Bacterial host cells and the process for making a glycosylated protein.

Description

1 / 80 Bacterial host cells and the process for making a glycosylated protein. Separated from BR112020001815-5, filed on August 7, 2018. TECHNICAL FIELD

[001] The present invention relates to the field of recombinant nucleic acid manipulation and, specifically, to the precise removal of specific nucleic acid regions while ensuring that required portions of nucleic acid are not removed. Specifically, the present invention enables the removal of multiple unwanted genetic elements that may have been incorporated, for example, as selection markers, during recombinant manipulation of a genomic polynucleotide. The process uses short recombination sites, such as FRT sites, to flank a genetic element that is to be subsequently removed. The use of non-identical recombination sites to flank different genetic elements that are to be removed allows for the efficient removal of the intended genetic elements without loss of other elements of the genomic polynucleotide. FUNDAMENTALS

[002] Genetic manipulation of prokaryotic and eukaryotic organisms often involves the stable insertion of genetic elements into the genome so that a stable lineage with the required attribute can be generated. Alternatively, genetic manipulation can be used to remove unwanted elements of genetic material, optionally replacing the removed genetic material with other genetic inserts. Selection markers are usually introduced during genetic manipulation so that cells containing the correct genetic manipulation can be selected. However, it is useful to subsequently remove the markers once a manipulated host cell has... Petition 870260072914, dated 07 / 22 / 2026, page 11 / 209 2 / 80 has been correctly established, especially if the host cell is to be used for the manufacture of products for medical or veterinary use.

[003] The removal of unique genetic markers from a genome is known. Flp recombinase has been found to have a role in the inversion of yeast genetic material (Broach and Hicks (1980) Cell 21; 501-506, Broach et al (1982) Cell 29; 227-234). The potential of Flp recombinase has been investigated in E. coli (Cox (1983) PNAS 80; 4223-4227, Vetter et al (1983) PNAS 80; 7204-7288, Andrews et al (1985) Cell 40; 795-803) and roles for Flp recombinase in excision, inversion, translocation and insertion of genetic elements have been elucidated (Gronostajski and Sadowski (1985) J. Biol. Chem. 260; 12328-35). Flp recombinase produces recombination between Flp recombinase target sites (FRTs) which are genetic elements of approximately 48 bp. FRT sites have been used to flank a selection marker, allowing its subsequent excision from a yeast genome using Flp recombinase (Cregg and Madden (1989) Mol. Gen. Genet.219; 320-323) and a similar strategy was used to excise antibiotic resistance markers in E. coli (Cherepanov and Wackernagel (1995) Gene 158; 9-14).

[004] More complex genetic manipulation of prokaryotic and eukaryotic organisms requires the stable integration and / or removal of multiple genetic elements into / from the genome. For example, manipulation of E. coli to produce proteins bound to specific saccharides has been described (WO 09 / 104074, WO 11 / 60615, WO 11 / 138361). To achieve bioconjugate production in E. coli, it is necessary to introduce multiple genetic elements into the host cell, including one or more copies of several genes encoding the glycotransferases that are needed to assemble the required saccharide chain, a gene encoding an oligosaccharyltransferase such as PglB, a gene Petition 870260072914, dated 07 / 22 / 2026, page 12 / 209 3 / 80 encoding the required protein containing a glycosylation site and potentially additional genes encoding other enzymes, such as polymerases, co-polymerases, flippases, and / or enzymes to correctly 'decorate' the saccharide. It is also beneficial to remove specific genetic elements, such as lipopolysaccharide O-antigen ligase, native glycosyltransferase or oligosaccharyl transferase, flippases, polymerases, or co-polymerases. It would be beneficial to integrate several of these genes into the genome of the production cell and remove unwanted genes in order to facilitate production (WO 14 / 57109, WO 15 / 52344). However, this is difficult using standard methods (Datsenko and Wanner (2000) PNAS 97; 6640-5, Kuhlman and Cox (2010) 38; e92).In particular, it is difficult to remove multiple selection markers associated with multiple integrations because interference can occur between recombination sites flanking the selection markers, resulting in the excision of some of the required genetic material as well as selection markers. This is particularly problematic if the selection markers to be excised are positioned relatively close to each other in the genome. This problem is demonstrated in Figure 4.

[005] The present invention provides a solution to this problem by using pairs of identical recombination sites to flank each nucleic acid segment to be removed from the genome; wherein each identical pair of recombination sites is different from the pair of recombination sites flanking other nucleic acid segments to be removed from the genome.

[006] Consequently, a method is provided for removing at least two portions of nucleic acid from a genomic polynucleotide insert in a host cell, said method comprising the steps of: a) prepare a genomic polynucleotide comprising Petition 870260072914, dated 07 / 22 / 2026, page 13 / 209 4 / 80 a first nucleic acid insert that is flanked by a pair of first recombination sites in the same orientation that are identical to each other and have a first nucleic acid sequence; b) expose the genomic polynucleotide from step a) to a recombinase that recognizes early recombination sites so that identical recombination sites recombine, resulting in the excision of the first insert nucleic acid and one of the early recombination sites; c) insert into the genomic polynucleotide of step b) a second insert nucleic acid flanked by a pair of second recombination sites in the same orientation wherein the second recombination sites are identical to each other and have a second nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence; and d) expose the genomic polynucleotide from step c) to a recombinase that recognizes the second recombination sites so that identical recombination sites recombine, resulting in the excision of the second insert nucleic acid and one of the second recombination sites, but without removing any genomic polynucleotide sequence that is not flanked by identical recombination sites.

[007] Consequently, a method is also provided for removing at least two portions of nucleic acid from a genomic polynucleotide insert in a host cell, said method comprising the steps of: a) prepare a genomic polynucleotide comprising at least one first and one second insert nucleic acid, wherein i) a first insert nucleic acid is flanked by first recombination sites in the same orientation that are identical to each other and have a first nucleic acid sequence ii) the second Petition 870260072914, dated 07 / 22 / 2026, page 14 / 209 5 / 80 of the insert nucleic acid is flanked by second recombination sites in the same orientation that are identical to each other and have a second nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence; and iii) any additional recombination sites have a nucleic acid sequence that shares no more than 98% sequence identity with the first or second nucleic acid sequences; and b) exposing the genomic polynucleotide to a recombinase that recognizes the first and second recombination sites so that identical recombination sites recombine, resulting in the excision of the nucleic acid insert flanked by identical recombination sites, but without the removal of genomic polynucleotide that is not flanked by identical recombination sites.

[008] Without wishing to be limited by theory, the use of non-identical pairs of recombination sites favors recombination between identical pairs of recombination sites so that the expected nucleic acid segments are preferably removed.

[009] In a second aspect of the invention, a host cell comprising a genomic polynucleotide prepared by the method of the invention is provided.

[0010] In a third aspect of the invention, a host cell genomic polynucleotide is provided comprising a first recombination-engineered region and a second recombination-engineered region, wherein a unique first recombination site is adjacent to the first recombination-engineered region, and a unique second recombination site is adjacent to the second recombination-engineered region, wherein the first and second recombination sites have nucleotide sequences that share 90-98% identity with each other and with the Petition 870260072914, dated 07 / 22 / 2026, p. 15 / 209 6 / 80 nucleic acid sequence of any additional recombination sites present in the host cell genomic polynucleotide.

[0011] In a fourth aspect of the invention, a host cell is provided comprising a host cell genomic polynucleotide containing a first recombination engineered region and a second recombination engineered region, wherein a first recombination site scar is adjacent to the first recombination engineered region and a second recombination site scar is adjacent to the second recombination engineered region; wherein the first and second recombination site scars have different polynucleotide sequences that are less than 98% identical to each other and less than 98% identical to the polynucleotide sequence of any additional recombination site scar present in the host cell genomic polynucleotide.

[0012] In a fifth aspect of the invention, a process is provided for making a glycosylated protein comprising the steps of; a) cultivate the host cell of the invention under conditions appropriate for the production of glycosylated protein and b) Isolate the glycosylated protein from the culture.

[0013] In a sixth aspect of the invention, a prokaryotic genomic polynucleotide or a eukaryotic chromosome is provided comprising at least two recombination site scars adjacent to at least two recombination regions, wherein each recombination site scar has a different polynucleotide sequence.

[0014] In a seventh aspect of the invention, a process is provided for genetically engineering a host cell comprising the steps of: a) integrate a first polynucleotide cassette including a Petition 870260072914, dated 07 / 22 / 2026, page 16 / 209 7 / 80 first selection marker flanked by a first pair of recombination sites; b) remove the first selection marker by the action of a recombinase that recognizes the first pair of recombination sites; c) integrate a second polynucleotide cassette including a second selection marker flanked by a second pair of recombination sites; and d) remove the second selection marker by the action of a recombinase that recognizes the second pair of recombination sites; where the first pair of recombination sites have an identical nucleic acid sequence and the second pair of recombination sites have an identical nucleic acid sequence, and the first and second pairs of recombination sites share 9098% nucleic acid sequence identity.

[0015] In an eighth aspect of the invention, an engineered host cell obtainable by the process of the invention is provided. For example, an engineered host cell is optionally modified by the insertion of multiple copies of a particular gene or gene cluster. A particular locus for integration can be selected to optimize the expression level of different genes. Similarly, the integration of 2, 3, 4, 5, 6 or more copies of a gene or gene cluster at different loci can optimize the expression of the gene or gene cluster. Brief Description of the Figures

[0016] Figure 1 - Western blot on SDS PAGE loaded with periplasmic extracts. Left panel: detection with His antiserum. Right panel: detection with anti-33F antiserum. Lane 1 contains molecular weight markers, lane 2 contains strain 8661, lane 3 contains strain 10852, and lane 4 contains strain 10853. Petition 870260072914, dated 07 / 22 / 2026, p. 17 / 209 8 / 80

[0017] Figure 2 - Genomic organization scheme of the substituted wca and rfb clusters in strains 10175 and 10180.

[0018] Figure 3 - Colony PCR in selected clones derived from FLP-mediated resistance cassette removal from strains 10175 to 10180. Lanes 1, 14, and 27 contain 1kb of GeneRuler™ DNA ladder, lane 2 contains St10175 before removal, lanes 3 and 4 contain FRTwt, pattern B, lanes 5 and 6 contain FRTwt pattern E, lanes 7, 8, and 9 contain FRT3, pattern E, lanes 10, 11, and 12 contain FRT3, pattern D, lanes 13, 15, and 16 contain FRT10, pattern R, lanes 17 and 18 contain FRT10, pattern D, lanes 19 and 20 contain FRT10, pattern C, lanes 21 and 22 contain FRT13, pattern E, lane 23 contains FRT13, Pattern A, lanes 24 and 25 contain FRT13, Pattern D, lanes 26 and 28 contain FRT13, Pattern C, lane 29 contains FRT13, Pattern B, lanes 30, 31, 32 and 33 contain FRT14, lane 34 contains FRT14, Pattern D, lanes 35 and 36 contain FRT15, ​​Pattern E and lanes 37, 38 and 39 contain FRT15, ​​Pattern C.

[0019] Figure 4 - Preparative PCR in genomic DNA strains derived from FLP-mediated resistance cassette removal.

[0020] Figure 5 - Conceptual scheme demonstrating the advantage of using alternative FRT sites. Detailed Description

[0021] The invention provides a method of removing at least two insert nucleic acid portions from a genomic polynucleotide in a host cell, said method comprising the steps of: a) preparing a genomic polynucleotide comprising a first insert nucleic acid that is flanked by a pair of first recombination sites in the same orientation that are identical to each other and have a first nucleic acid sequence; b) expose the genomic polynucleotide from step a) to a recombinase that recognizes the early recombination sites so that identical recombination sites recombine resulting Petition 870260072914, dated 07 / 22 / 2026, page 18 / 209 9 / 80 in the excision of the first insert nucleic acid and one of the first recombination sites; c) insert into the genomic polynucleotide of step b) a second insert nucleic acid flanked by a pair of second recombination sites in the same orientation wherein the second recombination sites are identical to each other and have a second nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence; and d) expose the genomic polynucleotide from step c) to a recombinase that recognizes the second recombination sites so that identical recombination sites recombine, resulting in the excision of the second insert nucleic acid and one of the second recombination sites, but without removing any genomic polynucleotide sequence that is not flanked by identical recombination sites.

[0022] The invention also provides a method for removing at least two nucleic acid insert portions from a genomic polynucleotide in a host cell, said method comprising the steps of: a) prepare a genomic polynucleotide comprising at least one first and one second insert nucleic acid, wherein i) a first insert nucleic acid is flanked by a pair of first recombination sites in the same orientation that are identical to each other and have a first nucleic acid sequence ii) the second insert nucleic acid is flanked by a second pair of second recombination sites in the same orientation that are identical to each other and have a second nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence and iii) any additional recombination sites have a nucleic acid sequence that Petition 870260072914, dated 07 / 22 / 2026, page 19 / 209 10 / 80 shares no more than 98% sequence identity with the first or second nucleic acid sequences; and b) exposing the genomic polynucleotide to a recombinase that recognizes the first and second recombination sites so that identical recombination sites recombine, resulting in the excision of the nucleic acid insert flanked by identical recombination sites, but without the removal of genomic polynucleotide that is not flanked by identical recombination sites.

[0023] While not wishing to be limited by theory, the use of non-identical pairs of recombination sites favors recombination between identical pairs of recombination sites so that the expected sections of nucleic acid are preferably removed.

[0024] The term insert nucleic acid means a segment of nucleic acid that becomes integrated into a genomic polynucleotide as a result of genetic manipulation of the genomic polynucleotide. The insert nucleic acid is typically a segment of nucleic acid that becomes integrated into the genomic polynucleotide as an unintended consequence of the genetic recombination process rather than a segment of nucleic acid, for example, a gene to be expressed, which is the target of the genetic recombination to introduce. Thus, the insert nucleic acid is optionally a genetic marker such as an antibiotic resistance marker. Alternatively, the insert nucleic acid is a sequence inserted into the genomic polynucleotide to assist homologous recombination, for example, a runway.The purpose of the present invention is to provide an efficient method for removing insert nucleic acid from genomic polynucleotides effectively, once multiple genetic manipulations have been completed.

[0025] The term recombination sites means sequences in Petition 870260072914, dated 07 / 22 / 2026, page 20 / 209 11 / 80 both sides of the insert nucleic acid that allow its subsequent removal from the genomic polynucleotide by genetic recombination mediated by a recombinase. These are typically nucleotide sequences recognized by a recombinase, allowing the deletion of the intervening sequence after homologous recombination.

[0026] The term recombination-engineered region means a portion of the genomic polynucleotide that has been genetically engineered. This may involve the addition of a nucleic acid sequence, the deletion of a nucleic acid sequence, or the replacement of a nucleic acid sequence. The term genomic polynucleotide means a large piece of genetic material, for example, a eukaryotic chromosome or prokaryotic genetic material.

[0027] The term host cell means a prokaryotic or eukaryotic cell. Typically, the host cell has been genetically manipulated to contain new genetic material and / or to remove genetic material. In one embodiment, multiple genetic manipulations will be performed on the host cell, resulting in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 insert nucleic acids that can be effectively excised using the method of the invention.

[0028] The method of the invention can be used to remove insert nucleic acid after any form of prior genetic manipulation. For example, insert nucleic acid may be present in the genomic polynucleotide as a result of the addition of genetic material, removal of genetic material, or replacement of deleted genetic material with additional genetic material. The method of the invention is suitable for use with prokaryotic genomic polynucleotides, with plasmids, or with eukaryotic chromosomes.

[0029] In one embodiment, the first and second insert nucleic acids are selection markers, for example, markers of Petition 870260072914, dated 07 / 22 / 2026, page 21 / 209 12 / 80 selection markers are used to identify host cells in which the addition, removal, or replacement of genetic material has successfully occurred. In one embodiment, the selection markers are antibiotic resistance markers. In another embodiment, the selection markers encode proteins that confer antibiotic resistance, for example, ampicillin, kanamycin, chloramphenicol, spectinomycin, or gentamicin.

[0030] In one embodiment, the pair of recombination sites flanking the insert nucleic acid are identical to each other. In another embodiment, the pairs of recombination sites flanking an insert nucleic acid are in the same orientation. This allows efficient recombination to occur in the presence of a recombinase that recognizes the recombination site, resulting in the deletion of the insert nucleic acid and one of the recombination sites. Such recombination results in a single remaining recombination site; that is, as a recombination site scar on the genomic polynucleotide.

[0031] In one embodiment, the first and second pairs of recombination sites have nucleic acid sequences that share no more than 98%, 96%, 94%, 92%, 90%, 85%, 80%, 75%, or 70% identity. In one embodiment, the first and second pairs of recombination sites have nucleic acid sequences that share 70-98%, 75-98%, 80-98%, 85-98%, 90-98%, 92-98%, 94-98%, or 96-98% identity. In an appropriate embodiment, the first and second pairs of recombination sites share 90-98% identity between the sequence of the first recombination site and the sequence of the second recombination site.

[0032] In one embodiment, the first and second recognition sites share no more than 98%, 96%, 94%, 92%, 90%, 85%, 80%, 75%, or 70% identity with any other recombination site in the genomic polynucleotide. In one embodiment, the Petition 870260072914, dated 07 / 22 / 2026, p. 22 / 209 13 / 80 first and second recombination sites have nucleic acid sequences that share 70-98%, 75-98%, 80-98%, 85-98%, 90-98%, 92-98%, 94-98%, or 96-98% identity with any other recombination site present in the genomic polynucleotide. In an appropriate embodiment, the first and second pairs of recombination sites share 90-98% identity between the sequence of the first recombination site and the sequence of the second recombination site and any other recombination site present in the genomic polynucleotide.

[0033] In one embodiment, step a) prepares a genomic polynucleotide comprising a third insert nucleic acid that is flanked by a set of identical third recombination sites having a third nucleic acid sequence that shares no more than 98% 96%, 94%, 92% or 90% or 50%98%, 60%-98%, 70%-98%, 80%-98%, 85%-98% 90%-98%, 92%-98%, 94%-98%, or 96%-98% sequence identity with the nucleic acid sequence of the first or second recombination site or any additional recombination sites. In an appropriate embodiment, the third recombination site shares 90-98% identity with the first or second recombination site or any additional recombination sites.

[0034] In one embodiment, step a) prepares a genomic polynucleotide comprising a fourth insert nucleic acid that is flanked by a set of identical fourth recombination sites having a fourth nucleic acid sequence that shares no more than 98%, 96%, 94%, 92% or 90% or 50%-98%, 60%-98%, 70%-98%, 80%-98%, 85%-98%, 90%-98%, 92%-98%, 94%-98%, or 96%-98% sequence identity with the first nucleic acid sequence, second nucleic acid sequence, third nucleic acid sequence or the nucleic acid sequence of any sites of Petition 870260072914, dated 07 / 22 / 2026, p. 23 / 209 14 / 80 additional recombination. In an appropriate embodiment, the fourth recombination site shares 90-98% identity with the first or second recombination site or any additional recombination sites.

[0035] In one embodiment, step a) prepares a genomic polynucleotide comprising a fifth insert nucleic acid that is flanked by a set of identical fifth recombination sites having a fifth nucleic acid sequence that shares no more than 98%, 96%, 94%, 92%, or 90%, or 50%-98%, 60%-98%, 70%-98%, 80%-98%, 85%-98%, 90%-98%, 92%-98%, 94%-98%, or 96%-98% sequence identity with the first nucleic acid sequence, second nucleic acid sequence, third nucleic acid sequence, fourth nucleic acid, or the nucleic acid sequence of any additional recombination sites. In an appropriate embodiment, the fifth recombination site shares 90-98% identity with the first or second recombination site or any additional recombination sites.

[0036] In one embodiment, step a) prepares a genomic polynucleotide comprising a sixth insert nucleic acid that is flanked by a set of identical sixth recombination sites having a sixth nucleic acid sequence that shares no more than 98%, 96%, 94%, 92%, or 90%, or 50%-98%, 60%-98%, 70%-98%, 80%-98%, 85%-98%, 90%-98%, 92%-98%, 94%-98%, or 96%-98% sequence identity with the first nucleic acid sequence, second nucleic acid sequence, third nucleic acid sequence, fourth nucleic acid, fifth nucleic acid, or the nucleic acid sequence of any additional recombination sites. In an appropriate embodiment, the sixth recombination site shares 9098% identity with the first or second recombination site or any additional recombination sites. Petition 870260072914, dated 07 / 22 / 2026, p. 24 / 209 15 / 80

[0037] The principles for the design of FRT sites are described in Turan S, Kuehle J, Schambach A, Baum C, and Bode J (2010) J. Mol. Biol. 402; 52-69. For example, a typical FRT site consists of 48 base pairs comprising two inverted 13-bp repeats (a' and a) around an 8-bp spacer and a fourth repeat (b), separated by a 1-bp gap in the forward orientation to the adjacent repeat b—^ a' —^ spacer <—a

[0038] Variation is typically introduced in the spacer sequence. Preferably, the AT content of the spacer is above 75%. Preferably, changes are made to bp in at least one of the positions 2, 3, 4, 5, 6, or 7 of the spacer. Preferably, positions 1 and 8 of the spacer are unchanged. Preferably, no major interruptions are made in the 5'-polypyrimidine tracts.

[0039] In one embodiment, the genomic polynucleotide prepared in step a) comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 insert nucleic acids each flanked with identical pairs of recombination sites wherein each pair of recombination sites are different from other pairs of recombination sites, for example, a particular pair of recombination sites shares 90%-98% sequence identity with any other pair of recombination sites in the genomic polynucleotide.

[0040] In one embodiment, the recombination sites are 30-50, or 40-50 base pairs in length, preferably 48 base pairs in length. In one embodiment, the recombination sites are recognized by a recombinase. In one embodiment, the recombinase is preferably a FLP-recombinase. For example, the first and second recombination sites (as pairs or post-recombination, as scars) are different from each of the other flipase recognition target (FRT) sites or variant FRT sites.

[0041] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 25 / 209 16 / 80 FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[0042] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[0043] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[0044] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[0045] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[0046] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[0047] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 26 / 209 17 / 80 FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[0048] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0049] In one embodiment, the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO: 1) and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[0050] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[0051] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[0052] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[0053] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[0054] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[0055] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 27 / 209 The first FRT site is 18 / 80 with SEQ ID NO: 2, and the second recombination site is an FRT site with SEQ ID NO: 8.

[0056] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0057] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[0058] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 2 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[0059] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[0060] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[0061] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[0062] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[0063] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[0064] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0065] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 28 / 209 19 / 80 is an FRT site with SEQ ID NO: 3 and the second recombination site is an FRT site with SEQ ID NO: 10.

[0066] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[0067] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 3 and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[0068] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[0069] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[0070] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[0071] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[0072] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0073] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[0074] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[0075] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 29 / 209 The 20 / 80 FRT site has the sequence SEQ ID NO: 4, and the second recombination site is an FRT site with the sequence SEQ ID NO: 2.

[0076] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 4 and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[0077] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[0078] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[0079] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[0080] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0081] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[0082] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[0083] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[0084] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 5 and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[0085] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 30 / 209 21 / 80 is a FRT site with SEQ ID NO: 5 and the second recombination site is an FRT site with SEQ ID NO: 4.

[0086] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[0087] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[0088] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0089] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[0090] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 7 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[0091] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 7 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0092] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 7 and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[0093] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[0094] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[0095] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 31 / 209 The 22 / 80 FRT site has the sequence SEQ ID NO: 6, and the second recombination site is an FRT site with the sequence SEQ ID NO: 3.

[0096] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[0097] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 6 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[0098] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[0099] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 10.

[00100] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[00101] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[00102] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[00103] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[00104] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[00105] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 32 / 209 23 / 80 is a FRT site with sequence number 8 (SEQ ID): 8, and the second recombination site is an FRT site with sequence number 6 (SEQ ID): 6.

[00106] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 8 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[00107] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[00108] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[00109] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[00110] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[00111] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[00112] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[00113] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[00114] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 9 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[00115] In one embodiment, the first recombination site is a Petition 870260072914, dated 07 / 22 / 2026, p. 33 / 209 The 24 / 80 FRT site has sequence number 9, and the second recombination site is an FRT site with sequence number 10.

[00116] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 1.

[00117] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 2.

[00118] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 3.

[00119] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 4.

[00120] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 5.

[00121] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 6.

[00122] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 7.

[00123] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 8.

[00124] In one embodiment, the first recombination site is an FRT site having the sequence SEQ ID NO: 10 and the second recombination site is an FRT site having the sequence SEQ ID NO: 9.

[00125] In one embodiment, the first and second nucleic acids Petition 870260072914, dated 07 / 22 / 2026, page 34 / 209 25 / 80 of the insert are located in the genomic polynucleotide where the distance between the first and second insert nucleic acids is less than 500kb, 400kb, 300kb, 200kb, 150kb, 100kb, 75kb, 50kb, or 25kb.

[00126] In one embodiment, in step b) the genomic polynucleotide is exposed to recombinase by introducing a gene encoding FLP-recombinase into the host cell. The gene encoding FLP-recombinase is optionally introduced into the host cell in a plasmid under the control of an inducible promoter or a constitutive promoter. Where an inducible promoter is used, the pCP20 plasmid, having the nucleic acid sequence SEQ ID NO:13, can be used appropriately.

[00127] In one embodiment, the gene encoding FLP-recombinase has a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to the sequence with SEQ ID NO:12. In another embodiment, the FLP-recombinase has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence with SEQ ID NO:11.

[00128] In one embodiment, the insert nucleic acid encodes at least one selection marker. However, the method of the invention is particularly usable when at least 2, 3, 4, 5, or 6 insert nucleic acids each encode a selection marker. In these cases, multiple selection markers can be removed from a genomic polynucleotide in a single step without the removal of DNA regions between the selection markers by an unwanted recombination event. This benefit is particularly strong where the selection markers are located within 100, 75, 50, 25, 10, 5, or 2 kb of each other in the genomic polynucleotide.

[00129] In one embodiment, the genomic polynucleotide is from an Escherichia, Neisseria, Shigella, Klebsiella, Xanthomonas species, Petition 870260072914, dated 07 / 22 / 2026, page 35 / 209 26 / 80 Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, or C. perdidoridium. However, it is evident that the method of the invention can be used to remove multiple nucleic acids from the insert of a genomic polynucleotide from any organism, including eukaryotic and prokaryotic organisms, plants, insects, yeast, and mammalian organisms, including mice, rats, and rabbits. The method of the invention is appropriate where the genomic polynucleotide is from E. coli.

[00130] A further aspect of the invention is a genomic polynucleotide prepared by the method of the invention.

[00131] Following the implementation of the invention process, at least one first and one second region of the genomic polynucleotide are recombinantly manipulated and two nucleic acid deletions occur between pairs of identical recombination sites. This results in the loss of one recombination site per pair and the intervening nucleic acid.The result is a host cell genomic polynucleotide comprising a first recombination-engineered site and a second recombination-engineered site, wherein a single first recombination site is adjacent to the first recombination-engineered region, and a single second recombination site is adjacent to the second recombination-engineered region, wherein the first and second recombination sites have nucleotide sequences that share 90-98% identity with each other and with the nucleic acid sequence of any additional recombination sites present in the host cell genomic polynucleotide.

[00132] In one embodiment, the first and second recombination-engineered regions are regions where part of the host cell genome has been removed. In one embodiment, the first and Petition 870260072914, dated 07 / 22 / 2026, page 36 / 209 27 / 80 second recombination-engineered regions are sites where an additional nucleic acid segment of more than 20, 50, 100, 250, or 500 base pairs in length has been inserted. In one embodiment, the first and second recombination sites are recombination sites for a recombinase, for example, an FLP recombinase that has the amino acid sequence SEQ ID NO: 11.

[00133] In one embodiment, the first and second recombination sites are 30-50 or 40-50 base pairs in length, preferably 48 base pairs in length. In one embodiment, the first recombination site has a nucleic acid sequence of SEQ ID NO: 1-10. As described above, it is intended that any of the recombination sites of SEQ ID NO: 1-10 can be used with any other recombination site of SEQ ID NO: 1-10 so that recombination occurs between homologous pairs of recombination sites, but not between heterologous pairs of recombination sites. Preferred combinations have a first recombination site having a sequence selected from the group of SEQ ID NO: 1-6 used in combination with a second recombination site having a sequence selected from the group of SEQ ID NO: 1-6 (where the second recombination site is different from the first recombination site).

[00134] In one embodiment, the method of the invention results in a host cell comprising a host cell genomic polynucleotide containing a first recombination-engineered region and a second recombination-engineered region, wherein a first recombination site scar is adjacent to the first recombination-engineered region and a second recombination site scar is adjacent to the second recombination-engineered region; wherein the scars of the first and second recombination sites are adjacent to the first recombination-engineered region. Petition 870260072914, dated 07 / 22 / 2026, page 37 / 209 28 / 80 second recombination sites have different polynucleotide sequences that are less than 98% identical to each other and less than 98% identical to the polynucleotide sequence of any additional recombination site scar present in the host cell genomic polynucleotide.

[00135] In one embodiment, the first and second recombination engineered regions are regions in which part of the host cell genome has been removed. In another embodiment, the first and second recombination engineered regions are regions in which an additional nucleic acid segment of more than 20, 50, 100, 250, or 500 base pairs in length has been inserted.

[00136] In one embodiment, the first and second recombination sites are recombination sites for a recombinase, for example, an FLP recombinase, for example, an FLP recombinase having the amino acid sequence SEQ ID NO: 11.

[00137] In one embodiment, the first and second recombination sites are 30-50 or 40-50 base pairs in length, preferably 48 base pairs in length. In one embodiment, the first recombination site has a nucleic acid sequence with SEQ ID NO: 1-10. A second recombination site will have a different nucleic acid sequence that is optionally selected from SEQ ID NO: 1-10. Combinations of first and second recombination sites both with SEQ ID NO: 1-6 are preferred.

[00138] In one embodiment, the first and second recombination sites are separated by less than 500 kbases, 400 kbases, 300 kbases, 200 kbases, 150 kbases, 100 kbases, 75 kbases, 50 kbases, 25 kbases, 10 kbases, 5 kbases, 4 kbases, 3 kbases, 2 kbases, 1 kbase. The possibility of intervening nucleic acid being unintentionally deleted, if the method of the invention is not followed, is greater when the first and second recombination sites Petition 870260072914, dated 07 / 22 / 2026, page 38 / 209 29 / 80 are close together. Thus, the method of the invention is more advantageous where the first and second recombination sites are close together.

[00139] In one embodiment, genetic manipulations are performed on an E. coli genome, for example, E. coli strain W3110. The genetic manipulation optionally involves the removal of a wca colonic acid cluster and optionally replacing it with insert DNA, for example, a heterologous glycan cluster. The genetic manipulation optionally involves the deletion of a waaL gene and optionally replacing it with a pglB gene. The genetic manipulation optionally involves the deletion of at least part of an rfb cluster, for example, at least part of an O16 rfb cluster. In one embodiment, at least 1, 2, or all 3 of a waaL gene, at least part of an rfb cluster, and at least part of a wca colonic acid cluster are deleted.In one embodiment, at least 1, 2, or all 3, or one waaL gene, at least part of an rfb cluster, and at least part of a wca collagenic acid cluster are replaced by heterologous genes, optionally as described above.

[00140] The invention also discloses a process for making a glycosylated protein comprising the steps of; a) cultivate the host cell of the invention under conditions appropriate for the production of glycosylated protein and b) Isolate the glycosylated protein from the culture.

[00141] The production of engineered glycosylated proteins in bacterial host cells may require multiple manipulations of the host cell genomic polynucleotide in order to delete some host cell genes and incorporate heterologous genes encoding the proteins required to produce a planned glycosylated protein, for example, a bioconjugate. Multiple recombinant manipulations of the host cell genome may Petition 870260072914, dated 07 / 22 / 2026, page 39 / 209 30 / 80 introduce multiple genetic markers, which would be advantageous to remove. Thus, the processes of the invention are particularly applicable to the construction of a host cell that is subsequently used for the production of glycosylated proteins, for example, bioconjugates.

[00142] A further aspect of the invention is a prokaryotic genomic polynucleotide or a eukaryotic chromosome comprising at least two (for example 3, 4, 5, 6, 7, 8, 9 or 10) recombination site scars adjacent to at least two (for example 3, 4, 5, 6, 7, 8, 9 or 10) recombination regions, wherein each recombination site scar has a different polynucleotide sequence. Typically, the number of recombination site scars is equal to the number of recombination regions.

[00143] A further aspect of the invention is a process for genetically engineering a host cell comprising the steps of; a) integrate a first polynucleotide cassette including a first selection marker flanked by a first pair of recombination sites; b) remove the first selection marker by the action of a recombinase that recognizes the first pair of recombination sites; c) integrate a second polynucleotide cassette including a second selection marker flanked by a second pair of recombination sites; and d) remove the second selection marker by the action of a recombinase that recognizes the second pair of recombination sites; where the first pair of recombination sites have an identical nucleic acid sequence and the second pair of recombination sites have an identical nucleic acid sequence and the Petition 870260072914, dated 07 / 22 / 2026, p. 40 / 209 31 / 80 The first and second pairs of recombination sites share 9.098% nucleic acid sequence identity.

[00144] In one embodiment, the step b) and step d) recombinase is an FLP recombinase, for example, an FLP recombinase that has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[00145] A further aspect of the invention is an engineered host cell obtainable by the process of the invention.

[00146] All patent references or applications listed in this patent report are incorporated by reference herein.

[00147] The invention is further described in the following paragraphs: 1. A method for removing at least two portions of nucleic acid from an insert of a genomic polynucleotide in a host cell, said method comprising the steps of: a) prepare the genomic polynucleotide comprising a first insert nucleic acid flanked by a pair of first recombination sites in the same orientation that are identical to each other and have a first nucleic acid sequence; b) expose the genomic polynucleotide from step a) to a recombinase that recognizes early recombination sites so that identical recombination sites recombine, resulting in the excision of the first insert nucleic acid and one of the early recombination sites; c) insert into the genomic polynucleotide of step b) a second insert nucleic acid flanked by a pair of second recombination sites in the same orientation wherein the second recombination sites are identical to each other and have a second nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence; and Petition 870260072914, dated 07 / 22 / 2026, p. 41 / 209 32 / 80 d) expose the genomic polynucleotide from step c) to a recombinase that recognizes the second recombination sites so that identical recombination sites recombine, resulting in the excision of the second insert nucleic acid and one of the second recombination sites, but without removing any genomic polynucleotide sequence that is not flanked by identical recombination sites.

[00148] 2. A method for removing at least two nucleic acid insert portions from a genomic polynucleotide in a host cell, said method comprising the steps of: a) prepare the genomic polynucleotide comprising at least one first and one second insert nucleic acid, wherein i) a first insert nucleic acid is flanked by first recombination sites in the same orientation that are identical to each other and have a first nucleic acid sequence ii) the second insert nucleic acid is flanked by second recombination sites in the same orientation that are identical to each other and have a second nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence and iii) any additional recombination sites have a nucleic acid sequence that shares no more than 98% sequence identity with the first or second nucleic acid sequences; and b) exposing the genomic polynucleotide to a recombinase that recognizes the first and second recombination sites so that identical recombination sites recombine, resulting in the excision of the nucleic acid insert flanked by identical recombination sites, but without the removal of genomic polynucleotide sequence that is not flanked by identical recombination sites.

[00149] 3. The method of paragraph 1 or 2, wherein the genomic polynucleotide is a prokaryotic genomic polynucleotide or a plasmid. Petition 870260072914, dated 07 / 22 / 2026, page 42 / 209 33 / 80

[00150] 4. The method of paragraph 1 or 2 or 3 in which the genomic polynucleotide is a eukaryotic chromosome.

[00151] 5. The method of any of paragraphs 1-4 in which the first and second insert nucleic acids are selection markers.

[00152] 6. The method in paragraph 5 where the first and second insert nucleic acids are selection markers encoding proteins that confer resistance to ampicillin, kanamycin, chloramphenicol, spectinomycin or gentamicin.

[00153] 7. The method of any of paragraphs 2-6 in which step a) prepares a genomic polynucleotide comprising a third insert nucleic acid that is flanked by a set of identical third recombination sites having a third nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence, second nucleic acid sequence or the nucleic acid sequence of any additional recombination sites.

[00154] 8. The method in paragraph 7 wherein step a) prepares a genomic polynucleotide comprising a fourth insert nucleic acid that is flanked by a set of identical fourth recombination sites having a fourth nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence, second nucleic acid sequence, third nucleic acid sequence or the nucleic acid sequence of any additional recombination sites.

[00155] 9. The method in paragraph 8 in which step a) prepares a genomic polynucleotide comprising a fifth insert nucleic acid that is flanked by a set of identical fifth recombination sites having a fifth nucleic acid sequence that shares no more than 98% sequence identity with the Petition 870260072914, dated 07 / 22 / 2026, page 43 / 209 34 / 80 first nucleic acid sequence, second nucleic acid sequence, third nucleic acid sequence, fourth nucleic acid, or the nucleic acid sequence of any additional recombination sites.

[00156] 10. The method in paragraph 9 wherein step a) prepares a genomic polynucleotide comprising a sixth insert nucleic acid that is flanked by a set of identical sixth recombination sites having a sixth nucleic acid sequence that shares no more than 98% sequence identity with the first nucleic acid sequence, second nucleic acid sequence, third nucleic acid sequence, fourth nucleic acid, fifth nucleic acid or the nucleic acid sequence of any additional recombination sites.

[00157] 11. The method of any of paragraphs 2-5, wherein the genomic polynucleotide prepared in step a) comprises 3, 4, 5, 6, 7, 8, 9, or 10 insert nucleic acids each flanked with identical pairs of recombination sites wherein each pair of recombination sites shares 90%-98% sequence identity with any other pair of recombination sites

[00158] 12. The method of any of the paragraphs 1-11 wherein the recombination sites are 30-50 base pairs in length, preferably 48 base pairs in length.

[00159] 13. The method of any of the paragraphs 1-12 in which the recombination sites are recognized by a recombinase, preferably a FLP-recombinase.

[00160] 14. The method of any of the paragraphs 1-13 wherein the first and second insert nucleic acids are located in the genomic polynucleotide wherein the distance between the first and second insert nucleic acids is less than 100kb.

[00161] 15. The method of any of paragraphs 1-14 in which Petition 870260072914, dated 07 / 22 / 2026, p. 44 / 209 35 / 80 the first and second recombination sites are flippase recognition target (FRT) sites or variant FRT sites.

[00162] 16. The method in paragraph 15 where the first recombination site is an FRT site having the sequence 5'gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3' (SEQ ID NO:1).

[00163] 17. The method of any of the paragraphs 15-16 wherein the second recombination site is a variant FRT site having the sequence of any of the SEQ ID NO:2, 3, 4, 5 or 6.

[00164] 18. The method of any of the paragraphs 1-17 wherein in step b) the genomic polynucleotide is exposed to recombinase by introducing a gene encoding FLP-recombinase into the host cell.

[00165] 19. The paragraph 18 method in which the encoding gene FLP-recombinase has a nucleic acid sequence that is at least 80% identical to the sequence with SEQ ID NO:12.

[00166] 20. The method in paragraph 18 where the FLP-recombinase has an amino acid sequence at least 80% identical to the sequence in SEQ D NO:11.

[00167] 21. The method of any of the paragraphs 18-20, in which the gene encoding FLP-recombinase is introduced into the host cell on a plasmid under the control of an inducible promoter or a constitutive promoter.

[00168] 22. The method of paragraph 21 in which the plasmid contains an FLP-recombinase gene under the control of an inducible promoter.

[00169] 23. The method of paragraph 21 in which the plasmid is pCP20, having the nucleic acid sequence SEQ ID NO:12.

[00170] 24. The method of any of the paragraphs 1-23 in which the inert nucleic acid encodes at least one selection marker.

[00171] 25. The paragraph 24 method in which at least 2, 3, 4, 5 or 6 insert nucleic acids each encode a marker of Petition 870260072914, dated 07 / 22 / 2026, page 45 / 209 36 / 80 selection.

[00172] 26. The method of any of the paragraphs 1-25 wherein the genomic polynucleotide is of a species Escherichia, Neisseria, Shigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus or C. perdidoridium.

[00173] 27. The method in paragraph 26 where the genomic polynucleotide is from E. coli.

[00174] 28. A host cell comprising a genomic polynucleotide prepared by the method of any of paragraphs 1-27.

[00175] 29. A host cell genomic polynucleotide comprising a first recombination-engineered region and a second recombination-engineered region, wherein a single first recombination site is adjacent to the first recombination-engineered region, and a single second recombination site is adjacent to the second recombination-engineered region, wherein the first and second recombination sites have nucleotide sequences that share 90-98% identity with each other and optionally with the nucleic acid sequence of any additional recombination sites present in the host cell genomic polynucleotide.

[00176] 30. The host cell genomic polynucleotide of paragraph 29 in which the first and second recombination engineered regions are regions in which part of the host cell genome has been removed.

[00177] 31. The host cell genomic polynucleotide of paragraph 29 or 30 in which the first and second recombination engineered regions are regions in which an additional nucleic acid segment of above 20, 50, 100, 200, 300, 400 or 500 base pairs in length has been inserted. Petition 870260072914, dated 07 / 22 / 2026, page 46 / 209 37 / 80

[00178] 32. The host cell genomic polynucleotide of any of paragraphs 29-31 in which the first and second recombination sites and recombination sites for a recombinase.

[00179] 33. The host cell genomic polynucleotide of paragraph 32 in which the recombinase is an FLP recombinase.

[00180] 34. The host cell genomic polynucleotide of paragraph 33 in which the FLP recombinase has the amino acid sequence of SEQ ID NO: 11.

[00181] 35. The host cell genomic polynucleotide of any one of paragraphs 29-34 in which the first and second recombination sites are 30-50 base pairs in length, preferably 48 base pairs in length.

[00182] 36. The host cell genomic polynucleotide of paragraph 35 in which the first recombination site has a nucleic acid sequence of any one of SEQ ID NO: 1-10.

[00183] 37. A host cell comprising a host cell genomic polynucleotide containing a first recombination engineered region and a second recombination engineered region, wherein a first recombination site scar is adjacent to the first recombination engineered region and a second recombination site scar is adjacent to the second recombination engineered region; wherein the first and second recombination site scars have different polynucleotide sequences that are less than 98% identical to each other and optionally less than 98% identical to the polynucleotide sequence of any additional recombination site scar present in the host cell genomic polynucleotide.

[00184] 38. The host cell of paragraph 37 in which the first and second recombination-engineered regions are regions in Petition 870260072914, dated 07 / 22 / 2026, page 47 / 209 38 / 80 that part of the host cell's genome was removed.

[00185] 39. The host cell of paragraph 37 or 38 in which the first and second recombination engineered regions are regions in which an additional nucleic acid segment of above 20, 50, 100, 200, 300, 400 or 500 base pairs in length has been inserted.

[00186] 40. The host cell of any of paragraphs 3739 in which the first and second recombination sites are recombination sites for a recombinase.

[00187] 41. The host cell of paragraph 40 in which the recombinase is an FLP recombinase.

[00188] 42. The host cell of paragraph 41 in which the FLP recombinase has the amino acid sequence of SEQ ID NO: 11.

[00189] 43. The host cell of any of paragraphs 3742 in which the first and second recombination sites are 30-50 base pairs in length, preferably 48 base pairs in length.

[00190] 44. The host cell of paragraph 43 in which the first recombination site has a nucleic acid sequence of any one of SEQ ID NO: 1-10.

[00191] 45. The host cell of any of paragraphs 3744 in which the first and second recombination sites are separated by less than 100 kbases, 75 kbases, 50 kbases, 25 kbases, 10 kbases, 5 kbases, 4 kbases, 3 kbases, 2 kbases, or 1 kbase.

[00192] 46. The host cell of paragraph 45 in which the first and second recombination sites are separated by less than 5kbases.

[00193] 47. A prokaryotic genomic polynucleotide or a eukaryotic chromosome comprising at least two scars of Petition 870260072914, dated 07 / 22 / 2026, page 48 / 209 39 / 80 recombination sites adjacent to at least two recombination-engineered regions, where each recombination site scar has a different polynucleotide sequence.

[00194] 48. A process for genetically engineering a host cell comprising the steps of; a) integrate a first polynucleotide cassette including a first selection marker flanked by a first pair of recombination sites; b) remove the first selection marker by the action of a recombinase that recognizes the first pair of recombination sites; c) integrate a second polynucleotide cassette including a second selection marker flanked by a second pair of recombination sites; and d) remove the second selection marker by the action of a recombinase that recognizes the second pair of recombination sites; where the first pair of recombination sites have an identical nucleic acid sequence and the second pair of recombination sites have an identical nucleic acid sequence, and the first and second pairs of recombination sites share 9098% nucleic acid sequence identity.

[00195] 49. The process in paragraph 48 where the recombinase in step b) ed) is a FLP recombinase

[00196] 50. The process in paragraph 49 in which the recombinase of FLP has an amino acid sequence that is at least 80% identical to SEQ ID NO:11.

[00197] 51. An engineered host cell obtainable by the process of any of paragraphs 48 - 50.

[00198] 52. An engineered host cell comprising unique copies of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 sites of Petition 870260072914, dated 07 / 22 / 2026, p. 49 / 209 40 / 80 recombination in the host cell genomic polynucleotide, where each recombination site has a nucleotide sequence that is less than 98% identical to the other recombination sites.

[00199] 53. The engineered host cell of paragraph 52 in which at least 2 recombination sites are FRT sites.

[00200] 54. The engineered host cell of paragraph 52 or claim 53 in which the at least 2 recombination sites are separated by less than 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 3 kb or 1 kb in the host cell genomic polynucleotide.

[00201] 55. An engineered Gram-negative host cell in which at least part of a native rfb cluster and at least part of a wca collagenic acid cluster have been deleted while keeping an rfb cluster promoter intact.

[00202] 56. The engineered Gram-negative host cell of paragraph 53 in which a waaL gene is also deleted.

[00203] 57. The engineered Gram-negative host cell of paragraph 53 or 54 wherein the Gram-negative host cell is E. coli.

[00204] 58. The engineered Gram-negative host cell of any of paragraphs 53-55 wherein at least part of the native rfb cluster is replaced with a heterologous glycan cluster.

[00205] 59. The engineered Gram-negative host cell of any of paragraphs 53-56 in which the waaL gene is replaced with a pglB gene.

[00206] 60. The host cell of any one of paragraphs 28 or 37-46 or 51-59 wherein the host cell is engineered to express a) an oligosaccharyltransferase, for example, PglB or PglL; b) a heterologous glycan cluster, for example, an rfb cluster or a cluster of genes encoding glycosyltransferases required to synthesize a polysaccharide Petition 870260072914, dated 07 / 22 / 2026, page 50 / 209 41 / 80 capsular; and a protein containing a glycosylation site recognized by oligosaccharyltransferase, for example, an optimized consensus sequence disclosed in WO 06 / 119987 (claim 1)

[00207] 61. A process for making a glycosylated protein comprising the steps of: i) cultivate the host cell of paragraph 60 under appropriate conditions for the production of glycosylated protein and ii) isolate the glycosylated protein from the culture.

[00208] In order to better understand this invention, the following examples are presented. These examples are for illustrative purposes only and should not be interpreted as limiting the scope of the invention in any way. EXAMPLES Example 1: Use of two pairs of alternative FRTs during strain construction for the production of capsular polysaccharide conjugate of S. pneumoniae serotype 33F.

[00209] Strain stGVXN8661 is a derivative of Escherichia coli W3110 that contains several genomic modifications involving the use of FRTwt such that single FRTwt was present in three positions in the genomic DNA, adjacent to recombinant event sites.

[00210] Additional genomic manipulation was used to delete additional genes from stGVXN8661 while keeping the rest of the genome intact. The selection marker needs to be removed in order to allow further modification of the strain.

[00211] First steps regarding the construction of pDOC plasmids for use in the deletion. p3910 and p3911 were prepared as follows. An insert was generated resulting from an assembly PCR using two PCR products and oligonucleotide pairs for cloning HR2 and the clmR cassette into the donor pDOC-C plasmid. A PCR product was generated from pKD3 (GenBank: Petition 870260072914, dated 07 / 22 / 2026, page 51 / 209 42 / 80 AY048742.1) using oligonucleotides encoding a clmR cassette and FRTwt sites, and another was the 3' homology region derived from PCR of W3110 genomic DNA with oligonucleotides. The assembled DNA was cut using Bam HI / Eco RI and cloned into the same sites on pDOCC, resulting in p482. A PCR product of the 5' homology region was then generated using W3110 chromosomal DNA and oligonucleotides, cut with Bam HI and Spe I, and cloned into the Spe I / Bam HI sites of p482, resulting in p562. The nucleotide sequence of a multiple cloning site obtained by annealing 5'-phosphorylated oligonucleotides was cloned via Nhe I and Bam HI onto p562, resulting in p1043. A kanamycin resistance cassette (kanR) flanked by two FRT3 sites was synthesized and cloned on pUC57 (GenBank: Y14837.1) by Genewiz LCC, resulting in p3268. The NdeI / BstBI fragment of p3268 containing FRT3-kanR-FRT3 was cloned onto p1043, replacing FRTwt-clmRFRTwt, resulting in p3602.The 5' homology region of p1043 and p3602 was replaced by cloning via Spe I / Nhe I, a new 5' homology region of 1276-bp, resulting in p3910 and p3911, respectively.

[00212] p3910 and p3911 encode the 5' and 3' homology regions with an MCS and an inverted clmR resistance cassette flanked by two FRTwt sites, and a kanR resistance cassette flanked by two FRT3 sites, respectively between them. The resulting plasmids were the donor plasmid for deletion of the selected genomic sequence and its replacement with FRTwt-clmR-FRTwt or with FRT3-kanR-FRT3.

[00213] For deletion, a helper plasmid is required. A pTKRED variant (GenBank: GU327533.1) p2824 was used.

[00214] Deletions and selection. Two parallel deletion procedures were performed on strain stGVXN8661. The two procedures differ in the use of p3910 or p3911 as plasmids. Petition 870260072914, dated 07 / 22 / 2026, page 52 / 209 43 / 80 donors, and for the resistance applied for selection: chloramphenicol when p3910 was used and kanamycin when p3911 was used. The stGVXN8661 strain was co-transformed with p2824 and the donor plasmid via electroporation. Due to the temperature-sensitive replication phenotype of p2824, resulting cells were cultured at 30°C at all times in LB supplemented with spectinomycin for p2824 selection and with chloramphenicol or kanamycin for p3910 and p3911 selection, respectively. Plasmids were inserted into acceptor cells to allow expression of the enzymes encoded in the helper plasmid in the presence of the donor plasmid DNA within the same cell.

[00215] Next, the insertion procedure was performed. The newly transformed strains were cultured in TSB medium in the presence of ampicillin and spectinomycin at 30°C on a 5 mL scale overnight at 180 rpm. 50 μL of the dense culture were transferred to a new tube containing 1 mL TSB supplemented with spectinomycin and chloramphenicol or kanamycin. The new culture was then cultured at 180 rpm for 2 hours at 30°C, the cells were centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was replaced with TSB medium supplemented with spectinomycin, 0.2% arabinose (w / v), and 1 mM IPTG. The medium composition supports helper plasmid selection and recombinase and Sce I endonuclease expression to allow insertion. The cells were resuspended and incubated at 30°C for 3 hours at 180 rpm. 50 μL of this culture were used to inoculate 1 mL of TSB supplemented with 0.2% arabinose (w / v) and 1 mM IPTG, which was cultured overnight at 30°C at 180 rpm.The absence of resistance at this stage intensified the loss of the helper plasmid.

[00216] 0.5 mL of the culture was placed in TSB plates supplemented with clm or kan, depending on the donor plasmid used (for DNA insert selection) and 10% (w / v) sucrose (for Petition 870260072914, dated 07 / 22 / 2026, page 53 / 209 44 / 80 counter-selection against the donor plasmid) and incubated at 37°C overnight (to select for the loss of the temperature-sensitive helper plasmid).

[00217] A 'carpet' of cells appeared for both procedures. Streaks were made on TSB plates supplemented with clm or kan, depending on the donor plasmid used, and incubated again at 37°C overnight.

[00218] To sort the resulting colonies for the correct insertion phenotype, single colonies from the streaked stripes were placed on replica plates over supplemented LB plates with spec, amp, or clm when p3910 was used, or over supplemented LB plates with spec, amp, or kan when p3911 was used. Colonies resistant to clm or kan (for the presence of the insert) but sensitive to amp and spec (for the absence of donor and helper plasmids) were further analyzed for insertion.

[00219] To confirm that the strain had lost the substituted DNA originating from W3110, and contained the DNA insert, colony PCR was performed. Candidate colonies with the correct phenotype were taken and subjected to a colony PCR test. Three PCRs were performed. i) One PCR amplifies the 5' region of the inserted DNA only if recombination occurred correctly. The oligonucleotides used are 4897 / 3233 for integration with p3910 and 4897 / 4363 for integration with p3911. ii) One PCR amplifies the 3' region of the inserted DNA only if recombination occurred correctly. Oligonucleotides used are 3315 / 3208 for integration with p3910 and 4364 / 3208 for integration with p3911. iii) A PCR amplifies the genomic region that was replaced, meaning that the correctly engineered strain should not give any product while the unengineered strain should. Oligonucleotides used are 3213 / 3208. Several clones of both integrations showed the correct PCR pattern (PCR ie ii positive, Petition 870260072914, dated 07 / 22 / 2026, page 54 / 209 45 / 80 PCR iii negative). The resulting strains were designated st8661 Δ / FRTwt- clmR-FRTwt (st10851) when p3910 was used as a donor plasmid and st8661 Δ:^T3-kanR-FRT3 (st10852) when p3911 was used as a donor plasmid.

[00220] The next step is the removal of antibiotic resistance from the integrated strains to obtain a 'marker-free' deletion of wbbIL. The two strains obtained were transformed with the temperature-sensitive pCP20 plasmid expressing FLP recombinase [1] and placed on supplemented LB plates with ampicillin to select pCP20. Plates were incubated overnight at 30°C to allow plasmid replication. 5 mL of LB cultures were inoculated with stripes from the plates and cultured overnight at 42°C to ensure pCP20 loss. Serial dilutions of the overnight cultures were placed on LB plates. Single colonies were replicated on supplemented LB plates with ampicillin, chloramphenicol, or without antibiotics when derived from st8661 Δ::FRTwt-clmR-FRTwt, or on supplemented LB plates with ampicillin, kanamycin, or without antibiotics when derived from st8661 Δ:^T3-kanR-FRT3.In both cases, 100% of the colonies grew only on antibiotic-free plates, indicating that, in both situations, the resistance cassette was removed by the pCP20-encoded FLP recombinase.

[00221] In order to understand if the DNA removed from FLP was limited to the resistance cassette inserted flanked by FRT, a colony PCR was performed. The 4897 / 2174 oligonucleotides used result in a 2781-bp product if the resistance is removed and the border regions are present. No bands are expected if the region between the newly inserted FRT and the FRTwt present in the upstream wca locus is lost. The current band was observed only when the resistance was removed from Δ:^T3-kanR-FRT3, indicating that the Petition 870260072914, dated 07 / 22 / 2026, page 55 / 209 46 / 80 FRT cross-reactivity does not occur between the FRTwt site of the wca locus and the newly introduced FRT3. Conversely, when FRTwt is introduced, cross-reactivity with the other FRTwt sites present in the wca locus causes loss of genomic material between the two sites. The resulting kanamycin resistance cassette strain st8661 &:FRT3-kanR-FRT3 (st10852) is called st10853.

[00222] To verify if the production of Sp33F glycoconjugate by strains st10852 and st10853 is comparable to that observed in st8661, the following experiment was performed. Strains st8661, st10852, and st10853 were transformed via electroporation with plasmids 3914, encoding the carrier protein, rcsA from E. coli K30, chain length regulator, wzy, all under the inducible IPTG promoter, and with plasmid 3750, encoding constitutively expressed genes wchA, and wciB genes for wzy from cluster 33F. Production cells were inoculated into 5 mL of TB-dev medium supplemented with 10 mM MgCl2, spectinomycin, and tetracycline and cultured overnight at 37°C in stationary phase. Cells were then diluted to an OD600 of 0.05 in 50 mL TBdev containing 10 mM MgCb, spectinomycin, tetracycline, and 0.01 mM IPTG. After 6 hours, 0.09 mM IPTG was added to the cultures, which were then cultured overnight at 37°C.IPTG directs the expression of elements encoded in p3814 (including the carrier protein and rcsA, which directs the expression of the 33F capsular polysaccharide cluster at the wca locus), and the genome-integrated pglB. Cells were then harvested by centrifugation and periplasmic cell extracts were prepared using the Lysozyme method [2]. The periplasmic extracts (normalized to OD600) were separated by SDS PAGE and analyzed by immunoblotting after electrotransfer (Figure 1). Detection with anti-His antiserum (left panel) and anti-33F antiserum (right panel) both show a clear ladder-like pattern between 70 and 170 kDa for all. Petition 870260072914, dated 07 / 22 / 2026, page 56 / 209 47 / 80 samples, strongly indicative of glycoproteins consisting of carrier protein and 33F polysaccharide. The quantity and quality of the glycoconjugate obtained from st10852 and st10853 is comparable to that observed in st8661. This indicates that the genes upstream of the deleted region are still present and active.

[00223] Strain st10853 was used for bioreactor-scale production of bioconjugate 33F. In addition, the strain's genome was further engineered via an analogous procedure, and a final resistance-free strain was obtained. Example 2: Systematic study on the use of alternative FRT sites for contemporaneous excision of adjacent resistance cassettes.

[00224] A series of E. coli W3110 derivatives were constructed, differing only in the presence of alternative FRT sequences. First, the O16 rfb cluster was replaced by a gentamicin resistance cassette gntR, in the same orientation as the replaced cluster, followed by a chloramphenicol resistance cassette clmR in the opposite orientation and enclosed between two FRTwt sites. Second, six parallel homologous recombinations were performed in order to replace the wca cluster of colanic acid with a kanamycin resistance cassette kanR in the opposite orientation of the replaced cluster, enclosed between two FRTwt sites, FRT3, FRT10, FRT13, FRT14, and FRT15, ​​resulting in strains 10175, 10176, 10177, 10178, 10179, and 10180, respectively.

[00225] The six strains are able to grow in media containing kanamycin, gentamicin, and chloramphenicol. Figure 2 describes the genetic organization of the wca and rfb loci in the six strains.

[00226] In order to evaluate the degree of cross-reactivity of the FRTwt site with alternative FRT sites, a resistance cassette removal protocol was applied to the six strains. The strains were Petition 870260072914, dated 07 / 22 / 2026, page 57 / 209 48 / 80 transformed with the temperature-sensitive pCP20 plasmid expressing FLP recombinase [1] and placed on supplemented LB plates with ampicillin to select pCP20. Plates were incubated overnight at 30°C to allow plasmid replication. 5 mL of LB cultures were inoculated with stripes from the plates and cultured overnight at 37°C to ensure pCP20 loss. Serial dilutions of the dense cultures were placed on LB plates. Sixty colonies isolated by recombination were replicated on supplemented LB plates with ampicillin, kanamycin, chloramphenicol, gentamicin, or no antibiotics and cultured overnight at 37°C.

[00227] In the case of cross-reactivity between the FRT sites flanking the chloramphenicol cassette and those flanking the kanamycin resistance cassette, loss of resistance to kanamycin, gentamicin, and chloramphenicol is expected. In the case of lack of cross-reactivity, resistance to gentamicin should be retained, while resistance to kanamycin and chloramphenicol should be lost. Persistence of kanamycin resistance can be explained by suboptimal efficacy of the FRT sites flanking the corresponding cassette. Persistence of chloramphenicol resistance can be explained either by suboptimal efficacy of the FRTwt pair, or by retention of pCP20, which is resistant to both ampicillin and chloramphenicol. In the latter scenario, concomitant persistence of ampicillin resistance is expected.

[00228] The resistance pattern of the replicated clones was observed and is summarized in Table 1. In general, five different phenotypic patterns were observed, ignoring the ampicillin resistance situation: pattern A: the clone is resistant to kanamycin, gentamicin, and chloramphenicol, indicating a complete lack of FLP recombinase activity in both FRT pairs; pattern B: no resistance, indicating non-specific cross-reactivity between the two FRT pairs; Petition 870260072914, dated 07 / 22 / 2026, page 58 / 209 49 / 80 pattern C: resistance to kanamycin and gentamicin, indicating defective FLP recombinase activity on the FRT pair flanking kanR; pattern D: resistance to chloramphenicol and gentamicin, indicating either defective FLP recombinase activity on the FRTwt pair flanking clmR or correct specific removal of kanR and clmR without cross-reaction between the FRT pairs flanking clmR and kanR, but persistence of pCP20 plasmid; pattern E: resistance to gentamicin only, indicating correct specific removal of kanR and clmR without cross-reaction between the FRT pairs flanking clmR and kanR. Table 1. Resistance patterns observed after removal of FLP-mediated resistance in six different strains. Number of colonies per antibiotic plate. Total: 60 colonies per FRT. Number of colonies belonging to resistance pattern. Total: 60 colonies per FRT 0 0 4 4 52 87 to 93 FRT13 20 10 19 59 1 1 9 18 31 52 to 82 FRT14 5 0 1 60 0 0 0 1 59 98 to 100 FRT15 0 14 0 60 0 0 14 0 46 77 a) Percentage of colonies with only gentamicin cassette left.

[00229] Pattern A (no resistance removed) was observed only for one clone when strain 10178, where kanR is flanked by Petition 870260072914, dated 07 / 22 / 2026, page 59 / 209 50 / 80 FRT13 sites were used. Pattern B (all resistances removed) was almost exclusively observed for strain 10175, where kanR is flanked by FRTwt, representing 93% of clones for resistance removal in this strain. The only exception is a clone derived from strain 10178, where FRT13 flanks kanR. Pattern E (only gentamicin resistance left) was always observed in more than 50% of cases for all strains with alternative FRT sites flanking kanR, while only 7% of clones derived from strain 10175 (FRTwt flanking both clmR and kanR) show this pattern. Pattern C (kanamycin and gentamicin resistance left) was observed in a few cases when FRT10, FRT13, and FRT15 flank kanR. This may indicate a slightly lower efficiency of FLP recombinase acting on these specific FRT sites. Pattern D (resistance to gentamicin and chloramphenicol left) was observed in several cases when FRT3, FRT10, FRT13, and FRT14 flanked kanR.With the exception of one clone derived from st10176 (FRT3), all clones exhibiting pattern D are also resistant to ampicillin, suggesting a high probability that the chloramphenicol-resistant phenotype is due to the persistence of pCP20 rather than a defective removal of clmR.

[00230] These results show that DNA loss between neighboring FRTwt pairs is highly probable (93% of cases), while the probability decreases significantly if one of the two FRTwt pairs is replaced by a pair of alternative FRT sites. Excision of gentamicin resistance was observed in only one case in 300 when either of the alternative FRT sites was flanking kanR, underscoring the specificity of the FLP-catalyzed reaction. The percentage of correct genetic pattern (only the remaining gentamicin cassette) when alternative FRT sites were used can be inferred from the phenotype. The phenotypic pattern E can only be Petition 870260072914, dated 07 / 22 / 2026, page 60 / 209 51 / 80 is explained by the genetic scenario in which a correct specific removal of both cassettes occurred, while the phenotypic pattern D can be explained by the same (only when ampicillin resistance is also present) or by the lack of clmR excision. Thus, clones belonging to the phenotypic pattern E represent the minimum possible number of clones in which the correct specific removal of both clmR and kanR occurred without loss of gntR, while clones belonging to the pattern E + D represent the maximum possible number of clones in which this genetic organization exists. Table 1 summarizes the percentage of clones with the correct genetic pattern, taking these considerations into account.

[00231] To confirm the genetic organization of the clusters after removal of FLP-mediated resistance, a colony PCR was performed on selected clones belonging to different phenotypic patterns for each strain tested. The use of 3206 / 3208 oligonucleotides results in a 7922 bp product if no resistance has been removed, a 3388 bp product if the entire genomic region between the two FRT sites has been removed, a 6990 bp product if only clmR is excised, a 6550 bp product if only kanR is excised, and a 5618 bp product if the desired pattern is achieved in which only gntR remains. All tested clones belonging to pattern D show the band corresponding to the excision of both clmR and kanR, and not just the excision of kanR. The product lengths observed for clones showing unequivocal resistance patterns (A, B, C, or E) correspond to the only possible inferred genetic pattern, with the following exceptions.Two of the four clones showing pattern E derived from strains in which FRTwt flanks both clmR and kanR were tested, but no PCR product was observed. Four clones belonging to pattern E of strain 10179 (FRT14) were tested and one of them was not. Petition 870260072914, dated 07 / 22 / 2026, page 61 / 209 52 / 80 showed no PCR product. The only colony belonging to pattern A, when alternative FRT sites were used, derives from the strain containing FRT13 and shows a length adjustment product with the removal of only clmR (6990 bp), instead of the expected band of 7922 bp observed in the control, when no resistance is removed. A clone derived from the strain with FRT13 showed phenotypic pattern C, but the PCR shows a length band corresponding to the removal of both clmR and kanR in reverse (Figure 3).

[00232] A clone derived from the strain carrying only FRTwt with pattern B, a clone derived from the strain carrying FRT3 with pattern E, a clone derived from the strain carrying FRT10 with pattern E, a clone derived from the strain carrying FRT10 with pattern C, a clone derived from the strain carrying FRT13 with pattern E, a clone derived from the strain carrying FRT13 with pattern B, a clone derived from the strain carrying FRT14 with pattern E, and a clone derived from the strain carrying FRT15 with pattern E were stored and named 10247, 10248, 10249, 10250, 10251, 10252, 10253, 10254, respectively. For these 8 strains, the genome was isolated and a PCR using 3206 / 3208 oligonucleotides was performed. The PCR products were purified and sequenced. The lengths obtained from the product (Figure 4) and the sequencing results further confirm the expected genomic organization.In the only strain where a complete removal of genomic material between the two FRT pairs is observed, when using an alternative FRT site (FRT13, strain 10525), the only FRT site left is FRTwt.

[00233] This experiment proves that using alternative FRT sites is a valid and effective approach for excising resistance cassettes from neighboring and near genomic regions of an existing FRTwt site without loss of enclosed DNA. Petition 870260072914, dated 07 / 22 / 2026, page 62 / 209 53 / 80 Example 3: Use of alternative FRT sites during strain development for additional production of capsular polysaccharide conjugate serotype S. pneumoniae s

[00234] Strain stGVXN9876 is a derivative of Escherichia coli W3110 that contains several genomic modifications involving the use of FRTwt such that single copies of FRTwt were present at three positions in the genomic DNA, adjacent to recombinant event sites.

[00235] The aim of the genomic manipulation was to add copies of glycosyltransferases from a glycan cluster of S. pneumoniae and gneimerase from C. jejuni.

[00236] The first steps regarding the construction of pDOC plasmids for use in replacement. p3408 was prepared as follows. A PCR product of the 5' homology region (containing 1.2 kb upstream of the first gene of the wca cluster, wza) was obtained from the E. coli W3110 genome using oligonucleotides, and cloned into the Eco RI / Xho I sites of pDOC-C, resulting in p693. A PCR product of the 3' homology region (containing 1.2 kb downstream of the last gene of the wca cluster, wcaM) was obtained from the E. coli W3110 genome using oligonucleotides and cloned into the Bcu I / Nhe I sites of p693, resulting in p699. A multiple cloning site was cloned into the Asc I / Bam HI sites of p699, resulting in p3259. Plasmid 3914 was obtained from Genewiz LCC as a gene synthesis service.The PCR product of p3914 with oligonucleotides 4110 / 4111, containing a kanamycin resistance cassette kanR, flanked by two FRT13 sites, was cloned into the Hind III site of p3259, resulting in p3306. Plasmid 3256 encodes genes encoding S. pneumoniae glycosyltransferases originating from PCR in the S. pneumoniae glycan cluster under the control of the synthetic promoter J23114 and followed by the transcriptional terminator rrnb T2. This expression cassette was... Petition 870260072914, dated 07 / 22 / 2026, page 63 / 209 54 / 80 was amplified and cloned into Pac I / Xma I sites of p3375, in the opposite direction relative to kanR. Plasmid 207, encoding previously amplified gne from the Campylobacter jejuni genome, was used as a template for a PCR. The resulting amplicon contains the synthetic promoter J23100, added with an oligonucleotide, gne upstream, and was cloned into the SbfI / XmaI site of p3375, resulting in p3408.

[00237] p3408 encodes the 5' and 3' homology regions for insertion into wca clusters and, between them, in the opposite orientation, the following elements: J23114 promoter, S. pneumoniae glycosyltransferase genes, rrnb T2 terminator, J23100 promoter, gne.

[00238] For the replacement, strain 9876 was co-transformed with pTKRED (GenBank: GU327533.1) and the donor plasmid p3408 via electroporation. Due to the temperature-sensitive replication phenotype of pTKRED, resulting cells were cultured at 30°C at all times in LB supplemented with spectinomycin for pTKRED selection and with kanamycin for p3408 selection. Plasmids were inserted into acceptor cells to allow expression of the enzymes encoded in the helper plasmid in the presence of the donor plasmid DNA within the same cell.

[00239] Next, the insertion procedure was performed. The newly transformed strain was cultured in TSB medium in the presence of kanamycin and spectinomycin at 30°C on a 5 mL scale overnight at 180 rpm. 50 μL of the dense culture were transferred to a new tube containing 1 mL TSB supplemented with spectinomycin and kanamycin. The new culture was then cultured at 180 rpm for 2 hours at 30°C, the cells were centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was replaced with TSB medium supplemented with spectinomycin, 0.2% arabinose (w / v), and 1 mM IPTG. The medium composition supports helper plasmid selection and recombinase and SceI endonuclease expression to allow insertion. The cells were then re-placed in Petition 870260072914, dated 07 / 22 / 2026, page 64 / 209 55 / 80 suspension and further incubated at 30°C for 3 hours at 180 rpm. 0.5 mL of the culture was placed on TSB plates supplemented with kan (for selection of the DNA insert) and 10% (w / v) sucrose (for counter-selection against the donor plasmid) and incubated at 37°C overnight (to select for the loss of the temperature-sensitive auxiliary plasmid). A 'carpet' of cells appeared. Streaks were made on TSB plates supplemented with kan and incubated at 37°C overnight.

[00240] To sort the resulting colonies for the correct insertion phenotype, single colonies from the streaked stripes were placed on replica plates over LB plates supplemented with spec, amp, or kan. Colonies resistant to kan (for presence of the insert) but sensitive to amp and spec (for absence of donor and helper plasmids) were further analyzed for insertion.

[00241] To confirm that the strain had lost the substituted DNA originating from W3110, and contained the DNA insert, colony PCR was performed. Candidate colonies with the correct phenotype were taken and subjected to a colony PCR test. Two PCRs were performed. i) One PCR uses oligonucleotides 3206 / 4195 and amplifies the 5' region of the inserted DNA only if recombination occurred correctly. ii) One PCR uses oligonucleotides 3081 / 3957 and amplifies the 3' region of the inserted DNA only if recombination occurred correctly. Several clones from the integration showed the correct PCR pattern (positive PCR ie ii). The resulting strain was designated st10084.

[00242] The next step is the removal of antibiotic resistance from the integrated strain. Strain 10084 was transformed with the temperature-sensitive pCP20 plasmid expressing an FLP recombinase [1] and placed on ampicillin-supplemented LB plates to select pCP20. Plates were incubated overnight at 30°C to allow plasmid replication. 5 mL of LB cultures were inoculated with stripes from the plates and cultured overnight at 42°C to ensure Petition 870260072914, dated 07 / 22 / 2026, page 65 / 209 56 / 80 pCP20 loss. Serial dilutions of overnight cultures were placed on LB plates. 60 single colonies were replicated on LB plates supplemented with ampicillin, kanamycin, or no antibiotics. All colonies grew on plates without antibiotic, 5 colonies grew on kanamycin plates (resistance cassette was not excised), 14 colonies grew on ampicillin plates (pCP20 was retained).

[00243] In order to confirm that the loss of kanamycin resistance is due to cassette excision, and that no genomic material except the kanamycin resistance cassette was lost, a colony PCR was performed. Using oligonucleotides 3081 and 3957, the following is expected: i. A 1513-bp band if the kanamycin cassette is removed and the DNA bordering the FRT sites is not removed; ii. A 2879-bp band if the kanamycin cassette was not removed; iii. No PCR product if the DNA region between the FRT13 site and the FRTwt site present in the rfb O16 locus was excluded from the loop.

[00244] 12 colonies with the correct resistance pattern (no resistance to ampicillin and kanamycin) were tested by colony PCR, and all of them showed the expected 1513-bp band if the kanamycin resistance cassette is excised and the DNA between the FRT13 and FRTwt sites is intact. As a control, the strain before resistance removal showed the expected 2879-bp band.

[00245] The use of two pairs of alternative FRT sites (FRT13 for wca locus replacement, FRTwt for rfb locus replacement) allowed for double marker-free integration without DNA loss at these two adjacent loci. The resulting strain was named 10085. Example 4: Use of alternative FRT sites to introduce additional recombinant changes in a strain already containing single copies of FRTwt.

[00246] Strain stLMTB11280 is a derivative of Escherichia coli Petition 870260072914, dated 07 / 22 / 2026, p. 66 / 209 57 / 80 W3110 contains several genomic modifications involving the use of FRTwt, such that copies of FRTwt are present in multiple positions in the genomic DNA, adjacent to recombinant event sites. Two unique copies of FRTwt were present, as well as a pair of FRTwt sequences flanking an a.chloramphenicol resistance cassette.

[00247] Additional genomic manipulations were performed to add a copy of genetically engineered clustering to the genome.

[00248] The donor pDOC plasmid pLMTB4184 encodes the 5' and 3' homology regions for the rfbD to wbbL gene substitution of the O16 antigen cluster. Between them, in the same orientation, is a transcription unit encoding seven genes of interest followed by a kanamycin resistance cassette in the opposite orientation flanked by two FRT3 sites.

[00249] For the replacement, strain 11280 was co-transformed with pTKRED (GenBank: GU327533.1) and the donor plasmid p4184 via electroporation. Due to the temperature-sensitive replication phenotype of pTKRED, resulting cells were cultured at 30°C at all times in TSB supplemented with 10 mM MgCl, spectinomycin for pTKRED selection, and kanamycin for p4184 selection. Plasmids were inserted into acceptor cells to allow expression of the enzymes encoded in the helper plasmid in the presence of the donor plasmid DNA within the same cell.

[00250] Next, the insertion procedure was performed. The newly transformed strain was cultured in 10 mM MgCl2 TSB medium in the presence of kanamycin and spectinomycin at 30°C on a 5 mL scale overnight at 180 rpm. 50 μL of the dense culture were transferred to a new tube containing 1 mL of TSB supplemented with spectinomycin and kanamycin. The new culture was then cultured at 180 Petition 870260072914, dated 07 / 22 / 2026, p. 67 / 209 The cells were centrifuged at 58 / 80 rpm for 2 hours at 30°C, then centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was replaced with TSB medium supplemented with kan, 10 mM MgCb, 0.2% arabinose (w / v), and 1 mM IPTG. The medium composition supports helper plasmid selection and recombinase and Sce I endonuclease expression to allow insertion. The cells were resuspended and incubated at 30°C for 4 hours at 180 rpm. The cells were centrifuged at 4000 rpm for 15 minutes at 4°C and then resuspended in 1 mL TSB MgCh 0.2% arabinose, 1 mM IPTG, and incubated at 30°C for 1 hour. The dense culture was then placed in TSB plates supplemented with kan (for selection of the DNA insert) and 10% (w / v) sucrose (for counter-selection against the donor plasmid) and incubated at 37°C overnight (to select for loss of the temperature-sensitive helper plasmid). A 'carpet' of cells appeared.Stripes were made on TSB plates supplemented with kan and 10% (w / v) sucrose and incubated at 37°C overnight.

[00251] To sort the resulting colonies for the correct insertion phenotype, single colonies from the streaked stripes were placed on replica plates over LB plates supplemented with spec, amp, or kan. Colonies resistant to kan (for presence of the insert) but sensitive to amp and spec (for absence of donor and helper plasmids) were further analyzed for insertion.

[00252] To confirm that the strain had lost the substituted DNA originating from W3110, and contained the DNA insert, colony PCR was performed. Candidate colonies with the correct phenotype were taken and subjected to a colony PCR test. Three PCRs were performed. i) One PCR uses 2449 / 5210 oligonucleotides and amplifies the 5' region of the inserted DNA only if recombination occurred correctly. ii) One PCR uses 546 / 1237 oligonucleotides and amplifies the 3' region of the inserted DNA only if recombination occurred correctly. iii) One Petition 870260072914, dated 07 / 22 / 2026, pp. 68 / 209 59 / 80 PCR uses 3454 / 3455 oligonucleotides which yield a product only if a target locus has not been engineered, meaning unsuccessful recombination. Several clones from the integration show the correct PCR pattern (PCR ie ii positive, PCR iii negative). The resulting strain was designated stLMTB11339.

[00253] The next step is the removal of antibiotic resistance to chloramphenicol (ACE group) and kanamycin from the integrated strain. Strain 11339 was transformed with the temperature-sensitive pCP20 plasmid expressing FLP recombinase [1] and placed on LB plates supplemented with ampicillin to select pCP20. Plates were incubated overnight at 30°C to allow plasmid replication. 5 mL of LB cultures were inoculated with stripes from the plates and cultured overnight at 42°C to ensure pCP20 loss. Serial dilutions of the overnight cultures were placed on LB plates. 60 single colonies were replicated on LB plates supplemented with ampicillin, kanamycin, chloramphenicol, or no antibiotics.Nine colonies did not grow on plates without antibiotic, no colonies grew on plates with kanamycin, 15 colonies grew on chloramphenicol (resistance cassette on ACE was not excised), 19 colonies grew on plates with ampicillin (pCP20 was retained). A total of 41 colonies showed the correct resistance pattern (growth only on LB plates without antibiotic).

[00254] In order to confirm that the loss of resistance is due to cassette excision, and that no genomic material except the resistance cassette was lost, two colony PCRs were performed. 1) Kanamycin cassette removal. Using oligonucleotides 3376 and 1265 is expected: i. A 900-bp band if the kanamycin cassette is removed and the DNA bordering the FRT sites is not removed; ii. A 1945-bp band if the kanamycin cassette has not been removed; iii. No PCR product if the DNA region between the FRT3 site and the site Petition 870260072914, dated 07 / 22 / 2026, pp. 69 / 209 60 / 80 FRTwt present at the wca locus was excluded from the circuit. 2) Removal of the chloramphenicol cassette. Using oligonucleotides 3376 and 3495 is expected: i. A 2023-bp band if the chloramphenicol cassette is removed and the DNA bordering the FRT sites is not removed; ii. A 2991-bp band if the chloramphenicol cassette has not been removed.

[00255] 8 colonies with the current resistance pattern (no resistance to ampicillin, chloramphenicol, and kanamycin) were tested by colony PCR, and all showed the expected 900-bp band if the kanamycin resistance cassette is excised and the DNA between the FRT13 and FRTwt sites is intact. Only 4 of the 8 colonies showed the expected 2023-bp band after removal of the chloramphenicol cassette, while the other 4 showed no signal in the PCR. As a control, the strain before resistance removal showed the expected 2991 and 1945-bp bands for the chloramphenicol and kanamycin cassettes, respectively.

[00256] The use of two pairs of alternative FRT sites (FRT13 for wca locus replacement, FRTwt for rfb locus replacement) allowed for double marker-free integration without DNA loss at these two adjacent loci. Furthermore, using two different selection markers allowed for the simultaneous excision of the chloramphenicol resistance cassette from the ECA cluster of wca and the kanamycin resistance cassette from the rfb O16 cluster. The resulting strain was named 11340. Example 5: Preparation of a strain free of unwanted genetic elements through the use of alternative FRT sites.

[00257] Strain stLMTB10502 is a derivative of Escherichia coli W3110 in which the following genes have been deleted: i. waaL, replaced by an FRTwt site; ii. rfb O16 cluster from rfbD to wbbL, replaced by an FRT3 site.

[00258] The goal of genomic manipulation was to delete the Petition 870260072914, dated 07 / 22 / 2026, pp. 70 / 209 61 / 80 collagenic acid cluster wca while keeping intact the short genomic region (2525 bp) between the aforementioned cluster and the rfbD gene (the second gene of the rfb O16 cluster), so that a strain devoid of unwanted sugar clusters can be used as a starting point for other homologous recombinations. Maintaining a genomic region between the collagenic acid and the O16 antigen clusters is crucial because i. it contains the O16 antigen cluster promoter which is exploited for the expression of the inserted elements and ii. the strain can be further engineered using donor pDOCs for the replacement of the O16 antigen cluster as the homologous regions are maintained.

[00259] The donor plasmid pDOC pLMTB3385 encodes the 5' and 3' homology regions for the wza to wcaM gene substitution of the collagenic acid cluster. Between them, in the opposite orientation, was a kanamycin resistance cassette flanked by FRT15 sites.

[00260] For the replacement, strain 11502 was co-transformed with pTKRED (GenBank: GU327533.1) and the donor plasmid p3385 via electroporation. Due to the temperature-sensitive replication phenotype of pTKRED, resulting cells were cultured at 30°C at all times in TSB supplemented with spectinomycin for pTKRED selection and with kanamycin for p3385 selection. Plasmids were inserted into acceptor cells to allow expression of the enzymes encoded in the helper plasmid in the presence of the donor plasmid DNA within the same cell.

[00261] Next, the insertion procedure was performed. The newly transformed strain was cultured in TSB medium in the presence of kanamycin and spectinomycin at 30°C on a 5 mL scale overnight at 180 rpm. 50 μL of the dense culture were transferred to a new tube. Petition 870260072914, dated 07 / 22 / 2026, page 71 / 209 62 / 80 containing 1 mL of TSB supplemented with spectinomycin and kanamycin. The new culture was then grown at 180 rpm for 2 ha at 30°C, the cells were centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was replaced with TSB medium supplemented with kan, 10 mM MgCl2, 0.2% arabinose (w / v), and 1 mM IPTG. The medium composition supports helper plasmid selection and recombinase and SceI endonuclease expression to allow insertion. The cells were resuspended and further incubated at 30°C for 4 ha at 180 rpm. The cells were centrifuged at 4000 rpm for 15 minutes at 4°C and then resuspended in 1 mL of 0.2% TSB MgCl ara, 1 mM IPTG, and incubated at 30°C for 1 h.The dense culture was then placed on TSB plates supplemented with kan (for selection of the DNA insert) and 10% (w / v) sucrose (for counterselection against the donor plasmid) and incubated at 37°C overnight (to select for the loss of the temperature-sensitive helper plasmid). A 'carpet' of cells appeared. Streaks were made on TSB plates supplemented with kan and 10% (w / v) sucrose and incubated at 37°C overnight.

[00262] To sort the resulting colonies for the correct insertion phenotype, single colonies from the streaked stripes were placed on replica plates over LB plates supplemented with spec, amp, or kan. Colonies resistant to kan (for the presence of the insert) but sensitive to amp and spec (for the absence of donor and helper plasmids) were further analyzed for insertion. 11 out of 60 clones tested had the correct pattern, while the remainder showed persistent ampicillin resistance.

[00263] To confirm that the strain had lost the substituted DNA originating from W3110, and contained the DNA insert, colony PCR was performed. Candidate colonies with the correct phenotype were taken and subjected to a colony PCR test. Three PCRs were performed. i) Petition 870260072914, dated 07 / 22 / 2026, page 72 / 209 63 / 80 i) A PCR using oligonucleotides 3206 / 4363 amplifies the 5' region of the inserted DNA only if recombination occurred correctly. ii) A PCR using oligonucleotides 4364 / 3975 amplifies the 3' region of the inserted DNA only if recombination occurred correctly. iii) A PCR using oligonucleotides 3872 / 3957 yielded a product only if a target locus was not engineered, signifying unsuccessful recombination. All integration clones showed the correct PCR pattern (PCR ie ii positive, PCR iii negative). The resulting strain was designated stLMTB10605.

[00264] The next step is the removal of the kanamycin resistance cassette from the integrated strain. Strain 10605 was transformed with the temperature-sensitive pCP20 plasmid expressing an FLP recombinase [1] and placed on ampicillin-supplemented LB plates to select pCP20. Plates were incubated overnight at 30°C to allow plasmid replication. 5 mL of LB cultures were inoculated with stripes from the plates and cultured overnight at 42°C to ensure pCP20 loss. Serial dilutions of the overnight cultures were placed on LB plates. 10 single colonies were replicated on supplemented LB plates with ampicillin, kanamycin, or no antibiotics. 2 colonies showed the correct resistance pattern (growth only on antibiotic-free LB plates) and were tested by colony PCR.

[00265] In order to confirm that the loss of resistance is due to cassette excision, and that no genomic material, except the resistance cassettes, was lost, a colony PCR was performed using oligonucleotides 3206 and 3957, annealing off the FRT15 sites flanking the kanamycin resistance. A 423-bp band is expected if the kanamycin cassette is removed and the DNA bordering the FRT sites is not removed; a 2862-bp band is expected if the kanamycin cassette has not been removed; no product of Petition 870260072914, dated 07 / 22 / 2026, page 73 / 209 64 / 80 PCR is expected if the DNA region between the FRT15 and FRT3 sites present in the rfb locus is excluded from the loop. Both colonies tested showed the expected pattern from the removal of the correct cassette.

[00266] The use of two pairs of alternative FRT sites (FRT15 for wca locus replacement, FRT3 for rfb locus replacement) allowed for obtaining a double marker-free deletion without DNA loss at these two adjacent loci. This is the first evidence of lack of cross-reactivity between FRT3 and FRT15 sites. The resulting strain was named 10651.

[00267] The complete or partial ECA wca cluster (wzzE to wecG) was then removed from strain 10651, resulting in strains 10739 and 10740 respectively, which can be used as general starting strains for the development of specific saccharide bioconjugate production derivatives. Example 6: Use of alternative FRT sites during strain development to allow integration of homologous gene clusters.

[00268] Strain stLMTB10739 was used as a starting strain for the integration of two highly homologous gene clusters.

[00269] In the first genetic manipulation, the wca collagenic acid cluster was replaced by a heterologous glycan cluster. The donor plasmid pDOC pLMTB2941 encodes the 5' and 3' homology regions for the wca cluster replacement. Between them, in the same orientation, a heterologous gene cluster was followed by a chloramphenicol resistance cassette in the opposite orientation flanked by two FRTwt sites.

[00270] For replacement, strain 10739 was co-transformed with pTKRED (GenBank: GU327533.1) and the donor plasmid p2941 via electroporation. Due to the temperature-sensitive replication phenotype Petition 870260072914, dated 07 / 22 / 2026, page 74 / 209 65 / 80 of pTKRED, resulting cells were cultured at 30°C throughout in TBdev supplemented with spectinomycin for pTKRED selection and with ampicillin for p2941 selection. Plasmids were inserted into acceptor cells to allow expression of the enzymes encoded in the helper plasmid in the presence of donor plasmid DNA within the same cell.

[00271] Next, the insertion procedure was performed. The newly transformed strain was cultured in TBdev medium in the presence of chloramphenicol and spectinomycin at 30°C on a 5 mL overnight scale at 180 rpm. 50 μL of the dense culture were transferred to a new tube containing 2 mL of TBdev supplemented with spectinomycin and chloramphenicol. The new culture was then cultured at 180 rpm for 3 hours at 30°C, the cells were centrifuged at 4000 rpm for 5 minutes at 4°C, and the supernatant was replaced with 2 mL of TBdev medium supplemented with spectinomycin, 0.2% arabinose (w / v), and 1 mM IPTG. The medium composition supports helper plasmid selection and recombinase and Sce I endonuclease expression to allow insertion. The cells were resuspended and incubated at 30°C for 4 hours at 180 rpm. The cells were centrifuged at 4000 rpm for 5 minutes at 4°C and then resuspended in 2 mL of TBdev and incubated at 37°C for 1 hour.The dense culture was then placed in TBdev plates supplemented with clm (for selection of the DNA insert) and 10% (w / v) sucrose (for counter-selection against the donor plasmid) and incubated at 37°C overnight (to select for loss of the temperature-sensitive helper plasmid). A 'carpet' of cells appeared. Streaked streaks were made in TSB plates supplemented with kan and 10% (w / v) sucrose and incubated at 37°C overnight.

[00272] To sort the resulting colonies for the correct insertion phenotype, 120 unique colonies from the striped stripes were placed in Petition 870260072914, dated 07 / 22 / 2026, pp. 75 / 209 66 / 80 replicate plates on LB plates supplemented with spec, amp, or clm. Colonies resistant to clm (for the presence of the insert) but sensitive to amp and spec (for the absence of donor and helper plasmids) were further analyzed for insertion. 119 out of 120 colonies were resistant to clm and sensitive to amp and spec.

[00273] To confirm that the strain had lost the substituted DNA originating from W3110, and contained the DNA insert, colony PCR was performed. Candidate colonies with the correct phenotype were taken and subjected to a colony PCR test. Three PCRs were performed. i) One PCR uses 1822 / 3050 oligonucleotides and amplifies the 5' region of the inserted DNA only if recombination occurred correctly. ii) One PCR uses 1366 / 746 oligonucleotides and amplifies the 3' region of the inserted DNA only if recombination occurred correctly. iii) One PCR uses 3967 / 3969 oligonucleotides that amplify part of the inserted genome. 21 out of the 21 clones tested from the integration showed the correct PCR pattern (PCR i, ii, and iii are positive).

[00274] 10 clones were tested for functionality. All clones tested acquired the ability to express heterologous genes. One high-performing clone was selected and named stLMTB10867.

[00275] As an additional step, a second gene cluster with high homology to the first heterologous gene cluster was inserted into the rfb locus of the O16 antigen, which is constitutively expressed. Given a long homology stretch between the wca-integrated cluster and the second glycan gene cluster, it is essential to maintain chloramphenicol resistance pressure during this second homologous recombination procedure. In this way, it was possible to select the desired recombination event because, if the homology stretch were used as the 5' recombination region, the chloramphenicol resistance cassette would be excised. Petition 870260072914, dated 07 / 22 / 2026, page 76 / 209 67 / 80 In this case, the donor plasmid is pDOC p3952, encoding the 5' and 3' homology regions for the rfb cluster replacement. Between these, in the same orientation, is the second gene cluster, followed by a kanamycin resistance cassette in the opposite orientation flanked by two FRT3 sites.

[00276] For the replacement, strain 10867 was co-transformed with pTKRED (GenBank: GU327533.1) and the donor plasmid p3952 via electroporation. Due to the temperature-sensitive replication phenotype of pTKRED, resulting cells were cultured at 30°C at all times in LB supplemented with spectinomycin for pTKRED selection and with kanamycin for p3952 selection. Plasmids were inserted into acceptor cells to allow expression of the enzymes encoded in the helper plasmid in the presence of the donor plasmid DNA within the same cell.

[00277] Next, the insertion procedure was performed. The newly transformed strain was cultured in TBdev medium in the presence of kanamycin and spectinomycin at 28°C on a 5 mL overnight scale at 180 rpm. 50 μL of the dense culture were transferred to a new tube containing 2 mL of TBdev supplemented with spectinomycin and chloramphenicol. The new culture was then cultured at 180 rpm for 3 hours at 30°C, the cells were centrifuged at 4000 rpm for 5 minutes at 4°C, and the supernatant was replaced with 2 mL of TBdev medium supplemented with spectinomycin, 0.2% arabinose (w / v), and 1 mM IPTG. The medium composition supports helper plasmid selection and recombinase and Sce I endonuclease expression to allow insertion. The cells were resuspended and incubated at 30°C for 4 hours at 180 rpm. 50 µL of the culture were used to inoculate 2 mL TBdev with 0.2% arabinose (w / v) and 1 mM IPTG, which were cultured overnight at 30°C.The following day, the culture was placed at 37°C for 1 hour and then placed in TBdev plates supplemented with kan (for... Petition 870260072914, dated 07 / 22 / 2026, page 77 / 209 68 / 80 DNA insert selection), clm (for selection of the desired recombination event), and 10% (w / v) sucrose (for counter-selection against the donor plasmid) and incubated at 37°C overnight (to select for the loss of the temperature-sensitive helper plasmid). A 'carpet' of cells appeared. Streaked streaks were made on TBdev plates supplemented with clm, kan, and 10% (w / v) sucrose and incubated at 37°C overnight.

[00278] To sort the resulting colonies for the correct insertion phenotype, 60 unique colonies from the streaked stripes were placed on replica plates over LB plates supplemented with spec, amp, or clm+kan. Colonies resistant to clm and kan (for the presence of the insert and with the correct recombination pattern), but sensitive to amp and spec (for the absence of donor and helper plasmids) were further analyzed for insertion. 55 out of the 60 colonies showed the desired resistance pattern.

[00279] Candidate colonies with the correct phenotype were taken and subjected to a colony PCR test. Two PCRs were performed. i) One PCR uses 3204 / 3940 oligonucleotides and amplifies the 5' region of the inserted DNA only if recombination occurred correctly, and genetic material between the wca and rfb loci was not lost. ii) One PCR uses 548 / 1237 oligonucleotides and amplifies the 3' region of the inserted DNA only if recombination occurred correctly. iii) One PCR uses 3967 / 3969 oligonucleotides that amplify part of the inserted genome. 30 colonies were first screened for PCR i. 3 positive colonies were found. PCRs ii and iii were performed only on these colonies and resulted as positive for all of them.

[00280] The three clones were tested for functionality (enzyme expression). All clones tested acquired the ability to express the expected enzymes, specifically from Petition 870260072914, dated 07 / 22 / 2026, pp. 78 / 209 69 / 80 RFB clustering (see below for explanation). A high-performance clone was selected and named stLMTB10883.

[00281] The next step is the removal of antibiotic resistance to chloramphenicol (wca cluster of cholanic acid) and kanamycin (integration mentioned above in O16 cluster) from the integrated strain. Strain 10883 was transformed with the temperature-sensitive pCP20 plasmid expressing FLP recombinase [1] and placed on supplemented LB plates with ampicillin to select pCP20. Plates were incubated overnight at 30°C to allow plasmid replication. 5 mL of LB cultures were inoculated with stripes from the plates and cultured overnight at 42°C to ensure pCP20 loss. Serial dilutions of the overnight cultures were placed on LB plates. 20 single colonies were replicated on LB plates supplemented with ampicillin, kanamycin, chloramphenicol, or no antibiotics. All colonies showed the correct resistance pattern (growth only on LB plates without antibiotic).

[00282] In order to confirm that the loss of resistance is due to cassette excision, and that no genomic material except the resistance cassette was lost, two colony PCRs were performed. i. Kanamycin cassette removal using oligonucleotides 3966 and 1237 that bind outside FRT3 sites; ii. Chloramphenicol cassette removal using oligonucleotides 3929 and 1231 that bind outside FRTwt sites. The three screened colonies had the expected pattern from the correct removal of the kanamycin cassette. Two of them were tested for chloramphenicol PCR and also resulted in the expected pattern. One of the two confirmed clones resulting from this kanamycin / chloramphenicol removal was named stLMTB10900.

[00283] The use of two pairs of alternative FRT sites (FRT13 for rfb locus replacement, FRTwt for wca locus replacement) allowed for double marker-free integration without DNA loss. Petition 870260072914, dated 07 / 22 / 2026, pp. 79 / 209 70 / 80 in these two adjacent loci. In this particular case, simultaneous removal was essential as the persistence of the chloramphenicol cassette during the insertion of the second copy of the cluster and the kanamycin resistance cassette was strictly necessary for selection for the correct recombination event.

[00284] Strains 27_0048 10739, 10867, 10883, and 10900 were tested and compared for functionality by obtaining competent cells and transforming them with different sets of plasmids. 5 mL of TBdev 10 mM MgCl2 supplemented with appropriate antibiotics were inoculated with 10 μL of the recovery suspension and cultured overnight at 37°C. 50 mL of TBdev 10 mM MgCl2 and antibiotic main cultures were inoculated at OD600 0.1 and shaken at 37°C. Induction was performed at OD600 0.8 to 1 with 1 mM IPTG and 0.1% arabinose, when necessary. Cultures were grown overnight at 37°C. A volume corresponding to 2 OD was collected, resuspended in 100 μL of Lammli buffer, and heated for 10 minutes at 95°C. Proteinase K was added and incubated at 55°C for one hour, followed by 10 minutes at 70°C for inactivation. Samples were thoroughly vortexed and centrifuged. A volume corresponding to 0.4 OD was loaded onto an SDSPAGE gel.After cycling, the sample was transferred onto a membrane, and Western blotting was used to measure the expressed enzymes. The wca-encoded cluster relies on rcsA expression for its own expression, while the rfb-encoded cluster is active, but biosynthesis requires wchA. The plasmid combinations used were selected to understand if both clusters are active. It was observed that both integrated clusters are functional: the presence of wchA in strains 10883 and 10900 results in the production of antiserum-reactive species, while the addition of rcsA activates the wca-integrated cluster, resulting in antiserum-reactive species. Petition 870260072914, dated 07 / 22 / 2026, pp. 80 / 209 71 / 80 antiserum. Example 7: Usable sequences for performing insert DNA deletion. SEQ ID NO: 1 FRTwt 5'GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACT TC-3' SEQ ID NO: 2 FRT3 5'GAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACT TC-3' SEQ ID NO: 3 FRT10 5'GAAGTTCCTATTCCGAAGTTCCTATTCACTAGAATGTATAGGAACT TC-3' SEQ ID NO: 4 FRT13 5GAAGTTCCTATTCCGAAGTTCCTATCCATATAAGTATAGGAACT TC-3' SEQ ID NO: 5 FRT14 5′GAAGTTCCTATTCCGAAGTTCCTATTCTATCAGAAGTATAGGAACT TC-3' SEQ ID NO: 6 FRT15 5'GAAGTTCCTATTCCGAAGTTCCTATTCTTAGGAGTATAGGAACT TC-3' SEQ ID NO: 7 FRT5 5' GAAGTTCCTATTCCGAAGTTCCTATTCACTAGAATGTATAGGAACT Petition 870260072914, of 22 / 07 / 2026, p. 81 / 209 72 / 80 TC - 3' SEQ ID NO: 8 FRT11 5'GAAGTTCCTATTCCGAAGTTCCTATTCTGAACTAAGTATAGGAACT TC - 3' SEQ ID NO: 9 FRT12 5'GAAGTTCCTATTCCGAAGTTCCTATTCTTTCTGAAGTATAGGAACT TC - 3' SEQ ID NO: 10 FRT16 5'GAAGTTCCTATTCCGAAGTTCCTATTCTCCGGCAGTATAGGAAC TTC - 3' SEQ ID NO: 11 FLP recombinase MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMITHN GTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPA WEFTIIPYYGQKHSDITDIVSSLQLQFESSEEADKGNSHSKKMLKAL LSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLATFINCGRFSDIKN VDPKSFKLVQNKYLGVIIQCLVTEKTSVSRHIYFFSARGRIDPLVYLD EFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKKKNAPY SIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVART TYTHQITAIPDHYFALVSRYYDPISKEMIALKDETNPIEEWQHIEQLK GSAEGSIRYPAWNGIISQEVLDYLSYINRRI SEQ ID NO: 12 FLP recombinase ATGCCACAATTTGATATTATGTAAAACACCACCTAAGGTGCTTG TTCGTCAGTTTGTGGAAAGGTTTGAAAGACCTTCAGGTGAGAAAAT AGCATTATGTGCTGCTGAACTAACCTATTTATGTTGGATGATTACA CATAACGGAACAGCAATCAAGAGAGCCATTCATGAGCTATAAT ACTATCATAAGCAATTCGCTGAGTTTCGATATTGTCAATAAATCACT CCAGTTTAAATACAAGACGCAAAAAGCAACAATTCTGGAAGCCTCA Petition 870260072914, of 22 / 07 / 2026, p. 82 / 209 73 / 80 TTAAAGAAATTGATTCCTGCTTGGGAATTTACAATTATTCCTTACTA TGGACAAAAACATCAATCTGATATCACTGATATTGTAAGTAGTTTG CAATTACAGTTCGAATCATCGGAAGAAGCAGATAAGGGAAATAGC CACAGTAAAAAAATGCTTAAAGCACTTCTAAGTGAGGGTGAAAGC ATCTGGGAGATCACTGAGAAAATACTAAATTCGTTTGAGTATACTT CGAGATTTACAAAAACAAAAACTTTATACCAATTCCTCTTCCTAGCT ACTTTCATCAATTGTGGAAGATTCAGCGATATTAAGAACGTTGATC CGAAATCATTTAAATTAGTCCAAAATAAGTATCTGGGAGTAATAAT CCAGTGTTTAGTGACAGAGACAAAGACAAGCGTTAGTAGGCACAT ATACTTCTTTAGCGCAAGGGGTAGGATCGATCCACTTGTATATTTG GATGAATTTTTGAGGAATTCTGAACCAGTCCTAAAACGAGTAAATA GGACCGGCAATTCTTCAAGCAATAAACAGGAATACCAATTATTAAA AGATAACTTAGTCAGATCGTACAATAAAGCTTTGAAGAAATGCG CCTTATTCAATCTTTGCTATAAAAAATGGCCCAAAATCTCACATTG GAAGACATTTGATGACCTCATTTCTTTCAATGAAGGGCCTAACGGA GTTGACTAATGTTGTGGGAAATTGGAGCGATAAGCGTGCTTCTGC CGTGCCAGGACAACGTATACTCATCAGATAACAGCAATACCTGA TCACTACTTCGCACTAGTTTCTCGGTACTATGCATATGATCCAATA TCAAAGGAAATGATAGCATTGAAGGATGAGACTAATCCAATTGAG GAGTGGCAGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGAAG CATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCATCCTACATAAATAGACGCATA SEQ ID NO: 13 pCP20 contains FLP gene GAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTT GAAGGATCAGATCACGCATCTTCCCGAACGCAGACCGTTCCGT GGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAA AGCTCTCATCAACCGTGGCTCCCTCACTTTGGCTGGGATGGGG GGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGG CAGACCTCAGCGCCACAGGTGCGGTTGCTGGCGCTAACCGTTTTT ATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTTA Petition 870260072914, of 22 / 07 / 2026, p. 83 / 209 74 / 80 CCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAG AAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAAC CAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCG ACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTT ATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCA ATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCA GTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCA CAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTG TCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAG AAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCA CCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTT AGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGA ATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCA GAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACA AGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGC CATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTG AATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTA AAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATT GAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTG GGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAG CTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGC CGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCG GTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTT CCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTG CCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAG TGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGG GGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTTGT TTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGT TGGGCGCCTGTAGTGCCATTTACCCCCATTCACTGCCAGAGCCGT Petição 870260072914, de 22 / 07 / 2026, pág. 84 / 209 75 / 80 GAGCGCAGCGAACTGAATGTCACGAAAAAGACAGCGACTCAGGT GCCTGATGGTCGGAGACAAAAGGAATATTCAGCGATTTGCCCGAG CTTGCGAGGGTGCTACTTAAGCCTTTAGGGTTTTAAGGTCTGTTTT GTAGAGGAGCAAACAGCGTTTGCGACATCCTTTTGTAATACTGCG GAACTGACTAAAGTAGTGAGTTATACACAGGGCTGGGATCTATTC TTTTTATCTTTTTTTATTCTTTCTTTATTCTATAAATTATAAC CACTTG AATATAAACAAAAAAAACACACAAAGGTCTAGCGGAATTTACAGAG GGTCTAGCAGAATTTACAAGTTTTCCAGCAAAGGTCTAGCAGAATT TACAGATACCCACAACTCAAAGGAAAAGGACTAGTAATTATCATTG ACTAGCCCATCTCAATTGGTATAGTGATTAAAATCACCTAGACCAA TTGAGATGTATGTCTGAATTAGTTGTTTTCAAAGCAAATGAACTAG CGATTAGTCGCTATGACTTAACGGAGCATGAAACCAAGCTAATTTT ATGCTGTGTGGCACTACTCAACCCCACGATTGAAAACCCTACAAG GAAAGAACGGACGGTATCGTTCACTTATAACCAATACGTTCAGAT GATGAACATCAGTAGGGAAAATGCTTATGGTGTATTAGCTAAAGCA ACCAGAGAGCTGATGACGAGAACTGTGGAAATCAGGAATCCTTTG GTTAAAGGCTTTGAGATTTTCCAGTGGACAAACTATGCCAAGTTCT CAAGCGAAAAATTAGAATTAGTTTTTAGTGAAGAGATATTGCCTTA TCTTTTCCAGTTAAAAAAATTCATAAAATATAATCTGGAACATGTTA AGTCTTTTGAAAACAAATACTCTATGAGGATTTATGAGTGGTTATTA AAAGAACTAACACAAAAGAAAACTCACAAGGCAAATATAGAGATTA GCCTTGATGAATTTAAGTTCATGTTAATGCTTGAAAATAACTACCAT GAGTTTAAAAGGCTTAACCAATGGGTTTTGAAACCAATAAGTAAAG ATTTAAACACTTACAGCAATATGAAATTGGTGGTTGATAAGCGAGG CCGCCCGACTGATACGTTGATTTTCCAAGTTGAACTAGATAGACAA ATGGATCTCGTAACCGAACTTGAGAACAACCAGATAAAAATGAATG GTGACAAAATACCAACAACCATTACATCAGATTCCTACCTACATAA CGGACTAAGAAAAACACTACACGATGCTTTAACTGCAAAAATTCAG CTCACCAGTTTTGAGGCAAAATTTTTGAGTGACATGCAAAGTAAGT ATGATCTCAATGGTTCGTTCTCATGGCTCACGCAAAAACAACGAAC Petição 870260072914, de 22 / 07 / 2026, pág. 85 / 209 76 / 80 CACACTAGAGAACATACTGGCTAAATACGGAAGGATCTGAGGTTC TTATGGCTCTTGTATCTATCAGTGAAGCATCAAGACTAACAAACAA AAGTAGAACAACTGTTCACCGTTACATATCAAAGGGAAAACTGTCC ATATGCACAGATGAAAACGGTGTAAAAAAGATAGATACATCAGAG CTTTTACGAGTTTTTGGTGCATTTAAAGCTGTTCACCATGAACAGA TCGACAATGTAACAGATGAACAGCATGTAACACCTAATAGAACAG GTGAAACCAGTAAAACAAAGCAACTAGAACATGAAATTGAACACCT GAGACAACTTGTTACAGCTCAACAGTCACACATAGACAGCCTGAA ACAGGCGATGCTGCTTATCGAATCAAAGCTGCCGACAACACGGGA GCCAGTGACGCCTCCCGTGGGGAAAAAATCATGGCAATTCTGGAA GAAATAGCGCCTGTTTCGTTTCAGGCAGGTTATCAGGGAGTGTCA GCGTCCTGCGGTTCTCCGGGGCGTTCGGGTCATGCAGCCCGTAA TGGTGATTTACCAGCGTCTGCCAGGCATCAATTCTAGGCCTGTCT GCGCGGTCGTAGTACGGCTGGAGGCGTTTTCCGGTCTGTAGCTC CATGTTCGGAATGACAAAATTCAGCTCAAGCCGTCCCTTGTCCTG GTGCTCCACCCACAGGATGCTGTACTGATTTTTTTCGAGACCGGG CATCAGTACACGCTCAAAGCTCGCCATCACTTTTTCACGTCCTCCC GGCGGCAGCTCCTTCTCCGCGAACGACAGAACACCGGACGTGTA TTTCTTCGCAAATGGCGTGGCATCGATGAGTTCCCGGACTTCTTC CGGATTACCCTGAAGCACCGTTGCGCCTTCGCGGTTACGCTCCCT CCCCAGCAGGTAATCAACCGGACCACTGCCACCACCTTTTCCCCTGGCATGAAATTTAACTATCATCCCGCGCCCCCTGTTCCCTGACAG CCAGACGCAGCCGGCGCAGCTCATCCCCGATGGCCATCAGTGCG GCCACCACCTGAACCCGGTCACCGGAAGACCACTGCCCGCTGTT CACCTTACGGGCTGTCTGATTCAGGTTATTTCCGATGGCGGCCAG CTGACGCAGTAACGGCGGTGCCAGTGTCGGCAGTTTTCCGGAAC GGGCAACCGGCTCCCCCAGGCAGACCCGCCGCATCCATACCGCC AGTTGTTTACCCTCACAGCGTTCAAGTAACCGGGCATGTTCATCAT CAGTAACCCGTATTGTGAGCATCCTCTCGCGTTTCATCGGTATCAT TACCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACT Petição 870260072914, de 22 / 07 / 2026, pág. 86 / 209 77 / 80 AAACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTT AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAA CTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC GTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT GCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGG TCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGT ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACA TGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGA ATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACAC GGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGC TGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGAT CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA AATGTTGAATACTCACTACTCTTCCTTTTTCAATATTATTGAAGCATT TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTT AGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCGAAAAG TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTA TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTTTATAAACCGTGGAGCGGGCAAATACTGAGCTGATGAGCAATTTCCCGT. CACCAGTGCCCTTCTGATGAAGCGTCAGCACGACGTTCCTGTCCA CGGTACGCCTGCGGCCAAATTTGATTCCTTTCAGCTTTGCTTCCT Petition 870260072914, de 22 / 07 / 2026, pág. 87 / 209 78 / 80 GTCGGCCCTCATTCGTGCGCTCTAGGATCCTCTACGCCGGACGC ATCGTGGCCGGCATCACCGGCGCTGAGGTCTGCCTCGTGAAGAA GGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGC CAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGT GGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTC TGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTA ATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAA ACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAA AACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCC CCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGAC GACTGAATCCGGTGAGAATGGCAGAATAGGAACTTCGGAATAGGA ACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGC GAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTG CGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGC GTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGAT GAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGG GGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATG CTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCATAGTACCGAGAAACTAGTGCGA AGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCT GGCCACGGCAGAAGCACTTATCGCTCCAATTTCCCACAACATT AGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAA TGTCTTCCAATGTGAGATTTTGGGCCATTTTTAGCAAAGATTG AATAAGGCGCATTTTTCTTCAAAGCTTTTATTGTACGATCTGACTAA GTTATCTTTTAATATTGGTATTCCTGTTTATTGCTTGAAGAATTGC CGGTCCTATTTACTCGTTTAGGACTGGTCCAGAATCCTTCAAAATTCAAATTCAA TTCATCCAAATACAAGTGGATCGATCCTACCCCTTGCGCTAAAG Petition 870260072914, of 22 / 07 / 2026, p. 88 / 209 79 / 80 AAGTATATGTGCCTACTAACGCTTGTCTTTGTCTCTGTCACTAAAC ACTGGATTATTACTCCCAGATACTTATTTTGGACTAATTTAAATGAT TTCGGATCAACGTTCTTAATATCGCTGAATCTTCCACAATTGATGA AAGTAGCTAGGAAGAGGAATTGGTATAAAGTTTTTGTTTTTGTAAA TCTCGAAGTATACTCAAACGAATTTAGTATTTTCTCAGTGATCTCC CAGATGCTTTCACCCTCACTTAGAAGTGCTTTAAGCATTTTTTTACT GTGGCTATTTCCCTTATCTGCTTCTTCCGATGATTCGAACTGTAAT TGCAAACTACTTACAATATCAGTGATATCAGATTGATGTTTTTGTCC ATAGTAAGGAATAATTGTAAATTCCCAAGCAGGAATCAATTTCTTTA ATGAGGCTTCCAGAATTGTTGCTTTTTGCGTCTTGTATTTAAACTG GAGTGATTTATTGACAATATCGAAACTCAGCGAATTGCTTATGATA GTATTATAGCTCATGAATGTGGCTCTCTTGATTGCTGTTCCGTTAT GTGTAATCATCCAACATAAATAGGTTAGTTCAGCAGCACATAATGC TATTTTCTCACCTGAAGGTCTTTCAAACCTTTCCACAAACTGACGA ACAAGCACCTTAGGTGGTGTTTTACATAATATATCAAATTGTGGCA TACAACCTCCTTAGTACATGCAACCATTATCACCGCCAGAGGTAAA ATAGTCAACACGCACGGTGTTAGATATTTATCCCTTGCGGTGATAG ATTTAACGTATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGC TTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAA TGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGAT GGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGC GTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGT ATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGT ACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCT TAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAAC CAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAA TTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGG AATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGA TTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAA Petição 870260072914, de 22 / 07 / 2026, pág. 89 / 209 80 / 80 ACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCC ACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGG GAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATC GGCAAGGTGTTCTGGTCGGCGCATAGCTGATAACAATTGAGCAAG AATCTGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGC TCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTG ATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCA GCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATAC CTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGC ATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCAT AAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCA TTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCA TCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCG ACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGT TGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATAT GGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTT TATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGA GATTTT Petição 870260072914, de 22 / 07 / 2026, pág. 90 / 209

Claims

1 / 3 CLAIMS 1. Bacterial host cell, characterized in that it comprises a host cell genomic polynucleotide containing a first recombination engineered region and a second recombination engineered region, wherein a first recombination site scar is adjacent to the first recombination engineered region and a second recombination site scar is adjacent to the second recombination engineered region; wherein: - the first and second recombination site scars have different polynucleotide sequences that are 70%-98% identical to each other and optionally 70%-98% identical to the polynucleotide sequence of any additional recombination site scar present in the host cell genomic polynucleotide;- The first and second engineered recombinant regions involve at least two of i) a deletion of a waaL gene and / or ii) a deletion of at least part of a wca colonic acid cluster and / or iii) a deletion of at least part of an rfb cluster; and - the host cell is engineered to express a) an oligosaccharyltransferase, for example, PglB or PglL; b) a heterologous glycan cluster, for example, an rfb cluster or a cluster of genes encoding glycosyltransferases necessary to synthesize a capsular polysaccharide; and a protein containing a glycosylation site recognized by the oligosaccharyltransferase.

2. Bacterial host cell, according to claim 1, characterized in that the first and second recombination sites are recombination sites for a recombinase, for example, an FLP recombinase.

3. Bacterial host cell, according to Petition 870260072914, dated 07 / 22 / 2026, page 91 / 209 2 / 3 claim 1, characterized in that the first recombination site has a nucleic acid sequence of any one of SEQ ID NO: 1-10.

4. Bacterial host cell, according to any one of claims 1 to 3, characterized in that the first and second recombination sites are separated by less than 100, 75, 50, 25, 10 or 5 kbases.

5. Engineered bacterial host cell, characterized in that it comprises unique copies of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 recombination sites in the host cell genomic polynucleotide, wherein each recombination site has a nucleotide sequence that is 70%-98% identical to the other recombination sites, wherein the host cell is engineered to express a) an oligosaccharyltransferase, for example, PglB or PglL; b) a heterologous glycan cluster, for example, an rfb cluster or a cluster of genes encoding glycosyltransferases necessary to synthesize a capsular polysaccharide; and a protein containing a glycosylation site recognized by the oligosaccharyltransferase.

6. Engineered bacterial host cell according to claim 5, characterized in that at least 2 recombination sites are FRT sites.

7. Engineered bacterial host cell, according to claim 5 or 6, characterized in that the at least 2 recombination sites are separated by less than 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 3 kb or 1 kb in the host cell genomic polynucleotide.

8. Process for making a glycosylated protein, characterized in that it comprises the steps of: (i) cultivating the bacterial host cell, as defined in claim 1 or 5, under conditions appropriate for the production of glycosylated protein; and (ii) isolating the glycosylated protein from the culture.

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