pg of a suicide vector termed pPuroPtet-I-Scel. Control transformations without primer or without plasmid were carried out in parallel. After the electroporation pulse, cells recovered at 37°C for two h before inoculating one-fifth of the transformation into a 75-cm2 flask containing 25 ml of Hayflick supplemented with puromycin. Flasks were incubated at 37°C for 5 days; at 1 day post-inoculation, aTc was added into the medium to induce the expression of I-Scel. In this way, cells have a window of time of 24 hto incorporate the oligo and delete the scar before expressing the endonuclease to eliminate clones carrying the restriction site incorporated at the scar. After this incubation survivor cells were scraped from the flask and seeded onto non-selective Hayflick-agar plates. Grown colonies were picked from 96-well plates filled with 200 pl of non-selective Hayflick; once expanded, genomic DNA was extracted as described above, and the excision of the selector plasmid from the edited area by PCR.

Rational design and screening ofPcum inducible systems with Venus reporter

In the last years, a novel inducible system based on the regulatory components (i.e., CymR transcription factor and its operator sequence CuO) of the cmt operon from Pseudomonas putida has been successfully adapted to different organisms (Choi et al., Appl Environ Microbiol, 2010; Choi et al., Appl Environ Microbiol, 2006; Kaczmarczyk et al., Appl Environ Microbiol, 2013; Horbal et al., Appl Microbiol Biotechnol, 2014; Seo et al., Appl Microbiol Biotechnol, 2019) including mammalian cells (Mullick. et al., BMC Biotechnol, 2006). Given its apparent portability and the fact that the system responds to cumate, a non-toxic, inexpensive and carbon-source independent compound, we decided to adapt this system to M. pneumoniae . Based on this system we rationally designed different cumate responsive constructs for Mycoplasma and tested their performance driving the expression of a reporter gene (i.e. the coding sequence of venus fluorescent protein).

In all the designs the CymR coding gene (i.e. the repressor of the system) is placed under control of SynMyco regulatory region (RR), a synthetic sequence that promotes efficient transcription and translation of coding sequences in different Mycoplasma species. To drive the expression of the reporter gene we used three different sequences derived from PVeg, a strong constitutive promoter of B. subtilis already used in the cumate inducible system available for this strain (Fig. 12A). Pcuml design was based on the WT sequence of PVeg, which has been described to carry two different binding sites for RNA polymerase (RNApol) (Fig. 12A). Based on the almost constitutive behaviour of the Pcuml design (Fig. 12B), we generated PCum2, in which the RNApol binding site more distant to the CuO was removed (Fig. 12A). Finally, given the limited strength of Pcum2 design at the induced condition (Fig. 12B), we generated Pcum2.1, a derivative of PCum2 in which few nucleotides were changed to increase the affinity of RNApol complex towards this sequence (Fig. 12A).

For the screening of the different inducible systems, transposons carrying the venus coding gene coupled to the Pcum designs or to the previously available Ptet system were transformed into WT strain. The resulting strains were inoculated together in 96-well plates filled with Hayflick medium supplemented with the corresponding doses of cumate (4 pM, 20 pM and 100 pM) or aTc (2 ng/ml, 10 ng/ml and 50 ng/ml) as inducer, as indicated. In addition, the WT strain (that did not carry the venus coding gene) was included as a control strain to determine autofluorescence. Each strain and inducer dose were assessed in three different wells (i.e. biological replicates). After 48 h of growth, medium was removed from the wells and washed twice with PBS to minimize interference of the medium with fluorescence measurements. The absorbance and fluorescence values were measured using Tecan I- control 1.9.17.0 Infinite 200. The settings were determined for optimal gain, 25 flashes and 20 ps of integration time. The fluorescence settings were .ex = 514 nm and /.cm = 574 nm, whereas absorbance was determined at /. = 600 nm. All fluorescence levels were normalized by absorbance levels. To summarize all the results, we calculated the leakiness and the inducibility of all the inducible systems. Leakiness was determined after dividing the fluorescent signal in the absence of inducer for each strain, by that observed in the WT strain. Inducibility was calculated after dividing the fluorescence signal observed in the optimal inducer concentration, by that obtained in the absence of inducer for each strain. The values obtained for these two parameters in each inducible system were assessed for statistical significance using a one-way anova Tukey’s test.

Vector backbone excision mediated by cumate induction

Clones carrying pLoxPuroCreVcre selector vector inserted at the edited area were grown in 24-well plates in Hayflick medium supplemented with cumate at 100 pM final concentration. Additional medium compositions based on plain Hayflick, Hayflick supplemented with puromycin, or Hayflick supplemented with puromycin and cumate were included as controls (Fig. 13C). When the cells were grown and reached confluency, genomic DNA was extracted using MasterPure DNA purification kit (Lucigen) following manufacturer’s instructions. For the screening around 30 ng the gDNA prep were used as template for the PCR reaction. Products of the PCR were subjected to an electrophoretic analysis to estimate the size of the amplified products. To further confirm the intended modifications, these products were cleaned using QIAquick PCR purification kit and sequenced by Sanger method.

Funding acknowledgement

Carlos Pinero Lambea is supported by a “Programa Torres Quevedo” (PTQ) contract PTQ2020-011048 funded by MCIN/AEI/10. 13039/501100011033 and NextGenerationEU/PRTR.

Claims

1. An oligonucleotide modification system for genetic modification of a bacterium, wherein said bacterium comprises an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein, and wherein the oligonucleotide modification system comprises: a first nucleotide arrangement comprising:

- the recognition sequence for said first site-specific recombinase (RSA), and

2. Hie oligonucleotide modification system according to claim 1, wherein the ssDNA annealing protein is a third recombinase, preferably a GP35 recombinase, more preferably a GP35 recombinase having an amino acid sequence which is at least 65% identical, preferably at least 75% identical, more preferably at least 85% identical, most preferably at least 95% identical to the amino acid sequence of GP35 from Bacillus subtilis bacteriophage SPP1 as defined by SEQ ID NO: 1 based on the total length of the amino acid sequence of the recombinase.

3. The oligonucleotide modification system according to claim 1 or 2, wherein the second nucleotide arrangement further comprises a nucleotide sequence that encodes at least one gene product that allows for phenotypic selection of bacteria that comprise the second nucleotide arrangement.

4. The oligonucleotide modification system according to any one of the preceding claims, wherein the second nucleotide arrangement further comprises a nucleotide sequence encoding the first recombinase (RSA) and/or a nucleotide sequence encoding the second recombinase (RSB), preferably wherein each recombinase is operably linked to a promoter sequence.

5. The oligonucleotide modification system according to any one of the preceding claims, wherein at least the first recombinase (RSA) or the second recombinase (RSB) is operably linked to an inducible operator sequence, preferably wherein the second recombinase (RSB) is operably linked to an inducible operator sequence.

6. The oligonucleotide modification system according to any one of the preceding claims, wherein at least one of the recombinases is operably linked to a cumate responsive inducible operator sequence (CuO), preferably having a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% to SEQ ID NO: 6 or SEQ ID NO: 10.

7. The oligonucleotide modification system according to any one of the preceding claims, wherein the first nucleotide arrangement further comprises an intron-encoded endonuclease restriction site sequence located

- between the RSA recognition sequence and the stretch of at least 5 contiguous nucleotides of genomic sequence (HA1) of the bacterium to be modified (located) 5’ of the RSA recognition sequence, or

8. Use of an oligonucleotide modification system as defined in any one of the preceding claims for genetic modification of a bacterium, preferably wherein the genetic modification is an introduction, replacement, or deletion of one- or more nucleotide sequences.

9. A method to genetically modify a bacterium, the method comprising the steps of:

- transforming a culture of said bacterium comprising an ssDNA annealing protein or an oligonucleotide encoding said ssDNA annealing protein with (i) an oligonucleotide modification system as defined in any one of claims 1 to 7 and (ii) a first recombinase (RSA),

- selecting bacteria from the culture comprising a genomic sequence wherein the second nucleotide arrangement is integrated, - expressing a second recombinase (RSB) in the bacterium, thereby removing the second nucleotide arrangement from the genomic sequence of the bacterium.

10. The method according to claim 9, wherein the first recombinase (RSA) and/or the second recombinase (RSB) is encoded in a nucleotide sequence of a third nucleotide arrangement.

11. The method according to claim 9 or 10, wherein the first and second recombinase are encoded in the second nucleotide arrangement, preferably wherein the second nucleotide arrangement is a plasmid.

12. The method according to claim 11, wherein the method comprises temporal regulation of the expression of the first and/or the second recombinase, preferably wherein the method comprises temporal regulation of expression of the second recombinase.

13. The method according to any one of claims 9 to 12, wherein the method comprises a further step of contacting said bacterium with a nucleotide arrangement consisting of HA1 and HA2, and contacting said bacterium with an intron-encoded endonuclease, preferably wherein the intron-encoded endonuclease is a I-Scel endonuclease.

14. The method according to claim 13, which is a method wherein the resulting bacterium does not contain any site-specific recombinase scar sequence in its genomic sequence.

15. A genetically modified bacterium obtained by the method defined in any one of claims 9 to 14.