Processes to make neosaxitoxin and analogues thereof

Abstract

The present invention relates to processes to make neosaxitoxin, and analogues and variants thereof, and intermediates in the production of neosaxitoxin in recombinant host cells. Neosaxitoxin and the analogues and variants thereof may be used in the production of pharmaceutical compositions.

Claims

1. A process for producing a neosaxitoxin analogue or variant, the process comprising the steps: (A) contacting the substrates: (i)S-adenosylmethionine, (ii) arginine (iii) acetyl-CoA, malony-CoA or propionyl-CoA, and (iv) carbamoyl phosphate, with Sxt A, B, D, G, H, I, S, T, U, V, W and X polypeptides, in a reaction medium, wherein the reaction medium additionally comprises a PPTase, and optionally (B) isolating and/or purifying a neosaxitoxin analogue or variant from the reaction medium, wherein the neosaxitoxin analogue or variant is: (I) gonyautoxin 1 or gonyautoxin 4, and the substrates (i)-(iv) are additionally contacted with Sxt DIOX and Sxt SUL; (II) saxitoxin, and the substrates (i)-(iv) are not contacted with Sxt X; (III) gonyautoxin 2 or gonyautoxin 3, and the substrates (i)-(iv) are additionally contacted with Sxt DIOX and Sxt SUL, and the substrates (i)-(iv) are not contacted with Sxt X; (IV) gonyautoxin 5, and the substrates (i)-(iv) are additionally contacted with Sxt N, and the substrates (i)-(iv) are not contacted with Sxt X; or (V) Lyngbya wollei toxin, and the substrates (i)-(iv) are additionally contacted with Sxt ACT, and the substrates (i)-(iv) are not contacted with Sxt X.

2. A process for producing a neosaxitoxin analogue or variant in a host cell, the process comprising the steps: (A) culturing a host cell which comprises nucleic acid molecules encoding the Sxt polypeptides A, B, D, G, H, I, S, T, U, V, W and X in a culture medium in the presence of the substrates: (i)S-adenosylmethionine, (ii) arginine (iii) acetyl-CoA, malony-CoA or propionyl-CoA, and (iv) carbamoyl phosphate, and wherein the host cells do not comprise nucleic acid molecules encoding one or more of Sxt polypeptides Q, R and ORF24, under conditions which are suitable for the production of a neosaxitoxin analogue or variant; and optionally (B) isolating and/or purifying the neosaxitoxin analogue or variant from the host cells or from the culture medium, wherein the neosaxitoxin analogue or variant is: (I) gonyautoxin 1 or gonyautoxin 4, and the host cell additionally comprises nucleic acid molecules encoding Sxt DIOX and Sxt SUL; (II) saxitoxin, and the host cell does not comprise a nucleic acid molecule encoding Sxt X; (III) gonyautoxin 2 or gonyautoxin 3, and the host cell additionally comprises nucleic acid molecules encoding Sxt DIOX and Sxt SUL, and the host cell does not comprise a nucleic acid molecule encoding Sxt X; (IV) gonyautoxin 5, and the host cell additionally comprises a nucleic acid molecule encoding Sxt N, and the host cell does not comprise a nucleic acid molecule encoding Sxt X; or (V) Lyngbya wollei toxin, and the host cell additionally comprises a nucleic acid molecule encoding Sxt ACT, and the host cell does not comprise a nucleic acid molecule encoding Sxt X.

3. A process as claimed in claim 2, wherein the host cell comprises an additional nucleic acid molecule encoding a phosphopantetheinyl transferase (PPTase).

4. A process as claimed in claim 2, wherein the host cells do not comprise nucleic acid molecules encoding one or more of the Sxt polypeptides C, J and K.

5. A process as claimed in claim 1, wherein the substrates are not contacted with one or more of the Sxt polypeptides Q, R and ORF24.

6. A process as claimed in claim 2, wherein the host cells do not comprise nucleic acid molecules encoding any of the said Sxt polypeptides in one or more of (a)-(c): (a) C, Q, R and ORF24; (b) L, Q, R and ORF 24; or (c) J, K, L, Q, R and ORF 24.

7. A process as claimed in claim 2, wherein the host cells do not comprise nucleic acid molecules encoding one or more of Sxt polypeptides F, M and P.

8. A process as claimed in claim 2, wherein the host cells additionally comprise nucleic acid molecules encoding one or more of Sxt polypeptides C, E, J, K, L, and R.

9. A process as claimed in claim 2, wherein the host cells do not comprise nucleic acid molecules encoding one or more of Sxt polypeptides F, M, N, O, P, Y, Z, ORF3, ORF4, ORF29, ORF34, OMPR or HISA, wherein the neosaxitoxin analogue or variant is: (I) gonyautoxin 1 or gonyautoxin 4, and the substrates (i)-(iv) are additionally contacted with Sxt DIOX and Sxt SUL, and/or the host cell additionally comprises nucleic acid molecules encoding Sxt DIOX and Sxt SUL; (II) saxitoxin, and the substrates (i)-(iv) are not contacted with Sxt X and/or the host cell does not comprise a nucleic acid molecule encoding Sxt X; (III) gonyautoxin 2 or gonyautoxin 3, and the substrates (i)-(iv) are additionally contacted with Sxt DIOX and Sxt SUL, and the substrates (i)-(iv) are not contacted with Sxt X and/or the host cell additionally comprises nucleic acid molecules encoding Sxt DIOX and Sxt SUL, and the host cell does not comprise a nucleic acid molecule encoding Sxt X; or (V) Lyngbya wollei toxin, and the substrates (i)-(iv) are additionally contacted with Sxt ACT, and the substrates (i)-(iv) are not contacted with Sxt X and/or the host cell additionally comprises a nucleic acid molecule encoding Sxt ACT, and the host cell does not comprise a nucleic acid molecule encoding Sxt X.

10. A process as claimed in claim 2, wherein the host cell is a recombinant prokaryotic host cell, where the prokaryotic cell is an E. coli cell.

11. A process as claimed in claim 2, wherein the host cell is a heterotroph.

12. A process as claimed in claim 2, wherein the host cell is a recombinant yeast cell.

13. A process as claimed in claim 2, wherein the neosaxitoxin analogue or variant, or a pharmaceutically acceptable salt thereof, is formulated into a pharmaceutical composition.

14. A process as claimed in claim 13, wherein the formulating step comprises admixing isolated or purified neosaxitoxin analogue or variant with one or more pharmaceutically-acceptable carriers, adjuvants and/or excipients.

15. A host cell which comprises nucleic acid molecules coding for the Sxt polypeptides A, B, D, G, H, I, S, T, U, V, W and X, wherein the host cell does not comprise nucleic acid molecules coding for: (i) one or more of the Sxt polypeptides C, J or K; (ii) one or more of the Sxt polypeptides Q, R and ORF24; (iii) one or more of the Sxt polypeptides C, Q, R and ORF24; (iv) one or more of the Sxt polypeptides L, Q, R and ORF 24; (v) one or more of the Sxt polypeptides J, K, L, Q, R and ORF 24; or (vi) one or more of the Sxt polypeptides F, M and P, wherein the host cell: (I) additionally comprises nucleic acids encoding Sxt DIOX and Sxt SUL; (II) does not comprise a nucleic acid encoding Sxt X; (III) additionally comprises nucleic acids encoding Sxt DIOX and Sxt SUL, and does not comprise a nucleic acid encoding Sxt X; (IV) additionally comprises a nucleic acid encoding Sxt N, and does not comprise a nucleic acid encoding Sxt X; or (V) additionally comprises a nucleic acid encoding Sxt ACT, and does not comprise a nucleic acid encoding Sxt X.

16. A host cell as claimed in claim 15, wherein the host cell 1) does not comprise nucleic acid molecules coding for one or more of the Sxt polypeptides F, M and P and 2) comprises nucleic acid molecules coding for one or more Sxt polypeptides selected from the group consisting of Sxt C, E, J, K, L, and/or R.

17. A host cell as claimed in claim 15, wherein the host cell does not comprise nucleic acid molecules coding for one or more of the Sxt polypeptides selected from the group consisting of F, M, N, O, P, Y, Z, ORF3, ORF4, ORF29, ORF34, OMPR or HISA, wherein the host cell: (I) additionally comprises nucleic acids encoding Sxt DIOX and Sxt SUL; (II) does not comprise a nucleic acid encoding Sxt X; (III) additionally comprises nucleic acids encoding Sxt DIOX and Sxt SUL, and does not comprise a nucleic acid encoding Sxt X; or (V) additionally comprises a nucleic acid encoding Sxt ACT, and does not comprise a nucleic acid encoding Sxt X.

18. A host cell as claimed in claim 15, wherein the host cell is a recombinant prokaryotic host cell, where the prokaryotic host cell is an E. coli cell or where the host cell is a recombinant yeast cell.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Previous biochemical pathway for the production of saxitoxin which was proposed by Kellmann (2008).

(2) FIGS. 2-3: Two revised biochemical pathways for the production of neosaxitoxin.

(3) In FIG. 2, the pathway involves the use of a 6-5-5-membered ring intermediate.

(4) In FIG. 3, the pathway involves the use of a 5-5-5-membered ring intermediate which gets converted to a 6-5-5-ring system.

(5) FIGS. 4A-B: Proposed biochemical pathways showing where in the pathways the N-1 hydroxyl group is introduced in the production of neosaxitoxin. The —R group indicates where the hydroxylation may occur.

(6) FIG. 5: SDS-PAGE of SxtA after IMAC. Lane 1: protein solution containing SxtA (147 kDa); the chaperons, DnaK and GroES, (70 kDa and 60 kDa, respectively); Sfp, (27 kDa). Lane 2: Ladder Precision Plus Protein Standards Dual Color (BioRad).

(7) FIG. 6: MALDI-TOF-TOF analysis of phosphopantetheinylation of the SxtA-ACP domain. Purified SxtA (a) and SxtA co-expressed with Sfp(b) were analysed by MALDI-TOF-TOF with respective masses of 14,694 Da and 15,034 Da. The difference of 340 Da corresponds to the attachment of a phosphopantetheinyl arm.

(8) FIG. 7: Liquid chromatography-mass spectroscopy of E. coli transformant methanolic extracts. a) Total ion current chromatograms of both induced (dashed line) and non-induced (solid line) extracts. b) Extracted ion chromatograms for m/z=187.06 of both induced (dashed line) and non-induced (solid line) extracts. c) ProgenesisQl statistical analysis comparing the expression of the molecular ion at m/z 187.06 between induced (square) and non-induced (circle) extracts. d) Mass spectrum of induced extract.

(9) FIG. 8: Scheme for the insertion of the PPTase gene construct into the mannitol operon of E. coli.

(10) FIGS. 9A and 9B: (a) sxtI integrated into the lactose operon, kanamycin cassette removed by flippase recombination. (b) sxt2 integrated into the maltose operon, kanamycin cassette removed by flippase recombination. (c) sxt3 (all genes) integrated into the xylose operon, kanamycin cassette removed by flippase recombination. (d) sxt4 integrated into the melobiose operon, kanamycin cassette not removed.

(11) FIG. 10: Detection of intermediate 8 in cell extract of E. coli BL21(DE3) T3PPTase NSX3v1.

(12) FIG. 11: MS/MS spectra of Intermediate 8 in cell extract of E. coli BL21(DE3) T3PPTase NSX3v1.

(13) FIG. 12: Accurate mass LC-MS analysis of neosaxitoxin standard solution (100 nM). Selected ion monitoring chromatogram of m/z 316.13639 (400 mmu window) is shown in the upper plot, and MS spectrum across the peak, and theoretic spectrum are shown in the middle and lower plots, respectively. The retention time (RT), manually integrated peak area (MA), signal to noise ratio (SN) is given in the upper plot. The measured mass, chemical composition, and mass error (ppm) is given in the middle and lower plots.

(14) FIG. 13: Accurate mass LC-MS analysis of extract from E. coli BL21(DE3) sfp NSX3v3. Selected ion monitoring chromatogram of m/z 316.13639 (400 mmu window) is shown in the upper plot, and MS spectrum across the peak, and theoretic spectrum are shown in the middle and lower plots, respectively. The retention time (RT), manually integrated peak area (MA), signal to noise ratio (SN) is given in the upper plot. The measured mass, chemical composition, and mass error (ppm) is given in the middle and lower plots.

(15) FIG. 14: Recovery of intermediate 1 from E. coli BL21(DE3) pVB-sxtABC by various methods of cell lysis. LC-MS analysis was carried out in SIM mode (m/z 187.15534 with a 400 mmu mass window). The instrument response is given as peak area (counts per second).

(16) FIG. 15: Production levels of neoSTX in percent relative to E. coli sfp NSX3v4 (sfp sxt123 V4). The estimated concentration of neoSTX in the extract of sfp sxt123 V4 was 2.8 nM.

(17) FIG. 16: Concentration the sodium channel blocking toxin neoSTX from E. coli BL21(DE3) sfp (negative control strain), and BL21(DE3) sfp NSX3v3 grown in shaker flask cultures in 50 ml TB medium. Each bar represents a biological replicate. Error bars represent the standard deviation of 4 replicates MNBA assays of the same extract. All samples were extracted by weak cation exchange solid phase extraction prior to analysis from a 1.2 g cell pellet. The concentration is given in nmol neoSTX per liter. The average concentration of neoSTX in the extract was 38.74 nM (±7.8 standard deviation).

(18) FIG. 17: Production of intermediate 1 by E. coli BL21(DE3) sfp pVB-sxtA. sfp pVB: pVB vector without insert. sfp nat 8: pVB vector with native sxtA codon usage and 8 bp spacer between RBS and start codon. sfp nat 10: pVB vector with native sxtA codon usage and 10 bp spacer between RBS and start codon. sfp syn 8: pVB vector with synthetic sxtA gene that was codon usage-optimised for expression in E. coli with and 8 bp spacer between RBS and start codon. sfp syn 10: pVB vector with synthetic sxtA gene that was codon usage-optimised for expression in E. coli with and 10 bp spacer between RBS and start codon.

(19) FIG. 18: Production of Intermediate 8 and neoSTX in E. coli strains. T3PPT: E. coli T3PPTase NSX3V1. NsPPT: E. coli T3PPTase NSX3V1 pET28b-NsPPT, coding for the Nodularia spumigena phosphopantetheinyl transferase. T3Ala18T3PPTase: E. coli T3PPTase NSX3V1 pET30b-Ala18T3PPTase, coding for T3PPTase that had the first 18 N-terminal amino acids removed to improve solubility during expression. Cultures were induced with 0.2 mM IPTG, and 1 mM toluic acid, where indicated.

(20) FIG. 19: LC chromatogram, MS spectra, MS/MS fragmentation (from top to bottom) of sulfotransferase assay of SxtN using saxitoxin as a substrate.

(21) FIG. 20: LC chromatogram, MS spectra, MS/MS fragmentation (from top to bottom) of dioxygenase assay (SxtDIOX) using STX as substrate. Peaks that indicate hydroxy-STX production in the assay (right), not present in the control (left) are indicated by arrows.

EXAMPLES

(22) The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

(23) The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1: Production of SxtA

(24) Subcloning of sxtA and sfp in pET28b

(25) PCR amplifications of sxtA from Cylindrospermopsis raciborskii T3 and sfp from Bacillus subtilis were performed from genomic DNA using Velocity polymerase (Bioline). The manufacturer's protocols were followed, with an annealing temperature of 55° C. and extension times of 4 min and 1 min for sxtA and sfp, respectively (sxtA primers: forward, 5′-GGGCTTTCATATGTTACAAAAGATTAA-3′ (SEQ ID NO: 81) and reverse, 5′-AAAGTATGCGGCCGCATGCTTGAGTAT-3′ (SEQ ID NO: 82); sfp primers: forward 5′-GGCATCCATGGGCAAGATTTACGGAA-3′ (SEQ ID NO: 83) and reverse, 5′-GGCATCTCGAGTTATAAAAGCGCTTCG-3′ (SEQ ID NO: 84)). PCR amplicons were purified (DNA clean and concentrator 5× kit, ZymoResearch) and digested with NdeI/NotI (sxtA) or NcoI/XhoI (sfp; New England Biolabs). Digested products were purified and ligated into pET-28b (Novagen), cut with the same enzymes, and purified via agarose gel electrophoresis (DNA recovery kit, ZymoResearch). After transformation in electrocompetent Escherichia coli GB2005 cells, positive clones were screened by PCR of the purified plasmid (PureLink Miniprep, Invitrogen) with the universal primers T7 promoter and T7 terminator. The inserts were sequenced (Ramaciotti Center at UNSW, Australia) for verification.

(26) For co-expression with sxtA, the sfp gene was amplified from pET28b::sfp using Velocity polymerase (Bioline) with primers for the T7 promoter and T7 terminator (5′-GGTTAAGATCTGAAATTAATACGACTC-3′ (SEQ ID NO: 85), 5′-TTTTAAGATCTTTTCAGCAAAAAACCC-3′ (SEQ ID NO: 86)). The manufacturer's protocols were followed with an annealing temperature of 55° C. and an extension time of 1 min. Amplicons were purified (DNA clean and concentrator 5× kit, ZymoResearch) and digested with BglII (New England Biolabs). Digested products were purified and ligated into a pET28b::sxtA cut with the same enzyme, and purified as described previously. After transformation in electrocompetent Escherichia coli GB2005 cells, the positive clones were screened by PCR of the purified plasmid (PureLink Miniprep, Invitrogen). The insert was sequenced (Ramaciotti Center at UNSW, Australia) for verification.

(27) Expression of sxtA with Sfp and Purification of Holo-SxtA

(28) For expression of sxtA, 0.5 mL of overnight E. coli BL21 (DE3) transformants containing pET28b::sxtA,sfp and pRARE plasmids (Invitrogen) was subcultured in 50 mL Lysogeny Broth (LB) medium supplemented with 50 μg.Math.mL.sup.−1 kanamycin and 30 μg.Math.mL.sup.−1 chloramphenicol and incubated at 30° C. under agitation (200 rpm) until an optical density of 0.8-1.0 at 600 nm. Cultures were then induced with 200 μM isopropyl β-D-thiogalactoside (IPTG) and incubated overnight at 18° C. with agitation and pelleted by centrifugation (4000 rpm, at 4° C. for 20 min, Hitachi CR22GIII centrifuge, R10A5 rotor). The cell pellet was frozen until further purification.

(29) Pelleted cells were resuspended in 10 mL of lysis buffer (20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 20 mM imidazole), and lysed by sonication (Branson Digital Sonifier M450, 3 mm probe, 30% of amplitude, 3 min at 4° C. with cycles of 15 s power on and 59 s off). The resulting suspension was centrifuged (20 000 rpm, at 4° C. for 60 min, Hitachi CR22GIII centrifuge, R20A2 rotor), and the supernatant was loaded on a Ni-affinity column (1 mL HiTrap column, fitted on an AKTApurifier, GE Healthcare), equilibrated with 20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 20 mM imidazole (butter A). After injection, the column was washed (35 mL of buffer A, 1 mL.Math.min.sup.−1) and the proteins were eluted using 20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 500 mM imidazole (buffer B) and a stepwise gradient of 0% to 20% buffer B in 20 min, followed by 10 min at 20%, then a linear gradient from 20% to 100% in 20 min, and a final wash at 100% for 10 min. The collected fractions (1 mL, detection at 280 nm) were analyzed by 10% polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE), and the fractions containing the pure protein were pooled. The protein solution was desalted and concentrated via centrifugal filter (Amicon Ultra-4 centrifugal filter unit 100 k) with 50 mM HEPES-150 mM NaCl pH7.4 and then glycerol was added to a final concentration of 10% (w/v). Protein concentration was determined by using protein assay kit (Bio-Rad) and stored at −80° C.

(30) Cloning of the Acyl Carrier Protein Domain of sxtA for Expression Alone and with Sfp

(31) The acyl carrier protein of sxtA (sxtA-ACP) coding gene was amplified using Velocity polymerase (Bioline) (primers: forward 5′-ATATCCATGGGACCTGGTGATCGCAAAGGA-3′ (SEQ ID NO: 87) and reverse 5′-TATCTCGAGAGTGTTGATTTCGTTGGCTG-3′ (SEQ ID NO: 88)). Manufacturer protocols were followed with an annealing temperature of 54° C. and an extension time of 1.5 min. PCR amplicons were purified, digested, and ligated into pET-28b using the same method as described for sfp. After transformation in electrocompetent Escherichia coli GB2005 cells, the positive clones were screened by with universal primers T7 promoter and T7 terminator and sequenced as described previously. For co-expression with sfp, the gene was cloned in pET28b::sxtA-ACP plasmid, as described previously.

(32) Expression and Purification of Apo- and Holo-sxtA-ACP

(33) For expression of sxtA-ACP, 10 mL of overnight E. coli BL21 (DE3) transformants containing pET28b::sxtA-ACP and pET28b::sxtA-ACP,sfp plasmids were grown in 1 L Lysogeny broth (LB) supplemented with 50 μg.Math.mL.sup.−1 kanamycin and 30 μg.Math.mL.sup.−1 chloramphenicol and incubated at 37° C. under agitation (200 rpm) until the induction with 100 μM IPTG, as described above. Cells were collected by centrifugation (4000 rpm, at 4° C. for 20 min, Hitachi CR22GIII centrifuge, R10A5), resuspended in 15 mL of Lysis buffer, and disrupted by sonication (Branson Digital Sonifier M450, 3 mm probe, 30% of amplitude, 3 min at 4° C. with cycles of 1 s power on followed by 4 s off). The resulting suspension was centrifuged (20 000 g, at 4° C. for 60 min, Hitachi CR22GIII centrifuge, R20A2 rotor), and the supernatant was loaded on a Ni-affinity column (1 mL HiTrap column, fitted on an AKTApurifier, GE Healthcare) previously equilibrated with 20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 20 mM imidazole (buffer A). After injection, the column was washed (35 mL of buffer A, 1 mL.Math.min.sup.−1) and the proteins were eluted using 20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 500 mM imidazole (buffer B) and a linear gradient from 0% to 20% B in 20 min, then 10 min at 20%, then a linear gradient from 20% to 100% in 20 min, and a final wash at 100% for 20 min. The collected fractions (1 mL, detection at 280 nm) were analyzed by 15% polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE), and fractions containing the pure protein were pooled together. The protein solution was desalted and concentrated using centrifucation filtration (Amicon Ultra-15 centrifugal filter unit 3 k) with 50 mM HEPES-150 mM NaCl, pH 7.4, freezed in liquid nitrogen and stored at −80° C. Protein concentration was determined by using protein assay kit (Bio-Rad).

(34) SxtA was successfully detected after purification on IMAC and SDS-PAGE (FIG. 5). A protein at the expected size of 143 kDa was purified with a yield of 0.5 mg.Math.L-1 of culture, and the identity was confirmed to be SxtA by trypsinolysis.

(35) MALDI-TOF-TOF Mass Spectrometry Analysis of Apo- and Holo-sxtA-ACP

(36) Protein mass was detected by MALDI-TOF-TOF mass spectrometry. For matrix preparation, 10 mg of 3 5-dimethoxy-4-hydroxyl cinnamic acid was added into 1 mL 80% acetonitrile with 0.1% TFA, and 1 mL of matrix was mixed with 1 μl of protein sample on the surface of a MALDI target plate, followed by analysis by Bruker ultrafleXtreme MALDI-TOF/TOF with a YAG laser. Data acquisition was performed in the positive ion mode and the instrument calibrated immediately prior to each analysis. Analysis was performed in the linear delayed extraction mode acquiring 100 averaged spectra. The results are shown in FIG. 6.

(37) Extraction of the 4-Amino-3-Oxo-Guanidinoheptane

(38) BL21 (DE3) transformants containing pRARE and pET28b::sxtA,sfp were resuspended in methanol acidified with 0.1% of acid acetic. The cells were disrupted by sonication (Branson Digital Sonifier M450, 3 mm probe, 30% of amplitude, 2 min at 4° C. with cycles of 5 s power on and 15 s off). The resulting suspension was centrifuged (20,000 rpm, at 4° C. for 30 min, Hitachi CR22GIII centrifuge, R20A2 rotor). The supernatant was dried under rotary evaporation and store at −20° C. until further analysis.

(39) Mass Spectroscopy Analyses of 4-Amino-3-Oxo-Guanidinoheptane

(40) To determine the biosynthetic products of SxtA, intracellular metabolites of E. coli cells containing pET28b::sxtA,sfp and pRARE were chemically extracted and analysed by LC-MS.

(41) Samples were resuspended in 95:5 acetonitrile:water (typically 1 mL) and transferred to HPLC vials for mass spectrometric analysis. LC-MS analysis was performed on 10 μL of sample using a Dionex U3000 UHPLC interfaced to a Q-Exactive Plus (ThermoFisher Scientific) via a heated electrospray interface. Ionisation was performed in positive mode under default source conditions (as suggested by the manufacturer's Tune software). Samples were injected onto a Waters BEH HILIC column (2.1×100 mm, 1.9 μm). Chromatographic conditions were as in Turner et al. with no modifications.

(42) The mass spectrometer was run in data dependent analysis mode, with six MS/MS spectra being collected after every full scan mass spectrum. Inclusion lists of toxins and metabolites from the literature where used to prioritise their detections.

(43) Comparison of the mass spectra between induced cells and non-induced controls showed the presence of a molecular ion at 1.1 min of m/z 187.06 [M+H]+, corresponding to the protonated mass of the AOGH(C.sub.8H.sub.18N.sub.4O). Secondary ion fragmentation of this molecular ion resulted in a fragmentation pattern closely resembling that of AOGH. The experiment was realised in eight replicates and mass data were analysed by Progenesis 01 to quantify the difference of expression between induced and non-induced extraction. The putative AOGH compound was observed to be overexpressed in the induced cells, compared to non-induced controls (Figure), with a maximum fold change equal to infinity (entirely absent in negative controls) and an ANOVA p-value of 1.1×10.sup.−16.

(44) The compound at 187.06 Da mass was purified and analysed NMR confirmed the structure of the AOGH.

Example 2: sxtI Fragment

(45) The following three open reading frames (ORFs) from C. raciborskii T3 were placed in a nucleotide fragment labelled “sxt1”: sxtA, sxtB and sxtC. Each ORF was codon optimised for expression in E. coli by Life Technology using GeneArt software.

(46) EcoRI restriction sites were added at both ends of the fragment. In addition, homologous regions of 50 bp to the lacZ (H1) and lacA (H2) genes were added to the 5′- and 3′-end, respectively, for integration by RED ET mediated recombination into the E. coli genome. Downstream of H1, a terminator was added to prevent readthrough, followed by the transcription factor gene xylS and its Ps2 promoter. (XylS is the transcription factor that is required for transcription from the Pm promoter, after binding to its inducer, toluic acid.) This was followed by a terminator and then Pm promoter for the transcription of the downstream sxt genes. Upstream of each ORF, a ribosomal binding site (RBS) was added. Finally, a kanamycin resistance marker with two flanking FRT sites for excision by flippase-recombinase (GeneBridges), and homology arm H2 were added, including a terminal EcoRI site.

Example 3: sxt2 Fragment

(47) The following sxt genes were included in the “sxt2” fragment: sxtD, sxtE, sxtG, sxtH, sxtI, sxtJ, sxtK, sxtL. Each ORF was codon optimised for expression in E. coli by Life Technology using GeneArt software.

(48) The sxt2 fragment was designed to have terminal EcoRI sites, homology arms for recombination with the malE (H1) and malG (H2) genes of E. coli, and a Pm promoter upstream of the sxt genes. This fragment also contained the kanR resistance cassette with FRT sites like fragment sxt1.

Example 4: sxt3 Fragment

(49) Three variants of sxt3 fragment were designed. The following sxt genes were included in the “sxt3” fragment, variant 1: sxtQ, sxtR, orf24, sxtS, sxtT, sxtU, sxtV, sxtW, sxtX. The following sxt genes were included in the “sxt3” fragment, variant 2: sxtQ, sxtR, sxtS, sxtT, sxtU, sxtV, sxtW, sxtX. The following sxt genes were included in the “sxt3” fragment, variant 3: sxtS, sxtT, sxtU, sxtV, sxtW, sxtX. Each ORF was codon optimised for expression in E. coli by Life Technology using GeneArt software.

(50) Each variant of the sxt3 fragment was designed to have terminal EcoRI sites, homology arms for recombination with the xylF (H1) and xylB (H2) genes of E. coli, and a Pm promoter upstream of the sxt genes. Each variant of this fragment also contained the kanR resistance cassette with FRT sites like fragment sxt1.

Example 5: sxt4 Fragment

(51) The following sxt genes were included in the “sxt4” fragment: sxtF, sxtP, sxtM. Each ORF was codon optimised for expression in E. coli by Life Technology using GeneArt software.

(52) The sxt4 fragment was designed to have terminal EcoRI sites, homology arms for recombination with the melR (H1) and melB (H2) genes of E. coli, and a Pm promoter upstream of sxt genes. This fragment also contained the kanR resistance cassette with FRT sites like fragment sxt1.

Example 6: Creation of E. coli BL21(DE3) PPTase

(53) It is desirable to use a phosphopantetheinyl transferase (PPTase) to convert the apo-form of the polyketide synthase, SxtA, into its holo-form.

(54) The entire genomes of Raphidiopsis brooki D9 and Cylindrospermopsis racoborskii CS-505 have been sequenced and the sequences deposited in GenBank. Each of these strains only contain a single PPTase gene. Specific PCR primers were designed based on the D9 and CS-505 PPTase genes to PCR amplify and sequence the PPTase gene from C. raciborskii T3.

(55) A synthetic gene construct was designed for integration of the C. raciborskii T3 PPTase gene into the maltose operon of E. coli BL21(DE3). The PPTase gene was codon optimised for expression in E. coli and the construct was fitted with a T7 promoter and a beta-lactamase gene for selection for ampicillin resistance. The start codon was also changed to ATG.

(56) The gene construct was then integrated into the maltose operon of E. coli BL21(DE3) (Life Technology) using the Quick & Easy E. coli Gene Deletion Kit following the manufacturer's instructions (Gene Bridges, catalog No. K006), and as depicted in FIG. 8. Correct integration and absence of unintended mutations was verified by sequencing.

Example 7: Production of 4-Amino-3-Oxo-Guanidinoheptane by E. coli BL21(DE3) PPTase pSxt1

(57) The sxtI fragment was produced by Life Technology inside a pMA-RQ (ampR) plasmid backbone. This plasmid will be called pSxt1 herein. The pSxt1 plasmid was transformed into E. coli BL21(DE3) PPTase, which carries the phosphopantetheinyl transferase from Cylindrospermopsis raciborskii T3 in its genome. The PPTase gene is under T7 promoter control (IPTG-inducible), and the sxtA, B, and C genes on pSxt1 are under Pm promoter control (m-toluoic acid inducible).

(58) The goal of this experiment was to measure the expression and activity of the sxtA gene in E. coli at three different temperatures, 5° C., 19° C., and 30° C., at constant inducer concentrations (IPTG (0.5 mM) and m-toluic acid (1 mM)). The experimental strain was E. coli BL21(DE3) PPTase pSxt1, and the negative control was E. coli BL21 (DE3) (parent strain). Enzymatic product of only SxtA was expected, as the substrates for SxtB and SxtC are the enzymatic products of other enzymes, such as SxtG, which are not encoded on pSxt1.

(59) Seed-cultures of BL21(DE3) (parent strain) and BL21(DE3) PPTase pSxt1 were grown overnight at 37° C. in, respectively, LB and LB 50 μg/ml ampicillin and kanamycin. The following day, triplicates volumes of 200 ml LB medium with kanamycin 50 μg/ml were inoculated each with 2 ml seed culture of E. coli BL21(DE3) PPTase pSxt1, and incubated at 30° C. until the culture reached an OD.sub.600 of ˜0.6.

(60) For the negative control, triplicate volumes of 200-ml LB without antibiotics were inoculate each with 2 ml seed culture from E. coli BL21(DE3) and incubated at 30° C. to an OD.sub.600 of ˜0.6. All cultures were then transferred to the corresponding temperature, and acclimatised for 30 minutes. Subsequently, IPTG (0.5 mM final concentration) and m-toluic acid (1 mM final concentration) was added to each culture, which were then incubated for 48 hours at 5° C., and for 18 hours at 19° C., and at 30° C.

(61) Samples for SDS-PAGE as well as for RT-PCR were taken before induction, and at the end of the experiment. After 18 hours induction, the cultures were harvested by centrifugation 7.500 rpm for 12 min. The cell pellets were frozen until extraction for LC-MS analysis

(62) Extraction

(63) The cell pellets were resuspended in 1 ml water and sonicated on ice (output control 4, duty cycle 50%, 7 minutes in total in cycles of 2-3 minutes). After sonication, 4 ml acetonitrile was added, the samples were vortexed, and centrifuged to remove cell debris and other particulates.

(64) LC-MS/MS Analysis

(65) LC-MS analysis was carried out at the Hormone laboratory, Haukeland University Hospital on a Waters Xevo TQ-S coupled to an iclass Acquity UPLC that was fitted with a 50×2.1 mm Acquity BEH amide column. Mobile phase A consisted of 10 mM ammonium formate pH 3.0 and mobile phase B consisted of 95% acetonitrile with 10 mM ammonium formate pH 3.0. The flow-rate was 0.4 ml per min, and the column temperature was held at 40° C. A gradient over 3 min was applied from 85% B to 70% B. The injection volume was always 1 μl.

(66) Analytes were ionised in positive mode by electrospray ionisation. The following mrm transitions were measured. Arginine 175.05-.fwdarw.60.00, 175-.fwdarw.70.02, 4-amino-3-oxo-guanidinoheptane: 187.1-.fwdarw.170.1, 187.1-.fwdarw.128.1, 187.1-.fwdarw.110.1, 187.1-.fwdarw.72.08, 187.1-.fwdarw.60.05, according to Tsuchiya et al. (2014) and Tsuchiya et al. (2015).

(67) A large arginine peak was detected in all culture treatments, as well as for pure arginine solution (data not shown), whereas a 4-amino-3-oxo-guanidinoheptane signal was absent from the arginine solution and all controls. Furthermore, a 4-amino-3-oxo-guanidinoheptane signal was absent from all E. coli BL21(DE3) PPTase pSxt1 cultures, apart from the culture induced at 19° C.

(68) This experiment demonstrated that SxtA could be expressed in a catalytically-active form in E. coli BL21(DE3) using a synthetic DNA construct. In this experiment, expression occurred only at a growth temperature of 19° C., and after induction with 0.5 mM IPTG and 1 mM toluic acid.

Example 8: Integration of Fragments sxtI to Sxt3 into Sugar Catabolising Operons of E. coli DE3(BL21) PPTase

(69) The sxtI construct was integrated into the lac operon of E. coli BL21(DE3) PPTase as follows:

(70) The synthetic DNA construct, sxt1, was PCR amplified using 2 μl template DNA, 0.4 μM primers, Phusion DNA polymerase in a 50 μl reaction volume.

(71) The PCR cycles were as follows: 98 C for 1 min, 30 cycles: 98 C for 5 sec, 72 C for 2 min, 72 C for 5 min, then hold at 4° C.

(72) The PCR product (8019 bp) was then used for RED ET mediated recombination, after its purification by gel electrophoresis and gel extraction. Prior to recombination, E. coli BL21(DE3) PPTase was transformed with the plasmid pRED (GeneBridges) by electroporation, and transcription of pRED encoded recombinases was induced according to the manufacturer's instructions. Subsequently, the transformed strain was electroporated with the PCR product described above (2.5 kV, 200 Ohm, 25 μF, 150 ng PCR product in 5 μl), followed by recombination into the lac operon.

(73) The electroporated cells were plated onto LB agar with 15 μg/ml kanamycin, and grown overnight at 37° C. Resulting clones were picked and plated onto MacConkey (with lactose) agar with 15 μg/ml kanamycin, and grown for at least 24 h at 37° C. Resulting white colonies were isolated into pure culture, and tested by PCR and sequencing for correct and mutation-free integration of fragment sxtI into the lac operon.

(74) Upon confirmation, a clone was chosen to remove the kanamycin resistance cassette by flippase-recombination using plasmid p707, according to the manufacturer's protocol (GeneBridges). Resulting clones were screened for kanamycin resistance, and the successful removal of the kanamycin resistance cassette was verified by PCR. In addition, PCR was used to verify that the remainder of the sxtI fragment was intact.

(75) The resulting strain was then used for the integration of fragment sxt2 into the maltose operon, using the same approach. The resulting strain, where fragments sxtI and sxt2 were integrated, and after removal of the kanamycin resistance cassette, was used by the same approach to integrate each of three variants of sxt3 fragment into the xylose operon.

(76) Sxt3 Variant E. coli BL21(DE3) Strains

(77) TABLE-US-00005 Strain Genes deleted from Strain Designation sxt3 T3PPTase sxt1 sxt2 sxt3 v1 NSX3v1 none T3PPTase sxt1 sxt2 sxt3 v2 NSX3v2 orf24 T3PPTase sxt1 sxt2 sxt3 v3 NSX3v3 sxtQ, sxtR, and orf24

(78) The 4′-phosphopantetheinyl transferase gene sfp from Bacillus subtilis (Pfeifer B A, et al. (2001) Science 291:1790-2) was then used to replace the T3 PPTase in each of the above strains to produced additional strains.

(79) The resulting strains were then used for the integration of fragment sxt4 into the melobiose operon of E. coli BL21(DE3) T3PPTase and E. coli BL21(DE3) sfp. The kanamycin cassette was not removed. Diagrams of the sxtI-4 fragments are given in FIG. 9.

Example 9: Creation of Variant Strains of E. coli BL21(DE3) Sfp NSX3v3

(80) Strain E. coli BL21(DE3) sfp NSX3v3 was used as the parent strain to create the following strains where individual or sets of genes were deleted by homologous recombination methods.

(81) TABLE-US-00006 Table of E. coli BL21(DE3) strains and what genes they have integrated into dispensable sugar operons Strains Description Genes T3PPTase T3PPTase T3PPTase T3PPTase T3PPTase, sxt1 T3PPTase sxt1 sxtA, sxtB, sxtC T3PPTase T3PPTase, sxt1, sxt2 T3PPTase sxt1 sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, sxt2 sxtI, sxtJ, sxtK, sxtL T3PPTase T3PPTase, sxt1, sxt2, T3PPTase sxt1 sxt3; all genes of sxt3 sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, sxt2 present sxtI, sxtJ, sxtK, sxtL, sxtQ, sxtR, ORF24, sxt3 v1 sxtS, sxtT, sxtU, sxtV, sxtW, sxtX T3PPTase T3PPTase, sxt1, sxt2, T3PPTase sxt1 sxt3; sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, sxt2 orf24 was deleted from sxtI, sxtJ, sxtK, sxtL, sxtQ, sxtR, sxtS, sxt3 v2 sxt3 sxtT, sxtU, sxtV, sxtW, sxtX T3PPTase T3PPTase, sxt1, sxt2, T3PPTase sxt1 sxt3; sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, sxt2 sxtQ, sxtR and orf24 were sxtI, sxtJ, sxtK, sxtL, sxtS, sxtT, sxtU, sxt3 v3 deleted from sxt3 sxtV, sxtW, sxtX T3PPTase T3PPTase, sxt1, sxt2, T3PPTase sxt1 sxt3, sxt4; sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, sxt2 sxtQ, sxtR and orf24 were sxtI, sxtJ, sxtK, sxtL, sxtS, sxtT, sxtU, sxt3 v3 deleted from sxt3; kanR sxtV, sxtW, sxtX, sxtF, sxtP, sxtM sxt4 present sfp sfp, sxt1, sxt2, sxt3; sfp sxt123 V1 all genes of sxt3 present sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, (NSXv1) sxtI, sxtJ, sxtK, sxtL, sxtQ, sxtR, ORF24, sxtS, sxtT, sxtU, sxtV, sxtW, sxtX sfp sfp, sxt1, sxt2, sxt3: sfp sxt 123 V2 orf24 was deleted from sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, (NSXv2) sxt3 sxtI, sxtJ, sxtK, sxtL, sxtQ, sxtR, sxtS, sxtT, sxtU, sxtV, sxtW, sxtX sfp sfp, sxt1, sxt2, sxt3: sfp sxt 123 V3 sxtQ, sxtR and orf24 were sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, (NSXv3) deleted from sxt3 sxtI, sxtJ, sxtK, sxtL, sxtS, sxtT, sxtU, sxtV, sxtW, sxtX sfp sxt 123 V4 sfp, sxt1, sxt2, sxt3: sfp (NSXv4) sxtQ, sxtR and or124 were sxtA, sxtB, sxtD, sxtE, sxtG, sxtH, sxtI, deleted from sxt3; sxtC sxtJ, sxtK, sxtL, sxtS, sxtT, sxtU, sxtV, was deleted from sxt1 sxtW, sxtX sfp sxt 123 V5 sfp, sxt1, sxt2, sxt3: sfp (NSXv5) sxtQ, sxtR and orf24 were sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, deleted from sxt3; sxtL sxtI, sxtJ, sxtK, sxtS, sxtT, sxtU, sxtV, was deleted from sxt2 sxtW, sxtX sfp sxt 123 V6 sfp, sxt1, sxt2, sxt3: sfp (NSXv6) sxtQ, sxtR and orf24 were sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, deleted from sxt3; sxtJ, sxtI, sxtS, sxtT, sxtU, sxtV, sxtW, sxtX sxtK, sxtL were deleted from sxt2 sfp sxt 123 V7 sfp, sxt1, sxt2, sxt3, sfp (NSXv7) sxt4:sxtQ, sxtR and orf24 sxtA, sxtB, sxtC, sxtD, sxtE, sxtG, sxtH, were deleted from sxt3; sxtI, sxtJ, sxtK, sxtL, sxtS, sxtT, sxtU, kanR present sxtV, sxtW, sxtX, sxtF, sxtP, sxtM

Example 10: Use of LC-MS to Detect Neosaxitoxin and Intermediates

(82) LC-MS was used to detect neosaxitoxin and intermediates in the production of neosaxitoxin. LC-MS analysis was performed on a ThermoFisher Scientific Q Exative Hybrid Quadrupole mass spectrometer that was fitted with an ESI II ion source, and coupled online to a Dionex 3000 Ultimate RSLC Ultrapressure liquid chromatography instrument.

(83) Mass/Charge Ratios (m/z) of Possible Saxitoxin Analogues and Intermediate Metabolites that Might by Produced by E. coli BL21(DE3) NSX3 Strains

(84) TABLE-US-00007 m/z [M + H].sup.+ Chemical Compound adduct Composition Intermediate 1 187.15534 C.sub.8H.sub.19ON.sub.4 Intermediate 2 229.17714 C.sub.9H.sub.21ON.sub.6 Intermediate 3 211.16657 C.sub.9H.sub.19N.sub.6 Intermediate 4 209.14584 C.sub.9H.sub.17N.sub.6 Intermediate 5 207.13527 C.sub.9H.sub.15N.sub.6 Intermediate 6 223.13019 C.sub.9H.sub.15ON.sub.6 Intermediate 7 223.13090 C.sub.9H.sub.15ON.sub.6 Intermediate 8 225.14584 C.sub.9H.sub.17ON.sub.6 Intermediate 9 268.15165 C.sub.10H.sub.18O.sub.2N.sub.7 dcSTX 257.13566 C.sub.9H.sub.17O.sub.3N.sub.6 dcneoSTX 273.13058 C.sub.9H.sub.17O.sub.4N.sub.6 STX 300.14148 C.sub.10H.sub.18O.sub.4N.sub.7 neoSTX 316.13639 C.sub.10H.sub.18O.sub.5N.sub.7 STX-15N4 304.12962 C.sub.10H.sub.18O.sub.4N.sub.3-.sup.15N.sub.4

Example 11: Detection of Intermediate 1 and Intermediate 8 in E. coli Extract

(85) E. coli BL21(DE3) T3PPtase NSX3v1 was grown in 20 ml LB broth in a 100 ml flask at 19° C. with shaking (200 rpm). The culture was induced with 0.2 mM IPTG, 1 mM toluic acid, when it reached an OD.sub.600 of 0.9, and grown for 18 hours to an OD.sub.600=5.

(86) The cells were harvested by centrifugation, and extracted in 800 μl of 0.1 M acetic acid in methanol. The lysate was centrifuged, and the supernatant diluted 1:5 with acetonitrile prior to LC-MS analysis.

(87) Intermediates 1 and 8 were detected in the extract. The spectra from Intermediate 8 are shown in FIGS. 10 and 11.

Example 12: Production of Neosaxitoxin

(88) A seed culture of E. coli sfp NSX3v3 was prepared in 50 ml Hi YE medium, and incubated overnight at 30° C. (225 rpm). A New Brunswick BioFlo 110 fermentor (2 L volume) equipped a heater sleeve, water cooling, a pH and dissolved oxygen probe was set up for a fermentation experiment according to the manufacturer's instructions. The fermentor was filled with 2 L Hi-YE medium base, and autoclaved. Stock solutions of yeast extract, magnesium chloride, glucose and glycerol were aseptically added to the fermenter after autoclavation. In addition, 2 ml sterile solution of 5% antifoam 204 (Sigma Aldrich) was added to the fermenter. The medium was inoculated with 20 ml overnight seed-culture, which had an OD.sub.600 of 5.71. The OD.sub.600 of the starting culture was measured at 0.15. The culture was incubated at 19° C. with an initial agitation at 50 rpm, which was increased to 600 rpm after 26 hours incubation. The culture was purged with pressurised air at a flow-rate of 2 l/hour. The pH and dissolved oxygen levels were continuously monitored, and the growth was measured off-line after taking samples. The pH (pH 7.1) was stable during the entire experiment, as well as the dissolved oxygen level (100%). The culture was induced with 0.5 mM m-toluic acid and 0.2 mM IPTG after 22 hours incubation (OD.sub.600=10.6), and incubated for a further 22 hours. Sterile glucose solution (400 g/I) was fed to the culture at a flow-rate of 2.9 ml/min between 24 and 26.3 hours of the experiment (total 11.4 g), and 1.8 ml of a sterile 1 M magnesium sulfate solution was added at 26.3 hours of the experiment in an attempt to stimulate growth and production of neoSTX. The experiment was terminated after 45 hours, when the culture was harvested by centrifugation (5000×5, 30 minutes at 4° C.). The cell pellet was washed twice with 50 ml ice-cold MQ water (5.000×g, 10 min, 4° C.), weighed, and stored at −20° C. until analysis. The total biomass obtained was 23.77 g.

(89) The cells from the fermentation experiments were lysed by boiling in 0.1% formic acid, and the extract was diluted 1:5 with acetonitrile with 0.1% formic acid prior to LC-MS analysis (see FIGS. 12 and 13). The content of neoSTX in the pellet was also analysed by a mouse neuroblastoma assay MS.

Example 13: Optimisation of Cell Lysis to Extract Intermediates

(90) The native genes sxtA, sxtB, sxtC were cloned into pVB vector (Vectrons Biosolutions), and the resulting vector pVB-sxtABC was transformed into E. coli BAP1. BAP1 pVB-sxtABC was grown in 10 ml TB medium at 19° C. with shaking at 200 rpm for 24 hours. The cells were harvested by centrifugation, and washed twice with ice-cold MQ water, the pellets were weighed, and stored at −20° C. until analysis.

(91) The pellets were resuspended (1:3 wet weight:volume) in various lysis solutions:

(92) 1. 50% methanol with 25 mM sodium hydroxide

(93) 2. 50% methanol with 0.5% ammonium hydroxide

(94) 3. 50% methanol with 0.1% formic acid

(95) 4. 0.1 mM hydrochloric acid

(96) 5. 0.1% formic acid

(97) 6. 0.5% ammonium hydroxide

(98) Treatments 1, 2 and 3 were sonicated for 2 minutes using a Branson Sonifier 250, equipped with a microprobe (output 2, duty cycle 50%). Treatments 3, to 5 were boiled for 5 minutes in a boiling water batch. The extracts were centrifuged for 10 minutes at 18000×g (4° C). 250 μl supernatant were mixed with 750 μl acetonitrile with 0.4% formic acid, and centrifuged (10 minutes at 18000×g, 4° C.). The supernatant was transferred to autosampler vials for LC-MS analysis of Intermediate 1 (see FIG. 14).

(99) 5 minutes boiling in 0.1% formic acid was regarded as the most suitable method, as it did not cause interference with HILIC LC-MS, and produced a minimal amount of precipitation during extraction.

Example 14: Optimal Strain and Production Levels

(100) Seed-cultures of the E. coli strains were prepared in 10 ml LB broth and grown overnight at 30° C. with shaking at 200 rpm. The following strains were compared for neoSTX production levels:

(101) E. coli BL21(DE3) sfp

(102) E. coli BL21(DE3) T3PPTase sxt1 sxt2

(103) E. coli BL21(DE3) T3PPTase NSX3v1

(104) E. coli BL21(DE3) sfp NSX3v1

(105) E. coli BL21(DE3) sfp NSX3v2

(106) E. coli BL21(DE3) sfp NSX3v3

(107) E. coli BL21(DE3) sfp NSX3v4

(108) E. coli BL21(DE3) sfp NSX3v5

(109) E. coli BL21(DE3) sfp NSX3v6

(110) E. coli BL21(DE3) sfp NSX3v7

(111) 30 ml Terrific Broth (TB) with 0.4% glycerol and buffered to pH 6.8 with 89 mM phosphate buffer were inoculated with 300 μl overnight seed-culture to an approximate OD.sub.600 of 0.05. Cultures were incubated at 30° C. with shaking at 225 rpm until they reached an OD.sub.600 of 0.4. The cultures were then transferred to 19° C. (with shaking 225 rpm), and grown to an OD.sub.600 of 0.5. For induction of sfp and sxt genes, 0.05 mM IPTG (0.05 mM final concentration) and toluic acid (0.5 mM final concentration) were added to the cultures, which incubated at 19° C. with shaking (225 rpm) for a further 48 hours. Each strain was grown in triplicate sub-cultures. 25 ml of each culture was harvested by centrifugation at 3000×g for 10 min at 4° C. Aliquots of 1 nil supernatant was stored at −20° C., and the remaining supernatant discarded. The cell pellets were washed with twice with respectively 25 and 5 ml ice-cold MQ water (3000×g for 10 min at 4° C.). Cell pellets were weighed and stored at −20° C.

(112) Toxin Extraction from Cell Pellets

(113) Bacterial cell pellet was resuspended in 0.1% formic acid at a ratio of 1:4 (wwt:vol) and boiled for 5 minutes. The extract was briefly cooled on ice, centrifuged at 16.000×g for 10 minutes, and the supernatant collected. For LC-MS analysis, the supernatant was diluted with 90% acetonitrile:10% methanol:0.1% formic acid containing 25 nM saxitoxin-.sup.15N.sub.4 internal standard at a ratio of 1:5. The sample was centrifuged (16.000×g for 10 minutes, 4° C.) and the supernatant transferred to autosampler vials for LC-MS analysis.

(114) Toxin Extraction from Growth Media

(115) Growth media (200 μl) was acidified with 2 μl 10% formic acid, boiled for 5 minutes, cooled on ice, and centrifuged at 16000×g for 10 minutes at 4° C. The supernatant was transferred to a new tube, and 40 μl internal standard was added (25 nM saxitoxin-.sup.15N.sub.4 in 10% methanol 90% acetonitrile and 0.1% formic acid). The tube was centrifuged at 16000×g 4° C. for 10 minutes, and the supernatant transferred to an HPLC auto-sampler vial for LC-MS analysis. The results are shown in FIG. 15.

Example 15: Mouse Neuroblastoma Assay (MNBA) for the Detection of Sodium Channel Blocking Toxins

(116) E. coli Cultures for MNBA

(117) Seed-cultures of the E. coli strains BL21(DE3) sfp and BL21(DE3) sfp NSX3v3 were prepared in 10 ml LB broth and grown overnight at 30° C. with shaking at 200 rpm. 50 ml Terrific Broth (TB) with 0.4% glycerol and buffered to pH 6.8 with 89 mM phosphate buffer were inoculated with 500 μl overnight seed-culture to an approximate OD.sub.600 of 0.05. Cultures were incubated at 30° C. with shaking at 225 rpm until they reached an OD.sub.600 of 0.4. The cultures were then transferred to 19° C. (with shaking 225 rpm), and grown to an OD.sub.600 of 0.5. For induction of sfp and sxt genes, 0.05 mM IPTG (0.05 mM final concentration) and toluic acid (0.5 mM final concentration) were added to the cultures, which incubated at 19° C. with shaking (225 rpm) for a further 48 hours. Each strain was grown in triplicate sub-cultures. Cultures were harvested by centrifugation at 2500×g for 10 min at 4° C. The cell pellets were washed with twice with respectively 25 ice-cold MQ water (2500×g for 10 min at 4° C.). Cell pellets were weighed and stored at −20° C.

(118) Cell Extraction for MNBA

(119) Bacterial cells were extracted by weak cation exchange solid phase extraction (WCX-SPE) for the MNBA, using Accell Plus CM cartridges (360 mg, 1.1 ml, WAT010910, Waters) according to the following protocol.

(120) 1.2 g cell pellet was resuspended in 3.6 ml 0.15% formic acid and lysed by boiling for 5 minutes. The cell extract was cleared by centrifugation at 16.000×g for 10 minutes.

(121) Columns were conditioned with 3.6 ml methanol, followed by 3.6 ml 0.15% formic acid. 3 ml sample was loaded, and the column was washed with 1.8 ml MQ water, followed by 1.8 ml acetonitrile. The column was then dried, and the sample eluted in 2×3.6 ml methanol with 5% formic acid. The eluate was dried by vacuum centrifugation, and the sample reconstituted in 200 μl 0.01% formic acid.

(122) Mouse Neuroblastoma Assay for Sodium Channel Blocking Toxins

(123) A mouse neuroblastoma assay was used (as described by Humpage et al. (2007). Environ. Toxicol. Chem. 26:1512-9), using the mouse neuro-2a cell line (CCL131).

(124) A calibrator curve for the MNBA assay was prepared with and without biological matrix using a certified reference material for neoSTX (CRM-NEO-c, lot 2009-02-18, 65.6 μM in 3 mM HCl). The results are shown in FIG. 16.

Example 16: Immunochemical Detection of Neosaxitoxin by ELISA

(125) A saxitoxin ELISA Kit (Abraxis PN 52255B, Microtiter Plate 96T) was used to detect neoSTX produced in E. coli. The kit employs polyclonal saxitoxin antibodies, which have 1.3% cross-reactivity to neoSTX. Cultures of E. coli T3PPTase NSX3v3 were prepared and cell lysates obtained. Samples were extracted by solid phase extraction on SampliQ silica columns (Agilent PN 5982-2211, 1 ml, 100 mg) Jansson D and Astot C (2015)m J Chromatogr A 1417:41-8). Extracted and evaporated samples were dissolved in 100 μl sample buffer provided by the STX ELISA kit. A 1:1000 dilution of E. coli T3PPTase NSX3v3 extract in sample buffer was also prepared. The assay was calibrated by a 2 point standard curve using Std 0 as the blank, Std 1 (0.0668 nM STX) and STd 5 (1.3365 nM STX) provided by the kit. The reference sample was used to estimate the accuracy of the STX ELISA kit for neoSTX, whereas the recovery of neoSTX during SPE was estimated based on the ratio of the extracted reference sample versus the reference sample. Results of the assay are shown in the Table below. The estimated accuracy was 119%, whereas the recovery during SPE was 92%. The assay detected an equivalent of 1.16 nM STX in the cell extract of E. coli T3PPTase NSX3v3. Converted to neoSTX, this amounts to 89.1 nM, and a yield of approximately 223 pmol neoSTX per liter of E. coli culture.

(126) STX ELISA. Std 0, Std 1, and Std 5 were provided by the STX ELISA kit. The ELISA assay was calibrated by 2 points (y=−0.4627+0.7597). The concentration of neoSTX was calculated on the basis of 1.3% cross reactivity of the STX antibody according to the manufacturer. ND: none detected. The recovery of neoSTX by SPE was 92%.

(127) TABLE-US-00008 neo STX Calculated STX STX Ratio equivalent Conc'n Sample ID (nM) (nM) ABS.sub.450 (ABS.sub.sample/ABS.sub.std0) (nM) (nM neoSTX) Std 0 0 1.8190 1.00000 ND Std 1 0.067 1.3257 0.72879 0.067 Std 5 1.337 0.2570 0.14129 1.337 Ref 100 0.0700 0.03848 1.559 119.901 Ref 100 0.1723 0.09474 1.437 110.548 Extract'd NSX3 0.4070 0.22375 1.158 89.101 cells NSX3 1.8740 1.03024 ND media NSX3 1.8870 1.03738 ND cells 1:1000 diluted

Example 17: Effect of Synthetic v. Native Gene and RBS Spacer

(128) A number of pVB constructs were made using the following elements:

(129) (i) the Pm promoter;

(130) (ii) the sxtA synthetic (codon-optimised for E. coli) or the sxt native gene; and

(131) (iii) a 8 or 10 bp spacer between the ribosome binding site (RBS) and start codon.

(132) The sequence of the Pm promoter with the 8 bp spacer between RBS and start codon of SxtA is shown below. The RBS and start codon are shown with capital letters, the spacer is underlined:

(133) TABLE-US-00009 (SEQ ID NO: 100) agtccagccttgcaagaagcggatacaggagtgcaaaaaatggctatctc tagaaaggcctaccccttaggctttatgcaacagaaacaataataatGGA GtcatgaacATG

(134) The sequence of the Pm promoter with the 10 bp spacer between RBS and start codon of SxtA is shown below. The RBS and start codon are shown with capital letters, the spacer is underlined:

(135) TABLE-US-00010 (SEQ ID NO: 101) agtccagccttgcaagaagcggatacaggagtgcaaaaaatggctatctc tagaaaggcctaccccttaggctttatgcaacagaaacaataataatGGA GtcatgaacatATG

(136) The four pVB-sxtA plasmids were independently transformed into E. coli BL21(DE3) sfp.

(137) Cultures of each of the four E. coli BL21(DE3) sfp with pVB-sxtA variants were prepared and cells were harvested. Cell pellets were extracted and the presence of Intermediate 1 was analysed by LC-MS. The results are shown in FIG. 17. It can be seen that significantly more Intermediate 1 was produced in the synthetic sxtA construct which contained a 10 bp spacer between the RBS and start codon, compared to the synthetic sxtA construct which contained the 8 bp spacer.

Example 18: Production of Intermediate 8 in the Presence of Various PPTases

(138) In this experiment, the following PPTases were compared:

(139) 1. T3PPT: E. coli BL21(DE3) T3PPTase NSX3v1

(140) 2. T3PPT: E. coli BL21(DE3) T3PPTase NSX3v1 pET28B-NsPPT

(141) (pET vector with phosphopantetheinyl transferase from Nodularia spumigena)

(142) 3. Ala18T3PPT: E. coli BL21(DE3) T3PPTase NSX3v1 pET30b-Ala18T3PPTase

(143) (T3PPTase where there first 18 amino acids were removed to increase solubility of expressed protein.)

(144) The strains were cultured for 18 hours in 20 ml LB broth at 19° C. with shaking 200 rpm. Strains with pET vector were grown in the presence of 50 μg/ml kanamycin. Cultures were either grown without inducer, or induced with 0.2 mM IPTG and 1 mM toluic acid at OD.sub.600 ca. 0.8. There was clear background expression of sxt and PPTase genes in the absence of inducer.

(145) Intermediate 8 and neoSTX were measured, and used as an indicator for the effectiveness of the PPTase. The results are shown in FIG. 18. The Ala18T3PPT gave the highest levels, but reduced levels after induction. This phenomenon may be due to inhibition of SxtA, when PPTase levels get too high, and occupy the enzyme.

Example 19: Production of Saxitoxin Analogues and Variants

(146) Saxitoxin may be converted to GTX-5 by SxtN (i.e. by sulfonation of the carbamoyl side-chain). Similarly, saxitoxin may be converted 11-hydroxy STX by SxtDIOX. This is a step preceding C-11 sulfonation to convert STX to GTX-2/3 (or neosaxitoxin to GTX-4/1).

(147) sxtN from Scytonema cf. crispum UCFS15 was cloned into an appropriate expression vector, and placed under the control of the IPTG-inducible T7 promoter, and provided with a hexa-histidine tag on its N- and C-terminus.

(148) sxtO from S. cf. crispum UCFS15 was cloned into an appropriate expression vector, and placed under the control of the IPTG-inducible T7 promoter, and provided with a hexa-histidine tag on its N- and C-terminus.

(149) The expression vectors were expressed in E. coli and the Sxt proteins were purified using No-NTA resin (Novagen). Purity of the proteins was assessed on SDS gels.

(150) The sulfotransferase activity of SxtN was tested using saxitoxin or GTX2. The results were determined using HPLC-MS/MS. Using saxitoxin as substrate, a peak at 2.13 min was identified that was not present in the control. This contained a major peak of m/z 380.10, which fragmented (LC-MS/MS) to m/z of 300.14 and 282.13, proposed to be GTX5 (shown in FIG. 19). In using GTX2 as a substrate, no difference in toxin presence was observed between the samples and the control.

(151) The dioxygenase activity of SxtDIOX was tested using saxitoxin. The results were determined using HPLC-MS/MS. The substrate was identified in both the assay and the control. Further, a peak of m/z 316.14>296.13 at 1.66 min that was not present in the control was identified in the assay, suggesting the presence of hydroxylated saxitoxin (FIG. 20).

(152) Sequences

(153) The accompanying Sequence Listing is fully incorporated herein as part of the description.

(154) TABLE-US-00011 SEQ ID NO: 91: Sequence of sxt1 fragment after integration into lactose operon. Flanking regions of xylose the operon at integration site are included. Open reading frames of XylS gene and sxt genes (A, B, C) are indicated by upper case letters. gtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcagtggagatgcccaagggcacttcg ggtcgaggaacccgacctgcattgggacgcggccacggagagcgcgggcaaacgccggcactatagccagtggag tttgtaaaacgctatttcagagcttggagagtgtctaagaaagccgggcgatgccaacccatcccttcttcggct aCGTTCGTAATCAAGCCACTTCCTTTTTGCATTGACGCAGGGTGTCGGAAGGCAACTCGCCGAACGCGCTCCTAT AGTTTTCAGCGAAGCGTCCCAAATGTAAGAAGCCGTAGTCTAGGGCTATCTCAGTTATACTACGCACATTGGCAC TGGGATCGTTCAAGCAGGCGCGGATGCTTTCGAGCTTGCGGTTGCGGATGTAGTTCTTCGGCGTGGTGCCGGCAT GCTTCTCGAACAAATTGTAGAGCGAGCGTGGACTCATCATCGCCAGCTCCGCTAACCGCTCAAGGCTGATATTCC GTTTGAGATTCTCCTCAATGAATTGAACGACTCGCTCGAAAGACGGGTTACCTTTGCTGAAAATTTCACGGCTGA CATTGCTGCCCAGCATTTCGAGCAGCTTGGAAGCGATGATCCCCGCATAGTGCTCTTGGACCCGAGGCATCGACT TTGTATGTTCCGCTTCGTCACAAACTAACCCGAGTAGATTGATAAAGCCATCGAGTTGCTGGAGATTGTGTCGCG CGGCGAAACGGATACCCTCCCTCGGCTTGTGCCAATTGTTGTCACTGCATGCCCGATCAAGGACCACTGAGGGCA ATTTAACGATAAATTTCTCGCAATCTTCTGAATAGGTCAGGTCGGCTTGGTCATCCGGATTGAGCAGCAATAGTT CGCCCGGCGCAAAATAGTGCTCCTGGCCATGGCCACGCCACAGGCAATGGCCTTTGAGTATTATTTGCAGATGAT AACAGGTCTCTAATCCAGGCGAGATTACCCTCACGCTACCGCCGTAGCTGATTCGACACAGGTCGAGGCATCCGA AGATTCTGTGGTGCAGCCTGCCTGCCGGGGGCCCGCCCTTGGGCAGGCGAATAGAGTGCGTACCGACATACTGGT TAACATAATCGGAGACTGCATAGGGCTCGGCGTGGACGAAGATCTGACTTTTCTCGTTCAATAAGCAAAAATCCA Tagttcacggttctcttattttaatgtgggctgcttggtgtgatgtagaaaggcgccaagtcgatgaaaatgcag gaattaattcgcagatcctggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcgg tctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaa acgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaa aggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatc cgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgcca ggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtagatcccagccttgcaagaagcggata caggagtgcaaaaaatggctatctctagaaaggcctaccccttaggctttatgcaacagaaacaataataatgga gtcatgaacATGCTGCAGAAAATCAATCGTTATACCCATGGTTTTGTTGCCGTTCCGGTTATTCTGGCATGTCGT GAAAAAGGTGTTTTTGAACTGCTGGCAGATGAAAGTCCGCTGAGCCTGAATCAGATGGTTGAACATCTGGGTGCC AATAGCGGTCATTTTCAGGTTGCACTGCGTATGCTGGAAAGTCTGCATTGGCTGAGCCGTAATAAAGAACTGAAA TATAGCCTGACCGCAGAAGCAGCAATTCATAACAAAATTAGCGAAGATATCCTGCAGCTGTATAATCTGCCGATT CAGAGCTATCTGGAAGGTAAACAGGGCAATCTGCTGGGTCGTTGGATTGAACGTAGCTGTCAGCTGTGGAATCTG GATAATCCGCTGATGGCAGATTTTCTGGATGGTCTGCTGGTTATTCCGCTGCTGCTGGCACTGCATAAACATAAC CTGCTGGCCGATTCTGAAGATAAACCGCTGCTGAGCAGCCTGAGCAGTACCGTTCAAGAAGAACTGGGTAAACTG TTTCTGCATCTGGGTTGGGCAGATCTGACAGCAGGTCGTCTGACCATTACCGAACTGGGTCGCTTTATGGGTGAA CGTGCACTGAATACCGCAATTGTTGCAAGCTATACCCCGATGCTGAGTCGTATTCATGATGTTCTGTTTGGTAAT TGCCTGAGCGTTTTTCAGCGTGATGCAAGCGGTCATGAACGTCATATTGATCGTACCCTGAATGTTATTGGTAGC GGTTTTCAGCACCAGAAATACTTTGCAGATCTGGAAGAAAGCATTCTGAGCGTGTTTAATCAGCTGCCGCTGGAA GAACAGCCGAAATACATTACCGATATGGGTTGTGGTGATGGCACCCTGCTGAAACGTGTTTGGGAAACCATTCAG TTTAAAAGCGCACGTGGTAAAGCACTGGAACAGTATCCGCTGCGTCTGATTGGTGTTGATTATAATGAAGCAAGC CTGAAAGCAACCACCCGTACCCTGGCAAGCCTGCCGCATCTGGTTCTGCAGGGTGATATTGGTAATCCGGAACAA ATGGTTCGTAGCCTGGAAGCACATGGCATTCATGATCCGGAAAATATTCTGCATATTCGCAGCTTTCTGGATCAC GATCGTCTGTTTATTCCGCCTCAGAAACGTAATGAACTGAAAGAACGTGCCCATCTGCCGTATCAGAGTGTTTGT GTTGATGATCAGGGTGAACTGATTCCTCCGCATGTTATGGTTCAGAGCCTGGTGGAACACCTGGAACGTTGGAGC CAGGTTGTTAATAAACATGGTCTGATGATTCTGGAAGTGCATTGTCTGGAACCGCGTGTTGTTTATCAGTTTCTG GATAAAAGCGAAAACCTGCACTTTGATGCATTTCAGGGTTTTAGCCAGCAGTATCTGGTTGAAGCCGAAGTTTTT CTGATGAGCGCAGCACAGGTTGGTCTGTTTCCGAAACTGGAACTGAGCAAACGTTATCCGAAAACCTTTCCGTTT ACCCGTATTACCCTGAACTATTTCGAAAAACGTCCGTACAAAATCAGCCATGCATATCTGAGCGATCTGCCTGCA CTGGTTGACCTGGAAGTTAAATGTTGGCCTGAGAATCTGCGTGCAAGCACCCATGAAATTCGTCGTCGTCTGGAA CTGAATCCGCAGGGTAACCTGGTTCTGATTATTGAAGATCAGATTATCGGTGCCATTTACAGCCAGACCATTACA AGCACCGAAGCCCTGGAAAATGTTAAATATGCACAGGTTCCGACCCTGCATACACCGCAGGGTTCAGTGATTCAG CTGCTGGCCCTGAACATTCTGCCGGAATTTCAGGCACGTGGTCTGGGCAATGAACTGCGTGATTTTATGCTGTAT TATTGCACCCTGAAAGGTGGTATTGAAAGCGTTGTTGGTGTTACCCGTTGTCGCAATTATGTGAATTATAGCCAG ATGCCGATGATGGAATATCTGAAACTGCATAATGAACAGCGTCAACTGCTGGATCCGATTGTTGGTTTTCATGTT AGCGGTGGTGCAGAAATTCGTGGCATTATTGCAAATTATCGTCCGGAAGATACAGATAATCTGGGTATGGGTATT CTGATCGAATATAACCTGCGTGATAGCGCACTGCATTCACCGGGTGATCGTAAAGGTCCGTATATCAATAGCGCA ATTGGTAGCCTGGTTCCGAAAGCGACCAGCGCAACCAAAGAAAACAAAACCGTTGCGGATCTGGTGAAAGAATGT ATTCTGAAAGTGATGGGTAGCCAGCGTCAGGCAGCATATGCACCGCAGCAGAAACTGCTGGACATGGGTCTGGAT AGCCTGGATCTGCTGGAACTGCAGACCCTGCTGGAAGAACGTCTGGGTATTAATCTGAGCGGCACCTTTTTTCTG CAAAAAAACACCCCGACCGCCATCATTACCTATTTTCAGAATCAGGTCGTGCAAGAGAAACAGAGTGATCTGGCA CCGCCTGTTGATAGCGCCAATGAAATCAATACACTGGAAAACGTTGTGAATCAGCAGAAAATTCCGCAGGTTACA CGTGTTGTTACCGAACAGCAGGGACGTAAAGTTCTGATTGATGGTCATTGGGTTATTGATTTTGCCAGCTGTAAT TATCTGGGCCTGGACCTGCATCCGAAAGTTAAAGAAGCAATTCCTCCGGCACTGGATAAATGGGGCACCCATCCG AGCTGGACCCGTCTGGTTGCAAGTCCGGCAATTTATGAGGAACTGGAAGAGGAACTGTCAAAACTGCTGGGTGTG CCGGATGTTCTGGTTTTTCCGGCAGTTACACTGCTGCAGATTGGTATTCTGCCTCTGCTGACCGGTAATAATGGT GTGATTTTTGGCGATATTGCAGCCCATCGTTGTATTTATGAAGCATGTTGTCTGGCCCAGCATAAAGGTGCACAG TTTATTCAGTATCGTCATAACGACCTGAATGATCTGGCCGAAAAACTGGCCAAATATCCGCCTGAACAGGTTAAA ATCATTGTGATCGATGGTGTGTATAGCATGAGTGCCGATTTCCCGGACCTGCCTGCATATGTTCATCTGGCAAAA GAATATAACGCCCTGATCTATATGGATGATGCACATGGCTTTGGCATTCTGGGTGAAAATCCGAGCAGCGATATG CCGTATGGTTATAAAGGTAATGGCATGGTGAACTACTTTGATCTGCGTTTTGCCGAAGATAACATCATTTATGTT GCAGGTCTGAGCAAAGCCTATAGCAGCTATGCAGCATTTCTGACCTGTGGTGATCGTCGTATTAAAACCAATTTT CGTAATGCATGGACCGCGATTTTTAGCGGTCCGAGTCCGGTTGCAAGCCTGGCCAGCGCACTGGCAGGTCTGCAG GTTAATCGTCAAGAAGGTGAACAGCTGCGCAAACAAATCTATCATCTGACACATAAACTGGTTACCCAGGCTCGT GCCATTGGTTTTGAAGTTGATAATTATGGTTATGTGCCGATTGTGGGTGTTCTGGTGGGTGATGCACAGCATATG ATTGATGTGTGCCAACTGCTGTGGGAATATGGTATCCTGATTACCCCTGCAATTTTTCCGATTGTGCCGCTGAAT AAATCAGCACTGCGTTTTAGCATTACCGCAGCAAATACCGAAGAAGAAATTGATCAGGCCATCAAAAGTCTGAAA GCAGTTTGGGACCTGCTGCAAAAACGTAAAGCCCTGCCGTGTAAACAAGAAGAAAATATCCTGAAACATTGAgaa ggagatatacatATGACCCATGTTGCCCTGGAACAGGCAATTGCAAAAGTTCCGCGTAGCATTCAGAGCGAACTG CGTACCATTCTGGCACAGCATGCAGTTATTGATAGCAGCGTTGTGGCAAGCTGGATTGATCGTCTGGGCACCAAT ATTAGTACCCTGATGATCCAGCTGCTGCCGGTTGCAGCAACCTATGCACGTGTTCCGATTAGCCAGTTTTATGTT GGTGCCATTGCACTGGGCAAACCGCAGAGTAAAAATCAGCTGGGTAGCGGCACCCTGTATTTTGGTGCAGATATG GAATTTGTTGGTCAGGCACTGAGCTTTAGCGTTCATGCAGAACAGAGCGCCACCATTAATGCCTGGCTGCATGGC GAAACCGGACTGCAGGCACTGGCAATCCATGAAGCACCGTGTGGTTATTGTCGCCAGTTTCTGTATGAAATGGCA ACCGTGAATCAGAATTTTGTGCTGCTGGTGAAAAGCAATGAAAGCCAGCCGGAACAGACCTATACCAGCAACAAA CTGCCGCATTTTCTGCCTGAACCGTTTGGTCCAGCCGATCTGGGTCTGACCGGTGGCCTGATGCAGACCGTGTTT CACGATCTGGAAACCTATAGCACCGATGATGTTGTTCTGGCAGCACTGAGTGCAGCAAATCAGAGTTATGCACCG TATACCAAAAACTTTGCCGGTGTTGCACTGAAAGATAGTCATGGTAACATTTTTACAGGTCGCTATGCCGAAAAC GCAGCATTTAATAGCAGCATGAGCCCGATGGAAAGCGCACTGACCTTTATGAATATGAATCGTTATTCACAGAGC CTGTTCGATATTTGTGATGCAGTTCTGGTAGAAGTGGAAACCGGTATTAGTCAGCGTCCGGTTACCGAAGCCTTT CTGAGTAGCATTGCACCGAAAGTGAAACTGCGCTATGCACCGGCAACCCCGAGCAGTAACAAACTGTGAgaagga gatatacatATGTTTCAGACCAAAAGCTATTATAGCGTCGTTGGCCTGGAAACCGAACTGATTAAAGGTAAATTC TTCATGAGCAACGAACTGACCAATGAACAGGTGTTTAAACTGGTGTGCATGGAAGTGATTGAAAAAATGGGTTTT GCACACTTTCCGCCTATTATCCTGGTTTATGAAATGACCAATTCCGGCTTTGTTGATTGGTGCGAGCAGATGGTT TTTGTGGATGATAAAGGCAAACTGGATGAGGGCGAAAAATTTCTGCTGGATTGGATGCGTCGTAATGTGGGTAAT TTTGATCTGATTCGCGAACTGATGCCGGTGGCAGAACGCCTGGAAATGAAAATGCGTAGCTAActcggtaccaaa ttccagaaaagaggcctcccgaaaggggggccttttttcgttttggtcccgaagttcctattctctagaaagtat aggaacttcgaccgcctgtctatttctcttacggttccaacatccatataggccgcaatt SEQ ID NO: 92: Sequence of sxt3 fragment version 1 integrated into the xylose operon, which contains all genes. Flanking regions of xylose the operon at integration site are included. sxt open reading frames (Q, R, orf24, S, T, U, V, W, X) are indicated by upper case letters. ttgcatttccttgagccttatccgacttgtcagtcggataaggctttttactttgtctcaggcagttgagctac gagcctgaagcgttgttggtgcgttttatcatgcctggcgggtaggtcggataaggcgttcacgccgcatccga caaccacgcagcgttacctgatgtgacgccgacaattctcatcatcgctacaacatgacctcgctatttacatc gcgatactcttttggcgtcgtgtcatatgcttttttaaaaacagagtagaaatattgcagcgatggataaccgc acatttgcgatatctcattgatcgacaaggtggttgaaatcagcagactgcgcgctttctccagcttctcggca tgaatcatggcatggatggtttcacccacctcttctttaaaacgcttctcaagattggagcgcgagatcccgac cgcatccagtacctgatccactttaatccctttacaggcgtgattacgaatgtaatgcatggcctgaataacgg cgggatcggtcagcgagcgataatctgttgagcgccgttcaatgacgcgaactggtgggaccaaaattcgctgt agcggcatttcttctttatctaataatcgatgcaacagttttgccgcctgatagcccatttgccgcgcgccctg agcgaccgaagaaagggcgacacgcgacagatagcgggtcagttcttcgttatcgatgccaatcacgcataatt tttccggtacgggaatatgtagatgttcacatacttgcagaatatgccgcgctcgggcgtcagtaacggcaata atcccggtttgcggtggtagcgtttgtagccagtctgccagccgattttgcgcgtgttgccagttctctggcgc ggtttctaacccctgataaaccactccgcgatacttttcttcggcgacaagctgacgaaatgcatattcgcgct cagtggcccaacgtttgccgcttgattccggaagaccataaaaagcaaagcggttaacgcctttctcttttaaa tgcaaaaatgcgctttcaaccagcgcatagttatcggtggcaatgtaatgaacgggtgggtaactttctgcaag gtgatacgagccgccaaccccaacaatggggacgtcgacatcagccagcgcttgctcgatctgtttgtcgtcga agtcggcaatgacgccatctcctaaccagtccttgattttatcaatgcgggcgcggaaatcttcttcaatgaaa atatcccattccgattgtgacgcctgtaaatattcccctacgccttctactacctgccggtcataggctttatt ggcattgaacagtaatgtgatgcggtgacgtttagtaaacatggttcttttcctgctgaatcatgcaaaaactc aaaaccggtaatacgtaaccggctttgagaaaatttttatcaaaatcaagaacggcgtttggttgcggagtcca tccatactgccagcaacagaatcgcacctttaacgatatactgccagaaggtcggtacatccatcatactcatg ccgttatccagtgaagccatgataaatgcccccattactgctccggcaacgcttcccacaccgccagccaggct ggtgccgccaatcacgcatgctgcaattgcgtccagttcggcgatatttcccgcagaaggtgaaccagcgccaa gtcgagaactaaggattaatccggcgatggctaccattaatccgttaatcgcgaacacggcaagtttggtgcgt tcaacgttaatcccggagagacgtgctgcttccagattgccgccgatggcataaatgcgtcgtccaaatgccgt ccgcgtttccataaacattccgccgagtaacagcaacgtcagcagcagaacaggagtgggaacgccacggtaat cattcaacagccagattgcgcctaatacgatgatagcggttaaagcctggcgaccgactactgcggtagaggcc ggagactgcaaacccaaagcctgacggcgcattcttccgcgccattgccaaccaacaaaagccattaagccaag cgcgccaatgatgaagccagtgctggcaggtagatagctttgcccaatttgtgacatcgcggcgctggtggggg aaacagtcgtgccgttggtgatgccaatgagtatgccgcgaaatgccaacatgcccgcgagggtgacaataaat gaagggactttgcggtacgcgacccaccatccgttccaggcaccgagaagcagtcccagaaccaacgtcacaat gatggtaagtggcaaaggccagcctaaccagacgtcacaaatcgccgcgacgccacctaatagccccatcattg agccgacggaaaggtcgatttcagcagaaattatgacgaacaccattcctaccgcgaggatgccggtaatcgcg gtctggcgtaacaggttggagacgttacgggcgcttaagtaggcaccatcggtggtccaggtaaagaacagcat gattgcgatgatagctgcaatcatcacgaagacctgcaaattcagtgatttcagcccggagaagctaccggatg tcggtacggccaatttcacttcagacggattgcttttcgacatgatgttcgctcctcaatgcggcttccatcac ctgctcctgagtcaggttatgatttatcaggttggcttttagtttcccttcatgcatcaccagtacacgatcgc taaggccgagcacttcaggtaattcggaagagatgacaataacggcaataccctgctggacgagttggttaatt aatttgtagatctcgtatttcgcgccaatatcgatacccctggtgggttcatcaagaatgagaatgcgcgggtt aagtaacagacagcgagcgaggatcgctttttgctgattgccgccgctcaaacgtccaatagcaaggtcggggg acgacgttttaactttgagttgctggattgattccagaatacatttttgctctgccgcgtcatcaagctggcta atgccaccggtaaatttattgagtcggcgagggtaatatttttaccaaccgccattaccggaacgatgccgtcg cgctttctgtcttcgggtaccatcgcaatcccctgggcgatggcttgctgacagttacgaatatctacctgttt gccatcaatataaatttttccttcccattgtccgggccacacgccaaacaggcactgaatggtctcggtacgtc cggcaccaacgagtccggcaatacccagtatttcgccacgtttcagggaaaacgagacatcattaactcgttta atatgacgattgaccggatgccatgccgtcagatgttcaatacgtaatatttcatctccggtggtatgtggttc attagggtaaagcgcggttaactctcgcccgaccatcatggtgataatatcgtcttcactcattccggcagcat cacgcgtaccaatgtgctgtccgtcgcgaataacgcaaatcgtatcggaaatcgctttgacttcgttgagtttg tgcgaaatataaatacaggcgataccgtgctgttgtagatcgcgaataatatccagtaaaaccgacgtttcctg ctcagttaatgaggctgtcggttcatcgagaattaacaagcgcacctgtttattaagtgccttggcaatttcaa ccagttgttgttgcccaagccctaaatcgccaacgcgggtatcaggtgaaatggataaactgacctgtgcgagc agcttctgacagcgtagcgtcatcaggtcataatccataatgccattgtgggttatttcgttacccaggaagat attttccagcacggtcaattctttcaccagggccaattcctgatgaatgatggcgatacctttgcgttcggtat cgcggatgtgactcgcctgaatctcttctcccgcaaaaataatttcgccttcgtaggagccatggggataaata ccacacagcactttcatcagcgttgatttaccagacccattttccccacaaagtgagacgatttcgccagcatt caaccgcaagcagacgttatcaatcgccttcacactgccgaaggttttggtaatgttcttcatttcaagtagat aaggcataacgactccacctaagccaattcattcacgcggcatggagagaaatcacgcccccgctccgcgccgg gcgtaacgcttacagctcgctctctttgtggaatccgtctttaattaccgtatctttgatgttgtttttattca catcgatcggtgtcaggaggcgggaggggacatctttcaggccattattcagtgaggtccagccttgcaagaag cggatacaggagtgcaaaaaatggctatctctagaaaggcctaccccttaggctttatgcaacagaaacaataa taatggagtcatgaacatATGGTGATTAAAAACCTGTGTCCGGATGGTGTTACCCCGATTTGGAATAAAAGCCA GATGGAAAGCAGCCTGCTGGAAGAATGTCTGCCTGCATGGGTTCGTACCAGCTATAGCACCTTTGTTGAAACCA TTAGCGATAGCGCATTTCCGTGTTTTTGGGGCACCATTGGTGAACAGAAAGGTATGATTCGTTATCTGATTGTT AGCAGCCTGACCGATCCGATTCTGGTTGAACATACCCTGGAAGGTATCTACAAATATATCGATGAAGTGAACGA AAACGAACTGCTGCAGCATGAAAATGCAGATCTGCTGACCCTGGTTATCTTTTTTCCGCCTGAACCGACCGTTC TGACCGTTGAAGAATATGCAGGTCAGGCATTTGATTTTCTGAATGCACTGCATAGCCTGGATGCAGTTAGCTGT CCGTGTCATTGGAGCGCAGATCCGCAGAGCGCAAATTGGAGCTATAGCCTGGGTGGTTGTGCACTGTTTGTTAG CGTTAGCACACCGGCAAATCAGAAACGTCGTAGCCGTCATCTGGGTAGCGGTATGACCTTTGTTATTACACCGG TTGAAGTGCTGCTGAATAAACATGGTGGTGAAAACAGCAGCATTTTTCGTCGTGTTCGTGAATATGATGGTATT CCGCCTCATCCGAATCTGCTGATTATGCCTGGTAATGGTAAAGTGGGTAATGAACTGACCGTGCAGGTTCTGCC GGATAATAATGATAGCGAAATCAGCTTCGATTTTCAGTATAAATTCAAAGATTGagaaggagatatacatATGA CCATCCAGATTGTGCAGCATAACCTGGAATATAGCTTTGTGACCCCGAAAGAAACCAGCGATTTTGTTGAACGT ACCATGAGCGTTTTTGATCAGGCATATCCGAAATTTCTGATCCATGATGTTTGGGCAGATCCGGCAAGCCTGGC CCTGTTTGAAATTTATCCGGAATTTCAGTTTGGTCTGGTGGAAGCAACCACCCAGCTGATGATTGCACAGGGTA ATTGTATTCCGCTGACCTATGAAAGCCGTTTTGATGAACTGCCGGATGAAGGTTGTGATTGGGCACTGGCAAAA TGGCTGGAAGATCGCGAACAGAATCGTCTGCCGAATGCCCTGTGTGTTGTGAGCATTAGCATCCTGCCGGAATA TCAGGGTAAAAATCTGAGCCAGTATCTGATCGGCTATATGAAAGAACTGGCACAGTATCATGGTCTGAATAGCC TGATTATGGCAGCACGTCCGAGCCTGAAATATCTGTATCCGCTGATTCCGATTGAACGCTATATTACCTGGCGT GATAAAAACGGCCTGATTTTTGATCCGTGGCTGCGTGTTAATGTTAAACATGGCGCAAAAATTGCCGGTATCTG CTTTAAAAGCACCACCATTAATGATACCATTGATGGTTGGGAGGATCGTGTTGGTATGCGTTTTCCGGAAACCG GTGATTATATCATTCCGAAAGGTCTGGTTCCGGTGAAAATTGATTATCCGAATAACATGGGCATCTACATCGAA CCGAATATCTGGCTGTATTATGATCTGGACTGAgaaggagatatacatATGATCAACATCGAACAGTTTCGCCA AGAAATCGAAGATTGGATTATTAACGTTGTCAGCATTCCGAACCCGCTGACCGGTAATTTTCCTCCGTGTCCGT ATGCAAAAGCAGCATGGCTGAATAATCGTGTTAGCGTGCGTTGGTTTCATGGTCCGGAACTGCCTGAACTGCTG ATGGAACAAATTCGTACATGGAACAACGATTTCGAGATGGTGATTTTTGGTTGCGATCCTCAGAATCTGGATGC ACAGCGTCTGGAACGTTATATCACCAAAGCAAATTATGTGCTGCCCGAATATGACCTGGTTGCACTGGGTAGCC ATCCGGATAAACAGTATGTTGGTGATGATGCCGAAAATGTGAACAACGTGATTATTACCCATCCGAAATATGTT CTGGCAAGCGTTCAGAGCTTTAGCCAGCTGCAAGAGGCAAGTGATGAGCTGCTGCGTCTGGGTTATTTCCAGTA TTGGTCAGCAGAAAAACTGGCCGAAATGAAAAGCGAACGTGCAAGCCATAATCTGAGCAGCATTCAGCGTAAAA ATAGCTATCGTATTATCCCGACCAACCATTGAgaaggagatatacatATGCTGACCGCAGAACAGAAACAGGCA TATACCAATGATGGCTATTTTACCGTGGAAGAAGCAGTTCCGAAAGCACTGATTGAAGAAATTCGCCATGAAGT GGAACTGATCACCGAGCAGAAACGTGGTGGTGTGCTGGCAGGCGATTATGAATGGTGGTCAGAACACACCATTC CGGATCCGGTTCGTTATCAGAAAATTATCCAGCGTCTGCTGGAACTGCCGACCGTTATGGGTCCGGTTCAGGCC CTGATTGGTAGCGATATTTTTCTGTTAATTACCGACCTGGCAATTATTCGTGCAGGCACCGGTTATATTGCATG GCATCAGGATCATGGCTATGTTGTTGAAGTTCTGAACGCCCTGGCAAGCATGAGCAAAAATGAGCTGAATGATG ATGCACTGCGCCTGCTGGTGCCGGTTGCAAATCAGGCAATGGTGTTTATTACCATCTATCTGCAGGATACCGAT AACACCATGGGCACCATGCGTGTGATTCCGAGCAGCCATCAGTGGGAACATAGTCTGGATAGCAGCAGCGCCAA TTCACTGAATGCAGAAATTTGTCTGAGCCTGCCTGGTGGTGCAGCAATGTTTTATACCCCGACCGTTTGGCATA CCGCAGCAGCAAATACCAGCATTACCGATTATCGTATGCTGACGCTGATCTTCACCAAAAACAACATTAAACCG CTGCTGGTTGATGCCCTGAAACGTATTATTTGAgaaggagatatacatATGACAACCACCGATCCGATCCTGAT TAATAACTGGCATGTTGTGGCAAATGTCGAGGATTGTAAACCGGGTAGCATTACCCGTAGCCGTTTACTGGGTG TTAAACTGGTTCTGTGGCGTAGCTATGAACAGAATAGCCCGATTCAGGTTTGGCTGGATTATTGTCCGCATCGT GGTGTTCCGCTGAGCATGGGTGAAATTACCAATAATACCCTGGTTTGTCCGTATCATGGCTGGCGTTATAATGA AGCAGGTAAATGTATTCAGATTCCGGCACATCCGGGTATGGTTCCGCCTGCAAGCGCAGAAGCACGTACCTATC ATAGCCAAGAACGTTATGGTCTGGTTTGGGTTTGTCTGGGTGATCCGGTTAATGATATTCCGTCATTTCCGGAA TGGGATGATCCGAATTATCACAAAACCTACACCAAAAGCTATCTGATTAAAGCAAGCGCCTTTCGCGTTATGGA TAATTCACTGGATGTTAGCCATTTTCCGTTTATTCATGATGGCTGGCTGGGCGATCGTAACTATACCAAAGTGG AAGAATTTGAAGTGAAACTGGATAAAGATGGTCTGACGATGGGCAAATATCAGTTTCAGACCAGCCGTATTGTG AGCCATATTGAAGATGATAGCTGGGTGAATTGGTTTCGTCTGAGCCATCCGCTGTGTCAGTATTGTGTTAGCGA AAGTCCGGAAATGCGTATTGTTGATCTGATGACCATTACGCCGATTGATGAAGAAAATAGCGTTCTGCGCATGC TGATCATGTGGAATGGTTATGAAACCCTGGAAAGCAAAATGCTGACAGAGTATGATGAAACGATCGAACAGGAT ATTCGTATTCTGCATGCCCAGCAGCCGGTGCGTCTGCCGCTGCTGACACCGAAGCAGATTAATACCCAGCTGTT TAGCCATGAAATTCATGTTCCGAGCGATCGTTGTACCCTGGCATATCGTCGTTGGCTGAAACAACTGGGTGTGA CCTATGGTGTTTGTTGAgaaggagatatacatATGGCAGGTAAACTGGATGGTAAGGTTGCAATTATTACCGGT GCAAGCAGCGGTATTGGTGAAGCCACCGCATTTGCACTGGCAGCAGAAGGTGCAAAAGTTGCAATTGCAGCCCG TCGTGCAGAACTGTTACATGCACTGGCCAAACGTATTGAAGCAAGCGGTGGTCAGGCACTGCCGATTGTTACCG ATATCACCGATGAAAGCCAGGTTAATCATCTGGTTCAGAAAACCAAAGTTGAACTGGGTCATGTTGATATCCTG GTGAATAATGCAGGTATTGGCGTTTTTGGTGCAATCGATACCGGTAATCCGGCAGATTGGCGTCGTGCATTTGA TGTTAATGTGCTGGGTGTTCTGTATGCAATTCATGCAGTTCTGCCTTTACTGAAAGCACAGAAAAGCGGTCATA TTGTGAATATTAGCAGCGTGGATGGTCGTATTGCACAGAGCGGTGCAGTTGTTTATAGCGCAGCAAAAAGCGGT GTTAATGCCCTGAGCGAAGCACTGCGTCAAGAAGTGAGCCTGGATAATATTCGTGTGACCATTATTGAACCGGG TCTGGTAGATACCCCGTTTAATGATCTGATTAGTGATCCGATTACCAAACAGCTGAGCAAAGAACAGCTGTCAA CCATTACTCCGCTGCAGAGCGAAGATATTGCACGTGCCATTATCTATGCAGTTACCCAGCCGGATCATGTTAAC GTTAATGAAATTCTGATTCGTCCGACCGCAGAGGATAATTGAgaaggagatatacatATGAACCTGACCCTGAA CAAAGAAGAAAAACAGCTGCTGACGGCATATAGCGGCACCGAACTGCAGCTGACAGCAGATGTTCTGGTTATTG GTGGTGGTCCGGCAGCCGCATGGGCAGCTTGGGCAGCAGGCGCACAGGGTGTGAAAGTTATTATTGTGGATAAA GGTTTTCTGGGCACCAGCGGTGCCGCAGCCGCAAGCGGTAATAGCGTTATGGCACCGTCACCGGAAAATTGGGA AAAAGATGTGAGCGAATGTTACAGCAAAGGTAATAATCTGGCAAATCTGCGTTGGATTGAACGTGTTATTGAAA AAGCCTGGCTGTCACTGCCGCTGGTTGAAGATTGGGGTTATCGTTTTCCTAAAGAAAATGGTGAAAGCGTGCGT CAGAGCTATTATGGTCCTGAATATATGCGTGTTCTGCGGAAAAATCTGCTGCGCGTTGGTGTTCAGATCTTTGA TCAGTCACCGGCACTGGAATTACTGCTGGCACAGGATGGTAGCGTTGCCGGTGCACGTGGTGTGCAGCGTCAGA ATCATCGTACATATACCGTTCGTGCCGGTGCCGTTGTTCTGGCCAATGGTGGTTGCGCATTTCTGAGTAAAGCA CTGGGTTGTAATACCAATACCGGTGATGGTCTGTTAATGGCAGTTGAAGCCGGTGGTGAACTGAGCAGTATGGA AGCCAGCAGCCATTATACCATTAGCACCGCCTTTAATGCAACCGTTACCCGTGCAGCTCCGTTTTATTGGGCAA GCTATACCGATGAAGCTGGCAATGATCTGGGTGGCTATATTAACGGTCGTCGTGATCCGAGCTTTCTGCCGAAC GCACTGCTGAAAGGTCCGGTTTATGCACGTCTGGATCGTGCAACACCGGAAATTCAGGCGCTGGTAGAAAAAAG CCATTTTATTGCATTTCTGCCGTACAAGAAAGCCGGTATTGATCCGTATACCGAACGTGTTCCGGTTACCCTGG TGCTGGAAGGCACCGTGCGTGGCACCGGTGGTATTCGCATTGTTAATGATTCATGTGGCACCAAAGTTCCGGGA CTGTATGCAGCGGGTGATGCAGCAAGCCGTGAATTTCTGGCAGGCATTGCCAGCGGTGGTGATGGACCGAATGC AGCATGGGCAATTTCAACCGGTCAGTGGGCAGGCGAAGGTGCAGCAGCCTTTGCAAAAAGTCTGGGTGCACATG TTCATGAACGCGTTGTTCGTCCGGCAGGCCAGGCAGGTCTGCGTAGTCAGTATCCGGGTAGCGAAACCTTTGAT AGTGAAGCAGTTGTTCGTGGCGTTCAGGCAGAAATGTTTCCGCTGGAAAAAAACTATCTGCGCTGTGAACAGGG ACTGCTGGATAGCCTGGCAAAACTGGAAATGCTGTGGCAGCAGGTTCAGGGTAATCCGAAACAGGATACAGTTC GTGATCTGGAATTTTCACGTCGTGCGGCAGCACTGGTTAGCGTGGCACGTTGGGCATATTTTAGCGCACTGCAT CGTAAAGAAACCCGTAGCGAACATATCCGTATTGATTACCCGGAAACGGATCCGAATCAACTGTATTATCAGGC AACCGGTGGCCTGGAACGTCTGTGGGTGCGTCGTGATTGGGTTAAAGATGCAAGCGCCACCCCTCCGGTGCTGA CCACCTGAgaaggagatatacatATGATTGAACTGGTGAGCCATAAGCTGTGCATTAATTGTAATGTTTGTGTT CAGGTGTGCCCGACCAATGTTTTTGATGCAGTGCCGAATCAGCCTCCGGCAATTGCACGCCAAGAAGATTGTCA GACCTGTTTTATTTGTGAAGCATATTGTCCTGCAGATGCGCTGTATGTTGCACCGCAGAGCCATACCAATGTTG CAGTTAACGAAGATGATTTAATCGACAGCGGCATTATGGGTGAATATCGTCGCATTCTGGGTTGGGGCTATGGT CGTAAAAACAATAGCGAACTGGATACCGACCATAAACTGCGTCTGTTTGAATGAgaaggagatatacatATGTC ATTTCAGAAATTTGTGCAAGAAGCAGCCTATAAAGTCGCACCGTTTAAACCGAATCGTTTTGCCAAAATTAGCG AGCGTGAAGATAAATGTGCAATTCCGGTTCCGGCATGGCGTGCACTGCTGGCCAATCGTGACCTGTTTACCTGG AAAGGTATTCCGTTTCTGAAAGGTTGTACCGAAATTGCACTGTATAGCATGCTGCTGTATGAACTGCGTCCGAA AACGATTATTGAAATTGGTGCGCTGAGCGGTGGTAGCGCAATTTGGCTGGCAGATCATCTGGAACTGTTTCAGA TTGAAGGTTGCGTGTATTGCATTGATATTGATCTGTCTCTGCTGGACGAAAAAGCAAAAACCGATAGCCGTGTT CATTTTCTGGAAGGTGATTGCAATAATATGGGTGCAATTATGTCAAGCGAGCTGCTGAGTGGTCTGGCACATCC TTGGCTGATTGTTGAAGATGCACATGCAAATGCCGTTGGTGTGGTTGAATATTTTCACGAAAACGGTCTGAAAA GTGGCGATTACCTGATCGTGGAAGATACCAATAAAACAATGTGGGAACTGGATCGCGAAGAACTGGACCGTGAT GACCTGGATGAACAAGAACTGATCGAAAAAGGTGAGCAGAAATTAGCAGAACTGAAAAGCTGGCTGATGCTGCA TGAGAATGAATATCTGATAGATACCTACTATCAGGATATGTATGGCTATAATGGTAGCCGTAATTGGAACAGCA TTCTGAAACGTGTGGAAAAGAACTTTTAAtctaactaaaaacaccctaacgggtgttttttcttttctggtctc cccgaagttcctattctctagaaagtataggaacttcgctggattgggccgcgaaattaaccggcctgagcaat gtcccagctttaatcgctgcagctcaacaggctgatgaaagtgccgagccagtttggtttctgccttatctttc cggcgagcgtacgccacacaataatccccaggcgaagggggttttctttggtttgactcatcaacatggcccca atgaactggcgcgagcagtgctggaaggcgtgggttatgcgctggcagatggcatggatgtcgtgcatgcctgc ggtattaaaccgcaaagtgttacgttgattgggggcggggcgcgtagtgagtactggcgtcagatgctggcgga tatcagcggtcagcagctcgattaccgtacgggaggggatgtggggccagcactgggcgcagcaaggctggcgc agatcgcggcgaatccagagaaatcgctcattgaattgttgccgcaactaccgttagaacagtcgcatctacca gatgcgcagcgttatgccgcttatcagccacgacgagaaacgttccgtcgcctctatcagcaacttctgccatt aatggcgtaaacgttatcccctgcctgaccgggtgggggataattcacatctatatatctcagtaattaattaa tatttagtatgaatttattctgaaaatcatttgttaatggcatttttcagttttgtctttcgttggttactcgt aatgtatcgctggtagatatggagatcgtt SEQ ID NO: 93: Sequence of sxt3 fragment version 2 integrated into the xylose operon, which has orf24 deleted. Flanking regions of the xylose operon at integration site are included. sxt open reading frames (Q, R, S, T, U, V, W, X are indicated by upper case letters. ggtgtcaggaggcgggaggggacatctttcaggccattattcagtgaggtccagccttgcaagaagcggataca ggagtgcaaaaaatggctatctctagaaaggcctaccccttaggctttatgcaacagaaacaataataatggag tcatgaacatATGGTGATTAAAAACCTGTGTCCGGATGGTGTTACCCCGATTTGGAATAAAAGCCAGATGGAAA GCAGCCTGCTGGAAGAATGTCTGCCTGCATGGGTTCGTACCAGCTATAGCACCTTTGTTGAAACCATTAGCGAT AGCGCATTTCCGTGTTTTTGGGGCACCATTGGTGAACAGAAAGGTATGATTCGTTATCTGATTGTTAGCAGCCT GACCGATCCGATTCTGGTTGAACATACCCTGGAAGGTATCTACAAATATATCGATGAAGTGAACGAAAACGAAC TGCTGCAGCATGAAAATGCAGATCTGCTGACCCTGGTTATCTTTTTTCCGCCTGAACCGACCGTTCTGACCGTT GAAGAATATGCAGGTCAGGCATTTGATTTTCTGAATGCACTGCATAGCCTGGATGCAGTTAGCTGTCCGTGTCA TTGGAGCGCAGATCCGCAGAGCGCAAATTGGAGCTATAGCCTGGGTGGTTGTGCACTGTTTGTTAGCGTTAGCA CACCGGCAAATCAGAAACGTCGTAGCCGTCATCTGGGTAGCGGTATGACCTTTGTTATTACACCGGTTGAAGTG CTGCTGAATAAACATGGTGGTGAAAACAGCAGCATTTTTCGTCGTGTTCGTGAATATGATGGTATTCCGCCTCA TCCGAATCTGCTGATTATGCCTGGTAATGGTAAAGTGGGTAATGAACTGACCGTGCAGGTTCTGCCGGATAATA ATGATAGCGAAATCAGCTTCGATTTTCAGTATAAATTCAAAGATTGagaaggagatatacatATGACCATCCAG ATTGTGCAGCATAACCTGGAATATAGCTTTGTGACCCCGAAAGAAACCAGCGATTTTGTTGAACGTACCATGAG CGTTTTTGATCAGGCATATCCGAAATTTCTGATCCATGATGTTTGGGCAGATCCGGCAAGCCTGGCCCTGTTTG AAATTTATCCGGAATTTCAGTTTGGTCTGGTGGAAGCAACCACCCAGCTGATGATTGCACAGGGTAATTGTATT CCGCTGACCTATGAAAGCCGTTTTGATGAACTGCCGGATGAAGGTTGTGATTGGGCACTGGCAAAATGGCTGGA AGATCGCGAACAGAATCGTCTGCCGAATGCCCTGTGTGTTGTGAGCATTAGCATCCTGCCGGAATATCAGGGTA AAAATCTGAGCCAGTATCTGATCGGCTATATGAAAGAACTGGCACAGTATCATGGTCTGAATAGCCTGATTATG GCAGCACGTCCGAGCCTGAAATATCTGTATCCGCTGATTCCGATTGAACGCTATATTACCTGGCGTGATAAAAA CGGCCTGATTTTTGATCCGTGGCTGCGTGTTAATGTTAAACATGGCGCAAAAATTGCCGGTATCTGCTTTAAAA GCACCACCATTAATGATACCATTGATGGTTGGGAGGATCGTGTTGGTATGCGTTTTCCGGAAACCGGTGATTAT ATCATTCCGAAAGGTCTGGTTCCGGTGAAAATTGATTATCCGAATAACATGGGCATCTACATCGAACCGAATAT CTGGCTGTATTATGATCTGGACTGAgaaggagatatacatATGCTGACCGCAGAACAGAAACAGGCATATACCA ATGATGGCTATTTTACCGTGGAAGAAGCAGTTCCGAAAGCACTGATTGAAGAAATTCGCCATGAAGTGGAACTG ATCACCGAGCAGAAACGTGGTGGTGTGCTGGCAGGCGATTATGAATGGTGGTCAGAACACACCATTCCGGATCC GGTTCGTTATCAGAAAATTATCCAGCGTCTGCTGGAACTGCCGACCGTTATGGGTCCGGTTCAGGCCCTGATTG GTAGCGATATTTTTCTGTTAATTACCGACCTGGCAATTATTCGTGCAGGCACCGGTTATATTGCATGGCATCAG GATCATGGCTATGTTGTTGAAGTTCTGAACGCCCTGGCAAGCATGAGCAAAAATGAGCTGAATGATGATGCACT GCGCCTGCTGGTGCCGGTTGCAAATCAGGCAATGGTGTTTATTACCATCTATCTGCAGGATACCGATAACACCA TGGGCACCATGCGTGTGATTCCGAGCAGCCATCAGTGGGAACATAGTCTGGATAGCAGCAGCGCCAATTCACTG AATGCAGAAATTTGTCTGAGCCTGCCTGGTGGTGCAGCAATGTTTTATACCCCGACCGTTTGGCATACCGCAGC AGCAAATACCAGCATTACCGATTATCGTATGCTGACGCTGATCTTCACCAAAAACAACATTAAACCGCTGCTGG TTGATGCCCTGAAACGTATTATTTGAgaaggagatatacatATGACAACCACCGATCCGATCCTGATTAATAAC TGGCATGTTGTGGCAAATGTCGAGGATTGTAAACCGGGTAGCATTACCCGTAGCCGTTTACTGGGTGTTAAACT GGTTCTGTGGCGTAGCTATGAACAGAATAGCCCGATTCAGGTTTGGCTGGATTATTGTCCGCATCGTGGTGTTC CGCTGAGCATGGGTGAAATTACCAATAATACCCTGGTTTGTCCGTATCATGGCTGGCGTTATAATGAAGCAGGT AAATGTATTCAGATTCCGGCACATCCGGGTATGGTTCCGCCTGCAAGCGCAGAAGCACGTACCTATCATAGCCA AGAACGTTATGGTCTGGTTTGGGTTTGTCTGGGTGATCCGGTTAATGATATTCCGTCATTTCCGGAATGGGATG ATCCGAATTATCACAAAACCTACACCAAAAGCTATCTGATTAAAGCAAGCGCCTTTCGCGTTATGGATAATTCA CTGGATGTTAGCCATTTTCCGTTTATTCATGATGGCTGGCTGGGCGATCGTAACTATACCAAAGTGGAAGAATT TGAAGTGAAACTGGATAAAGATGGTCTGACGATGGGCAAATATCAGTTTCAGACCAGCCGTATTGTGAGCCATA TTGAAGATGATAGCTGGGTGAATTGGTTTCGTCTGAGCCATCCGCTGTGTCAGTATTGTGTTAGCGAAAGTCCG GAAATGCGTATTGTTGATCTGATGACCATTACGCCGATTGATGAAGAAAATAGCGTTCTGCGCATGCTGATCAT GTGGAATGGTTATGAAACCCTGGAAAGCAAAATGCTGACAGAGTATGATGAAACGATCGAACAGGATATTCGTA TTCTGCATGCCCAGCAGCCGGTGCGTCTGCCGCTGCTGACACCGAAGCAGATTAATACCCAGCTGTTTAGCCAT GAAATTCATGTTCCGAGCGATCGTTGTACCCTGGCATATCGTCGTTGGCTGAAACAACTGGGTGTGACCTATGG TGTTTGTTGAgaaggagatatacatATGGCAGGTAAACTGGATGGTAAGGTTGCAATTATTACCGGTGCAAGCA GCGGTATTGGTGAAGCCACCGCATTTGCACTGGCAGCAGAAGGTGCAAAAGTTGCAATTGCAGCCCGTCGTGCA GAACTGTTACATGCACTGGCCAAACGTATTGAAGCAAGCGGTGGTCAGGCACTGCCGATTGTTACCGATATCAC CGATGAAAGCCAGGTTAATCATCTGGTTCAGAAAACCAAAGTTGAACTGGGTCATGTTGATATCCTGGTGAATA ATGCAGGTATTGGCGTTTTTGGTGCAATCGATACCGGTAATCCGGCAGATTGGCGTCGTGCATTTGATGTTAAT GTGCTGGGTGTTCTGTATGCAATTCATGCAGTTCTGCCTTTACTGAAAGCACAGAAAAGCGGTCATATTGTGAA TATTAGCAGCGTGGATGGTCGTATTGCACAGAGCGGTGCAGTTGTTTATAGCGCAGCAAAAAGCGGTGTTAATG CCCTGAGCGAAGCACTGCGTCAAGAAGTGAGCCTGGATAATATTCGTGTGACCATTATTGAACCGGGTCTGGTA GATACCCCGTTTAATGATCTGATTAGTGATCCGATTACCAAACAGCTGAGCAAAGAACAGCTGTCAACCATTAC TCCGCTGCAGAGCGAAGATATTGCACGTGCCATTATCTATGCAGTTACCCAGCCGGATCATGTTAACGTTAATG AAATTCTGATTCGTCCGACCGCAGAGGATAATTGAgaaggagatatacatATGAACCTGACCCTGAACAAAGAA GAAAAACAGCTGCTGACGGCATATAGCGGCACCGAACTGCAGCTGACAGCAGATGTTCTGGTTATTGGTGGTGG TCCGGCAGCCGCATGGGCAGCTTGGGCAGCAGGCGCACAGGGTGTGAAAGTTATTATTGTGGATAAAGGTTTTC TGGGCACCAGCGGTGCCGCAGCCGCAAGCGGTAATAGCGTTATGGCACCGTCACCGGAAAATTGGGAAAAAGAT GTGAGCGAATGTTACAGCAAAGGTAATAATCTGGCAAATCTGCGTTGGATTGAACGTGTTATTGAAAAAGCCTG GCTGTCACTGCCGCTGGTTGAAGATTGGGGTTATCGTTTTCCTAAAGAAAATGGTGAAAGCGTGCGTCAGAGCT ATTATGGTCCTGAATATATGCGTGTTCTGCGGAAAAATCTGCTGCGCGTTGGTGTTCAGATCTTTGATCAGTCA CCGGCACTGGAATTACTGCTGGCACAGGATGGTAGCGTTGCCGGTGCACGTGGTGTGCAGCGTCAGAATCATCG TACATATACCGTTCGTGCCGGTGCCGTTGTTCTGGCCAATGGTGGTTGCGCATTTCTGAGTAAAGCACTGGGTT GTAATACCAATACCGGTGATGGTCTGTTAATGGCAGTTGAAGCCGGTGGTGAACTGAGCAGTATGGAAGCCAGC AGCCATTATACCATTAGCACCGCCTTTAATGCAACCGTTACCCGTGCAGCTCCGTTTTATTGGGCAAGCTATAC CGATGAAGCTGGCAATGATCTGGGTGGCTATATTAACGGTCGTCGTGATCCGAGCTTTCTGCCGAACGCACTGC TGAAAGGTCCGGTTTATGCACGTCTGGATCGTGCAACACCGGAAATTCAGGCGCTGGTAGAAAAAAGCCATTTT ATTGCATTTCTGCCGTACAAGAAAGCCGGTATTGATCCGTATACCGAACGTGTTCCGGTTACCCTGGTGCTGGA AGGCACCGTGCGTGGCACCGGTGGTATTCGCATTGTTAATGATTCATGTGGCACCAAAGTTCCGGGACTGTATG CAGCGGGTGATGCAGCAAGCCGTGAATTTCTGGCAGGCATTGCCAGCGGTGGTGATGGACCGAATGCAGCATGG GCAATTTCAACCGGTCAGTGGGCAGGCGAAGGTGCAGCAGCCTTTGCAAAAAGTCTGGGTGCACATGTTCATGA ACGCGTTGTTCGTCCGGCAGGCCAGGCAGGTCTGCGTAGTCAGTATCCGGGTAGCGAAACCTTTGATAGTGAAG CAGTTGTTCGTGGCGTTCAGGCAGAAATGTTTCCGCTGGAAAAAAACTATCTGCGCTGTGAACAGGGACTGCTG GATAGCCTGGCAAAACTGGAAATGCTGTGGCAGCAGGTTCAGGGTAATCCGAAACAGGATACAGTTCGTGATCT GGAATTTTCACGTCGTGCGGCAGCACTGGTTAGCGTGGCACGTTGGGCATATTTTAGCGCACTGCATCGTAAAG AAACCCGTAGCGAACATATCCGTATTGATTACCCGGAAACGGATCCGAATCAACTGTATTATCAGGCAACCGGT GGCCTGGAACGTCTGTGGGTGCGTCGTGATTGGGTTAAAGATGCAAGCGCCACCCCTCCGGTGCTGACCACCTG AgaaggagatatacatATGATTGAACTGGTGAGCCATAAGCTGTGCATTAATTGTAATGTTTGTGTTCAGGTGT GCCCGACCAATGTTTTTGATGCAGTGCCGAATCAGCCTCCGGCAATTGCACGCCAAGAAGATTGTCAGACCTGT TTTATTTGTGAAGCATATTGTCCTGCAGATGCGCTGTATGTTGCACCGCAGAGCCATACCAATGTTGCAGTTAA CGAAGATGATTTAATCGACAGCGGCATTATGGGTGAATATCGTCGCATTCTGGGTTGGGGCTATGGTCGTAAAA ACAATAGCGAACTGGATACCGACCATAAACTGCGTCTGTTTGAATGAgaaggagatatacatATGTCATTTCAG AAATTTGTGCAAGAAGCAGCCTATAAAGTCGCACCGTTTAAACCGAATCGTTTTGCCAAAATTAGCGAGCGTGA AGATAAATGTGCAATTCCGGTTCCGGCATGGCGTGCACTGCTGGCCAATCGTGACCTGTTTACCTGGAAAGGTA TTCCGTTTCTGAAAGGTTGTACCGAAATTGCACTGTATAGCATGCTGCTGTATGAACTGCGTCCGAAAACGATT ATTGAAATTGGTGCGCTGAGCGGTGGTAGCGCAATTTGGCTGGCAGATCATCTGGAACTGTTTCAGATTGAAGG TTGCGTGTATTGCATTGATATTGATCTGTCTCTGCTGGACGAAAAAGCAAAAACCGATAGCCGTGTTCATTTTC TGGAAGGTGATTGCAATAATATGGGTGCAATTATGTCAAGCGAGCTGCTGAGTGGTCTGGCACATCCTTGGCTG ATTGTTGAAGATGCACATGCAAATGCCGTTGGTGTGGTTGAATATTTTCACGAAAACGGTCTGAAAAGTGGCGA TTACCTGATCGTGGAAGATACCAATAAAACAATGTGGGAACTGGATCGCGAAGAACTGGACCGTGATGACCTGG ATGAACAAGAACTGATCGAAAAAGGTGAGCAGAAATTAGCAGAACTGAAAAGCTGGCTGATGCTGCATGAGAAT GAATATCTGATAGATACCTACTATCAGGATATGTATGGCTATAATGGTAGCCGTAATTGGAACAGCATTCTGAA ACGTGTGGAAAAGAACTTTTAAtctaactaaaaacaccctaacgggtgttttttcttttctggtctccccgaag ttcctattctctagaaagtataggaacttcgctggattgggccgcgaaattaaccggcctgagcaatgtcccag ctttaat SEQ ID NO: 94: Sequence of sxt3 fragment version 3 integrated into the xylose operon, which has sxtQ, sxtR and orf24 deleted. Flanking regions of the xylose operon at integration site are included. sxt open reading frames (S, T, U, V, W, X) are indicated by upper case letters. ggtgtcaggaggcgggaggggacatctttcaggccattattcagtgaggtCCAGCCTTGCAAGAAGCGGATACA GGAGTGCAAAAAATGGCTATCTCTAGAAAGGCCTACCCCTTAGGCTTTATGCAACAGAAACAATAATAATGGAG TCATGAACATGCTGACCGCAGAACAGAAACAGGCATATACCAATGATGGCTATTTTACCGTGGAAGAAGCAGTT CCGAAAGCACTGATTGAAGAAATTCGCCATGAAGTGGAACTGATCACCGAGCAGAAACGTGGTGGTGTGCTGGC AGGCGATTATGAATGGTGGTCAGAACACACCATTCCGGATCCGGTTCGTTATCAGAAAATTATCCAGCGTCTGC TGGAACTGCCGACCGTTATGGGTCCGGTTCAGGCCCTGATTGGTAGCGATATTTTTCTGTTAATTACCGACCTG GCAATTATTCGTGCAGGCACCGGTTATATTGCATGGCATCAGGATCATGGCTATGTTGTTGAAGTTCTGAACGC CCTGGCAAGCATGAGCAAAAATGAGCTGAATGATGATGCACTGCGCCTGCTGGTGCCGGTTGCAAATCAGGCAA TGGTGTTTATTACCATCTATCTGCAGGATACCGATAACACCATGGGCACCATGCGTGTGATTCCGAGCAGCCAT CAGTGGGAACATAGTCTGGATAGCAGCAGCGCCAATTCACTGAATGCAGAAATTTGTCTGAGCCTGCCTGGTGG TGCAGCAATGTTTTATACCCCGACCGTTTGGCATACCGCAGCAGCAAATACCAGCATTACCGATTATCGTATGC TGACGCTGATCTTCACCAAAAACAACATTAAACCGCTGCTGGTTGATGCCCTGAAACGTATTATTTGAGAAGGA GATATACATATGACAACCACCGATCCGATCCTGATTAATAACTGGCATGTTGTGGCAAATGTCGAGGATTGTAA ACCGGGTAGCATTACCCGTAGCCGTTTACTGGGTGTTAAACTGGTTCTGTGGCGTAGCTATGAACAGAATAGCC CGATTCAGGTTTGGCTGGATTATTGTCCGCATCGTGGTGTTCCGCTGAGCATGGGTGAAATTACCAATAATACC CTGGTTTGTCCGTATCATGGCTGGCGTTATAATGAAGCAGGTAAATGTATTCAGATTCCGGCACATCCGGGTAT GGTTCCGCCTGCAAGCGCAGAAGCACGTACCTATCATAGCCAAGAACGTTATGGTCTGGTTTGGGTTTGTCTGG GTGATCCGGTTAATGATATTCCGTCATTTCCGGAATGGGATGATCCGAATTATCACAAAACCTACACCAAAAGC TATCTGATTAAAGCAAGCGCCTTTCGCGTTATGGATAATTCACTGGATGTTAGCCATTTTCCGTTTATTCATGA TGGCTGGCTGGGCGATCGTAACTATACCAAAGTGGAAGAATTTGAAGTGAAACTGGATAAAGATGGTCTGACGA TGGGCAAATATCAGTTTCAGACCAGCCGTATTGTGAGCCATATTGAAGATGATAGCTGGGTGAATTGGTTTCGT CTGAGCCATCCGCTGTGTCAGTATTGTGTTAGCGAAAGTCCGGAAATGCGTATTGTTGATCTGATGACCATTAC GCCGATTGATGAAGAAAATAGCGTTCTGCGCATGCTGATCATGTGGAATGGTTATGAAACCCTGGAAAGCAAAA TGCTGACAGAGTATGATGAAACGATCGAACAGGATATTCGTATTCTGCATGCCCAGCAGCCGGTGCGTCTGCCG CTGCTGACACCGAAGCAGATTAATACCCAGCTGTTTAGCCATGAAATTCATGTTCCGAGCGATCGTTGTACCCT GGCATATCGTCGTTGGCTGAAACAACTGGGTGTGACCTATGGTGTTTGTTGAGAAGGAGATATACATATGGCAG GTAAACTGGATGGTAAGGTTGCAATTATTACCGGTGCAAGCAGCGGTATTGGTGAAGCCACCGCATTTGCACTG GCAGCAGAAGGTGCAAAAGTTGCAATTGCAGCCCGTCGTGCAGAACTGTTACATGCACTGGCCAAACGTATTGA AGCAAGCGGTGGTCAGGCACTGCCGATTGTTACCGATATCACCGATGAAAGCCAGGTTAATCATCTGGTTCAGA AAACCAAAGTTGAACTGGGTCATGTTGATATCCTGGTGAATAATGCAGGTATTGGCGTTTTTGGTGCAATCGAT ACCGGTAATCCGGCAGATTGGCGTCGTGCATTTGATGTTAATGTGCTGGGTGTTCTGTATGCAATTCATGCAGT TCTGCCTTTACTGAAAGCACAGAAAAGCGGTCATATTGTGAATATTAGCAGCGTGGATGGTCGTATTGCACAGA GCGGTGCAGTTGTTTATAGCGCAGCAAAAAGCGGTGTTAATGCCCTGAGCGAAGCACTGCGTCAAGAAGTGAGC CTGGATAATATTCGTGTGACCATTATTGAACCGGGTCTGGTAGATACCCCGTTTAATGATCTGATTAGTGATCC GATTACCAAACAGCTGAGCAAAGAACAGCTGTCAACCATTACTCCGCTGCAGAGCGAAGATATTGCACGTGCCA TTATCTATGCAGTTACCCAGCCGGATCATGTTAACGTTAATGAAATTCTGATTCGTCCGACCGCAGAGGATAAT TGAGAAGGAGATATACATATGAACCTGACCCTGAACAAAGAAGAAAAACAGCTGCTGACGGCATATAGCGGCAC CGAACTGCAGCTGACAGCAGATGTTCTGGTTATTGGTGGTGGTCCGGCAGCCGCATGGGCAGCTTGGGCAGCAG GCGCACAGGGTGTGAAAGTTATTATTGTGGATAAAGGTTTTCTGGGCACCAGCGGTGCCGCAGCCGCAAGCGGT AATAGCGTTATGGCACCGTCACCGGAAAATTGGGAAAAAGATGTGAGCGAATGTTACAGCAAAGGTAATAATCT GGCAAATCTGCGTTGGATTGAACGTGTTATTGAAAAAGCCTGGCTGTCACTGCCGCTGGTTGAAGATTGGGGTT ATCGTTTTCCTAAAGAAAATGGTGAAAGCGTGCGTCAGAGCTATTATGGTCCTGAATATATGCGTGTTCTGCGG AAAAATCTGCTGCGCGTTGGTGTTCAGATCTTTGATCAGTCACCGGCACTGGAATTACTGCTGGCACAGGATGG TAGCGTTGCCGGTGCACGTGGTGTGCAGCGTCAGAATCATCGTACATATACCGTTCGTGCCGGTGCCGTTGTTC TGGCCAATGGTGGTTGCGCATTTCTGAGTAAAGCACTGGGTTGTAATACCAATACCGGTGATGGTCTGTTAATG GCAGTTGAAGCCGGTGGTGAACTGAGCAGTATGGAAGCCAGCAGCCATTATACCATTAGCACCGCCTTTAATGC AACCGTTACCCGTGCAGCTCCGTTTTATTGGGCAAGCTATACCGATGAAGCTGGCAATGATCTGGGTGGCTATA TTAACGGTCGTCGTGATCCGAGCTTTCTGCCGAACGCACTGCTGAAAGGTCCGGTTTATGCACGTCTGGATCGT GCAACACCGGAAATTCAGGCGCTGGTAGAAAAAAGCCATTTTATTGCATTTCTGCCGTACAAGAAAGCCGGTAT TGATCCGTATACCGAACGTGTTCCGGTTACCCTGGTGCTGGAAGGCACCGTGCGTGGCACCGGTGGTATTCGCA TTGTTAATGATTCATGTGGCACCAAAGTTCCGGGACTGTATGCAGCGGGTGATGCAGCAAGCCGTGAATTTCTG GCAGGCATTGCCAGCGGTGGTGATGGACCGAATGCAGCATGGGCAATTTCAACCGGTCAGTGGGCAGGCGAAGG TGCAGCAGCCTTTGCAAAAAGTCTGGGTGCACATGTTCATGAACGCGTTGTTCGTCCGGCAGGCCAGGCAGGTC TGCGTAGTCAGTATCCGGGTAGCGAAACCTTTGATAGTGAAGCAGTTGTTCGTGGCGTTCAGGCAGAAATGTTT CCGCTGGAAAAAAACTATCTGCGCTGTGAACAGGGACTGCTGGATAGCCTGGCAAAACTGGAAATGCTGTGGCA GCAGGTTCAGGGTAATCCGAAACAGGATACAGTTCGTGATCTGGAATTTTCACGTCGTGCGGCAGCACTGGTTA GCGTGGCACGTTGGGCATATTTTAGCGCACTGCATCGTAAAGAAACCCGTAGCGAACATATCCGTATTGATTAC CCGGAAACGGATCCGAATCAACTGTATTATCAGGCAACCGGTGGCCTGGAACGTCTGTGGGTGCGTCGTGATTG GGTTAAAGATGCAAGCGCCACCCCTCCGGTGCTGACCACCTGAGAAGGAGATATACATATGATTGAACTGGTGA GCCATAAGCTGTGCATTAATTGTAATGTTTGTGTTCAGGTGTGCCCGACCAATGTTTTTGATGCAGTGCCGAAT CAGCCTCCGGCAATTGCACGCCAAGAAGATTGTCAGACCTGTTTTATTTGTGAAGCATATTGTCCTGCAGATGC GCTGTATGTTGCACCGCAGAGCCATACCAATGTTGCAGTTAACGAAGATGATTTAATCGACAGCGGCATTATGG GTGAATATCGTCGCATTCTGGGTTGGGGCTATGGTCGTAAAAACAATAGCGAACTGGATACCGACCATAAACTG CGTCTGTTTGAATGAGAAGGAGATATACATATGTCATTTCAGAAATTTGTGCAAGAAGCAGCCTATAAAGTCGC ACCGTTTAAACCGAATCGTTTTGCCAAAATTAGCGAGCGTGAAGATAAATGTGCAATTCCGGTTCCGGCATGGC GTGCACTGCTGGCCAATCGTGACCTGTTTACCTGGAAAGGTATTCCGTTTCTGAAAGGTTGTACCGAAATTGCA CTGTATAGCATGCTGCTGTATGAACTGCGTCCGAAAACGATTATTGAAATTGGTGCGCTGAGCGGTGGTAGCGC AATTTGGCTGGCAGATCATCTGGAACTGTTTCAGATTGAAGGTTGCGTGTATTGCATTGATATTGATCTGTCTC TGCTGGACGAAAAAGCAAAAACCGATAGCCGTGTTCATTTTCTGGAAGGTGATTGCAATAATATGGGTGCAATT ATGTCAAGCGAGCTGCTGAGTGGTCTGGCACATCCTTGGCTGATTGTTGAAGATGCACATGCAAATGCCGTTGG TGTGGTTGAATATTTTCACGAAAACGGTCTGAAAAGTGGCGATTACCTGATCGTGGAAGATACCAATAAAACAA TGTGGGAACTGGATCGCGAAGAACTGGACCGTGATGACCTGGATGAACAAGAACTGATCGAAAAAGGTGAGCAG AAATTAGCAGAACTGAAAAGCTGGCTGATGCTGCATGAGAATGAATATCTGATAGATACCTACTATCAGGATAT GTATGGCTATAATGGTAGCCGTAATTGGAACAGCATTCTGAAACGTGTGGAAAAGAACTTTTAATCTAACTAAA AACACCCTAACGGGTGTTTTTTCTTTTCTGGTCTCCCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGc tggattgggccgcgaaattaaccggcctgagcaatgtcccagctttaat SEQ ID NO: 95: Sequence of sxt1 fragment, integrated into the lactose operon after seam-less deletion of sxtC. Flanking regions of the lactose operon at integration site are included. sxt open reading frames are indicated by upper case letters. atgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaact taatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccc aacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctgg ctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcc catctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgtt actcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaac tcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcagtggagatgcccaagggc acttcgggtcgaggaacccgacctgcattgggacgcggccacggagagcgcgggcaaacgccggcactatagcc agtggagtttgtaaaacgctatttcagagcttggagagtgtctaagaaagccgggcgatgccaacccatccctt cttcggctacgttcgtaatcaagccacttcctttttgcattgacgcagggtgtcggaaggcaactcgccgaacg cgctcctatagttttcagcgaagcgtcccaaatgtaagaagccgtagtctagggctatctcagttatactacgc acattggcactgggatcgttcaagcaggcgcggatgctttcgagcttgcggttgcggatgtagttcttcggcgt ggtgccggcatgcttctcgaacaaattgtagagcgagcgtggactcatcatcgccagctccgctaaccgctcaa ggctgatattccgtttgagattctcctcaatgaattgaacgactcgctcgaaagacgggttacctttgctgaaa atttcacggctgacattgctgcccagcatttcgagcagcttggaagcgatgatccccgcatagtgctcttggac ccgaggcatcgactttgtatgttccgcttcgtcacaaactaacccgagtagattgataaagccatcgagttgct ggagattgtgtcgcgcggcgaaacggataccctccctcggcttgtgccaattgttgtcactgcatgcccgatca aggaccactgagggcaatttaacgataaatttctcgcaatcttctgaataggtcaggtcggcttggtcatccgg attgagcagcaatagttcgcccggcgcaaaatagtgctcctggccatggccacgccacaggcaatggcctttga gtattatttgcagatgataacaggtctctaatccaggcgagattaccctcacgctaccgccgtagctgattcga cacaggtcgaggcatccgaagattctgtggtgcagcctgcctgccgggggcccgcccttgggcaggcgaataga gtgcgtaccgacatactggttaacataatcggagactgcatagggctcggcgtggacgaagatctgacttttct cgttcaataagcaaaaatccatagttcacggttctcttattttaatgtgggctgcttggtgtgatgtagaaagg cgccaagtcgatgaaaatgcaggaattaattcgcagatcctggcggatgagagaagattttcagcctgatacag attaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctga ccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtaggga actgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggt gaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggc gggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcg tagatcccagccttgcaagaagcggatacaggagtgcaaaaaatggctatctctagaaaggcctaccccttagg ctttatgcaacagaaacaataataatggagtcatgaacatgctgcagaaaatcaatcgttatacccatggtttt gttgccgttccggttattctggcatgtcgtgaaaaaggtgtttttgaactgctggcagatgaaagtccgctgag cctgaatcagatggttgaacatctgggtgccaatagcggtcattttcaggttgcactgcgtatgctggaaagtc tgcattggctgagccgtaataaagaactgaaatatagcctgaccgcagaagcagcaattcataacaaaattagc gaagatatcctgcagctgtataatctgccgattcagagctatctggaaggtaaacagggcaatctgctgggtcg ttggattgaacgtagctgtcagctgtggaatctggataatccgctgatggcagattttctggatggtctgctgg ttattccgctgctgctggcactgcataaacataacctgctggccgattctgaagataaaccgctgctgagcagc ctgagcagtaccgttcaagaagaactgggtaaactgtttctgcatctgggttgggcagatctgacagcaggtcg tctgaccattaccgaactgggtcgctttatgggtgaacgtgcactgaataccgcaattgttgcaagctataccc cgatgctgagtcgtattcatgatgttctgtttggtaattgcctgagcgtttttcagcgtgatgcaagcggtcat gaacgtcatattgatcgtaccctgaatgttattggtagcggttttcagcaccagaaatactttgcagatctgga agaaagcattctgagcgtgtttaatcagctgccgctggaagaacagccgaaatacattaccgatatgggttgtg gtgatggcaccctgctgaaacgtgtttgggaaaccattcagtttaaaagcgcacgtggtaaagcactggaacag tatccgctgcgtctgattggtgttgattataatgaagcaagcctgaaagcaaccacccgtaccctggcaagcct gccgcatctggttctgcagggtgatattggtaatccggaacaaatggttcgtagcctggaagcacatggcattc atgatccggaaaatattctgcatattcgcagctttctggatcacgatcgtctgtttattccgcctcagaaacgt aatgaactgaaagaacgtgcccatctgccgtatcagagtgtttgtgttgatgatcagggtgaactgattcctcc gcatgttatggttcagagcctggtggaacacctggaacgttggagccaggttgttaataaacatggtctgatga ttctggaagtgcattgtctggaaccgcgtgttgtttatcagtttctggataaaagcgaaaacctgcactttgat gcatttcagggttttagccagcagtatctggttgaagccgaagtttttctgatgagcgcagcacaggttggtct gtttccgaaactggaactgagcaaacgttatccgaaaacctttccgtttacccgtattaccctgaactatttcg aaaaacgtccgtacaaaatcagccatgcatatctgagcgatctgcctgcactggttgacctggaagttaaatgt tggcctgagaatctgcgtgcaagcacccatgaaattcgtcgtcgtctggaactgaatccgcagggtaacctggt tctgattattgaagatcagattatcggtgccatttacagccagaccattacaagcaccgaagccctggaaaatg ttaaatatgcacaggttccgaccctgcatacaccgcagggttcagtgattcagctgctggccctgaacattctg ccggaatttcaggcacgtggtctgggcaatgaactgcgtgattttatgctgtattattgcaccctgaaaggtgg tattgaaagcgttgttggtgttacccgttgtcgcaattatgtgaattatagccagatgccgatgatggaatatc tgaaactgcataatgaacagcgtcaactgctggatccgattgttggttttcatgttagcggtggtgcagaaatt cgtggcattattgcaaattatcgtccggaagatacagataatctgggtatgggtattctgatcgaatataacct gcgtgatagcgcactgcattcaccgggtgatcgtaaaggtccgtatatcaatagcgcaattggtagcctggttc cgaaagcgaccagcgcaaccaaagaaaacaaaaccgttgcggatctggtgaaagaatgtattctgaaagtgatg ggtagccagcgtcaggcagcatatgcaccgcagcagaaactgctggacatgggtctggatagcctggatctgct ggaactgcagaccctgctggaagaacgtctgggtattaatctgagcggcaccttttttctgcaaaaaaacaccc cgaccgccatcattacctattttcagaatcaggtcgtgcaagagaaacagagtgatctggcaccgcctgttgat agcgccaatgaaatcaatacactggaaaacgttgtgaatcagcagaaaattccgcaggttacacgtgttgttac cgaacagcagggacgtaaagttctgattgatggtcattgggttattgattttgccagctgtaattatctgggcc tggacctgcatccgaaagttaaagaagcaattcctccggcactggataaatggggcacccatccgagctggacc cgtctggttgcaagtccggcaatttatgaggaactggaagaggaactgtcaaaactgctgggtgtgccggatgt tctggtttttccggcagttacactgctgcagattggtattctgcctctgctgaccggtaataatggtgtgattt ttggcgatattgcagcccatcgttgtatttatgaagcatgttgtctggcccagcataaaggtgcacagtttatt cagtatcgtcataacgacctgaatgatctggccgaaaaactggccaaatatccgcctgaacaggttaaaatcat tgtgatcgatggtgtgtatagcatgagtgccgatttcccggacctgcctgcatatgttcatctggcaaaagaat ataacgccctgatctatatggatgatgcacatggctttggcattctgggtgaaaatccgagcagcgatatgccg tatggttataaaggtaatggcatggtgaactactttgatctgcgttttgccgaagataacatcatttatgttgc aggtctgagcaaagcctatagcagctatgcagcatttctgacctgtggtgatcgtcgtattaaaaccaattttc gtaatgcatggaccgcgatttttagcggtccgagtccggttgcaagcctggccagcgcactggcaggtctgcag gttaatcgtcaagaaggtgaacagctgcgcaaacaaatctatcatctgacacataaactggttacccaggctcg tgccattggttttgaagttgataattatggttatgtgccgattgtgggtgttctggtgggtgatgcacagcata tgattgatgtgtgccaactgctgtgggaatatggtatcctgattacccctgcaatttttccgattgtgccgctg aataaatcagcactgcgttttagcattaccgcagcaaataccgaagaagaaattgatcaggccatcaaaagtct gaaagcagtttgggacctgctgcaaaaacgtaaagccctgccgtgtaaacaagaagaaaatatcctgaaacatt gagaaggagatatacatatgacccatgttgccctggaacaggcaattgcaaaagttccgcgtagcattcagagc gaactgcgtaccattctggcacagcatgcagttattgatagcagcgttgtggcaagctggattgatcgtctggg caccaatattagtaccctgatgatccagctgctgccggttgcagcaacctatgcacgtgttccgattagccagt tttatgttggtgccattgcactgggcaaaccgcagagtaaaaatcagctgggtagcggcaccctgtattttggt gcagatatggaatttgttggtcaggcactgagctttagcgttcatgcagaacagagcgccaccattaatgcctg gctgcatggcgaaaccggactgcaggcactggcaatccatgaagcaccgtgtggttattgtcgccagtttctgt atgaaatggcaaccgtgaatcagaattttgtgctgctggtgaaaagcaatgaaagccagccggaacagacctat accagcaacaaactgccgcattttctgcctgaaccgtttggtccagccgatctgggtctgaccggtggcctgat gcagaccgtgtttcacgatctggaaacctatagcaccgatgatgttgttctggcagcactgagtgcagcaaatc agagttatgcaccgtataccaaaaactttgccggtgttgcactgaaagatagtcatggtaacatttttacaggt cgctatgccgaaaacgcagcatttaatagcagcatgagcccgatggaaagcgcactgacctttatgaatatgaa tcgttattcacagagcctgttcgatatttgtgatgcagttctggtagaagtggaaaccggtattagtcagcgtc cggttaccgaagcctttctgagtagcattgcaccgaaagtgaaactgcgctatgcaccggcaaccccgagcagt aacaaactgtgactcggtaccaaattccagaaaagaggcctcccgaaaggggggccttttttcgttttggtccc gaagttcctattctctagaaagtataggaacttc SEQ ID NO: 96: Sequence of sxt4 fragment integrated into the melobiose operon. Flanking regions of the melobiose operon at integration site are included. sxt open reading frames are indicated by upper case letters. aagcctgccgtcagggcaatatcgagaatacttttatcggtatcgctcagCCAGCCTTGCAAGAAGCGGATACA GGAGTGCAAAAAATGGCTATCTCTAGAAAGGCCTACCCCTTAGGCTTTATGCAACAGAAACAATAATAATGGAG TCATGAACATATGGAAACCACGAGCAAAAAATTCAAAAGCGATCTGATTCTGGAAGCACGTGCAAGCCTGAAAC TGGGTATTCCGCTGGTTATTAGCCAGATGTGTGAAACCGGTATTTATACCGCAAATGCAGTTATGATGGGTCTG CTGGGCACCCAGGTTCTGGCAGCCGGTGCTCTGGGTGCACTGGCATTTCTGACCCTGCTGTTTGCATGTCATGG TATTCTGAGCGTTGGTGGTAGCCTGGCAGCGGAAGCATTTGGTGCAAACAAAATTGATGAAGTTAGCCGTATTG CAAGCGGTCAGATTTGGCTGGCAGTTACCCTGAGCCTGCCTGCAATGCTGCTGCTGTGGCATGGTGATACCATT CTGCTGTTATTTGGTCAAGAAGAAAGCAACGTTCTGCTGACCAAAACCTATCTGCATAGCATTCTGTGGGGTTT TCCGGCAGCACTGAGTATTCTGACACTGCGTGGTATTGCCAGCGCACTGAATGTTCCGCGTCTGATTACCATTA CCATGCTGACCCAGCTGATTCTGAATACCGCAGCAGATTATGTTCTGATCTTTGGTAAATTTGGTCTGCCGCAG CTGGGTCTGGCAGGTATTGGTTGGGCAACCGCACTGGGTTTTTGGGTTAGCTTTACCCTGGGTCTGATCCTGCT GATTTTTAGCCTGAAAGTGCGTGATTATAAACTGTTTCGTTATCTGCACCAGTTCGACAAGCAGATCTTTGTGA AAATCTTTCAGACCGGTTGGCCGATGGGTTTTCAGTGGGGTGCAGAAACAGCACTGTTTAATGTTACCGCATGG GTTGCAGGTTATCTGGGCACCGTTACCCTGGCAGCACATGATATTGGTTTTCAGACAGCAGAACTGGCAATGGT TATCCCGCTGGGTGTTGGTAATGTTGCAATGACCCGTGTTGGTCAGAGCATTGGTGAAAAAAATCCACTGGGTG CCCGTCGTGTTGCAAGCATTGGTATTACCATTGTTGGTATTTATGCCAGCATTGTTGCCCTGGTTTTTTGGCTG TTTCCOTATCAGATTGCAGGCATTTATCTGAACATTAATAACCCGGAAAACATTGAAGCCATCAAAAAAGCCAC CACCTTTATTCCACTGGCAGGTCTGTTTCAGATGTTTTATAGCATTCAGATCATTATCOTTGOTGCGCTGGTTG GTCTGCGTGATACCTTTGTTCCGGTTAGCATGAATCTGATTGTTTGGGGTCTGGGTTTAGCAGGTAGCTATTTT ATGGCAATTATTCTGGGTTGGGGTGGTATTGGTATCTGGCTGGCCATGGTTCTGAGTCCGCTGCTGAGCGCAGT TATTCTGACCGTTCGTTTTTATCGCGTGATTGATAATCTGCTGGCCAACAGTGATGATATGCTGCAGAATGCAA GCGTTACCACCCTGGGATGAGAAGGAGATATACATATGAAACGTCTGACGCTGCTGATCATTGCAGGTATTCTG TCAGTTAGCACCTTTCTGTGTATTACACCGGTTGCACTGGCCAATATTACCGATTATTATCTGAAAAACGAGAA ACTGAGCGGTCAGTTTAGCGTTCCGGTGAATCTGTCTGTTGGTGTTCGTTTTGCACATCGTAGCAGCTATGCAA CCGCAATTAACTTTCCGACCGGTCTGGATGCAGATAGCGTTGCAGTTGGTGATTTTAACAGCGATAGCAAACTG GATCTGGCCGTTACCAATTGGTTTGATAACAATGTTAGCGTGCTGCTGGGTAATGGCAATGGCAGCTTTGGTGC AGCAACCAATTTTCCGGTTGGCACCAATCCGGTTTTTGTTGTTACCGGTGATGTTAATGGTGACAGTAAACTGG ATTTAGCCGTGGCAAATTTTAGCAGCAATAATGTTTCAGTTCTGCTGGGAAACGGTAATGGTTCTTTTGGCGCA GCCACAAACTTTAGCGTTGGTACAAATCCGTATAGCGTGGCCATTGGTGATGTGAATAATGATAGTGAACTGGA CCTGGCATTTACGAACTGGTTCGATAATAAAGTTCTGGTGCTGTTAGGCAATGGTAATGGCTCGTTTGGTGCCG CAAGCTCATTTCCGGTGGATACCTATAGCATTAGCGTTGCGATTGCAGATTTCAACTCAGATTCTAAATTAGAC CTGGCGATCACCAATTGGGTGTCAAATAATGTGAGTGTGTTACTGGGGAATGGTAACGGTAGTTTTGGAGCTGC GACAAATTTTCCTGTGGGTACAAACCCGATTTTTGTGGCAACCGGTGACGTGAATGGCGATTCTAAGCTGGACT TAGCAGTTGCAAATACCAGCTCTAATAACGTTAGCGTTCTGTTAGGTAACGGGAACGGCTCATTCGGTGCTGCC ACGAATTTTCCAGCAGGCACCAACCCGTATAGTGTTGCAATTCGCGACGTTAACGGTGATAGCAAATTAGATTT AGCGGTGACCAACTATAGCAGCAACAACGTGAGTGTTCTGCCAGGCAACGGTAACGGATCATTTGGTATTGCGA CCAACTTTCCAGTAGGTACGAATCCGGAAAGCATTGCAATTGCCGATTTTAATGGGGATTCCAAGTTAGATCTG GCAGTGACAAATAGCGGTAACAATAATGTAAGCATACTGCTGAATAACTTTCAGGGTCTGCCGAAAAACAAGAT TTGAGAAGGAGATATACATATGACCAATACCGAACGTGGTCTGGCCGAAATTACCAGCACCGGTTATAAAAGCG AACTGCGTAGCGAAGCCCGTGTTAGCCTGCAGCTGGCAATTCCTCTGGTTCTGGTTGAAATTTGTGGCACCAGC ATTAATGTTGTTGATGTTGTGATGATGGGTTTACTGGGTACACAAGTGTTAGCAGCGGGTGCCCTGGGAGCAAT TGCCTTCCTGAGCGTTAGCAATACCTGCTATAATATGCTGCTGAGTGGTGTTGCAAAAGCAAGCGAAGCCTTTG GAGCCAATAAAATCGATCAGGTTTCACGTATTGCCTCAGGCCAGATTTGGTTAGCCCTGACCCTGTCATTACCA GCCATGCTGTTACTGTGGTATATGGATACCATCCTGGTTCTGTTTGGTCAGGTTGAAAGCAATACCCTGATTGC GAAAACATACCTGCATTCAATTGTGTGGGGCTTTCCTGCCGCAGTTGGTATCCTGATTCTGCGTGGCATAGCAA GTGCAGTTAACGTTCCTCAGCTGGTTACCGTGACCATGCTGGTTGGCCTGGTGCTGAATGCACCGGCTAATTAT GTGCTGATGTTCGGCAAATTCGGTTTACCGGAATTAGGCCTGGCTGGCATTGGCTGGGCCAGCACACTGGTGTT TTGGATTAGTTTTCTGGTTGGTGTTGTGCTGCTGATATTTTCACCGAAAGTTCGCGACTACAAACTGTTCCGCT ATTTACATCAGTTTGATCGTCAGACCGTGGTTGAGATTTTTCAGACGGGCTGGCCTATGGGCTTCCTGCTGGGT GTGGAAAGCGTTGTTCTGAGCCTGACCGCATGGCTGACCGGCTATCTGGGTACAGTGACCTTAGCAGCCCATGA AATTGCAATCCAGACTGCCGAACTGGCGATTGTGATTCCGTTAGGTATTGGCAATGTTGCCGTTACCCGTGTGG GCCAGACAATCGGCGAAAAAAACCCGCTGGGAGCACGCCGTGCAGCCCTGATTGGCATTATGATTGGTGGCATT TATGCGAGCCTGGTTGCAGTGATTTTTTGGTTATTCCCTTATCAAATCGCAGGCCTGTACCTGAAAATTAACGA TCCGGAATCAATGGAAGCAGTTAAAACCGCAACAAACTTTCTGTTTTTAGCTGGCCTGTTCCAGTTTTTTCATA GCGTGCAGATTATTGTTGTGGGTGTTCTGATTGGCCTGCAGGATACCTTTATCCCTCTGCTGATGAATCTGGTG GGCTGGGGACTGGGCCTGGCGGTTTCCTATTATATGGGTATTATCCTGTGCTGGGGTGGCATGGGCATCTGGTT AGGTCTGGTACTGTCACCGCTGCTGTCAGGCCTGATCCTGATGGTGCGCTTTTATCAAGAAATTGCCAATCGCA TTGCGAATAGCGACGATGGCCAAGAAAGCATTAGCATTGATAATGTTGAAGAACTGAGCTAATAGACCAACCCC TTGCGGCCTCAATCGGGGGGGATGGGGTTTTTTGTCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGcaa ccgtctgctgaaggaagccatctgacacttaaagccatcgttgcgct SEQ ID NO: 97: Sequence of sxt2 fragment integrated into the maltose operon. Flanking regions of the maltose operon atintegration site are included. sxt open reading frames (D, E, G, H, I J, K, L) are indicated by upper case letters. ctgtgaactaaaccgaggtcatgtaaggaatttcgtgatgttgcttgcaaccagccttgcaagaagcggataca ggagtgcaaaaaatggctatctctagaaaggcctaccccttaggctttatgcaacagaaacaataataatggag tcatgaacatgattgataccattagcgttctgctgcgtgaatggaccgttatttttctgaccggtctggcattt tggctgtgggaaattcgtagtccgctgcatcagattgaatacaaagccaaatttttcaaagaactgggttgggc aggtatcagctttgtttttcgtattgtttatgcctatgttagcgtggccattatcaaactgctgagcagcctgt ttatgggtgaaagcgcaaattttgccggtgttatgtatgttccgctgtggctgcgtattattaccgcatatatt ctgcaggatctgaccgattatctgctgcatcgtaccatgcatagcaatcagtttctgtggctgacccataaatg gcatcatagcaccaaacagagttggtggctgagcggtaataaagatagctttaccggtggtctgctgtataccg ttaccgcactgtggtttccgctgctggatattccgagcgaagttatgagcgttgttgcagttcatcaggtgatt cataacaactggattcacctgaatgtgaaatggaatagctggctgggtattatcgaatggatttatgttacacc gcgtatccataccctgcatcatctggataccggtggtcgtaatctgagcagtatgtttacctttattgatcgtc tgtttggcacctatgtgtttccggaaaactttgatatcgaaaaaagcaaaaaccgcctggatgatcagagcgtt accgttaaaaccattctgggtttctgagaaggagatatacatATGCTGAAAGATTTTAACCAGTTCCTGATTCG TACCCTGGCATTTGTTTTTGCCTTTGGCATTTTTCTGACAACCGGTGTTGGTATTGCAAAAGCAGATTATCTGG TGAAAGGTGGCAAAATTACCAATGTTCAGAATACCAGCAGCAACGGTGATAATTATGCAGTTAGCATTAGCGGT GGTTTTGGTCCGTGTGCAGATCGTGTTATTATTCTGCCGACCAGCGGTGTTATTAATCGTGATATTCACATGCG TGGTTATGAAGCAGCACTGACCGCACTGAGCAATGGTTTTCTGGTTGATATCTATGATTATACCGGTAGCAGCT GTAGCAATGGTGGCCAGCTGACCATTACCAATCAGCTGGGTAAACTGATTAGCAATTGAgaaggagatatacat ATGACCAATCAGAACAACCAAGAGCTGGAAAATGATCTGCCGATTGCAAAACAGCCGTGTCCGGTTAATAGCTA TAATGAATGGGATACCCTGGAAGAAGTTATTGTTGGTAGCGTTGAAGGTGCAATGCTGCCTGCACTGGAACCGA TTAACAAATGGACCTTTCCGTTTGAAGAACTGGAAAGCGCACAGAAAATTCTGAGCGAACGTGGTGGTGTTCCG TATCCGCCTGAAATGATTACCCTGGCACATAAAGAACTGAACGAGTTTATTCATATCCTGGAAGCCGAAGGTGT TAAAGTTCGTCGTGTTAAACCGGTTGATTTTAGCGTTCCGTTTAGCACACCGGCATGGCAGGTTGGTAGCGGTT TTTGTGCAGCAAATCCGCGTGATGTTTTTCTGGTTATTGGCAACGAAATTATCGAAGCACCGATGGCAGATCGT AATCGTTATTTTGAAACCTGGGCATATCGCGAAATGCTGAAAGAATATTTTCAGGCAGGCGCAAAATGGACCGC AGCACCGAAACCGCAGCTGTTTGATGCACAGTATGATTTCAATTTTCAGTTTCCGCAGCTGGGTGAACCGCCTC GTTTTGTTGTTACCGAATTTGAACCGACCTTTGATGCAGCCGATTTTGTTCGTTGTGGTCGTGATATTTTTGGC CAGAAAAGCCATGTTACCAATGGTCTGGGTATTGAATGGCTGCAGCGTCATCTGGAAGATGAATATCGCATTCA TATCATCGAAAGCCATTGTCCGGAAGCACTGCATATTGATACCACCCTGATGCCGCTGGCACCGGGTAAAATTC TGGTTAATCCGGAATTTGTGGACGTGAATAAACTGCCGAAAATTCTGAAAAGCTGGGATATTCTGGTTGCACCG TATCCGAATCATATTCCGCAGAATCAGCTGCGTCTGGTTAGCGAATGGGCAGGTCTGAATGTTCTGATGCTGGA TGAAGAACGTGTGATCGTGGAAAAAAATCAAGAGCAGATGATCAAAGCCCTGAAAGATTGGGGTTTTAAACCGA TTGTTTGCCACTTCGAAAGCTATTATCCGTTTCTGGGTAGCTTTCATTGTGCAACCCTGGATGTTCGTCGTCGT GGCACCCTGCAGAGCTATTTTTGAgaaggagatatacatATGACGACCGCAGATCTGATTCTGATCAATAATTG GTATGTTGTGGCCAAGGTGGAAGATTGTAAACCGGGTAGCATTACCACCGCACTGCTGCTGGGTGTTAAACTGG TTCTGTGGCGTAGCCGTGAACAGAATAGCCCGATTCAGATTTGGCAGGATTATTGTCCGCATCGTGGTGTTGCA CTGAGCATGGGTGAAATTGTGAATAATACCCTGGTTTGTCCGTATCATGGTTGGCGTTATAATCAGGCAGGTAA ATGTGTTCATATTCCGGCACATCCGGATATGACCCCTCCGGCAAGCGCACAGGCAAAAATCTATCATTGTCAAG AACGTTATGGTCTGGTTTGGGTTTGTCTGGGTGATCCGGTTAATGATATTCCGAGTCTGCCGGAATGGGATGAT CCGAATTATCATAATACCTGCACCAAGAGCTACTTTATCCAGGCAAGCGCATTTCGTGTGATGGATAACTTTAT TGATGTGAGCCATTTTCCGTTTGTGCATGATGGTGGTCTGGGCGATCGTAATCATGCACAGATTGAAGAATTTG AGGTGAAAGTGGATAAAGACGGTATTAGCATTGGCAATCTGAAACTGCAGATGCCTCGTTTTAATAGCAGCAAT GAAGATGATAGCTGGACCCTGTATCAGCGTATTAGCCATCCGCTGTGTCAGTATTATATCACCGAAAGCAGCGA AATTCGTACAGCAGATCTGATGCTGGTTACCCCGATTGATGAAGATAATTCACTGGTTCGTATGCTGGTGACCT GGAATCGTAGCGAAATTCTGGAAAGCACCGTTCTGGAAGAATTTGATGAAACCATTGAACAGGATATCCCGATT ATTCATAGCCAGCAGCCTGCACGTCTGCCGCTGCTGCCGAGCAAGCAGATTAATATGCAGTGGCTGAGCCAAGA AATTCATGTTCCGAGCGATCGTTGTACCGTTGCATATCGTCGTTGGCTGAAAGAACTGGGCGTTACCTATGGTG TTTGTTGAgaaggagatatacatATGCAGATTCTGGGTATCAGCGCCTATTATCATGATAGCGCAGCAGCAATG GTTATTGATGGTGAAATTGTTGCAGCAGCACAAGAAGAACGTTTTAGCCGTCGTAAACATGATGCAGGTTTTCC GACCGGTGCAATTACCTATTGTCTGAAACAGGTTGGCACCAAACTGCAGTATATTGATCAGATCGTGTTCTATG ATAAACCGCTGGTGAAATTTGAACGTCTGCTGGAAACCTATCTGGCCTATGCACCGAAAGGTTTTGGTAGTTTT ATTACCGCAATGCCGGTGTGGCTGAAAGAGAAACTGTATCTGAAAACCCTGCTGAAAAAAGAACTGGCACTGCT GGGTGAATGTAAAGCAAGCCAGCTGCCTCCGCTGCTGTTTACCAGCCATCATCAGGCACATGCAGCAGCAGCAT TTTTTCCGAGCCCGTTTCAGCGTGCAGCAGTTCTGTGTCTGGATGGTGTTGGTGAATGGGCAACCACCAGTGTT TGGCTGGGTGAAGGTAATAAACTGACACCGCAGTGGGAAATTGATTTTCCGCATAGCCTGGGCCTGCTGTATAG CGCATTTACCTATTATACCGGCTTTAAAGTGAACAGCGGTGAGTATAAACTGATGGGTCTGGCACCGTATGGTG AACCGAAATATGTTGATCAGATTCTGAAACATCTGCTGGATCTGAAAGAAGATGGCACCTTTCGTCTGAACATG GATTATTTCAATTATACCGTTGGTCTGACCATGACCAACCATAAATTTCATAGCATGTTTGGTGGTCCGCCTCG TCAGGCAGAAGGTAAAATTAGCCAGCGTGATATGGATCTGGCAAGCAGCATTCAGAAAGTTACCGAAGAAGTGA TTCTGCGTCTGGCACGTACCATTAAGAAAGAATTAGGTGTTGAATACCTGTGTCTGGCAGGCGGTGTTGGTCTG AATTGTGTTGCAAATGGTCGTATTCTGCGTGAGAGCGATTTTAAAGATATTTGGATTCAGCCTGCAGCCGGTGA TGCAGGTAGCGCAGTTGGTGCAGCACTGGCAATTTGGCATGAATATCATAAAAAACCGCGTACCAGCACCGCAG GCGATCGTATGAAAGGTAGCTATCTGGGTCCGAGCTTTAGCGAAGCAGAAATTCTGCAGTTTCTGAACAGCGTG AATATTCCGTATCATCGTTGTGTGGATAATGAACTGATGGCACGTCTGGCGGAAATTCTGGATCAGGGTAATGT TGTTGGTTGGTTTAGCGGTCGTATGGAATTTGGTCCGCGTGCACTGGGTGGTCGTAGCATTATTGGTGATAGCC GTAGCCCGAAAATGCAGAGCGTTATGAATCTGAAAATCAAATATCGCGAAAGCTTCCGTCCGTTTGCACCGAGC GTTCTGGCAGAACGTGTTAGCGATTATTTTGATCTGGATCGTCCGAGCCCGTATATGCTGCTGGTTGCACAGGT TAAAGAAAATCTGCATATTCCGATGACCCAAGAACAGCATGAACTGTTTGGTATCGAAAAACTGAATGTTCCGC GTAGCCAGATTCCGGCAGTTACCCATGTTGATTATAGCGCACGTATTCAGACCGTTCATAAAGAAACCAATCCG CGTTATTATGAACTGATCCGTCATTTTGAAGCACGTACCGGTTGTGCAGTTCTGGTTAATACCAGCTTTAATGT TCGTGGTGAACCGATTGTGTGTACACCGGAAGATGCATATCGTTGTTTTATGCGTACCGAGATGGATTACCTGG TGATGGAAAATTTTCTGCTGGTGAAAAGCGAACAGCCTCGTGGTAATAGTGATGAAAGCTGGCAGAAAGAATTT GAGCTGGATTGAgaaggagatatacatATGGAACAAATTAAAGAACTGGATAAGAAAGGCCTGCGTGAATTTGG TCTGATTGGTGGTAGCATTGTTGCCGTTCTGTTTGGTTTTCTGCTGCCGGTTATTCGTCATCATAGCCTGAGCG TTATTCCGTGGGTTGTTGCAGGTTTTCTGTGGATTTGGGCAATTATTGCACCGACCACCCTGAGCTTTATCTAT CAGATTTGGATGCGTATTGGTCTGGTGCTGGGTTGGATTCAGACCCGTATTATTCTGGGTGTTCTGTTCTATAT TATGATTACCCCGATCGGTTTTATTCGTCGTCTGCTGAATCAGGATCCGATGACCCGTATTTTTGAACCGGAAC TGCCGACCTATCGTCAGCTGAGCAAAAGCCGTACCACCCAGAGCATGGAAAAACCGTTCTGAgaaggagatata catATGTTAAAAGACACCTGGGATTTTATCAAGGATATCGCAGGCTTTATCAAAGAACAGAAAAACTATCTGCT GATTCCGCTGATTATTACCCTGGTTAGCCTGGGTGCACTGATTGTTTTTGCACAGAGCAGCGCAATTGCACCGT TTATCTATACCCTGTTTTGAgaaggagatatacatATGAGCAACTTCAAAGGCAGCGTTAAAATTGCACTGATG GGCATTCTGATTTTTTGCGGTCTGATTTTTGGTGTGGCCTTTGTTGAAATTGGTCTGCGTATTGCAGGCATTGA ACATATTGCCTTTCATAGCATTGATGAACATCGTGGTTGGGTTGGTCGTCCGCATGTTAGCGGTTGGTATCGTA CCGAAGGTGAAGCACATATTCAGATGAATAGTGATGGTTTTCGTGATCGCGAACACATTAAAGTGAAACCGGAA AATACCTTTCGTATTGCCCTGCTGGGTGATAGCTTTGTTGAAAGCATGCAGGTTCCGCTGGAACAGAATCTGGC AGCAGTTATTGAAGGCGAAATTAGCAGCTGTATTGCACTGGCAGGTCGTAAAGCCGAAGTTATTAACTTTGGTG TTACCGGTTATGGCAOCGATCAAGAACTGATTACCCTGCGTGAAAAAGTGTGGGATTATAGTCCGGATATTGTT GTGCTGGATTTCTATACCGGTAACGATATTGTTGATAATAGCCGTGCACTGTCCCAGAAATTCTATCCGAATGA ACTGGGTAGCCTGAAACCGTTTTTTATCCTGCGTGATGGTAATCTGGTTGTTGATGCAAGCTTTATCAACACCG ATAACTATCGTAGCAAACTGACCTGGTGGGGTAAAACCTATATGAAAATCAAAGATCATAGCCGCATTCTGCAG GTCCTGAATATGGTTCGTGATGCACTGAATAATAGCAGCCGTGGTTTTAGCAGCCAGGCAATTGAAGAACCGCT GTTTAGTGATGGTAAACAGGATACCAAACTGAGCGGCTTCTTCGATATCTATAAACCGCCTACCGATCCGGAAT GGCAGCAGGCCTGGCAGGTTACCGAAAAACTGATTAGTAGCATGCAGCATGAAGTGACCGCCAAAAAAGCCGAT TTTCTGGTTGTTACCTTTGGCGGTCCGTTTCAGCGCGAACCGCTGGTTCGTCAGAAAGAAATGCAAGAACTGGG TCTGACCGATTGGTTTTATCCGGAAAAACGTATTACCCGTCTGGGTGAAGATGAAGGTTTTAGCGTGCTGAATC TGAGCCCGAATCTGCAGGTTTATAGCGAACAGAATAATGCCTGTCTGTATGGTTTTGATGATACCCAGGGTTGT GTTGGTCATTGGAATGCACTGGGTCATCAGGTTGCAGGTAAAATGATTGCAAGCAAAATTTGTCAGCAGCAGAT GCGTGAAAGCATTCTGCCGCATAAACATGATCCGAGCAGCCAGAGCAGCCCGATTACCCAGAGCGTTATTCAGT AAtactotaaccocatcggccgtottaggggttttttgtcgaagttcctattctotagaaagtataggaacttc gacctgtggggtgactttgccgccgctgccgtgatgtctgcattaccgatc SEQ ID NO: 98: Sequence of sxt2 fragment integrated into the maltose operon after deletetion of sxtL. Flanking regions of the maltose operon at integration site are included. sxt open reading frames are indicated by upper case letters. ctgtgaactaaaccgaggtcatgtaaggaatttcgtgatgttgcttgcaaccagccttgcaagaagcggataca ggagtgcaaaaaatggctatctctagaaaggcctaccccttaggctttatgcaacagaaacaataataatggag tcatgaacATGATTGATACCATTAGCGTTCTGCTGCGTGAATGGACCGTTATTTTTCTGACCGGTCTGGCATTT TGGCTGTGGGAAATTCGTAGTCCGCTGCATCAGATTGAATACAAAGCCAAATTTTTCAAAGAACTGGGTTGGGC AGGTATCAGCTTTGTTTTTCGTATTGTTTATGCCTATGTTAGCGTGGCCATTATCAAACTGCTGAGCAGCCTGT TTATGGGTGAAAGCGCAAATTTTGCCGGTGTTATGTATGTTCCGCTGTGGCTGCGTATTATTACCGCATATATT CTGCAGGATCTGACCGATTATCTGCTGCATCGTACCATGCATAGCAATCAGTTTCTGTGGCTGACCCATAAATG GCATCATAGCACCAAACAGAGTTGGTGGCTGAGCGGTAATAAAGATAGCTTTACCGGTGGTCTGCTGTATACCG TTACCGCACTGTGGTTTCCGCTGCTGGATATTCCGAGCGAAGTTATGAGCGTTGTTGCAGTTCATCAGGTGATT CATAACAACTGGATTCACCTGAATGTGAAATGGAATAGCTGGCTGGGTATTATCGAATGGATTTATGTTACACC GCGTATCCATACCCTGCATCATCTGGATACCGGTGGTCGTAATCTGAGCAGTATGTTTACCTTTATTGATCGTC TGTTTGGCACCTATGTGTTTCCGGAAAACTTTGATATCGAAAAAAGCAAAAACCGCCTGGATGATCAGAGCGTT ACCGTTAAAACCATTCTGGGTTTCTGAgaaggagatatacatATGCTGAAAGATTTTAACCAGTTCCTGATTCG TACCCTGGCATTTGTTTTTGCCTTTGGCATTTTTCTGACAACCGGTGTTGGTATTGCAAAAGCAGATTATCTGG TGAAAGGTGGCAAAATTACCAATGTTCAGAATACCAGCAGCAACGGTGATAATTATGCAGTTAGCATTAGCGGT GGTTTTGGTCCGTGTGCAGATCGTGTTATTATTCTGCCGACCAGCGGTGTTATTAATCGTGATATTCACATGCG TGGTTATGAAGCAGCACTGACCGCACTGAGCAATGGTTTTCTGGTTGATATCTATGATTATACCGGTAGCAGCT GTAGCAATGGTGGCCAGCTGACCATTACCAATCAGCTGGGTAAACTGATTAGCAATTGAgaaggagatatacat ATGACCAATCAGAACAACCAAGAGCTGGAAAATGATCTGCCGATTGCAAAACAGCCGTGTCCGGTTAATAGCTA TAATGAATGGGATACCCTGGAAGAAGTTATTGTTGGTAGCGTTGAAGGTGCAATGCTGCCTGCACTGGAACCGA TTAACAAATGGACCTTTCCGTTTGAAGAACTGGAAAGCGCACAGAAAATTCTGAGCGAACGTGGTGGTGTTCCG TATCCGCCTGAAATGATTACCCTGGCACATAAAGAACTGAACGAGTTTATTCATATCCTGGAAGCCGAAGGTGT TAAAGTTCGTCGTGTTAAACCGGTTGATTTTAGCGTTCCGTTTAGCACACCGGCATGGCAGGTTGGTAGCGGTT TTTGTGCAGCAAATCCGCGTGATGTTTTTCTGGTTATTGGCAACGAAATTATCGAAGCACCGATGGCAGATCGT AATCGTTATTTTGAAACCTGGGCATATCGCGAAATGCTGAAAGAATATTTTCAGGCAGGCGCAAAATGGACCGC AGCACCGAAACCGCAGCTGTTTGATGCACAGTATGATTTCAATTTTCAGTTTCCGCAGCTGGGTGAACCGCCTC GTTTTGTTGTTACCGAATTTGAACCGACCTTTGATGCAGCCGATTTTGTTCGTTGTGGTCGTGATATTTTTGGC CAGAAAAGCCATGTTACCAATGGTCTGGGTATTGAATGGCTGCAGCGTCATCTGGAAGATGAATATCGCATTCA TATCATCGAAAGCCATTGTCCGGAAGCACTGCATATTGATACCACCCTGATGCCGCTGGCACCGGGTAAAATTC TGGTTAATCCGGAATTTGTGGACGTGAATAAACTGCCGAAAATTCTGAAAAGCTGGGATATTCTGGTTGCACCG TATCCGAATCATATTCCGCAGAATCAGCTGCGTCTGGTTAGCGAATGGGCAGGTCTGAATGTTCTGATGCTGGA TGAAGAACGTGTGATCGTGGAAAAAAATCAAGAGCAGATGATCAAAGCCCTGAAAGATTGGGGTTTTAAACCGA TTGTTTGCCACTTCGAAAGCTATTATCCGTTTCTGGGTAGCTTTCATTGTGCAACCCTGGATGTTCGTCGTCGT GGCACCCTGCAGAGCTATTTTTGAgaaggagatatacatATGACGACCGCAGATCTGATTCTGATCAATAATTG GTATGTTGTGGCCAAGGTGGAAGATTGTAAACCGGGTAGCATTACCACCGCACTGCTGCTGGGTGTTAAACTGG TTCTGTGGCGTAGCCGTGAACAGAATAGCCCGATTCAGATTTGGCAGGATTATTGTCCGCATCGTGGTGTTGCA CTGAGCATGGGTGAAATTGTGAATAATACCCTGGTTTGTCCGTATCATGGTTGGCGTTATAATCAGGCAGGTAA ATGTGTTCATATTCCGGCACATCCGGATATGACCCCTCCGGCAAGCGCACAGGCAAAAATCTATCATTGTCAAG AACGTTATGGTCTGGTTTGGGTTTGTCTGGGTGATCCGGTTAATGATATTCCGAGTCTGCCGGAATGGGATGAT CCGAATTATCATAATACCTGCACCAAGAGCTACTTTATCCAGGCAAGCGCATTTCGTGTGATGGATAACTTTAT TGATGTGAGCCATTTTCCGTTTGTGCATGATGGTGGTCTGGGCGATCGTAATCATGCACAGATTGAAGAATTTG AGGTGAAAGTGGATAAAGACGGTATTAGCATTGGCAATCTGAAACTGCAGATGCCTCGTTTTAATAGCAGCAAT GAAGATGATAGCTGGACCCTGTATCAGCGTATTAGCCATCCGCTGTGTCAGTATTATATCACCGAAAGCAGCGA AATTCGTACAGCAGATCTGATGCTGGTTACCCCGATTGATGAAGATAATTCACTGGTTCGTATGCTGGTGACCT GGAATCGTAGCGAAATTCTGGAAAGCACCGTTCTGGAAGAATTTGATGAAACCATTGAACAGGATATCCCGATT ATTCATAGCCAGCAGCCTGCACGTCTGCCGCTGCTGCCGAGCAAGCAGATTAATATGCAGTGGCTGAGCCAAGA AATTCATGTTCCGAGCGATCGTTGTACCGTTGCATATCGTCGTTGGCTGAAAGAACTGGGCGTTACCTATGGTG TTTGTTGAgaaggagatatacatATGCAGATTCTGGGTATCAGCGCCTATTATCATGATAGCGCAGCAGCAATG GTTATTGATGGTGAAATTGTTGCAGCAGCACAAGAAGAACGTTTTAGCCGTCGTAAACATGATGCAGGTTTTCC GACCGGTGCAATTACCTATTGTCTGAAACAGGTTGGCACCAAACTGCAGTATATTGATCAGATCGTGTTCTATG ATAAACCGCTGGTGAAATTTGAACGTCTGCTGGAAACCTATCTGGCCTATGCACCGAAAGGTTTTGGTAGTTTT ATTACCGCAATGCCGGTGTGGCTGAAAGAGAAACTGTATCTGAAAACCCTGCTGAAAAAAGAACTGGCACTGCT GGGTGAATGTAAAGCAAGCCAGCTGCCTCCGCTGCTGTTTACCAGCCATCATCAGGCACATGCAGCAGCAGCAT TTTTTCCGAGCCCGTTTCAGCGTGCAGCAGTTCTGTGTCTGGATGGTGTTGGTGAATGGGCAACCACCAGTGTT TGGCTGGGTGAAGGTAATAAACTGACACCGCAGTGGGAAATTGATTTTCCGCATAGCCTGGGCCTGCTGTATAG CGCATTTACCTATTATACCGGCTTTAAAGTGAACAGCGGTGAGTATAAACTGATGGGTCTGGCACCGTATGGTG AACCGAAATATGTTGATCAGATTCTGAAACATCTGCTGGATCTGAAAGAAGATGGCACCTTTCGTCTGAACATG GATTATTTCAATTATACCGTTGGTCTGACCATGACCAACCATAAATTTCATAGCATGTTTGGTGGTCCGCCTCG TCAGGCAGAAGGTAAAATTAGCCAGCGTGATATGGATCTGGCAAGCAGCATTCAGAAAGTTACCGAAGAAGTGA TTCTGCGTCTGGCACGTACCATTAAGAAAGAATTAGGTGTTGAATACCTGTGTCTGGCAGGCGGTGTTGGTCTG AATTGTGTTGCAAATGGTCGTATTCTGCGTGAGAGCGATTTTAAAGATATTTGGATTCAGCCTGCAGCCGGTGA TGCAGGTAGCGCAGTTGGTGCAGCACTGGCAATTTGGCATGAATATCATAAAAAACCGCGTACCAGCACCGCAG GCGATCGTATGAAAGGTAGCTATCTGGGTCCGAGCTTTAGCGAAGCAGAAATTCTGCAGTTTCTGAACAGCGTG AATATTCCGTATCATCGTTGTGTGGATAATGAACTGATGGCACGTCTGGCGGAAATTCTGGATCAGGGTAATGT TGTTGGTTGGTTTAGCGGTCGTATGGAATTTGGTCCGCGTGCACTGGGTGGTCGTAGCATTATTGGTGATAGCC GTAGCCCGAAAATGCAGAGCGTTATGAATCTGAAAATCAAATATCGCGAAAGCTTCCGTCCGTTTGCACCGAGC GTTCTGGCAGAACGTGTTAGCGATTATTTTGATCTGGATCGTCCGAGCCCGTATATGCTGCTGGTTGCACAGGT TAAAGAAAATCTGCATATTCCGATGACCCAAGAACAGCATGAACTGTTTGGTATCGAAAAACTGAATGTTCCGC GTAGCCAGATTCCGGCAGTTACCCATGTTGATTATAGCGCACGTATTCAGACCGTTCATAAAGAAACCAATCCG CGTTATTATGAACTGATCCGTCATTTTGAAGCACGTACCGGTTGTGCAGTTCTGGTTAATACCAGCTTTAATGT TCGTGGTGAACCGATTGTGTGTACACCGGAAGATGCATATCGTTGTTTTATGCGTACCGAGATGGATTACCTGG TGATGGAAAATTTTCTGCTGGTGAAAAGCGAACAGCCTCGTGGTAATAGTGATGAAAGCTGGCAGAAAGAATTT GAGCTGGATTGAgaaggagatatacatATGGAACAAATTAAAGAACTGGATAAGAAAGGCCTGCGTGAATTTGG TCTGATTGGTGGTAGCATTGTTGCCGTTCTGTTTGGTTTTCTGCTGCCGGTTATTCGTCATCATAGCCTGAGCG TTATTCCGTGGGTTGTTGCAGGTTTTCTGTGGATTTGGGCAATTATTGCACCGACCACCCTGAGCTTTATCTAT CAGATTTGGATGCGTATTGGTCTGGTGCTGGGTTGGATTCAGACCCGTATTATTCTGGGTGTTCTGTTCTATAT TATGATTACCCCGATCGGTTTTATTCGTCGTCTGCTGAATCAGGATCCGATGACCCGTATTTTTGAACCGGAAC TGCCGACCTATCGTCAGCTGAGCAAAAGCCGTACCACCCAGAGCATGGAAAAACCGTTCTGAgaaggagatata catATGTTAAAAGACACCTGGGATTTTATCAAGGATATCGCAGGCTTTATCAAAGAACAGAAAAACTATCTGCT GATTCCGCTGATTATTACCCTGGTTAGCCTGGGTGCACTGATTGTTTTTGCACAGAGCAGCGCAATTGCACCGT TTATCTATACCCTGTTTTGAtactctaaccccatcggccgtcttaggggttttttgtcgaagttcctattctct agaaagtataggaacttcacctgtggggtgactttgccgccgctgccgtgatgtctgcattaccgatc SEQ ID NO: 99: Sequence of sxt2 fragment integrated into the maltose operon after deletetion of sxtJ, sxtK and sxtL. Flanking regions of the maltose operon at integration site are included. sxt open reading frames are indicated by upper case letters. ctgtgaactaaaccgaggtcatgtaaggaatttcgtgatgttgcttgcaaccagccttgcaagaagcggataca ggagtgcaaaaaatggctatctctagaaaggcctaccccttaggctttatgcaacagaaacaataataatggag tcatgaacATGATTGATACCATTAGCGTTCTGCTGCGTGAATGGACCGTTATTTTTCTGACCGGTCTGGCATTT TGGCTGTGGGAAATTCGTAGTCCGCTGCATCAGATTGAATACAAAGCCAAATTTTTCAAAGAACTGGGTTGGGC AGGTATCAGCTTTGTTTTTCGTATTGTTTATGCCTATGTTAGCGTGGCCATTATCAAACTGCTGAGCAGCCTGT TTATGGGTGAAAGCGCAAATTTTGCCGGTGTTATGTATGTTCCGCTGTGGCTGCGTATTATTACCGCATATATT CTGCAGGATCTGACCGATTATCTGCTGCATCGTACCATGCATAGCAATCAGTTTCTGTGGCTGACCCATAAATG GCATCATAGCACCAAACAGAGTTGGTGGCTGAGCGGTAATAAAGATAGCTTTACCGGTGGTCTGCTGTATACCG TTACCGCACTGTGGTTTCCGCTGCTGGATATTCCGAGCGAAGTTATGAGCGTTGTTGCAGTTCATCAGGTGATT CATAACAACTGGATTCACCTGAATGTGAAATGGAATAGCTGGCTGGGTATTATCGAATGGATTTATGTTACACC GCGTATCCATACCCTGCATCATCTGGATACCGGTGGTCGTAATCTGAGCAGTATGTTTACCTTTATTGATCGTC TGTTTGGCACCTATGTGTTTCCGGAAAACTTTGATATCGAAAAAAGCAAAAACCGCCTGGATGATCAGAGCGTT ACCGTTAAAACCATTCTGGGTTTCTGAgaaggagatatacatATGCTGAAAGATTTTAACCAGTTCCTGATTCG TACCCTGGCATTTGTTTTTGCCTTTGGCATTTTTCTGACAACCGGTGTTGGTATTGCAAAAGCAGATTATCTGG TGAAAGGTGGCAAAATTACCAATGTTCAGAATACCAGCAGCAACGGTGATAATTATGCAGTTAGCATTAGCGGT GGTTTTGGTCCGTGTGCAGATCGTGTTATTATTCTGCCGACCAGCGGTGTTATTAATCGTGATATTCACATGCG TGGTTATGAAGCAGCACTGACCGCACTGAGCAATGGTTTTCTGGTTGATATCTATGATTATACCGGTAGCAGCT GTAGCAATGGTGGCCAGCTGACCATTACCAATCAGCTGGGTAAACTGATTAGCAATTGAgaaggagatatacat ATGACCAATCAGAACAACCAAGAGCTGGAAAATGATCTGCCGATTGCAAAACAGCCGTGTCCGGTTAATAGCTA TAATGAATGGGATACCCTGGAAGAAGTTATTGTTGGTAGCGTTGAAGGTGCAATGCTGCCTGCACTGGAACCGA TTAACAAATGGACCTTTCCGTTTGAAGAACTGGAAAGCGCACAGAAAATTCTGAGCGAACGTGGTGGTGTTCCG TATCCGCCTGAAATGATTACCCTGGCACATAAAGAACTGAACGAGTTTATTCATATCCTGGAAGCCGAAGGTGT TAAAGTTCGTCGTGTTAAACCGGTTGATTTTAGCGTTCCGTTTAGCACACCGGCATGGCAGGTTGGTAGCGGTT TTTGTGCAGCAAATCCGCGTGATGTTTTTCTGGTTATTGGCAACGAAATTATCGAAGCACCGATGGCAGATCGT AATCGTTATTTTGAAACCTGGGCATATCGCGAAATGCTGAAAGAATATTTTCAGGCAGGCGCAAAATGGACCGC AGCACCGAAACCGCAGCTGTTTGATGCACAGTATGATTTCAATTTTCAGTTTCCGCAGCTGGGTGAACCGCCTC GTTTTGTTGTTACCGAATTTGAACCGACCTTTGATGCAGCCGATTTTGTTCGTTGTGGTCGTGATATTTTTGGC CAGAAAAGCCATGTTACCAATGGTCTGGGTATTGAATGGCTGCAGCGTCATCTGGAAGATGAATATCGCATTCA TATCATCGAAAGCCATTGTCCGGAAGCACTGCATATTGATACCACCCTGATGCCGCTGGCACCGGGTAAAATTC TGGTTAATCCGGAATTTGTGGACGTGAATAAACTGCCGAAAATTCTGAAAAGCTGGGATATTCTGGTTGCACCG TATCCGAATCATATTCCGCAGAATCAGCTGCGTCTGGTTAGCGAATGGGCAGGTCTGAATGTTCTGATGCTGGA TGAAGAACGTGTGATCGTGGAAAAAAATCAAGAGCAGATGATCAAAGCCCTGAAAGATTGGGGTTTTAAACCGA TTGTTTGCCACTTCGAAAGCTATTATCCGTTTCTGGGTAGCTTTCATTGTGCAACCCTGGATGTTCGTCGTCGT GGCACCCTGCAGAGCTATTTTTGAgaaggagatatacatATGACGACCGCAGATCTGATTCTGATCAATAATTG GTATGTTGTGGCCAAGGTGGAAGATTGTAAACCGGGTAGCATTACCACCGCACTGCTGCTGGGTGTTAAACTGG TTCTGTGGCGTAGCCGTGAACAGAATAGCCCGATTCAGATTTGGCAGGATTATTGTCCGCATCGTGGTGTTGCA CTGAGCATGGGTGAAATTGTGAATAATACCCTGGTTTGTCCGTATCATGGTTGGCGTTATAATCAGGCAGGTAA ATGTGTTCATATTCCGGCACATCCGGATATGACCCCTCCGGCAAGCGCACAGGCAAAAATCTATCATTGTCAAG AACGTTATGGTCTGGTTTGGGTTTGTCTGGGTGATCCGGTTAATGATATTCCGAGTCTGCCGGAATGGGATGAT CCGAATTATCATAATACCTGCACCAAGAGCTACTTTATCCAGGCAAGCGCATTTCGTGTGATGGATAACTTTAT TGATGTGAGCCATTTTCCGTTTGTGCATGATGGTGGTCTGGGCGATCGTAATCATGCACAGATTGAAGAATTTG AGGTGAAAGTGGATAAAGACGGTATTAGCATTGGCAATCTGAAACTGCAGATGCCTCGTTTTAATAGCAGCAAT GAAGATGATAGCTGGACCCTGTATCAGCGTATTAGCCATCCGCTGTGTCAGTATTATATCACCGAAAGCAGCGA AATTCGTACAGCAGATCTGATGCTGGTTACCCCGATTGATGAAGATAATTCACTGGTTCGTATGCTGGTGACCT GGAATCGTAGCGAAATTCTGGAAAGCACCGTTCTGGAAGAATTTGATGAAACCATTGAACAGGATATCCCGATT ATTCATAGCCAGCAGCCTGCACGTCTGCCGCTGCTGCCGAGCAAGCAGATTAATATGCAGTGGCTGAGCCAAGA AATTCATGTTCCGAGCGATCGTTGTACCGTTGCATATCGTCGTTGGCTGAAAGAACTGGGCGTTACCTATGGTG TTTGTTGAgaaggagatatacatATGCAGATTCTGGGTATCAGCGCCTATTATCATGATAGCGCAGCAGCAATG GTTATTGATGGTGAAATTGTTGCAGCAGCACAAGAAGAACGTTTTAGCCGTCGTAAACATGATGCAGGTTTTCC GACCGGTGCAATTACCTATTGTCTGAAACAGGTTGGCACCAAACTGCAGTATATTGATCAGATCGTGTTCTATG ATAAACCGCTGGTGAAATTTGAACGTCTGCTGGAAACCTATCTGGCCTATGCACCGAAAGGTTTTGGTAGTTTT ATTACCGCAATGCCGGTGTGGCTGAAAGAGAAACTGTATCTGAAAACCCTGCTGAAAAAAGAACTGGCACTGCT GGGTGAATGTAAAGCAAGCCAGCTGCCTCCGCTGCTGTTTACCAGCCATCATCAGGCACATGCAGCAGCAGCAT TTTTTCCGAGCCCGTTTCAGCGTGCAGCAGTTCTGTGTCTGGATGGTGTTGGTGAATGGGCAACCACCAGTGTT TGGCTGGGTGAAGGTAATAAACTGACACCGCAGTGGGAAATTGATTTTCCGCATAGCCTGGGCCTGCTGTATAG CGCATTTACCTATTATACCGGCTTTAAAGTGAACAGCGGTGAGTATAAACTGATGGGTCTGGCACCGTATGGTG AACCGAAATATGTTGATCAGATTCTGAAACATCTGCTGGATCTGAAAGAAGATGGCACCTTTCGTCTGAACATG GATTATTTCAATTATACCGTTGGTCTGACCATGACCAACCATAAATTTCATAGCATGTTTGGTGGTCCGCCTCG TCAGGCAGAAGGTAAAATTAGCCAGCGTGATATGGATCTGGCAAGCAGCATTCAGAAAGTTACCGAAGAAGTGA TTCTGCGTCTGGCACGTACCATTAAGAAAGAATTAGGTGTTGAATACCTGTGTCTGGCAGGCGGTGTTGGTCTG AATTGTGTTGCAAATGGTCGTATTCTGCGTGAGAGCGATTTTAAAGATATTTGGATTCAGCCTGCAGCCGGTGA TGCAGGTAGCGCAGTTGGTGCAGCACTGGCAATTTGGCATGAATATCATAAAAAACCGCGTACCAGCACCGCAG GCGATCGTATGAAAGGTAGCTATCTGGGTCCGAGCTTTAGCGAAGCAGAAATTCTGCAGTTTCTGAACAGCGTG AATATTCCGTATCATCGTTGTGTGGATAATGAACTGATGGCACGTCTGGCGGAAATTCTGGATCAGGGTAATGT TGTTGGTTGGTTTAGCGGTCGTATGGAATTTGGTCCGCGTGCACTGGGTGGTCGTAGCATTATTGGTGATAGCC GTAGCCCGAAAATGCAGAGCGTTATGAATCTGAAAATCAAATATCGCGAAAGCTTCCGTCCGTTTGCACCGAGC GTTCTGGCAGAACGTGTTAGCGATTATTTTGATCTGGATCGTCCGAGCCCGTATATGCTGCTGGTTGCACAGGT TAAAGAAAATCTGCATATTCCGATGACCCAAGAACAGCATGAACTGTTTGGTATCGAAAAACTGAATGTTCCGC GTAGCCAGATTCCGGCAGTTACCCATGTTGATTATAGCGCACGTATTCAGACCGTTCATAAAGAAACCAATCCG CGTTATTATGAACTGATCCGTCATTTTGAAGCACGTACCGGTTGTGCAGTTCTGGTTAATACCAGCTTTAATGT TCGTGGTGAACCGATTGTGTGTACACCGGAAGATGCATATCGTTGTTTTATGCGTACCGAGATGGATTACCTGG TGATGGAAAATTTTCTGCTGGTGAAAAGCGAACAGCCTCGTGGTAATAGTGATGAAAGCTGGCAGAAAGAATTT GAGCTGGATTGAtactctaaccccatcggccgtcttaggggttttttgtcgaagttcctattctctagaaagta taggaacttcacctgtggggtgactttgccgccgctgccgtgatgtctgcattaccgatc

(155) TABLE-US-00012 SEQUENCE LISTING FREE TEXT SEQ ID NO: 91  Sequence of sxt1 fragment after integration into E. coli lactose operon.  SEQ ID NO: 92  Sequence of sxt3 fragment version 1 integrated into the E. coli xylose operon.  SEQ ID NO: 93  Sequence of sxt3 fragment version 2 integrated into the E. coli xylose operon.  SEQ ID NO: 94  Sequence of sxt3 fragment version 3 integrated into the E. coli xylose operon.  SEQ ID NO: 95  Sequence of sxt1 fragment, Integrated into the E. coli lactose operon.  SEQ ID NO: 96  Sequence of sxt4 fragment integrated into the E. coli melobiose operon.  SEQ ID NO: 97  Sequence of sxt2 fragment integrated into the E. coli maltose operon.  SEQ ID NO: 98  Sequence of sxt2 fragment integrated into the maltose operon after deletion of sxtL. SEQ ID NO: 99  Sequence of sxt2 fragment integrated into the maltose operon.