The present invention pertains to a probiotic comprising a generally recognized as safe (GRAS) microbiological organism, which GRAS microbiolog ical organism comprises a food-grade expression vector, which vector com prises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100, wherein said GRAS microbiological organism is capable of ex pressing and secreting said soluble form of Amuc_1100 or said fragment thereof, as further defined in the claims. Methods for treating a disease in a pa tient, comprising oral administration of the probiotic as defined herein are also described, as well as methods of preparing the probiotic disclosed herein.

Background of the invention

In 2001 , the World Health organization (WHO) defined in a report pro biotics as live microorganisms that, "when administered in adequate amounts, confer a health benefit on the host." Following this definition, a working group of the Food and Agriculture Organization (FAO)/WHO issued the Guidelines for the Evaluation of Probiotics in Food in 2002. A consensus definition of the term probiotics, based on available information and scientific evidence, was adopted after the aforementioned joint expert consultation between the FAO of theUnited Nations and the WHO.

The National Center for Complementary and Integrative Health de scribe probiotics as live microorganisms that are intended to have health bene fits when consumed or applied to the body. They are usually provided in form of yoghurt and other fermented foods, dietary supplements, and beauty products. Some bacteria are considered to help in digesting food, destroy disease-causing cells, or produce vitamins. Administration of probiotics is intended to induce changes in the microbiome in the gut, often in order to promote growth of micro organisms which are considered beneficial over those which are considered det rimental. Another mode of action of probiotics is considered by interactions be tween the probiotic microorganism and the host.

Ottman et al. (PLOS ONE (2017), 12(3): e0173004; doi: 10.1371 /jour nal. phone.0173004) disclose that the gut symbiont Akkermansia muciniphilia is positively correlated with a lean physiology, reduced body weight gain, amelio ration or metabolic responses and restoration of gut barrier function by modula tion of mucus layer thickness. The authors identified some of these beneficial effects to be due to an outer membrane pili-like protein named Amuc_1100. When expressed in a non food-grade expression vector as a purification-tagged protein in the non-GRAS microorganism E. coli, and following its purification, the purified protein was found to be a strong TLR2 activator and inducer of inter alia IL-10. Ottman et al. finally suggest the use of gram-negative Akkermansia mu- ciniphilia as a probiotic.

Similarly, Plovier et al. (Nature Medicine 2016; doi: 10.10387nm.4236) report that a purified His-tagged form of the membrane pro tein Amuc_1100 from Akkermansia muciniphila (expressed in E. coli) or the pas teurized Akkermansia muciniphila bacterium improves metabolism in obese and diabetic mice. Plovier et al. conclude that either live or pasteurized A. Mucini phila (i.e. the bacterium) grown on synthetic medium are a promising therapeutic tool in the management of metabolic syndrome.

Toll-like receptor 2 (TLR2), also designated as CD282, is a receptor of the Toll-like receptor (TLR) family, which plays a fundamental role in the re- cognition of pathogen-associated molecular patterns (PAMPs) that are expres sed on infectious agents. Upon activation, TLRs mediate the production of cyto kines necessary for modulating the immune response. TLR2 is expressed most abundantly in peripheral blood leukocytes, and mediates host response to mainly gram-positive bacteria, and yeast via stimulation of NF-KB. However, TLR2 recognizes many bacterial, viral and fungal compounds, as well as certain endogenous substances. In the intestine, TLR2 regulates the expression of CYP1A1, an enzyme which is key in detoxication of certain carcinogenic sub stances. Recently, it was found that TLR2 is involved in the activation of regula tory T cells (Tregs), that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. TLR2 is also expressed by intestinal epithelial cells and subsets of lamina propria mononuclear cells in the gastrointestinal tract. TLR2 has been observed downregulated in human papillomavirus-positive neoplastic keratocyt- res derived from uterine cervical preneoplastic lesions. Thus, TLR2 is assumed to be associated with tumorigenesis.

Often the microorganisms in probiotic foods are the same or similar to the ones naturally abundant in the human body. In contrast thereto, prebiotics are non-digestable food components that selectively stimulate the growth or ac- tivity of certain microorganisms. The term synbiotics commonly refers to prod ucts that combine probiotics and prebiotics. Nguyen et al. (J. Agric. Food Chem. 2011, 59, 5617-5624) discloses a food-grade system for inducible gene expression in Lactobacillus plantarum.

In 2015, the global retail market value for probiotics was US$41 bil lion, including sales of probiotic dietary supplements, fermented dairy products, and yoghurt, the latter accounting for 75% of total consumption. In 2015 supp lements produced US$4 billion and their growth is projected to be as high as 37% globally by 2020. At the same time, consumption of probiotic yoghurt in China has increased by 20% per year since 2014.

There is an existing need in the art for new useful probiotics, which exhibit and combine beneficial health effects. Such probiotics may suitably be applied in the treatment of diseases, including obesity and diabetes.

Brief explanation of the invention

The aforementioned need is addressed by the present invention, which is characterized by improving the health benefit of a generally recognized as safe (GRAS) microbiological organism, by incorporating into said GRAS mi crobiological organism a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100, such that the GRAS microbiological organism is capable of ex- pressing and secreting said soluble form of Amuc_1100 or said fragment thereof.

The invention is particularly advantageous for embodiments, wherein the GRAS microbiological organism is selected from the group of organisms consisting of a gram-positive bacteria and a gram-negative bacteria. This is be- cause it is expected that the beneficial effects reported for Amuc_1100, in par ticular its Toll-like receptor 2 (TLR-2) agonistic activity, will further improve the beneficial health effects which are ascribed to the induction of TLR-2 by PAMPs found in the membrane of these microorganisms. A particular advantageous benefit is to be expected in embodiments, wherein the GRAS microbiological organism is a gram-positive bacteria belonging to the order of lactic acid bacte ria.

To the Applicant’s best knowledge, there is no suggestion in the prior art to express a soluble form of Amuc_1100, or a functionally equivalent soluble fragment thereof, in a probiotic GRAS microbiological organism, let alone in a GRAS microorganism of the embodiments described herein. Rather, prior to the present invention, it was suggested to use live or pasteurized Akkermansia muciniphila. However, in the context of the invention, the GRAS microorganism is not Akkermansia muciniphila. In the alternative, a His-tagged Amuc_1100 pro tein was produced in E. coli and used in purified form for research purposes. In contrast, in embodiments of the present invention, said soluble form of Amuc_1100 or a fragment of said soluble form of Amuc_1100 does not need to comprise such a purification tag, and need not to be purified.

Moreover, while food-grade expression systems are disclosed for pri mary use in organisms of the genus Lactobacillus, in embodiments these ex pression systems are used in genus other than Lactobacillus, where these food- grade expression vectors are also functional. In this context, in embodiments said food-grade expression vector carries the SH71rep replicon, which has a broad functionality. Usually, said food-grade expression vector may carry a food- grade selection marker, which provides prototrophy to an otherwise auxotroph GRAS microbiological organism. In embodiments, the marker is alanine race- mase (air).

In embodiments, the nucleic acid sequence in said food-grade ex pression vector encodes a soluble form of Amuc_1100 having an amino acid sequence with at least 80% identity to SEQ ID NO: 2 (Amuc_1100). In embodi ments, said nucleic acid sequence encodes for a fragment of said soluble form of Amuc_1100, which has a length of at least 100 and up to 286 amino acids. Said nucleic acid sequence may also be optimized for expression in the genus selected from the group of Lactobacillus, Akkermanisa, Bifidobacterium, Bacil lus, Brevibacillus, Lactococcus and Saccharomyces and/or wherein said nucleic acid sequence is optimized for expression in one of the genus of GRAS micro- biological organisms as disclosed herein, and/or wherein said nucleic acid se quence is optimized for expression in one of the species of GRAS microbiologi cal organisms as disclosed herein.

Hence, in embodiments said nucleic acid sequence has at least 70% identity to SEQ ID NO: 1 (Amuc_1100). One useful example of said food-grade expression vector is p3050alrAmuc_1100-sh71 (SEQ ID NO: 9) or p3050Alr_Amuc1100-sh71 with 5’UTR, 3’UTR and terminator (SEQ ID NO: 15).

In a further aspect, the present invention also pertains to a method of treating a disease in a patient, comprising the step of administering orally, intra- colonally, or rectally a probiotic of the present invention. In embodiments, the disease is selected from the group consisting of obesity, diabetes, hypercholes terolemia, and/or the patient is a human patient. Further provided is a method for preparing a prebiotic according to the present invention, wherein the method comprises the step of introducing a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a function- ally equivalent fragment of said soluble form of Amuc_1100, into a GRAS micro biological organism, such that said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof.

Other objectives, aspects, embodiments, details and advantages of the present invention will become apparent from the following figures, detailed description, examples, and dependent claims.

Brief description of the drawings

Figure 1 shows a vector map of p3050alarAmuc1100-sh71 (SEQ ID NO: 9). Figure 2 shows a vector map of p3050alarAmuc1100_alcA-al1b1-sh71

(SEQ ID NO: 11).

Figure 3 shows a vector map of p3050Alr_Amuc1100_sh71 with 5’UTR, 3’UTR and terminator (SEQ ID NO: 15).

Figure 4 shows a vector map of pRac-DAO-lactase-int-attP-Amuc-aldh- fruct (SEQ ID NO: 33).

Brief description of the sequences

The present disclosure is accompanied by a sequence listing, which forms part of the present disclosure in its entirety.

Nucleic acid sequence encoding Amuc_1100 without its signal sequence (aa 1- 30), (SEQ ID NO: 1): atcgtcaatt ccaaacgcag tgaactggac aaaaaaatca gcatcgccgc caaggaaatc 60 aagtccgcca atgctgcgga aatcactccg agccgatcat ccaacgaaga gctggaaaaa 120 gaactgaacc gctatgccaa ggccgtgggc agcctggaaa cggcctacaa gcccttcctt 180 gcctcctccg cgctggtccc caccacgccc acggcattcc agaatgaact gaaaacattc 240 agggattccc tgatctcctc ctgcaagaaa aagaacattc tcataacgga cacatcctcc 300 tggctcggtt tccaggttta cagcacccag gctccctctg ttcaggcggc ctccacgctg 360 ggttttgaat tgaaagccat caacagcctg gtcaacaaac tggcggaatg cggcctgtcc 420 aaattcatca aggtgtaccg cccccagctc cccattgaaa ccccggcgaa caatccggaa 480 gaatcggacg aagccgacca ggccccatgg actcccatgc ctctggaaat agccttccag 540 ggcgaccggg aaagtgtatt gaaagccatg aacgccataa ccggcatgca ggactatctg 600 ttcacggtca actccatccg tatccgcaac gaacggatga tgccccctcc catcgccaat 660 ccggcagccg ccaaacctgc cgcggcccaa cccgccacgg gtgcggcttc cctgactccg 720 gcggatgagg cggctgcacc tgcagccccg gccatccagc aagtcatcaa gccttacatg 780 ggcaaggagc aggtctttgt ccaggtctcc ctgaatctgg tccacttcaa ccagcccaag 840 gctcaggaac cgtctgaaga ttaa 864

Amino acid sequence of Amuc 1100 without its signal sequence (aa 1-30),

(SEQ ID NO: 2): I V N S K R S E L D K K I S I A A K E I K S A N A A E I T P S R S S N E E L E K E L N R Y A K A V G S L E T A Y K P F L A S S A L V P T T P T A F Q N E L K T F R D S L I S S C K K K N I L I T D T S S

Amuc_1100 D1-30 sequence optimized for Bifidobacterium (SEQ ID NO: 3): attgtgaact ccaagcgctc cgagctggac aagaagatca gcattgccgc taaggagatc 60 aagtccgcca atgctgccga gatcacgccc tccaggagca gcaacgagga gctggaaaag 120 gagctgaacc ggtatgccaa agcggtgggt agcctggaaa ccgcgtacaa acccttcctt 180 gcgtcctcgg cgctcgttcc gaccaccccg acggccttcc agaacgagct caagacgttc 240 cgcgactccc tcatctcgtc ctgcaagaag aagaacatcc tcatcaccga tacgagctcc 300 tggttgggct tccaggtgta ctccacccag gccccgtcgg tccaagccgc ctcgaccttg 360 ggcttcgaac tgaaggccat caactccctg gtgaacaagc tggccgaatg cgggctgtcc 420 aagttcatca aggtgtatcg tccgcagctc cccatcgaaa ccccggccaa caaccccgag 480 gaatccgacg aggccgatca ggcgccctgg accccgatgc ctctcgagat cgcctttcag 540 ggcgatcgcg agtccgtgct gaaggcgatg aacgccatca ccggcatgca ggactacctt 600 ttcacggtga acagcatccg catccggaac gagcgcatga tgccgccgcc gattgcgaat 660 ccggcggccg cgaaaccggc agctgcccaa ccggccactg gagcagccag cctgacccct 720 gcggacgagg cagccgctcc tgcagctccg gcgatccaac aggtcatcaa gccgtacatg 780 ggcaaggaac aggtgttcgt ccaggtttcc ctgaacctgg tccacttcaa ccagcccaaa 840 gcccaggaac cgtcggagga ctga 864 Amuc_1100 D1-30 sequence optimized for Bacillus species (SEQ ID NO: 4): attgtgaact caaaacggtc tgagttggac aagaaaatca gcatagctgc aaaagagatc 60 aaatccgcaa acgcagcaga aattacgccg tcaagaagtt ccaacgaaga gctggagaaa 120 gaactgaatc gctatgccaa agcggttgga tcacttgaaa cggcatacaa gccgtttctt 180 gcgagctctg cccttgtacc gacaacaccg acagcgttcc aaaacgaact gaaaacattt 240 cgtgacagcc ttatatcttc ctgcaagaag aagaacatcc tcatcactga tacaagctct 300 tggttaggct ttcaggtgta tagcacacaa gcaccttcag ttcaagcggc atcaacgtta 360 ggctttgagc tgaaagccat caattcgttg gtgaacaaac ttgcggaatg tggcttatcg 420 aagtttatca aagtctatcg tccgcaatta cccattgaaa ccccagcaaa taaccctgaa 480 gaatcggatg aggcggatca agccccttgg accccaatgc ctttggaaat tgcctttcag 540 ggtgatagag aatctgtttt aaaagccatg aatgcgatta ccggaatgca ggactatctg 600 ttcacggtca atagtattcg cattcgaaat gagaggatga tgccaccgcc gattgctaat 660 cctgcagccg ctaaaccagc tgctgctcaa ccggcaactg gagctgcaag tctgactcct 720 gcggatgaag cggctgctcc agctgcccct gcgattcaac aggtaatcaa accgtacatg 780 gggaaagaac aggtatttgt ccaggtttca ttgaatctcg tgcatttcaa tcagccgaaa 840 gcccaagaac ccagcgaaga ttaa 864

Amuc_1100 D1-30 sequence optimized for Brevibacillus species (SEQ ID NO: 5): atcgtcaata gcaaacgcag tgaactggac aagaaaatct ccattgccgc aaaagagatc 60 aaatccgcaa acgctgccga aatcactccc tctcgtagtt ctaacgagga actggagaaa 120 gaactgaatc gctatgctaa agccgtaggc tctctggaaa ccgcgtacaa accgtttctt 180 gcgtcctctg cattggtccc caccacaccg accgcgtttc agaatgagct gaaaaccttc 240 cgcgattctc tgatctcgag ctgcaagaag aagaacatcc tcatcaccga cacatcgtcc 300 tggttgggat tccaagtata ctccacgcaa gctccaagcg tacaagcggc atcgactctt 360 ggctttgagc tgaaagctat caactccctc gttaacaagc tcgcggagtg tggcctttcc 420 aaattcatca aggtgtatcg acctcagctg ccaatcgaaa ctccggctaa caaccctgaa 480 gaatccgatg aagcagatca agccccatgg actccgatgc cactggaaat cgcgtttcaa 540 ggtgaccgtg aatccgtact gaaagccatg aacgcaatca cggggatgca agactacttg 600 ttcacggtga actccattcg cattcgcaat gaacgcatga tgccacctcc aattgcgaat 660 cctgcagctg caaaaccagc tgcggcacaa cccgctacag gtgcggcatc cttgactccg 720 gcagacgaag ctgctgctcc agctgcgcct gcaatccagc aagtgatcaa accctatatg 780 ggcaaagaac aggttttcgt acaggtttcc ctgaatctgg tgcatttcaa ccaaccgaaa 840 gcgcaagaac cttccgaaga ttaa 864 Amuc_1100 D1-30 sequence optimized for Lactococcus species (SEQ ID NO: 6): atagttaaca gcaaacgatc agagttagac aagaaaattt caattgcagc aaaggagata 60 aaatctgcca atgctgctga gattactccc tctagaagtt caaacgaaga acttgagaaa 120 gaattgaata gatatgcgaa agcggttggt tcacttgaaa ccgcgtataa accgtttcta 180 gcgagttctg ccttagtacc aactacacca acggcatttc agaatgaact taaaactttt 240 agagacagct taatttcatc atgcaagaag aagaacatac ttattacaga tacctcatca 300 tggttaggat ttcaggttta tagtactcaa gctccttcag ttcaagccgc atcaacgttg 360 ggttttgagt tgaaagcgat taatagctta gtaaacaaac ttgctgaatg tgggttgagt 420 aaatttatca aagtctatag accgcaatta cctattgaaa ctcccgctaa taatccagaa 480 gaaagtgatg aagcagatca agcaccatgg acacctatgc ctttggaaat tgcctttcaa 540 ggagatcgag aaagtgtttt aaaagccatg aatgcaatta caggaatgca agattactta 600 ttcaccgtca attctattcg tatccgtaat gaacgcatga tgcctccacc tattgcaaat 660 cctgcagctg ctaaaccggc tgcagcacaa ccagctacag gtgcagcttc tctaacacca 720 gccgatgaag ctgctgctcc agctgcacca gccatacaac aggtaatcaa accttatatg 780 ggcaaagaac aagtgtttgt tcaagtgtct ttaaatttag ttcatttcaa tcaaccaaaa 840 gctcaagaac catcagaaga ttaa 864

Amuc_1100 D1-30 sequence optimized for Saccharomyces (SEQ ID NO: 7): attgttaatt ctaagagatc cgaactggac aagaaaatct cgattgcagc gaaggaaatc 60 aaatcggcta atgcagctga aatcactcct tcaaggtcta gtaacgagga attggagaaa 120 gaattgaaca gatatgctaa agcagttggt agcttggaaa cagcctataa accgttctta 180 gcatctagcg cattagttcc aaccactcca acagcgtttc agaatgaact gaaaacgttt 240 agagacagct tgattagttc ttgcaagaag aagaacatct tgataacaga caccagttca 300 tggttaggct ttcaagtata ctctactcaa gcaccatcag ttcaagctgc atccactttg 360 ggattcgagt taaaggccat aaactcactt gtgaacaaac ttgctgaatg tggtctatcc 420 aagttcatca aagtttacag accccagtta ccgattgaaa ctcccgcaaa taatcctgaa 480 gagtcagatg aagccgatca agctccttgg acacctatgc ctctagaaat tgcttttcag 540 ggtgatagag agagtgtatt gaaagcgatg aatgccatta caggtatgca agattaccta 600 tttaccgtaa attccattag gatacgtaac gagagaatga tgccaccacc aattgccaat 660 cctgctgcag ccaaacccgc tgccgctcaa ccagcgactg gagcagcatc tcttacgcca 720 gccgatgaag ctgcagctcc agctgctcct gccatacaac aggtgataaa accctatatg 780 gggaaagaac aggtctttgt ccaagtctcg ttgaatttag tgcatttcaa ccaaccaaag 840 gctcaagaac cgtctgagga ttaa 864

Alanine racemase (air) (SEQ ID NO: 8) atgcaagcgg caactgttgt gattaaccgc cgcgctctgc gacacaacct gcaacgtctt 60 cgtgaactgg cccctgccag taaaatggtt gcggtggtga aagcgaacgc ttatggtcac 120 ggtcttcttg agaccgcgcg aacgctcccc gatgctgacg cctttggcgt agcccgtctc 180 gaagaagctc tgcgactgcg tgcgggggga atcaccaaac ctgtactgtt actcgaaggc 240 ttttttgatg ccagagatct gccgacgatt tctgcgcaac attttcatac cgccgtgcat 300 aacgaagaac agctggctgc gctggaagag gctagcctgg acgagccggt taccgtctgg 360 atgaaactcg ataccggtat gcaccgtctg ggcgtaaggc cggaacaggc tgaggcgttt 420 tatcatcgcc tgacccagtg caaaaacgtt cgtcagccgg tgaatatcgt cagccatttt 480 gcgcgcgcgg atgaaccaaa atgtggcgca accgagaaac aactcgctat ctttaatacc 540 ttttgcgaag gcaaacctgg tcaacgttcc attgccgcgt cgggtggcat tctgctgtgg 600 ccacagtcgc attttgactg ggtgcgcccg ggcatcattc tttatggcgt ctcgccgctg 660 gaagatcgct ccaccggtgc cgattttggc tgtcagccag tgatgtcact aacctccagc 720 ctgattgccg tgcgtgagca taaagccgga gagcctgttg gttatggtgg aacctgggta 780 agcgaacgtg atacccgtct tggcgtagtc gcgatgggct atggcgatgg ttatccgcgc 840 gccgcgccgt ccggtacgcc agtgctggtg aacggtcgcg aagtaccgat tgtcgggcgc 900 gtggcgatgg atatgatctg cgtagactta ggtccacagg cgcaggacaa agccggggat 960 ccggtcattt tatggggcga aggtttgccc gtagaacgta tcgctgaaat gacgaaagtal020 agcgcttacg aacttattac gcgcctgact tcaagggtcg cgatgaaata cgtggattaal080 p3050Alr_Amuc1100_sh71 (SEQ ID NO: 9) atatgaaaaa atttaacttt aaaaccatgt tgctattagt tttggctagt tgtgtcttcg 60 gggtcgtcgt taacgtgact actagtcttg gaccacaaac cgcaatcacc gcccaggcct 120 ccaaggtcga catcgtcaat tccaaacgca gtgaactgga caaaaaaatc agcatcgccg 180 ccaaggaaat caagtccgcc aatgctgcgg aaatcactcc gagccgatca tccaacgaag 240 agctggaaaa agaactgaac cgctatgcca aggccgtggg cagcctggaa acggcctaca 300 agcccttcct tgcctcctcc gcgctggtcc ccaccacgcc cacggcattc cagaatgaac 360 tgaaaacatt cagggattcc ctgatctcct cctgcaagaa aaagaacatt ctcataacgg 420 acacatcctc ctggctcggt ttccaggttt acagcaccca ggctccctct gttcaggcgg 480 cctccacgct gggttttgaa ttgaaagcca tcaacagcct ggtcaacaaa ctggcggaat 540 gcggcctgtc caaattcatc aaggtgtacc gcccccagct ccccattgaa accccggcga 600 acaatccgga agaatcggac gaagccgacc aggccccatg gactcccatg cctctggaaa 660 tagccttcca gggcgaccgg gaaagtgtat tgaaagccat gaacgccata accggcatgc 720 aggactatct gttcacggtc aactccatcc gtatccgcaa cgaacggatg atgccccctc 780 ccatcgccaa tccggcagcc gccaaacctg ccgcggccca acccgccacg ggtgcggctt 840 ccctgactcc ggcggatgag gcggctgcac ctgcagcccc ggccatccag caagtcatca 900 agccttacat gggcaaggag caggtctttg tccaggtctc cctgaatctg gtccacttca 960 accagcccaa ggctcaggaa ccgtctgaag attaaaagct tcaaattaca gcacgtgttg 1020 ctttgattga tagccaaaaa gcagcagttg ataaagcaat tactgatatt gctgaaaaat 1080 tgtaatttat aaataaaaat caccttttag aggtggtttt tttatttata aattattcgt 1140 ttgatttcgc tttcgataga acaatcaaag cgagaataag gaagataaat cccataaggg 1200 cgggagcaga atgtccgaga ctaattcatg gatcgatttt ttattaaaac gtctcaaaat 1260 cgtttctgag acgttttagc gtttatttcg tttagttatc ggcataatcg ttaaaacagg 1320 cgttatcgta gcgtaaaagc ccttgagcgt agcgtgcttt gcagcgaaga tgttgtctgt 1380 tagattatga aagccgatga ctgaatgaaa taataagcgc agcgtccttc tatttcggtt 1440 ggaggaggct caagggagtt tgagggaatg aaattccctc atgggtttga ttttaaaaat 1500 tgcttgcaat tttgccgagc ggtagcgctg gaaaaatttt tgaaaaaaat ttggaatttg 1560 gaaaaaaatg gggggaaagg aagcgaattt tgcttccgta ctacgacccc ccattaagtg 1620 ccgagtgcca atttttgtgc caaaaacgct ctatcccaac tggctcaagg gtttgagggg 1680 tttttcaatc gccaacgaat cgccaacgtt ttcgccaacg ttttttataa atctatattt 1740 aagtagcttt attgttgttt ttatgattac aaagtgatac actaatttta taaaattatt 1800 tgattggagt tttttaaatg gtgatttcag aatcgaaaaa aagagttatg atttctctga 1860 caaaagagca agataaaaaa ttaacagata tggcgaaaca aaaaggtttt tcaaaatctg 1920 cggttgcggc gttagctata gaagaatatg caagaaagga atcagaataa aaaaaataag 1980 cgaaagctcg cgtttttaga aggatacgag ttttcgctac ttgtttttga taaggtaata 2040 tatcatggct attaaatact aaagctagaa attttggatt tttattatat cctgactcaa 2100 ttcctaatga ttggaaagaa aaattagaga gtttgggcgt atctatggct gtcagtcctt 2160 tacacgatat ggacgaaaaa aaagataaag atacatggaa tagtagtgat gttatacgaa 2220 atggaaagca ctataaaaaa ccacactatc acgttatata tattgcacga aatcctgtaa 2280 caatagaaag cgttaggaac aagattaagc gaaaattggg gaatagttca gttgctcatg 2340 ttgagatact tgattatatc aaaggttcat atgaatattt gactcatgaa tcaaaggacg 2400 ctattgctaa gaataaacat atatacgaca aaaaagatat tttgaacatt aatgattttg 2460 atattgaccg ctatataaca cttgatgaaa gccaaaaaag agaattgaag aatttacttt 2520 tagatatagt ggatgactat aatttggtaa atacaaaaga tttaatggct tttattcgcc 2580 ttaggggagc ggagtttgga attttaaata cgaatgatgt aaaagatatt gtttcaacaa 2640 actctagcgc ctttagatta tggtttgagg gcaattatca gtgtggatat agagcaagtt 2700 atgcaaaggt tcttgatgct gaaacggggg aaataaaatg acaaacaaag aaaaagagtt 2760 atttgctgaa aatgaggaat taaaaaaaga aattaaggac ttaaaagagc gtattgaaag 2820 atacagagaa atggaagttg aattaagtac aacaatagat ttattgagag gagggattat 2880 tgaataaata aaagcccccc tgacgaaagt cgaagggggc ttttattttg gtttgatgtt 2940 gcgattaata gcaatacgat tgcaataaac aaaaggatcc atgcaagcgg caactgttgt 3000 gattaaccgc cgcgctctgc gacacaacct gcaacgtctt cgtgaactgg cccctgccag 3060 taaaatggtt gcggtggtga aagcgaacgc ttatggtcac ggtcttcttg agaccgcgcg 3120 aacgctcccc gatgctgacg cctttggcgt agcccgtctc gaagaagctc tgcgactgcg 3180 tgcgggggga atcaccaaac ctgtactgtt actcgaaggc ttttttgatg ccagagatct 3240 gccgacgatt tctgcgcaac attttcatac cgccgtgcat aacgaagaac agctggctgc 3300 gctggaagag gctagcctgg acgagccggt taccgtctgg atgaaactcg ataccggtat 3360 gcaccgtctg ggcgtaaggc cggaacaggc tgaggcgttt tatcatcgcc tgacccagtg 3420 caaaaacgtt cgtcagccgg tgaatatcgt cagccatttt gcgcgcgcgg atgaaccaaa 3480 atgtggcgca accgagaaac aactcgctat ctttaatacc ttttgcgaag gcaaacctgg 3540 tcaacgttcc attgccgcgt cgggtggcat tctgctgtgg ccacagtcgc attttgactg 3600 ggtgcgcccg ggcatcattc tttatggcgt ctcgccgctg gaagatcgct ccaccggtgc 3660 cgattttggc tgtcagccag tgatgtcact aacctccagc ctgattgccg tgcgtgagca 3720 taaagccgga gagcctgttg gttatggtgg aacctgggta agcgaacgtg atacccgtct 3780 tggcgtagtc gcgatgggct atggcgatgg ttatccgcgc gccgcgccgt ccggtacgcc 3840 agtgctggtg aacggtcgcg aagtaccgat tgtcgggcgc gtggcgatgg atatgatctg 3900 cgtagactta ggtccacagg cgcaggacaa agccggggat ccggtcattt tatggggcga 3960 aggtttgccc gtagaacgta tcgctgaaat gacgaaagta agcgcttacg aacttattac 4020 gcgcctgact tcaagggtcg cgatgaaata cgtggattaa acacgttact aaagggaatg 4080 gagaccgggg cccttcaata gagttcttaa cgttaatccg aaaaaaacta acgttaatat 4140 taaaaaataa gatccgcttg tgaattatgt ataatttgat tagactaaag aataggagaa 4200 agtatgatga tatttaaaaa actttctcgt taagataggt tgttggtgag catgttatat 4260 acggatgtat cggtttcctt aatgcaaaat tttgttgcta tcttattaat ttttctatta 4320 tatagatata ttcaaagaaa gataacattt aaacggatca tattagatat tttaatagcg 4380 attatttttt caatattata tctgtttatt tcagatgcgt cattacttgt aatggtatta 4440 atgcgattag ggtggcattt tcatcaacaa aaagaaaata agataaaaac gactgataca 4500 gctaatttaa ttctaattat cgtgatccag ttattgttag ttgcggttgg gactattatt 4560 agtcagttta ccatatcgat tatcaaaagt gatttcagcc aaaatatatt gaacaatagt 4620 gcaacagata taactttatt aggtattttc tttgctgttt tatttgacgg cttgttcttt 4680 atattattga agaataagcg gactgaatta caacatttaa atcaagaaat cattgaattt 4740 tcgttagaaa aacaatattt tatatttata tttattttat ttatagtaat agaaattatt 4800 ttagcagttg ggaatcttca aggagtaaca gccacgatat tattaaccat tatcattatt 4860 ttttgtgtcc ttatcgggat gactttttgg caagtgatgc tttttttgaa ggcttattcg 4920 attcgccaag aagccaatga ccaattggtc cggaatcaac aacttcaaga ttatctagtc 4980 aatatcgaac agcagtacac cgaattacgg cgatttaagc atgattatca aaacatctta 5040 ttatcgttgg agagttttgc cgaaaagggc gatcagcaac agtttaaggc gtattaccaa 5100 gaattattag cacaacggcc aattcaaagt gaaatccaag gggcagtcat tgcacaactc 5160 gactacttga aaaatgatcc tattcgagga ttagtcattc aaaagttttt ggcagccaaa 5220 caggctggtg ttactttaaa attcgaaatg accgaaccaa tcgaattagc aaccgctaat 5280 ctattaacgg ttattcggat tatcggtatt ttattagaca atgcgattga acaagccgtt 5340 caagaaaccg atcaattggt gagttgtgct ttcttacaat ctgatggttt aatcgaaatt 5400 acgattgaaa atacggccag tcaagttaag aatctccaag cattttcaga gttaggctat 5460 tcaacgaaag gcgctggtcg ggggactggt ttagctaatg tgcaggattt gattgccaaa 5520 caaaccaatt tattcttaga aacacagatt gaaaatagaa agttacgaca gacattgatg 5580 attacggagg aaacttaatt tgtatcccgt ttatttatta gaggatgatt tacagcaaca 5640 agcgatttat cagcaaatta tcgcgaatac gattatgatt aacgaatttg caatgacttt 5700 aacatgcgct gccagtgata ctgagacatt gttggcggca attaaggatc agcaacgagg 5760 tttattcttt ttggatatgg aaattgagga taaccgccaa gccggtttag aagtggcaac 5820 taagattcgg cagatgatgc cgtttgcgca aattgtcttc attacaaccc acgaggaact 5880 gacattatta acgttagaac gaaaaatagc gcctttagat tacattctca aggaccaaac 5940 aatggctgaa atcaaaaggc aattgattga tgatctattg ttagctgaga agcaaaacga 6000 ggcggcagcg tatcaccgag aaaatttatt tagttataaa ataggtcctc gctttttctc 6060 attaccatta aaggaagttg tttatttata tactgaaaaa gaaaatccgg gtcatattaa 6120 tttgttagcc gttaccagaa aggttacttt tccaggaaat ttaaatgcgc tggaagccca 6180 atatccaatg ctctttcggt gtgataaaag ttacttagtt aacctatcta atattgccaa 6240 ttatgacagt aaaacacgga gtttaaaatt tgtagatggc agtgaggcaa aagtctcgtt 6300 ccggaaatca cgggaactag tggccaaatt aaaacaaatg atgtagcgcc tgcagcacgc 6360 caaatgatcc cagtaaaaag ccacccgcat ggcgggtggc tttttattag ccctagaagg 6420 gcttcccaca cgcatttcag cgccttagtg ccttagtttg tgaatcatag gtggtatagt 6480 cccgaaatac ccgtctaagg aattgtcaga taggcctaat gactggcttt tataatatga 6540 gataatgccg actgtacttt ttacagtcgg ttttctaatg tcactaacct gccccgttag 6600 ttgaagaagg tttttatatt acagctccag atctaccggt gggcccatat taacgtttaa 6660 ccgataaagt tgaacgttaa tatttttttt gcgcagaaat ggtaaattga agcataatag 6720 tcttgtaagg tatttagctg gctggcgtaa agtatgcttt ataaaataat atataggagt 6780 atgattc 6787 human aldehyde dehydrogenase 1B1 (UNIPROT SEQ: P30837; SEQ ID NO: 10):

V D R A V K A A R E A F R L G S P W R R M D A S E

R G R L L N L L A D L V E R D R V Y L A S L E T L D N G K P F Q E S Y A L D L D E V I K V Y R Y F A G W A D K W H G K T I P M D G Q H F C F T R H E P

V G V C G Q I I P W N F P L V M Q G W K L A P A L Q H V D V D K V A F T G S T E V G H L I Q K A A G D S N L K R V T L E L G G K S P S I V L A D A D M E H A V E Q C H E A L F F N M G Q C C C A G S R T F V E E S I Y N E F L E R T V E K A K Q R K V G N P F E L D T Q Q G P Q V D K E Q F E R V L G Y I Q L G Q K E G A K L L C G G E R F G E R G F F I K P T V F G G V Q D D M R I A K E E I F G P V Q P L F K F K K I E E V V E R A N N T R Y G L A A A V F T R D L D K A M Y F T Q A L Q A G T V W V N T Y N I V T C H T P F G G F K E S G N G R E L G E D G L K A Y T E V K T V T I K V P Q K N S p3050alarAmuc_1100_alcA-al1 b1 -sh71 (SEQ ID NO: 11) atatgaaaaa atttaacttt aaaaccatgt tgctattagt tttggctagt tgtgtcttcg 60 gggtcgtcgt taacgtgact actagtcttg gaccacaaac cgcaatcacc gcccaggcct 120 ccaaaggagg tatcgtcaat tccaaacgca gtgaactgga caaaaaaatc agcatcgccg 180 ccaaggaaat caagtccgcc aatgctgcgg aaatcactcc gagccgatca tccaacgaag 240 agctggaaaa agaactgaac cgctatgcca aggccgtggg cagcctggaa acggcctaca 300 agcccttcct tgcctcctcc gcgctggtcc ccaccacgcc cacggcattc cagaatgaac 360 tgaaaacatt cagggattcc ctgatctcct cctgcaagaa aaagaacatt ctcataacgg 420 acacatcctc ctggctcggt ttccaggttt acagcaccca ggctccctct gttcaggcgg 480 cctccacgct gggttttgaa ttgaaagcca tcaacagcct ggtcaacaaa ctggcggaat 540 gcggcctgtc caaattcatc aaggtgtacc gcccccagct ccccattgaa accccggcga 600 acaatccgga agaatcggac gaagccgacc aggccccatg gactcccatg cctctggaaa 660 tagccttcca gggcgaccgg gaaagtgtat tgaaagccat gaacgccata accggcatgc 720 aggactatct gttcacggtc aactccatcc gtatccgcaa cgaacggatg atgccccctc 780 ccatcgccaa tccggcagcc gccaaacctg ccgcggccca acccgccacg ggtgcggctt 840 ccctgactcc ggcggatgag gcggctgcac ctgcagcccc ggccatccag caagtcatca 900 agccttacat gggcaaggag caggtctttg tccaggtctc cctgaatctg gtccacttca 960 accagcccaa ggctcaggaa ccgtctgaag attaatactt gaaaaaaaaa aaccccgccc 1020 ctgacagggc ggggtttttt tttccattgt ggtgatcgtt ccgacatgct tgtctgcatg 1080 ggtttctgcg tgtcgggact caagtgatct ggggcttgat gcatgtggga cagcacgagg 1140 tagaggtgga aactgacata cgactccgtt acatgccccg tttaagcgct atgcgtatcg 1200 tgccgtctaa tcccgtgatg gagcgttatc aggcacagta cggactggat gccctcatgg 1260 cgaaccacaa acctcaggag ctccctacgt actgagctat ccgcgcattg cttcgcctca 1320 tagctaaacg ggcatgacac acaatccgac catactcagg aaaacgcttc cactgtacaa 1380 agaggtccac ttcatctgga gaggccctag gaggtatgct cagattcttg gcgcctcgcc 1440 ttcttagcct ccaaggacgt acagccagat attcaagtgc agcagctctt ccgagcccga 1500 ttctcaatcc ggatattccg tataaccaac tgttcattaa caacgagtgg caagacgcag 1560 taagcaagaa aacgtttccg acagtcaatc caactaccgg agaagtgatc ggccacgttg 1620 cagaaggtga tcgggccgat gtcgatcgtg cagttaaagc tgcgagagag gctttcaggc 1680 ttgggtcccc atggcggagg atggatgctt cggaacgtgg cagactgctc aatctgttag 1740 ctgatcttgt agagcgagat cgggtatatc tggcatctct ggaaacactg gacaatggga 1800 agccatttca ggaatcctat gcccttgatc tggatgaggt gattaaggtg tatcgctatt 1860 ttgctggctg ggcagataag tggcatggga aaacaatacc gatggacggc cagcactttt 1920 gctttaccag acatgaacct gttggagtat gtggtcaaat cataccctgg aactttccgc 1980 tggtaatgca aggctggaaa ttagcacccg cgttagcgac gggtaataca gtggtcatga 2040 aagtagctga gcaaacgccg ctttcagcct tgtatttagc ctctcttatc aaagaagctg 2100 gatttcctcc gggtgttgtt aacatcatta caggatacgg ccctacagct ggcgcggcaa 2160 tcgcgcaaca tgtggacgta gacaaagtcg cctttactgg ctcaaccgaa gtcgggcatc 2220 tgatccagaa agctgctggc gatagcaact tgaaacgcgt tacactggag ttaggaggaa 2280 aatctccgag tattgtctta gcggatgcag atatggaaca tgctgttgaa cagtgccatg 2340 aagccttatt cttcaacatg ggtcagtgct gttgtgcggg atctcgtacc tttgtggaag 2400 agtccattta caatgaattt ctggaacgta ccgttgagaa ggcgaaacaa cgcaaagtcg 2460 gaaatccgtt tgagctggac acgcaacaag gtccacaagt ggacaaagaa cagtttgaaa 2520 gagttttggg ctacattcag ctcggacaga aagaaggagc caagttactt tgcggaggcg 2580 aacgatttgg tgaacggggt ttcttcatca aaccaactgt ctttggtgga gtgcaggatg 2640 acatgaggat tgcgaaagaa gagattttcg gccctgtgca acctctgttc aaatttaaga 2700 aaatcgaaga agttgtggaa agagccaaca atacgcggta tggccttgcg gcggcagtct 2760 ttactcgcga tttagacaag gcgatgtact ttacgcaagc cttgcaggca gggacagttt 2820 gggtgaatac gtataacatt gttacatgtc acacaccttt tggaggcttt aaagagtcag 2880 ggaatggacg agaattgggc gaagatgggt tgaaagcata cactgaggtc aaaacagtca 2940 cgataaaagt accccagaag aattcgtaat acttgaaaaa aaaaaacccc gcccctgaca 3000 gggcggggtt ttttttcatg gatcgatttt ttattaaaac gtctcaaaat cgtttctgag 3060 acgttttagc gtttatttcg tttagttatc ggcataatcg ttaaaacagg cgttatcgta 3120 gcgtaaaagc ccttgagcgt agcgtgcttt gcagcgaaga tgttgtctgt tagattatga 3180 aagccgatga ctgaatgaaa taataagcgc agcgtccttc tatttcggtt ggaggaggct 3240 caagggagtt tgagggaatg aaattccctc atgggtttga ttttaaaaat tgcttgcaat 3300 tttgccgagc ggtagcgctg gaaaaatttt tgaaaaaaat ttggaatttg gaaaaaaatg 3360 gggggaaagg aagcgaattt tgcttccgta ctacgacccc ccattaagtg ccgagtgcca 3420 atttttgtgc caaaaacgct ctatcccaac tggctcaagg gtttgagggg tttttcaatc 3480 gccaacgaat cgccaacgtt ttcgccaacg ttttttataa atctatattt aagtagcttt 3540 attgttgttt ttatgattac aaagtgatac actaatttta taaaattatt tgattggagt 3600 tttttaaatg gtgatttcag aatcgaaaaa aagagttatg atttctctga caaaagagca 3660 agataaaaaa ttaacagata tggcgaaaca aaaaggtttt tcaaaatctg cggttgcggc 3720 gttagctata gaagaatatg caagaaagga atcagaataa aaaaaataag cgaaagctcg 3780 cgtttttaga aggatacgag ttttcgctac ttgtttttga taaggtaata tatcatggct 3840 attaaatact aaagctagaa attttggatt tttattatat cctgactcaa ttcctaatga 3900 ttggaaagaa aaattagaga gtttgggcgt atctatggct gtcagtcctt tacacgatat 3960 ggacgaaaaa aaagataaag atacatggaa tagtagtgat gttatacgaa atggaaagca 4020 ctataaaaaa ccacactatc acgttatata tattgcacga aatcctgtaa caatagaaag 4080 cgttaggaac aagattaagc gaaaattggg gaatagttca gttgctcatg ttgagatact 4140 tgattatatc aaaggttcat atgaatattt gactcatgaa tcaaaggacg ctattgctaa 4200 gaataaacat atatacgaca aaaaagatat tttgaacatt aatgattttg atattgaccg 4260 ctatataaca cttgatgaaa gccaaaaaag agaattgaag aatttacttt tagatatagt 4320 ggatgactat aatttggtaa atacaaaaga tttaatggct tttattcgcc ttaggggagc 4380 ggagtttgga attttaaata cgaatgatgt aaaagatatt gtttcaacaa actctagcgc 4440 ctttagatta tggtttgagg gcaattatca gtgtggatat agagcaagtt atgcaaaggt 4500 tcttgatgct gaaacggggg aaataaaatg acaaacaaag aaaaagagtt atttgctgaa 4560 aatgaggaat taaaaaaaga aattaaggac ttaaaagagc gtattgaaag atacagagaa 4620 atggaagttg aattaagtac aacaatagat ttattgagag gagggattat tgaataaata 4680 aaagcccccc tgacgaaagt cgaagggggc ttttattttg gtttgatgtt gcgattaata 4740 gcaatacgat tgcaataaac aaaaggatcc atgcaagcgg caactgttgt gattaaccgc 4800 cgcgctctgc gacacaacct gcaacgtctt cgtgaactgg cccctgccag taaaatggtt 4860 gcggtggtga aagcgaacgc ttatggtcac ggtcttcttg agaccgcgcg aacgctcccc 4920 gatgctgacg cctttggcgt agcccgtctc gaagaagctc tgcgactgcg tgcgggggga 4980 atcaccaaac ctgtactgtt actcgaaggc ttttttgatg ccagagatct gccgacgatt 5040 tctgcgcaac attttcatac cgccgtgcat aacgaagaac agctggctgc gctggaagag 5100 gctagcctgg acgagccggt taccgtctgg atgaaactcg ataccggtat gcaccgtctg 5160 ggcgtaaggc cggaacaggc tgaggcgttt tatcatcgcc tgacccagtg caaaaacgtt 5220 cgtcagccgg tgaatatcgt cagccatttt gcgcgcgcgg atgaaccaaa atgtggcgca 5280 accgagaaac aactcgctat ctttaatacc ttttgcgaag gcaaacctgg tcaacgttcc 5340 attgccgcgt cgggtggcat tctgctgtgg ccacagtcgc attttgactg ggtgcgcccg 5400 ggcatcattc tttatggcgt ctcgccgctg gaagatcgct ccaccggtgc cgattttggc 5460 tgtcagccag tgatgtcact aacctccagc ctgattgccg tgcgtgagca taaagccgga 5520 gagcctgttg gttatggtgg aacctgggta agcgaacgtg atacccgtct tggcgtagtc 5580 gcgatgggct atggcgatgg ttatccgcgc gccgcgccgt ccggtacgcc agtgctggtg 5640 aacggtcgcg aagtaccgat tgtcgggcgc gtggcgatgg atatgatctg cgtagactta 5700 ggtccacagg cgcaggacaa agccggggat ccggtcattt tatggggcga aggtttgccc 5760 gtagaacgta tcgctgaaat gacgaaagta agcgcttacg aacttattac gcgcctgact 5820 tcaagggtcg cgatgaaata cgtggattaa acacgttact aaagggaatg gagaccgggg 5880 cccttcaata gagttcttaa cgttaatccg aaaaaaacta acgttaatat taaaaaataa 5940 gatccgcttg tgaattatgt ataatttgat tagactaaag aataggagaa agtatgatga 6000 tatttaaaaa actttctcgt taagataggt tgttggtgag catgttatat acggatgtat 6060 cggtttcctt aatgcaaaat tttgttgcta tcttattaat ttttctatta tatagatata 6120 ttcaaagaaa gataacattt aaacggatca tattagatat tttaatagcg attatttttt 6180 caatattata tctgtttatt tcagatgcgt cattacttgt aatggtatta atgcgattag 6240 ggtggcattt tcatcaacaa aaagaaaata agataaaaac gactgataca gctaatttaa 6300 ttctaattat cgtgatccag ttattgttag ttgcggttgg gactattatt agtcagttta 6360 ccatatcgat tatcaaaagt gatttcagcc aaaatatatt gaacaatagt gcaacagata 6420 taactttatt aggtattttc tttgctgttt tatttgacgg cttgttcttt atattattga 6480 agaataagcg gactgaatta caacatttaa atcaagaaat cattgaattt tcgttagaaa 6540 aacaatattt tatatttata tttattttat ttatagtaat agaaattatt ttagcagttg 6600 ggaatcttca aggagtaaca gccacgatat tattaaccat tatcattatt ttttgtgtcc 6660 ttatcgggat gactttttgg caagtgatgc tttttttgaa ggcttattcg attcgccaag 6720 aagccaatga ccaattggtc cggaatcaac aacttcaaga ttatctagtc aatatcgaac 6780 agcagtacac cgaattacgg cgatttaagc atgattatca aaacatctta ttatcgttgg 6840 agagttttgc cgaaaagggc gatcagcaac agtttaaggc gtattaccaa gaattattag 6900 cacaacggcc aattcaaagt gaaatccaag gggcagtcat tgcacaactc gactacttga 6960 aaaatgatcc tattcgagga ttagtcattc aaaagttttt ggcagccaaa caggctggtg 7020 ttactttaaa attcgaaatg accgaaccaa tcgaattagc aaccgctaat ctattaacgg 7080 ttattcggat tatcggtatt ttattagaca atgcgattga acaagccgtt caagaaaccg 7140 atcaattggt gagttgtgct ttcttacaat ctgatggttt aatcgaaatt acgattgaaa 7200 atacggccag tcaagttaag aatctccaag cattttcaga gttaggctat tcaacgaaag 7260 gcgctggtcg ggggactggt ttagctaatg tgcaggattt gattgccaaa caaaccaatt 7320 tattcttaga aacacagatt gaaaatagaa agttacgaca gacattgatg attacggagg 7380 aaacttaatt tgtatcccgt ttatttatta gaggatgatt tacagcaaca agcgatttat 7440 cagcaaatta tcgcgaatac gattatgatt aacgaatttg caatgacttt aacatgcgct 7500 gccagtgata ctgagacatt gttggcggca attaaggatc agcaacgagg tttattcttt 7560 ttggatatgg aaattgagga taaccgccaa gccggtttag aagtggcaac taagattcgg 7620 cagatgatgc cgtttgcgca aattgtcttc attacaaccc acgaggaact gacattatta 7680 acgttagaac gaaaaatagc gcctttagat tacattctca aggaccaaac aatggctgaa 7740 atcaaaaggc aattgattga tgatctattg ttagctgaga agcaaaacga ggcggcagcg 7800 tatcaccgag aaaatttatt tagttataaa ataggtcctc gctttttctc attaccatta 7860 aaggaagttg tttatttata tactgaaaaa gaaaatccgg gtcatattaa tttgttagcc 7920 gttaccagaa aggttacttt tccaggaaat ttaaatgcgc tggaagccca atatccaatg 7980 ctctttcggt gtgataaaag ttacttagtt aacctatcta atattgccaa ttatgacagt 8040 aaaacacgga gtttaaaatt tgtagatggc agtgaggcaa aagtctcgtt ccggaaatca 8100 cgggaactag tggccaaatt aaaacaaatg atgtagcgcc tgcagcacgc caaatgatcc 8160 cagtaaaaag ccacccgcat ggcgggtggc tttttattag ccctagaagg gcttcccaca 8220 cgcatttcag cgccttagtg ccttagtttg tgaatcatag gtggtatagt cccgaaatac 8280 ccgtctaagg aattgtcaga taggcctaat gactggcttt tataatatga gataatgccg 8340 actgtacttt ttacagtcgg ttttctaatg tcactaacct gccccgttag ttgaagaagg 8400 tttttatatt acagctccag atctaccggt gggcccatat taacgtttaa ccgataaagt 8460 tgaacgttaa tatttttttt gcgcagaaat ggtaaattga agcataatag tcttgtaagg 8520 tatttagctg gctggcgtaa agtatgcttt ataaaataat atataggagt atgattc 8577

Terminator iGEM-part BBa_B1006 (SEQ ID NO: 12) aaaaaaaaac cccgcccctg acagggcggg gtttttttt

5'UTR (SEQ ID NO: 13) AGGAGGT

3'UTR (SEQ ID NO: 14)

TACTTGAA p3050Alr_Amuc1100_sh71 with 5’UTR, 3’UTR and terminator (SEQ ID NO: 15) atatgaaaaa atttaacttt aaaaccatgt tgctattagt tttggctagt tgtgtcttcg 60 gggtcgtcgt taacgtgact actagtcttg gaccacaaac cgcaatcacc gcccaggcct 120 ccaaaggagg tatcgtcaat tccaaacgca gtgaactgga caaaaaaatc agcatcgccg 180 ccaaggaaat caagtccgcc aatgctgcgg aaatcactcc gagccgatca tccaacgaag 240 agctggaaaa agaactgaac cgctatgcca aggccgtggg cagcctggaa acggcctaca 300 agcccttcct tgcctcctcc gcgctggtcc ccaccacgcc cacggcattc cagaatgaac 360 tgaaaacatt cagggattcc ctgatctcct cctgcaagaa aaagaacatt ctcataacgg 420 acacatcctc ctggctcggt ttccaggttt acagcaccca ggctccctct gttcaggcgg 480 cctccacgct gggttttgaa ttgaaagcca tcaacagcct ggtcaacaaa ctggcggaat 540 gcggcctgtc caaattcatc aaggtgtacc gcccccagct ccccattgaa accccggcga 600 acaatccgga agaatcggac gaagccgacc aggccccatg gactcccatg cctctggaaa 660 tagccttcca gggcgaccgg gaaagtgtat tgaaagccat gaacgccata accggcatgc 720 aggactatct gttcacggtc aactccatcc gtatccgcaa cgaacggatg atgccccctc 780 ccatcgccaa tccggcagcc gccaaacctg ccgcggccca acccgccacg ggtgcggctt 840 ccctgactcc ggcggatgag gcggctgcac ctgcagcccc ggccatccag caagtcatca 900 agccttacat gggcaaggag caggtctttg tccaggtctc cctgaatctg gtccacttca 960 accagcccaa ggctcaggaa ccgtctgaag attaatactt gaaaaaaaaa aaccccgccc 1020 ctgacagggc ggggtttttt ttcatggatc gattttttat taaaacgtct caaaatcgtt 1080 tctgagacgt tttagcgttt atttcgttta gttatcggca taatcgttaa aacaggcgtt 1140 atcgtagcgt aaaagccctt gagcgtagcg tgctttgcag cgaagatgtt gtctgttaga 1200 ttatgaaagc cgatgactga atgaaataat aagcgcagcg tccttctatt tcggttggag 1260 gaggctcaag ggagtttgag ggaatgaaat tccctcatgg gtttgatttt aaaaattgct 1320 tgcaattttg ccgagcggta gcgctggaaa aatttttgaa aaaaatttgg aatttggaaa 1380 aaaatggggg gaaaggaagc gaattttgct tccgtactac gaccccccat taagtgccga 1440 gtgccaattt ttgtgccaaa aacgctctat cccaactggc tcaagggttt gaggggtttt 1500 tcaatcgcca acgaatcgcc aacgttttcg ccaacgtttt ttataaatct atatttaagt 1560 agctttattg ttgtttttat gattacaaag tgatacacta attttataaa attatttgat 1620 tggagttttt taaatggtga tttcagaatc gaaaaaaaga gttatgattt ctctgacaaa 1680 agagcaagat aaaaaattaa cagatatggc gaaacaaaaa ggtttttcaa aatctgcggt 1740 tgcggcgtta gctatagaag aatatgcaag aaaggaatca gaataaaaaa aataagcgaa 1800 agctcgcgtt tttagaagga tacgagtttt cgctacttgt ttttgataag gtaatatatc 1860 atggctatta aatactaaag ctagaaattt tggattttta ttatatcctg actcaattcc 1920 taatgattgg aaagaaaaat tagagagttt gggcgtatct atggctgtca gtcctttaca 1980 cgatatggac gaaaaaaaag ataaagatac atggaatagt agtgatgtta tacgaaatgg 2040 aaagcactat aaaaaaccac actatcacgt tatatatatt gcacgaaatc ctgtaacaat 2100 agaaagcgtt aggaacaaga ttaagcgaaa attggggaat agttcagttg ctcatgttga 2160 gatacttgat tatatcaaag gttcatatga atatttgact catgaatcaa aggacgctat 2220 tgctaagaat aaacatatat acgacaaaaa agatattttg aacattaatg attttgatat 2280 tgaccgctat ataacacttg atgaaagcca aaaaagagaa ttgaagaatt tacttttaga 2340 tatagtggat gactataatt tggtaaatac aaaagattta atggctttta ttcgccttag 2400 gggagcggag tttggaattt taaatacgaa tgatgtaaaa gatattgttt caacaaactc 2460 tagcgccttt agattatggt ttgagggcaa ttatcagtgt ggatatagag caagttatgc 2520 aaaggttctt gatgctgaaa egggggaaat aaaatgacaa acaaagaaaa agagttattt 2580 gctgaaaatg aggaattaaa aaaagaaatt aaggacttaa aagagcgtat tgaaagatac 2640 agagaaatgg aagttgaatt aagtacaaca atagatttat tgagaggagg gattattgaa 2700 taaataaaag cccccctgac gaaagtcgaa gggggetttt attttggttt gatgttgcga 2760 ttaatagcaa tacgattgca ataaacaaaa ggatccatgc aagcggcaac tgttgtgatt 2820 aaccgccgcg ctctgcgaca caacctgcaa cgtcttcgtg aactggcccc tgccagtaaa 2880 atggttgcgg tggtgaaagc gaacgcttat ggtcacggtc ttcttgagac cgcgcgaacg 2940 ctccccgatg ctgacgcctt tggcgtagcc cgtctcgaag aagctctgcg actgcgtgcg 3000 gggggaatca ccaaacctgt actgttactc gaaggctttt ttgatgccag agatctgccg 3060 acgatttctg cgcaacattt tcataccgcc gtgcataacg aagaacagct ggctgcgctg 3120 gaagaggcta gcctggacga gccggttacc gtctggatga aactcgatac cggtatgcac 3180 cgtctgggcg taaggccgga acaggctgag gcgttttatc atcgcctgac ccagtgcaaa 3240 aacgttcgtc agccggtgaa tatcgtcagc cattttgcgc gcgcggatga accaaaatgt 3300 ggcgcaaccg agaaacaact cgctatcttt aatacctttt gcgaaggcaa acctggtcaa 3360 cgttccattg ccgcgtcggg tggcattctg ctgtggccac agtcgcattt tgactgggtg 3420 cgcccgggca tcattcttta tggcgtctcg ccgctggaag atcgctccac cggtgccgat 3480 tttggctgtc agccagtgat gtcactaacc tccagcctga ttgccgtgcg tgagcataaa 3540 gccggagagc ctgttggtta tggtggaacc tgggtaagcg aacgtgatac ccgtcttggc 3600 gtagtcgcga tgggctatgg cgatggttat ccgcgcgccg cgccgtccgg tacgccagtg 3660 ctggtgaacg gtcgcgaagt accgattgtc gggcgcgtgg cgatggatat gatctgcgta 3720 gacttaggtc cacaggcgca ggacaaagcc ggggatccgg tcattttatg gggcgaaggt 3780 ttgcccgtag aacgtatcgc tgaaatgacg aaagtaagcg cttacgaact tattacgcgc 3840 ctgacttcaa gggtcgcgat gaaatacgtg gattaaacac gttactaaag ggaatggaga 3900 ccggggccct tcaatagagt tcttaacgtt aatccgaaaa aaactaacgt taatattaaa 3960 aaataagatc cgcttgtgaa ttatgtataa tttgattaga ctaaagaata ggagaaagta 4020 tgatgatatt taaaaaactt tctcgttaag ataggttgtt ggtgagcatg ttatatacgg 4080 atgtatcggt ttccttaatg caaaattttg ttgctatctt attaattttt ctattatata 4140 gatatattca aagaaagata acatttaaac ggatcatatt agatatttta atagcgatta 4200 ttttttcaat attatatctg tttatttcag atgcgtcatt acttgtaatg gtattaatgc 4260 gattagggtg gcattttcat caacaaaaag aaaataagat aaaaacgact gatacagcta 4320 atttaattct aattatcgtg atccagttat tgttagttgc ggttgggact attattagtc 4380 agtttaccat atcgattatc aaaagtgatt tcagccaaaa tatattgaac aatagtgcaa 4440 cagatataac tttattaggt attttctttg ctgttttatt tgacggcttg ttctttatat 4500 tattgaagaa taagcggact gaattacaac atttaaatca agaaatcatt gaattttcgt 4560 tagaaaaaca atattttata tttatattta ttttatttat agtaatagaa attattttag 4620 cagttgggaa tcttcaagga gtaacagcca cgatattatt aaccattatc attatttttt 4680 gtgtccttat cgggatgact ttttggcaag tgatgctttt tttgaaggct tattcgattc 4740 gccaagaagc caatgaccaa ttggtccgga atcaacaact tcaagattat ctagtcaata 4800 tcgaacagca gtacaccgaa ttacggcgat ttaagcatga ttatcaaaac atcttattat 4860 cgttggagag ttttgccgaa aagggcgatc agcaacagtt taaggcgtat taccaagaat 4920 tattagcaca acggccaatt caaagtgaaa tccaaggggc agtcattgca caactcgact 4980 acttgaaaaa tgatcctatt cgaggattag tcattcaaaa gtttttggca gccaaacagg 5040 ctggtgttac tttaaaattc gaaatgaccg aaccaatcga attagcaacc gctaatctat 5100 taacggttat tcggattatc ggtattttat tagacaatgc gattgaacaa gccgttcaag 5160 aaaccgatca attggtgagt tgtgctttct tacaatctga tggtttaatc gaaattacga 5220 ttgaaaatac ggccagtcaa gttaagaatc tccaagcatt ttcagagtta ggctattcaa 5280 cgaaaggcgc tggtcggggg actggtttag ctaatgtgca ggatttgatt gccaaacaaa 5340 ccaatttatt cttagaaaca cagattgaaa atagaaagtt acgacagaca ttgatgatta 5400 cggaggaaac ttaatttgta tcccgtttat ttattagagg atgatttaca gcaacaagcg 5460 atttatcagc aaattatcgc gaatacgatt atgattaacg aatttgcaat gactttaaca 5520 tgcgctgcca gtgatactga gacattgttg gcggcaatta aggatcagca acgaggttta 5580 ttctttttgg atatggaaat tgaggataac cgccaagccg gtttagaagt ggcaactaag 5640 attcggcaga tgatgccgtt tgcgcaaatt gtcttcatta caacccacga ggaactgaca 5700 ttattaacgt tagaacgaaa aatagcgcct ttagattaca ttctcaagga ccaaacaatg 5760 gctgaaatca aaaggcaatt gattgatgat ctattgttag ctgagaagca aaacgaggcg 5820 gcagcgtatc accgagaaaa tttatttagt tataaaatag gtcctcgctt tttctcatta 5880 ccattaaagg aagttgttta tttatatact gaaaaagaaa atccgggtca tattaatttg 5940 ttagccgtta ccagaaaggt tacttttcca ggaaatttaa atgcgctgga agcccaatat 6000 ccaatgctct ttcggtgtga taaaagttac ttagttaacc tatctaatat tgccaattat 6060 gacagtaaaa cacggagttt aaaatttgta gatggcagtg aggcaaaagt ctcgttccgg 6120 aaatcacggg aactagtggc caaattaaaa caaatgatgt agcgcctgca gcacgccaaa 6180 tgatcccagt aaaaagccac ccgcatggcg ggtggctttt tattagccct agaagggctt 6240 cccacacgca tttcagcgcc ttagtgcctt agtttgtgaa tcataggtgg tatagtcccg 6300 aaatacccgt ctaaggaatt gtcagatagg cctaatgact ggcttttata atatgagata 6360 atgccgactg tactttttac agtcggtttt ctaatgtcac taacctgccc cgttagttga 6420 agaaggtttt tatattacag ctccagatct accggtgggc ccatattaac gtttaaccga 6480 taaagttgaa cgttaatatt ttttttgcgc agaaatggta aattgaagca taatagtctt 6540 gtaaggtatt tagctggctg gcgtaaagta tgctttataa aataatatat aggagtatga 6600 ttc 6603 Integrative vector pRac-int-attP-Amuc-aldh-fruct (SEQ ID NO: 16) agagtttttt gcgccgcttt agtgtttatt ttgtttaatt gagtgcgtaa agtttgaagc 60 aggcgttgag ttaacgcaag agtccgtaag cgtaacgcgc gttgcagcgc cgctgttgtt 120 tgttggatta tgaaagtcgc taattgaatg aaattattag tgcggcgagt ttttattttg 180 gctggcgccg cttgaagggc gtttaaggca atgaaattcc gagttgggtt taattttaaa 240 agttgttggc gattttgccg agtggcagtg cgggcaagat ttttgaaaag aatttggaat 300 ttggcaagaa gtggggcgaa cgcaagcgca ttttgttgcc gtattatgat ccgccgttga 360 gtgcggaatg tcagtttttg tgtcagaagc gcagtattcc gaccggcagt cgcgtttaag 420 gcgtttttca gagtccgacc aatcgccagc gctttcgcca gcgctttttg taaatttata 480 tttaagttgc gttgttgttg tttttgtaat tgcagagtga taccttgatt ttgtaaaatt 540 atttgattgg cgttttttaa atggttatta gtgaaagtaa gaagcgcgtt atgattagtt 600 tgaccaagga acaggataag aagttgaccg atatggcgaa gcagaagggc tttagtaaga 660 gtgcggttgc ggcgttggcg attgaagaat atgcgcgcaa ggaaagtgaa taaaagaagt 720 aagcgaaggc gcgcgttttt cgccgcattc gcgtttttgc gacctgtttt taataaggca 780 atattagttg gttgttgaat accaaggcgc gcaattttgg ctttttgttg tatccggata 840 gtattccgaa tgattggaag gaaaagttgg aaagtttggg cgttagtatg gcggttagtc 900 cgttgcatga tatggatgaa aagaaggata aggatacctg gaatagtagt gatgttattc 960 gcaatggcaa gcattataag aagccgcatt atcatgttat ttatattgcg cgcaatccgg 1020 ttaccattga aagtgttcgc aataagatta agcgcaagtt gggcaatagt agtgttgcgc 1080 atgttgaaat tttggattat attaagggca gttatgaata tttgacccat gaaagtaagg 1140 atgcgattgc gaagaataag catatttatg ataagaagga tattttgaat attaatgatt 1200 ttgatattga tcgctatatt accttggatg aaagtcagaa gcgcgaattg aagaatttgt 1260 tgttggatat tgttgatgat tataatttgg ttaataccaa ggatttgatg gcgtttattc 1320 gcttgcgcgg cgcggaattt ggcattttga ataccaatga tgttaaggat attgttagta 1380 ccaatagtag tgcgtttcgc ttgtggtttg aaggcaatta tcagtgtggc tatcgcgcga 1440 gttatgcgaa ggttttggat gcggaaaccg gcgaaattaa gtaacagacc aagaagaaga 1500 gttatttgtt gaagatgcgc aattaaaaga agaagttgcg cacctaaaag agtgttttga 1560 aggataccga aaagtggaag ttgaattaag ttcagcagta aatttattaa gaagaaggct 1620 tgttgaataa gtaaaagccg ccgtaacgca agagtaaggg cgcgtttatt ttggtttaat 1680 gttgtgatta ataacagtat gattgtaata agcagaggat ccctgtcaga ccaagtttac 1740 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 1800 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 1860 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 1920 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 1980 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 2040 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 2100 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 2160 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 2220 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 2280 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 2340 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 2400 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 2460 ggggggegga gcctatggaa aaggatcctt tgattcagaa actatgtggc aagcttaata 2520 ataaatctgt caaaataatt tattttgaca gattttttta tctaataatt aaaataatta 2580 tttcacaatg ttcacaagcg cttacaaaag aaaatagatt gacttatgct aaactgaata 2640 atgtaaaaag aattttacat ttaaaggaga cctattagtg taataggacg tgcttcaggc 2700 gtgttgcctg tacgcatgct gattcttcag caagactact acctcatgag agttatagac 2760 tcatggatct tgctttgaag ggttttgtac attataggct cctatcacat gctgaaccta 2820 tggcctatta cattttttta tatttcaagg aggaaaagac catgaccatt ggcaatttgc 2880 gcccggcgac cgttttgatt gatgaaaccg cgattttgca taatgttcag catgaagttg 2940 cgcgcttgaa gaagcagacc cagttgtttg cggttgttaa ggcggatgcg tatggccatg 3000 gcatgttgcg cgttgcgcat gttgcgaagg cggcgggcgc gagtggcttt tgtgttgcgt 3060 tgttggatga agcgttggat ttgcgcgcgg cgaattatac cgaaccggtt ttggttttgg 3120 gcattgttcc gagtcagtat gcggcgattg cggcggcgca gaccgttagt ttgccgttga 3180 gtagtgttga ttggttgaag caggcgttgc cggttttgga agcgcagccg gaattgccgc 3240 cgttgcgctt gcatattgcg ttggataccg gcatgggccg cattggcttt accgatgatc 3300 agaccttgtt ggatgcgatt gcgtttattc aggcgcatcc gaaggcgttt accattgaag 3360 gcatttttac ccattttgcg accgcggatg atttggatga tgcgtatttt aagcagcagg 3420 ttgcgaagtt taatcatatg gttgcgttgt tgccgcatcg cccgcgctat gttcatgtta 3480 gtaatagtgc gaccagtttg tggcatgcgg cgtgtaatgg caatatggtt cgctatggcg 3540 ttgcgattta tggcttgaat ccgagtggcg atgttattcc gaccaccccg tttccgttgg 3600 aaccggcgtt gagtttggaa agtgaattga ccttttgtaa gcaggttcat gcgggcgatg 3660 gcattagtta tggcgttacc tatcgcgcga ccggcgatga atttattggc accgttccgg 3720 ttggctatgc ggatggctgg ttgcgccgct tgcagggctt tcatgttttg gttgatggcc 3780 atccgtgtga aattgttggc cgcgtttgta tggatcagtt tatgattcgc ttgccgaagg 3840 cgtatccggc gggcaccaag gttgttttga ttggccagag tggcgatcag gaaattacct 3900 tgttggatgt tgcgaagtat agtcatacca ttcattatga aattgcgtgt aatttgacca 3960 gtcgcttgaa gcgccagagt attaatccga ttgcgcgcta attagactcg aggtccttat 4020 gtggaggttt ctgggcatgc gagcgcgttt cagcagagcg tgaattggcg cgttgaataa 4080 tcggcgtgta ttggccttgc ctgtgatgac cgggctttgg tcaatgcagt caaggtcctt 4140 acacgcaggt ttcggtgcca atgagcgcgt ttaaaaaaaa accccgcccc tgacagggcg 4200 gggttttttt tgaattccca tggcaaaatt atgaaaaagt ctgtcaattt tgtttcggcg 4260 aattgataat gtgttatact cacgtaatag gacgtgcttc aggcgtgttg cctgtacgca 4320 tgctgattct tcagcaagac tactacctca tgagagttat agactcatgg atcttgcttt 4380 gaagggtttt gtacattata ggctcctatc acatgctgaa cctatggcct attacatttt 4440 tttatatttc aaggaggaaa agaccatgaa gaagtttaat tttaagacca tgttgttgtt 4500 ggttttggcg agttgtgttt ttggcgttgt tgttaatgtt accaccagtt tgggcccgca 4560 gaccgcgatt accgcgcagg cgagtaagtt ggaaattagt agtacctgtg atgcgattgt 4620 taatagtaag cgcagtgaat tggataagaa gattagtatt gcggcgaagg aaattaagag 4680 tgcgaatgcg gcggaaatta ccccgagtcg cagtagtaat gaagaattgg aaaaggaatt 4740 gaatcgctat gcgaaggcgg ttggcagttt ggaaaccgcg tataagccgt ttttggcgag 4800 tagtgcgttg gttccgacca ccccgaccgc gtttcagaat gaattgaaga cctttcgcga 4860 tagtttgatt agtagttgta agaagaagaa tattttgatt accgatacca gtagttggtt 4920 gggctttcag gtttatagta cccaggcgcc gagtgttcag gcggcgagta ccttgggctt 4980 tgaattgaag gcgattaata gtttggttaa taagttggcg gaatgtggct tgagtaagtt 5040 tattaaggtt tatcgcccgc agttgccgat tgaaaccccg gcgaataatc cggaagaaag 5100 tgatgaagcg gatcaggcgc cgtggacccc gatgccgttg gaaattgcgt ttcagggcga 5160 tcgcgaaagt gttttgaagg cgatgaatgc gattaccggc atgcaggatt atttgtttac 5220 cgttaatagt attcgcattc gcaatgaacg catgatgccg ccgccgattg cgaatccggc 5280 ggcggcgaag ccggcggcgg cgcagccggc gaccggcgcg gcgagtttga ccccggcgga 5340 tgaagcggcg gcgccggcgg cgccggcgat tcagcaggtt attaagccgt atatgggcaa 5400 ggaacaggtt tttgttcagg ttagtttgaa tttggttcat tttaatcagc cgaaggcgca 5460 ggaaccgagt gaagattaat tagactcgag gtccttatgt ggaggtttct gggcatgcga 5520 gcgcgtttca gcagagcgtg aattggcgcg ttgaataatc ggcgtgtatt ggccttgcct 5580 gtgatgaccg ggctttggtc aatgcagtca aggtccttac acgcaggttt cggtgccaat 5640 gagcgcgttt aaaaaaaaac cccgcccctg acagggcggg gtttttttta acttatatga 5700 caattttggt acaggagtct tcaaaagtgg cacagaacca aagtgatgga aaaataagaa 5760 actgcttgct ttactttgcc tattaatgct ataatgaaaa tgtagaaaag atggacgtga 5820 aaccagttca tcaaaaaaag taaaggagac tgttcaaccg taataggacg tgcttcaggc 5880 gtgttgcctg tacgcatgct gattcttcag caagactact acctcatgag agttatagac 5940 tcatggatct tgctttgaag ggttttgtac attataggct cctatcacat gctgaaccta 6000 tggcctatta cattttttta tatttcaagg aggaaaagac catgaagaag tttaatttta 6060 agaccatgtt gttgttggtt ttggcgagtt gtgtttttgg cgttgttgtt aatgttacca 6120 ccagtttggg cccgcagacc gcgattaccg cgcaggcgag taagttggaa attagtagta 6180 cctgtgatgc gcgctatagt agtgcggcgg cgttgccgag tccgattttg aatccggata 6240 ttccgtataa tcagttgttt attaataatg aatggcagga tgcggttagt aagaagacct 6300 ttccgaccgt taatccgacc accggcgaag ttattggcca tgttgcggaa ggcgatcgcg 6360 cggatgttga tcgcgcggtt aaggcggcgc gcgaagcgtt tcgcttgggc agtccgtggc 6420 gccgcatgga tgcgagtgaa cgcggccgct tgttgaattt gttggcggat ttggttgaac 6480 gcgatcgcgt ttatttggcg agtttggaaa ccttggataa tggcaagccg tttcaggaaa 6540 gttatgcgtt ggatttggat gaagttatta aggtttatcg ctattttgcg ggctgggcgg 6600 ataagtggca tggcaagacc attccgatgg atggccagca tttttgtttt acccgccatg 6660 aaccggttgg cgtttgtggc cagattattc cgtggaattt tccgttggtt atgcagggct 6720 ggaagttggc gccggcgttg gcgaccggca ataccgttgt tatgaaggtt gcggaacaga 6780 ccccgttgag tgcgttgtat ttggcgagtt tgattaagga agcgggcttt ccgccgggcg 6840 ttgttaatat tattaccggc tatggcccga ccgcgggcgc ggcgattgcg cagcatgttg 6900 atgttgataa ggttgcgttt accggcagta ccgaagttgg ccatttgatt cagaaggcgg 6960 cgggcgatag taatttgaag cgcgttacct tggaattggg cggcaagagt ccgagtattg 7020 ttttggcgga tgcggatatg gaacatgcgg ttgaacagtg tcatgaagcg ttgtttttta 7080 atatgggcca gtgttgttgt gcgggcagtc gcacctttgt tgaagaaagt atttataatg 7140 aatttttgga acgcaccgtt gaaaaggcga agcagcgcaa ggttggcaat ccgtttgaat 7200 tggataccca gcagggcccg caggttgata aggaacagtt tgaacgcgtt ttgggctata 7260 ttcagttggg ccagaaggaa ggcgcgaagt tgttgtgtgg cggcgaacgc tttggcgaac 7320 gcggcttttt tattaagccg accgtttttg gcggcgttca ggatgatatg cgcattgcga 7380 aggaagaaat ttttggcccg gttcagccgt tgtttaagtt taagaagatt gaagaagttg 7440 ttgaacgcgc gaataatacc cgctatggct tggcggcggc ggtttttacc cgcgatttgg 7500 ataaggcgat gtattttacc caggcgttgc aggcgggcac cgtttgggtt aatacctata 7560 atattgttac ctgtcatacc ccgtttggcg gctttaagga aagtggcaat ggccgcgaat 7620 tgggcgaaga tggcttgaag gcgtataccg aagttaagac cgttaccatt aaggttccgc 7680 agaagaatag ttaattagac tcgaggtcct tatgtggagg tttctgggca tgcgagcgcg 7740 tttcagcaga gcgtgaattg gcgcgttgaa taatcggcgt gtattggcct tgcctgtgat 7800 gaccgggctt tggtcaatgc agtcaaggtc cttacacgca ggtttcggtg ccaatgagcg 7860 cgtttaaaaa aaaaccccgc ccctgacagg gcggggtttt ttttttgaca tttcttgaag 7920 aggttggtat aatagtaata ggacgtgctt caggcgtgtt gcctgtacgc atgctgattc 7980 ttcagcaaga ctactacctc atgagagtta tagactcatg gatcttgctt tgaagggttt 8040 tgtacattat aggctcctat cacatgctga acctatggcc tattacattt ttttatattt 8100 caaggaggaa aagaccatga agaagtttaa ttttaagacc atgttgttgt tggttttggc 8160 gagttgtgtt tttggcgttg ttgttaatgt taccaccagt ttgggcccgc agaccgcgat 8220 taccgcgcag gcgagtaagt tggaaattag tagtacctgt gatgcgacca ccagtgcgag 8280 tagtacccag attagtcaga ccaataccgg cagtcagccg aataatgaaa ccaccggcga 8340 aaccgcgcag agtagtgtta atagtaccgc gaccgcgagt agtagtagtg ttgcggattt 8400 gccgagtagt agtgatagta agagtagtat tggcagtacc attagtcagc cgaccgttga 8460 taagaaggaa accagtaaga gtgataccgc ggataatgat ttgaccaaga gtgttaccac 8520 cagtgatagt gataaggcgt tgccgaccag taagaccacc ttgccgacca gtaatgaaca 8580 ggttcagagt agtgttggcc agagtcagac cgatcagccg gcgagtagtg cgaccattgc 8640 gaccaatgcg gttaccagtg atgttagtca gaatgatcag tataatgaac cgtatcgcaa 8700 tcagtatcat tatagtagta gtcagaattg gattaatgat ccgaatggct tgttttatga 8760 tagtaagacc ggcttgtata atttgtatta tcagtataat ccggaaggca atcagtgggg 8820 caatatgagt tggggccatg cggttagtaa ggatttgatt aattggaccc aggaagatgt 8880 tgcgattccg atgttgcaga atcagggctg ggaagatttt acctatacca ataccaccgg 8940 cagtttgaag gataagggcg aagttcgcta tgttggcgtt ccgaccacca attggggcga 9000 tgcggatggc aagaaggcga tttttagtgg cagtattgtt gttgatacca ataatgttag 9060 tggcttgggc aaggatgcga ttttggcgtt ttataccgcg gattatcaga ttgcgacccg 9120 caagaatgat ggcgcggaag atggctgggg cacctggatt ggcttgaccg aaattcagga 9180 acagcatttg gcgtatagtt tggatggcgg caagaccttt attcagtata gtaaggatgg 9240 caatgcggcg aatccgcagg cgattattcc gaccagtatg aatcagggcg gcgatgcggc 9300 gaattttcgc gatccgagtg ttgtttatga tgcggttaat aagcagtatt atttgaccgt 9360 tgttagtggc cagcaggcgt tgatttataa gagtagtaat ttgttggatt ggacctatgc 9420 gagtaagatt gaacgcgaaa atgatgttgg caatggcgtt tgggaatgtc cgagtttggt 9480 tccgatgaag gttgcgggca ccaatgaaac caagtgggtt ttttgtatta gtgttcagca 9540 gggcgcgcat gcgaccggca gtggcatgca gtattatgtt ggcaatatga ccgcggatgg 9600 cacctgggtt ccggaaagta gtaagacctt gcagaatccg atgaccatgg atagtggcga 9660 agatttttat gcgggcattc cgtttagtaa tatgccggat ggccgcaccg ttatgttggc 9720 gtggcagagt aattggagtt atgttgatga agcgaagacc agtccgtgga gtggcaatat 9780 gaccttgccg cgcgaattga gtttgaagaa gaatgcggat accaccgatg gctatttgtt 9840 gaccaatacc gttgttaagg aaattgcgaa taatgaagaa gcgaatgtta ttaataaggc 9900 ggaaagtaat tttaccgtta cccgcagtga tgaacaggtt cagtatgaag gcaagcagta 9960 taagattagt gcgaccttta gttgggatga agcggataag ccgaagagtg ttggctttaa 10020 gttgcgcgtt agtgatgatc agaagtatga tatgattgtt ggctatgatt tgaccaccgg 10080 cttgttgtat gttcagcgct tgaataccgg cgaaccgaat atgggcgcgc cgcgcgataa 10140 gatgaatgcg accgttaatg cggatggcag tattaccatt accgtttatg ttgatgaaac 10200 cagtattgaa gcgtttgcga atgatggcga aaagagtatt acccagaatt tttttatgcg 10260 cccggaaaat attggcgatc aggcgaccac cggcgtttat gtttatagta atgatggcac 10320 caccaagatt agtgatttga ccattaatcc gattaccagt atttggaata gtaccggcca 10380 gttgaccgaa aagtttgttg atgaaaatgg caataccatt gcgagtgata agattcagac 10440 cggccgcgtt ggccagagtt ataccagtga aagtgcgacc attccgggct atgtttttgt 10500 taaggaaaat accgatcata ttaatagtaa tcagttgtat accacccaga atcagaccat 10560 tacctatacc tatcgcgcga gtcaggcgag tgttgttacc aaggatacca ccttggttgc 10620 gggcccgagt gcggcgtgga atgcggcgga taatttggtt ggcgcgaccg atgcggatgg 10680 caatgcgttg gcggttagtg atttgaccgt taccggcgcg gttgatccga agaccccggg 10740 cacctatacc gttacctata gttataccga tgcgaccggc agtaagatta gtaaggaagc 10800 gaccgttacc gttattgcga gtaaggcgga tattgttacc aaggataccg cgatggttgc 10860 gggcccgagt gcgacctgga atgcggttga taattttgtt gaagcgaccg gcgcggatgg 10920 caatgcgttg accttgagtg atttgaccgt taatggcgcg gttgatccga agaccccggg 10980 cacctatacc gttacctata gttataccga tgcgaccggc aataagatta gtaagaaggc 11040 gaccgttacc gttattgcga gtaaggcgga tattgttacc aaggatacca ccatggttgc 11100 gggcccgagt gcgacctgga atgcggcgga taatttggtt attgcgaccg atgcgaaggg 11160 caatgcgttg gcgttgagta atttgaccgt taccggcagt gttgatagta agaccccggg 11220 cacctatacc gttacctata gttataccga tgcggcgggc aataagatta gtaaggaagc 11280 gaccgttacc gttattgcga gtaaggcgga tattgttacc aaggatacca ccatggttgc 11340 gggcccgagt gcggcgtgga atgcggcgaa taatttggtt agtgcgaccg atgcggatgg 11400 caatgcgttg gcgatgagta atttgaccgt taccggcacc gttgatttga agacccaggg 11460 cacctatacc gttacctata cctataccga tgttgcgggc aataagatta gtaaggaagc 11520 gaccgttacc gttttgaccg aaaaggaaac caatattgaa gataataccg gcagtagtat 11580 tagtaatgat cgcgaaaatc cgccggcgag tattaccggc aagggcggcg atgatattca 11640 tcagaatgcg aagaccacca tgaccaagaa gaagaccgaa accttgccgc aggcgggcaa 11700 tcatgttaat gaattggcga ttgttttggg ccagatgatt ttggcgattt gtgttggcgg 11760 cattttgtgg ttgaagcgcc gcgttaagcg cgtttaatta gactcgaggt ccttatgtgg 11820 aggtttctgg gcatgcgagc gcgtttcagc agagcgtgaa ttggcgcgtt gaataatcgg 11880 cgtgtattgg ccttgcctgt gatgaccggg ctttggtcaa tgcagtcaag gtccttacac 11940 gcaggtttcg gtgccaatga gcgcgtttaa aaaaaaaccc cgcccctgac agggcggggt 12000 ttttttttct agaactagtg gatccaaaat aaaaagcgcc taccccaccg accaaagtga 12060 atgggtagac gcctaacaaa tactcggagc aacaaggctc tttgtataca catttttaca 12120 caggagggca ataatatggc ggtattcaag cgagctaacc gaaaaagtaa gccttgggga 12180 ttccagtatt catacaaagt ggatggcatc tccaagcaga aaacatcatt ttacaaaaca 12240 agaaaagaag ctaaggctgc tgaggcgaag tacctcgctt ctactggcgg atctgtaaaa 12300 atcgatccag tgatcacttt cgcagattgg tatgacaagt ggttgcatac ctacaagata 12360 cgttctgttt ccgaactgac gatgaccaag tatgcaactt cgggtacaat catcagaaac 12420 tacttcaaag accttaaatt aattgactta acgcgcatga tttatcaaca gtttattaac 12480 aactatattg atgacggtta cggccacaag cacgcaaggc aatcagtcca gaagctacat 12540 tcacacgctc atcaagcaat tatggccgca gcagacgaag gtttgattag gcgcgattat 12600 gccgctcatg cagaactggg tggtaccgca ggcagatcag aagacacaaa atttcttgaa 12660 gctgatcagt tcgagaaact gcgagattat gttgatcaat ttgccaaccc gcaacgaatt 12720 gctctcatga tggttcaaac ggccatatac tctggcgctc ggcttggaga aattggtggc 12780 ttaacgtggg aagatattga tgagaagaag agcaccatca gtatcgacaa gaccttcaag 12840 tacaggtttg tcattcgtaa cgcggatggt agctggccag accgtgaaaa agtcttcggt 12900 ccgaccaaga ctccttcaag tgttcgtact atcaaagtaa gcccagttct tatcgctagc 12960 ctccataagc tcatattggc tgacagaata aaagcgatta acaatccgta ccatttactg 13020 tttcttgggc cgaccggctt gccaatatat agcaatggtg tcaacaagga acttcgccgc 13080 gctctcaaac atcttggtat tgagcgtcct gggttcggtt tccacggatt gcggcacacg 13140 catggcagct acttgcttta taaaggcctt gacattcagt atgtatcaca tcgcctcgga 13200 cacgaaaacg ttggcattac caccaagatc tatacacatc tgctggatgc gatgacacag 13260 aagcaggacg agaaagcaat gaatgtgttg tgactaaaaa tcgaaccaga gaaagcggct 13320 caatgtcaac tgccacaagg tttacagcac acattcaatt ttcgatcacg aaccattttc 13380 ctaaaaaatc gcaatttcag gctatttggt tcgatgtggt tcgatggatt atatttttta 13440 ggggtttttc ggagttcaga taaatgcaag aatgccggtt taaagccatt tctgagcact 13500 aaaaaagacc ctctaggggg ctttgatacc ggtgatcggg gtatcacgga atgtatacgt 13560 actgatatga ttgcatttat gacaaaaagt ggttcgatgt ggttcgatgc ttcaaacgac 13620 agcgaccaac aacacatctc tatataatag gtagaaatag cttttaagag ttcagaaata 13680 tgggcacaca aactagtcat ggcagtattt tttattaaaa tgttagta 13728 Integrase (Lactobacillus phage A2) (SEQ ID NO: 17)

M G R R L T N T R S N K A L C I H I F T Q E G N N M A V F K R A N R K S K P W G F Q Y S Y K V D G I S K Q K T S F Y K T R K E A K A A E A K Y L A S T G G S V K I D P V I T F A D W Y D K W L H T Y K I R S V S E L T M T K Y A T S G T I I R N Y F K D L S T I S I D K T F K Y R F V I R N A D G S W P D R E K V F G P T K T P S S V R T I K V S P V L I A S L H K L I L A D R I K A I N N P Y H L L F L G P T G L P I Y S N G V N K E L R R A L K H L G I E R P G F G F H G L R H T H G S Y L L Y K G L D I Q Y V S H R L G H E N V G I T T K I Y T H L L D A M T Q K Q D E K A M N V L

SEQ ID NO: 18 - nucleotide sequence of pRac plasmid agagttttttgcgccgctttagtgtttattttgtttaattgagtgcgtaaagtttga agcaggcgttgagttaacgcaagagtccgtaagcgtaacgcgcgttgcagcgccgct gttgtttgttggattatgaaagtcgctaattgaatgaaattattagtgcggcgagtt tttattttggctggcgccgcttgaagggcgtttaaggcaatgaaattccgagttggg tttaattttaaaagttgttggcgattttgccgagtggcagtgcgggcaagatttttg aaaagaatttggaatttggcaagaagtggggcgaacgcaagcgcattttgttgccgt attatgatccgccgttgagtgcggaatgtcagtttttgtgtcagaagcgcagtattc cgaccggcagtcgcgtttaaggcgtttttcagagtccgaccaatcgccagcgctttc gccagcgctttttgtaaatttatatttaagttgcgttgttgttgtttttgtaattgc agagtgataccttgattttgtaaaattatttgattggcgttttttaaatggttatta gtgaaagtaagaagcgcgttatgattagtttgaccaaggaacaggataagaagttga ccgatatggcgaagcagaagggctttagtaagagtgcggttgcggcgttggcgattg aagaatatgcgcgcaaggaaagtgaataaaagaagtaagcgaaggcgcgcgtttttc gccgcattcgcgtttttgcgacctgtttttaataaggcaatattagttggttgttga ataccaaggcgcgcaattttggctttttgttgtatccggatagtattccgaatgatt ggaaggaaaagttggaaagtttgggcgttagtatggcggttagtccgttgcatgata tggatgaaaagaaggataaggatacctggaatagtagtgatgttattcgcaatggca agcattataagaagccgcattatcatgttatttatattgcgcgcaatccggttacca ttgaaagtgttcgcaataagattaagcgcaagttgggcaatagtagtgttgcgcatg ttgaaattttggattatattaagggcagttatgaatatttgacccatgaaagtaagg atgcgattgcgaagaataagcatatttatgataagaaggatattttgaatattaatg attttgatattgatcgctatattaccttggatgaaagtcagaagcgcgaattgaaga atttgttgttggatattgttgatgattataatttggttaataccaaggatttgatgg cgtttattcgcttgcgcggcgcggaatttggcattttgaataccaatgatgttaagg atattgttagtaccaatagtagtgcgtttcgcttgtggtttgaaggcaattatcagt gtggctatcgcgcgagttatgcgaaggttttggatgcggaaaccggcgaaattaagt aacagaccaagaagaagagttatttgttgaagatgcgcaattaaaagaagaagttgc gcacctaaaagagtgttttgaaggataccgaaaagtggaagttgaattaagttcagc agtaaatttattaagaagaaggcttgttgaataagtaaaagccgccgtaacgcaaga gtaagggcgcgtttattttggtttaatgttgtgattaataacagtatgattgtaata agcagaggatccctgtcagaccaagtttactcatatatactttagattgatttaaaa cttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacc aaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatc aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtag ccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttg gactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgt gagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggta agcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaggatcctttgattcagaaactatgtg gcaagcttaataataaatctgtcaaaataatttattttgacagatttttttatctaa taattaaaataattatttcacaatgttcacaagcgcttacaaaagaaaatagattga cttatgctaaactgaataatgtaaaaagaattttacatttaaaggagacctattagt gtaataggacgtgcttcaggcgtgttgcctgtacgcatgctgattcttcagcaagac tactacctcatgagagttatagactcatggatcttgctttgaagggttttgtacatt ataggctcctatcacatgctgaacctatggcctattacatttttttatatttca ag- gaggaaaagaccatgaccattggcaatttgcgcccggcgaccgttttgattgatg aaaccgcgattttgcataatgttcagcatgaagttgcgcgcttgaagaagcagaccc agttgtttgcggttgttaaggcggatgcgtatggccatggcatgttgcgcgttgcgc atgttgcgaaggcggcgggcgcgagtggcttttgtgttgcgttgttggatgaagcgt tggatttgcgcgcggcgaattataccgaaccggttttggttttgggcattgttccga gtcagtatgcggcgattgcggcggcgcagaccgttagtttgccgttgagtagtgttg attggttgaagcaggcgttgccggttttggaagcgcagccggaattgccgccgttgc gcttgcatattgcgttggataccggcatgggccgcattggctttaccgatgatcaga ccttgttggatgcgattgcgtttattcaggcgcatccgaaggcgtttaccattgaag gcatttttacccattttgcgaccgcggatgatttggatgatgcgtattttaagcagc aggttgcgaagtttaatcatatggttgcgttgttgccgcatcgcccgcgctatgttc atgttagtaatagtgcgaccagtttgtggcatgcggcgtgtaatggcaatatggttc gctatggcgttgcgatttatggcttgaatccgagtggcgatgttattccgaccaccc cgtttccgttggaaccggcgttgagtttggaaagtgaattgaccttttgtaagcagg ttcatgcgggcgatggcattagttatggcgttacctatcgcgcgaccggcgatgaat ttattggcaccgttccggttggctatgcggatggctggttgcgccgcttgcagggct ttcatgttttggttgatggccatccgtgtgaaattgttggccgcgtttgtatggatc agtttatgattcgcttgccgaaggcgtatccggcgggcaccaaggttgttttgattg gccagagtggcgatcaggaaattaccttgttggatgttgcgaagtatagtcatacca ttcattatgaaattgcgtgtaatttgaccagtcgcttgaagcgccagagtattaatc cgattgcgcgctaattagactcgaggtccttatgtggaggtttctgggcatgcgagc gcgtttcagcagagcgtgaattggcgcgttgaataatcggcgtgtattggccttgcc tgtgatgaccgggctttggtcaatgcagtcaaggtccttacacgcaggtttcggtgc caatgagcgcgtttaaaaaaaaaccccgcccctgacagggcggggttttttttgaat tcaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttg ggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagt tttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggc gcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactat tctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcc aacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccata ccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaa ctattaactggcgaactacttactctagcttcccggcaacaattaatagactggatg gaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggttt attgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcat tggtaatctagaactagtcatggcagtattttttattaaaatgttagta SEQ ID NO: 19 - amino acid sequence of beta-lactamase encoded by CDS of the pRac plasmid

MS IQHFRVALI PFFAAFCLPVFAHPETLVKVKDAEDQL GARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCG AVLSRIDAGQEQLGRRIHYSQN DLVEYSPVTEKHLTD GMTVRELCSAAITMSDNTAAN LLLTTIGGPKELTAFLH NMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTL RKLLTGELLTLASRQQLI DWMEADKVAGPLLRSALPA GWFIADKSGAGERGSRGI IAALGPDGKPSRIVVIYTTG SQATMDERNRQIAE IGASLIKHW

SEQ ID NO: 20 - nucleotide sequence of pEM40 plasmid aattcgagctcggtacccgatccgtcgacccgattcacaaaaaataggcacacgaaa aacaagttaagggatgcagtttatgcatcccttaacttacttattaaataatttata gctattgaaaagagataagaattgttcaaagctaatattgtttaaatcgtcaattcc tgcatgttttaaggaattgttaaattgattttttgtaaatattttcttgtattcttt gttaacccatttcataacgaaataattatacttttgtttatctttgtgtgatattct tgatttttttctacttaatctgataagtgagctattcactttaggtttaggatgaaa atattctcttggaaccatacttaatatagaaatatcaacttctgccattaaaagtaa tgccaatgagcgttttgtatttaataatcttttagcaaacccgtattccacgattaa ataaatctcattagctatactatcaaaaacaattttgcgtattatatccgtacttat gttataaggtatattaccatatattttataggattggtttttaggaaatttaaactg caatatatccttgtttaaaacttggaaattatcgtgatcaacaagtttattttctgt agttttgcataatttatggtctatttcaatggcagttacgaaattacacctctttac taattcaagggtaaaatggccttttcctgagccgatttcaaagatattatcatgttc atttaatcttatatttgtcattattttatctatattatgttttgaagtaataaagtt ttgactgtgttttatatttttctcgttcattataaccctctttaatttggttatatg aattttgcttattaacgattcattataaccacttattttttgtttggttgataatga actgtgctgattacaaaaatactaaaaatgcccatattttttcctccttataaaatt agtataattatagcacggtcgacggatcggggatccaaaataaaaagcgcctacccc accgaccaaagtgaatgggtagacgcctaacaaatactcggagcaacaaggctcttt gtatacacatttttacacaggagggcaataatatggcggtattcaagcgagctaacc gaaaaagtaagccttggggattccagtattcatacaaagtggatggcatctccaagc agaaaacatcattttacaaaacaagaaaagaagctaaggctgctgaggcgaagtacc tcgcttctactggcggatctgtaaaaatcgatccagtgatcactttcgcagattggt atgacaagtggttgcatacctacaagatacgttctgtttccgaactgacgatgacca agtatgcaacttcgggtacaatcatcagaaactacttcaaagaccttaaattaattg acttaacgcgcatgatttatcaacagtttattaacaactatattgatgacggttacg gccacaagcacgcaaggcaatcagtccagaagctacattcacacgctcatcaagcaa ttatggccgcagcagacgaaggtttgattaggcgcgattatgccgctcatgcagaac tgggtggtaccgcaggcagatcagaagacacaaaatttcttgaagctgatcagttcg agaaactgcgagattatgttgatcaatttgccaacccgcaacgaattgctctcatga tggttcaaacggccatatactctggcgctcggcttggagaaattggtggcttaacgt gggaagatattgatgagaagaagagcaccatcagtatcgacaagaccttcaagtaca ggtttgtcattcgtaacgcggatggtagctggccagaccgtgaaaaagtcttcggtc cgaccaagactccttcaagtgttcgtactatcaaagtaagcccagttcttatcgcta gcctccataagctcatattggctgacagaataaaagcgattaacaatccgtaccatt tactgtttcttgggccgaccggcttgccaatatatagcaatggtgtcaacaaggaac ttcgccgcgctctcaaacatcttggtattgagcgtcctgggttcggtttccacggat tgcggcacacgcatggcagctacttgctttataaaggccttgacattcagtatgtat cacatcgcctcggacacgaaaacgttggcattaccaccaagatctatacacatctgc tggatgcgatgacacagaagcaggacgagaaagcaatgaatgtgttgtgactaaaaa tcgaaccagagaaagcggctcaatgtcaactgccacaaggtttacagcacacattca attttcgatcacgaaccattttcctaaaaaatcgcaatttcaggctatttggttcga tgtggttcgatggattatattttttaggggtttttcggagttcagataaatgcaaga atgccggtttaaagccatttctgagcactaaaaaagaccctctagggggctttgata ccggtgatcggggtatcacggaatgtatacgtactgatatgattgcatttatgacaa aaagtggttcgatgtggttcgatgcttcaaacgacagcgaccaacaacacatctcta tataataggtagaaatagcttttaagagttcagaaatatgggcacacaagaccggca tgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgct cacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgccta atgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggg aaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggttt gcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcg gctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatc aggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccg taaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatca caaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatacca ggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacg ctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacga accccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaa cccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcag agcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggcta cactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaa aagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgat cttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggt catgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttt taaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaat cagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgact ccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgc aatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagcc agccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtc tattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaa cgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttc attcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaa aaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagt gttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgt aagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtat gcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatag cagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaag gatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact gate ttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaa tgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcct ttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatt tgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagt gccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcg tatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgaca catgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagaca agcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgc ggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacag atgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactg ttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaaggggg atgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttg taaaacgacggccagtg

SEQ ID NO: 21 - amino acid sequence of Integrase (Lactobacillus phage A2) encoded by CDS of the pEM40 plasmid

Atgggtagacgcctaacaaatactcggagcaacaaggctctttgtatacacattttt acacaggagggcaataatatggcggtattcaagcgagctaaccgaaaaagtaagcct tggggattccagtattcatacaaagtggatggcatctccaagcagaaaacatcattt tacaaaacaagaaaagaagctaaggctgctgaggcgaagtacctcgcttctactggc ggatctgtaaaaatcgatccagtgatcactttcgcagattggtatgacaagtggttg catacctacaagatacgttctgtttccgaactgacgatgaccaagtatgcaacttcg ggtacaatcatcagaaactacttcaaagaccttaaattaattgacttaacgcgcatg atttatcaacagtttattaacaactatattgatgacggttacggccacaagcacgca aggcaatcagtccagaagctacattcacacgctcatcaagcaattatggccgcagca gacgaaggtttgattaggcgcgattatgccgctcatgcagaactgggtggtaccgca ggcagatcagaagacacaaaatttcttgaagctgatcagttcgagaaactgcgagat tatgttgatcaatttgccaacccgcaacgaattgctctcatgatggttcaaacggcc atatactctggcgctcggcttggagaaattggtggcttaacgtgggaagatattgat gagaagaagagcaccatcagtatcgacaagaccttcaagtacaggtttgtcattcgt aacgcggatggtagctggccagaccgtgaaaaagtcttcggtccgaccaagactcct tcaagtgttcgtactatcaaagtaagcccagttcttatcgctagcctccataagctc atattggctgacagaataaaagcgattaacaatccgtaccatttactgtttcttggg ccgaccggcttgccaatatatagcaatggtgtcaacaaggaacttcgccgcgctctc aaacatcttggtattgagcgtcctgggttcggtttccacggattgcggcacacgcat ggcagctacttgctttataaaggccttgacattcagtatgtatcacatcgcctcgga cacgaaaacgttggcattaccaccaagatctatacacatctgctggatgcgatgaca cagaagcaggacgagaaagcaatgaatgtgttgtgactaaaaatcgaaccagagaaa gcggctcaatgtcaactgccacaaggtttacagcacacattcaattttcgatcacga accattttcctaaaaaatcgcaatttcaggctatttggttcgatgtggttcgatgga ttatattttttaggggtttttcggagttcagataaatgcaagaatgccggtttaaag ccatttctgagcactaaaaaagaccctctagggggctttgataccggtgatcggggt atcacggaatgtatacgtactgatatgattgcatttatgacaaaaagtggttcgatg tggttcgatgcttcaaacgacagcgaccaacaacacatctctatataataggtagaa atagcttttaagagttcagaaatatgggcacaca SEQ ID NO: 22 - Primer fwd aaactagtgg atccaaaata aaaagcgcct

SEQ ID NO: 23 - Primer rev aaactagttt gtgtgcccat atttctgaac tct

SEQ ID NO: 24 - Primer int-attP fwd cgggtacaat catcagaaac

SEQ ID NO: 25 - Primer int-attP rev ccttggtatt caacaaccaa

SEQ ID NO: 26 - nucleotide sequence of pRac-int-attP plasmid

SEQ ID NO: 27 - amino acid sequence of beta-lactamase encoded by CDS of the pRac-int-attP plasmid

SEQ ID NO: 28 - amino acid sequence of Integrase (Lactobacillus phage A2) encoded by CDS of the pRac-int-attP plasmid SEQ ID NO: 29 - synthetic linear fragment

SEQ ID NO: 30 - Propeptide Leu Glu lie Ser Ser Cys Asp Ala SEQ ID NO: 31 - Colony PCR primer fwd cccagaatca gaccattacc SEQ ID NO: 32 - Colony PCR primer rev ccttggtatt caacaaccaa

SEQ ID NO: 33 - nucleotide sequence of pRac-DAO-lactase-int-attP-Amuc- aldh-fruct vector

1 agagtttttt gcgccgcttt agtgtttatt ttgtttaatt gagtgcgtaa agtttgaagc 61 aggcgttgag ttaacgcaag agtccgtaag cgtaacgcgc gttgcagcgc cgctgttgtt 121 tgttggatta tgaaagtcgc taattgaatg aaattattag tgcggcgagt ttttattttg 181 gctggcgccg cttgaagggc gtttaaggca atgaaattcc gagttgggtt taattttaaa 241 agttgttggc gattttgccg agtggcagtg cgggcaagat ttttgaaaag aatttggaat 301 ttggcaagaa gtggggcgaa cgcaagcgca ttttgttgcc gtattatgat ccgccgttga 361 gtgcggaatg tcagtttttg tgtcagaagc gcagtattcc gaccggcagt cgcgtttaag 421 gcgtttttca gagtccgacc aatcgccagc gctttcgcca gcgctttttg taaatttata 481 tttaagttgc gttgttgttg tttttgtaat tgcagagtga taccttgatt ttgtaaaatt 541 atttgattgg cgttttttaa atggttatta gtgaaagtaa gaagcgcgtt atgattagtt 601 tgaccaagga acaggataag aagttgaccg atatggcgaa gcagaagggc tttagtaaga 661 gtgcggttgc ggcgttggcg attgaagaat atgcgcgcaa ggaaagtgaa taaaagaagt 721 aagcgaaggc gcgcgttttt cgccgcattc gcgtttttgc gacctgtttt taataaggca 781 atattagttg gttgttgaat accaaggcgc gcaattttgg ctttttgttg tatccggata 841 gtattccgaa tgattggaag gaaaagttgg aaagtttggg cgttagtatg gcggttagtc 901 cgttgcatga tatggatgaa aagaaggata aggatacctg gaatagtagt gatgttattc 961 gcaatggcaa gcattataag aagccgcatt atcatgttat ttatattgcg cgcaatccgg 1021 ttaccattga aagtgttcgc aataagatta agcgcaagtt gggcaatagt agtgttgcgc 1081 atgttgaaat tttggattat attaagggca gttatgaata tttgacccat gaaagtaagg 1141 atgcgattgc gaagaataag catatttatg ataagaagga tattttgaat attaatgatt 1201 ttgatattga tcgctatatt accttggatg aaagtcagaa gcgcgaattg aagaatttgt 1261 tgttggatat tgttgatgat tataatttgg ttaataccaa ggatttgatg gcgtttattc 1321 gcttgcgcgg cgcggaattt ggcattttga ataccaatga tgttaaggat attgttagta 1381 ccaatagtag tgcgtttcgc ttgtggtttg aaggcaatta tcagtgtggc tatcgcgcga 1441 gttatgcgaa ggttttggat gcggaaaccg gcgaaattaa gtaacagacc aagaagaaga 1501 gttatttgtt gaagatgcgc aattaaaaga agaagttgcg cacctaaaag agtgttttga 1561 aggataccga aaagtggaag ttgaattaag ttcagcagta aatttattaa gaagaaggct 1621 tgttgaataa gtaaaagccg ccgtaacgca agagtaaggg cgcgtttatt ttggtttaat 1681 gttgtgatta ataacagtat gattgtaata agcagaggat ccctgtcaga ccaagtttac 1741 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 1801 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg

1861 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc

1921 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag

1981 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt

2041 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac

2101 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc

2161 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt

2221 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt

2281 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc

2341 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt

2401 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca

2461 ggggggegga gcctatggaa aaggatcctt tgattcagaa actatgtggc aagcttaata

2521 ataaatctgt caaaataatt tattttgaca gattttttta tctaataatt aaaataatta

2581 tttcacaatg ttcacaagcg cttacaaaag aaaatagatt gacttatgct aaactgaata

2641 atgtaaaaag aattttacat ttaaaggaga cctattagtg taataggacg tgcttcaggc

2701 gtgttgcctg tacgcatgct gattcttcag caagactact acctcatgag agttatagac

2761 tcatggatct tgctttgaag ggttttgtac attataggct cctatcacat gctgaaccta

2821 tggcctatta cattttttta tatttcaagg aggaaaagac catgaccatt ggcaatttgc

2881 gcccggcgac cgttttgatt gatgaaaccg cgattttgca taatgttcag catgaagttg

2941 cgcgcttgaa gaagcagacc cagttgtttg cggttgttaa ggcggatgcg tatggccatg

3001 gcatgttgcg cgttgcgcat gttgcgaagg cggcgggcgc gagtggcttt tgtgttgcgt

3061 tgttggatga agcgttggat ttgcgcgcgg cgaattatac cgaaccggtt ttggttttgg

3121 gcattgttcc gagtcagtat gcggcgattg cggcggcgca gaccgttagt ttgccgttga

3181 gtagtgttga ttggttgaag caggcgttgc cggttttgga agcgcagccg gaattgccgc

3241 cgttgcgctt gcatattgcg ttggataccg gcatgggccg cattggcttt accgatgatc

3301 agaccttgtt ggatgcgatt gcgtttattc aggcgcatcc gaaggcgttt accattgaag

3361 gcatttttac ccattttgcg accgcggatg atttggatga tgcgtatttt aagcagcagg

3421 ttgcgaagtt taatcatatg gttgcgttgt tgccgcatcg cccgcgctat gttcatgtta

3481 gtaatagtgc gaccagtttg tggcatgcgg cgtgtaatgg caatatggtt cgctatggcg

3541 ttgcgattta tggcttgaat ccgagtggcg atgttattcc gaccaccccg tttccgttgg

3601 aaccggcgtt gagtttggaa agtgaattga ccttttgtaa gcaggttcat gcgggcgatg

3661 gcattagtta tggcgttacc tatcgcgcga ccggcgatga atttattggc accgttccgg

3721 ttggctatgc ggatggctgg ttgcgccgct tgcagggctt tcatgttttg gttgatggcc

3781 atccgtgtga aattgttggc cgcgtttgta tggatcagtt tatgattcgc ttgccgaagg

3841 cgtatccggc gggcaccaag gttgttttga ttggccagag tggcgatcag gaaattacct

3901 tgttggatgt tgcgaagtat agtcatacca ttcattatga aattgcgtgt aatttgacca 3961 gtcgcttgaa gcgccagagt attaatccga ttgcgcgcta attagactcg aggtccttat

4021 gtggaggttt ctgggcatgc gagcgcgttt cagcagagcg tgaattggcg cgttgaataa

4081 tcggcgtgta ttggccttgc ctgtgatgac cgggctttgg tcaatgcagt caaggtcctt

4141 acacgcaggt ttcggtgcca atgagcgcgt ttaaaaaaaa accccgcccc tgacagggcg

4201 gggttttttt tgaattctag tatttttcgg tcattttaac ttgctatttc ttgaaggtaa

4261 taggacgtgc ttcaggcgtg ttgcctgtac gcatgctgat tcttcagcaa gactactacc

4321 tcatgagagt tatagactca tggatcttgc tttgaagggt tttgtacatt ataggctcct

4381 atcacatgct gaacctatgg cctattacat ttttttatat ttcaaggagg aaaagaccat

4441 gtataccgaa aataccggca agcatcatcg caatggcttg ccggtttggt tgttgccgtt

4501 gttggttgtt attagttttt ggggcgttag tcagaatatt atggttgttg atgcgagtag

4561 tgttgatttg gaaattagta gtacctgtga tgcggagcca tcaccgggga cattgcctcg

4621 aaaggcaggt gtttttagcg acctgtcaaa ccaagaattg aaagcagttc actcgttttt

4681 gtggtcgaaa aaggaattga gactccaacc gtcaagtacg acgacgatgg ccaagaacac

4741 cgtattcctc atcgaaatgt tacttccgaa aaaataccat gtcctccggt ttttggacaa

4801 aggcgagcgg catcccgtga gggaagcacg ggccgtgatt ttttttggcg accaagaaca

4861 ccccaacgtc accgaatttg ctgtcggccc cttaccaggg ccatgttata tgcgtgcttt

4921 atctccccgc cctggatacc aaagctcttg ggcctctcga cccataagta ccgcggaata

4981 cgcccttcta taccacacgt tacaagaggc gactaaaccg ctccatcagt tctttctaaa

5041 taccacgggc ttttcatttc aagactgcca cgatcggtgt cttgctttca cagacgttgc

5101 tcccagaggc gttgcaagcg gacaaaggcg tagctggttg attattcagc gatacgtcga

5161 gggatatttt ttacacccga ccggcttaga attgctagtt gaccatggta gtacggacgc

5221 gggccactgg gcagtggagc aagtatggta caatggaaaa ttttacggat ctcctgagga

5281 gcttgctcga aagtatgccg acggtgaggt cgatgtagtc gtactagaag acccgttgcc

5341 gggtggaaag ggacacgata gtaccgaaga accaccttta ttttcttcac acaaacctcg

5401 tggcgatttt ccgagcccaa ttcatgtatc tggcccacgc ttggtacagc cgcacggacc

5461 tcgtttccgg cttgagggca atgcagtctt gtatggcgga tggtcatttg cgtttcggct

5521 gcgctcgtcc tcgggcttac aggtacttaa cgtgcatttc ggtggggaac gcatcgcata

5581 cgaggtctct gtgcaagagg cggtggcgct gtacggaggt catacgccag ctgggatgca

5641 gactaaatat ttggatgttg gttggggtct gggcagcgtc acacatgaat tggccccagg

5701 cattgattgc ccagaaactg ccaccttctt agatactttc cactactatg atgctgatga

5761 tccagtgcat tatcctagag cattgtgctt gttcgagatg ccaaccggcg tgcctttacg

5821 acgtcacttc aactccaact ttaagggggg tttcaatttt tatgccggtt tgaaagggca

5881 ggtgcttgtc ctgcgtacga caagtacggt ttataactat gattatattt gggattttat

5941 tttctatcca aatggggtta tggaggcgaa aatgcacgcg actggctatg tgcatgcaac

6001 attttataca ccggaggggc tgcgtcatgg gacgcgttta catacacatc ttattggcaa

6061 tattcatacc catttggttc attatcgcgt cgatttggat gtggcgggca ctaagaattc 6121 ctttcagaca cttcaaatga agcttgaaaa tatcactaac ccatggtcgc ctcgtcatcg

6181 agttgttcaa ccaacgttgg agcaaaccca atattcctgg gaacggcagg ccgcctttcg

6241 gtttaaacgc aagttgccga agtatttact gtttacgtcg cctcaagaga acccatgggg

6301 tcataagcgc acttatcggt tacagattca ttcaatggcg gatcaggttt taccgcctgg

6361 ctggcaggaa gaacaggcta taacctgggc ccgctatcct ttggcggtta ccaagtatcg

6421 cgaaagtgaa ctgtgttcat cgagtatcta tcatcagaat gatccttggc atccgcctgt

6481 tgtgtttgaa cagtttttgc ataataatga aaatatcgaa aatgaagatc tggttgcatg

6541 ggttactgtt ggttttctgc atattccgca ttcggaagat attccgaata ctgctacccc

6601 gggtaatagt gttgggtttc tgctgcgccc gtttaatttt tttccggaag atccgagtct

6661 ggcttcccgg gataccgtta ttgtttggcc gcgcgataat ggtccgaatt atgttcagcg

6721 gtggattccg gaagatcggg attgttccat gccgccgccg tttagttata atggtaccta

6781 tcgcccggtt taattagact cgaggtcctt atgtggaggt ttctgggcat gcgagcgcgt

6841 ttcagcagag cgtgaattgg cgcgttgaat aatcggcgtg tattggcctt gcctgtgatg

6901 accgggcttt ggtcaatgca gtcaaggtcc ttacacgcag gtttcggtgc caatgagcgc

6961 gtttaaaaaa aaaccccgcc cctgacaggg eggggttttt tttcttaaaa tatagtcata

7021 gaattagggc gtcggtttta aagtaatagg acgtgcttca ggcgtgttgc ctgtacgcat

7081 gctgattctt cagcaagact actacctcat gagagttata gactcatgga tcttgctttg

7141 aagggttttg tacattatag gctcctatca catgctgaac ctatggccta ttacattttt

7201 ttatatttca aggaggaaaa gaccatgtat accgaaaata ccggcaagca tcatcgcaat

7261 ggcttgccgg tttggttgtt gccgttgttg gttgttatta gtttttgggg cgttagtcag

7321 aatattatgg ttgttgatgc gagtagtgtt gatttggaaa ttagtagtac ctgtgatgcg

7381 tctgactggg agtcggaccg taactttatc tcgactgcgg gacctcttac caatgaccta

7441 ttgcataatc tatcaggact gctaggcgac caatcgtcga acttcgtggc eggggataaa

7501 gatatgtacg tgtgtcacca gcctctgcct acattcctac ctgagtactt ttcgtccttg

7561 catgcatctc agatcacgca ctataaagta tttctatcgt gggcacagtt gctcccagcg

7621 ggcagtacgc agaaccctga tgagaaaaca gtacaatgtt accgtcgcct tttaaaagca

7681 ttgaagacag cacggctaca acccatggtt atattgcacc atcagacact tccggcgtcg

7741 acactgcggc gaacagaagc attcgctgac ttatttgcgg actatgccac gtttgcgttt

7801 cacagcttcg gcgatttagt tggcatctgg ttcactttct ctgatttgga agaagttatt

7861 aaggagctgc cccatcaaga gagtcgagca tcacaactac agactctctc cgacgctcac

7921 cggaaggcct atgagatcta ccacgagagt tacgcgtttc agggtggcaa gctctctgtt

7981 gttttacgag ccgaagacat cccggaatta ctgttagagc cgccgatatc tgctcttgca

8041 caagacacag tcgacttttt gtccttagat ctctcctatg aatgccaaaa tgaagcgtcg

8101 ttacgccaaa aactttctaa attacagact attgagccaa aagtaaaggt attcatcttc

8161 aatttaaaat taccagattg ccctagcaca atgaaaaatc ccgcttcttt gcttttttcc

8221 ctttttgaag cgattaacaa ggatcaggtt ttaaccattg gattcgatat caatgaattc 8281 ctgagttgct catcatctag taagaagagt atgtcgtgtt cactgaccgg atccttggct

8341 ctgcaaccag atcagcaaca ggaccatgaa actactgata gttcgcccgc aagcgcctac

8401 cagcggatat gggaggcatt cgcaaaccaa tctcgagctg agcgcgacgc tttcttacag

8461 gacacgttcc cagaaggttt cctatggggg gcgtctacag gtgcgtttaa tgttgagggg

8521 ggatgggcgg aaggcgggag aggcgtatcc atctgggatc cgcgtcggcc attgaatacg

8581 accgagggcc aagccacttt agaggtagcc tctgacagtt atcacaaggt ggcttctgac

8641 gtggctctac tatgtggcct gagagctcag gtatataagt tcagcatttc atggagccgg

8701 atcttcccca tggggcacgg ctctagtcca tcgcttcctg gcgtagcgta ctataacaag

8761 ttgatagatc gattgcagga tgctggaatt gaaccaatgg ccacactctt ccactgggac

8821 ttgccacagg cattgcaaga tcacggaggg tggcaaaatg aatcggtggt ggatgcgttc

8881 ctcgactacg cagcgttctg cttttcgacc tttggggacc gcgtcaagtt gtgggtaacg

8941 tttcacgaac cttgggtcat gtcctatgca ggctacggga ctggccaaca tcccccaggc

9001 atttctgacc cgggcgtagc gtcttttaaa gtagcccacc ttgttctaaa agcacatgct

9061 cgcacgtggc atcattataa cagccatcat cgcccccaac aacaaggcca cgtcgggatc

9121 gttcttaact ccgattgggc agagccgctt tctcctgagc ggccggagga tttacgcgct

9181 tcagaacggt tcctccattt catgctcggg tggttcgccc acccggtctt tgtcgacgga

9241 gattatccag caacattgag aacacagata caacaaatga accgccagtg cagtcatcca

9301 gtcgctcaat taccggaatt tacagaagca gaaaagcagc tccttaaggg ttcggctgat

9361 tttcttggcc tttcacacta cacgagccgg ttaattagca atgctcccca aaacacctgt

9421 atcccaagct atgacacgat tggcggcttt tcacaacacg tcaatcacgt atggccacaa

9481 acaagctctt cctggatccg cgttgtcccg tggggcataa gacgattgct gcaatttgtg

9541 agcttggaat acacccgggg taaggtgccg atttatttag cgggcaatgg catgcccata

9601 ggtgaatcag aaaacttgtt cgacgattcc ttacgtgtcg actattttaa ccagtacatt

9661 aacgaggtat tgaaggcaat caaagaagac agtgtggacg tccgctctta tatcgcccgc

9721 tctcttattg atggtttcga aggaccctcc gggtattcgc aacgtttcgg gctgcaccac

9781 gttaacttta gcgactcttc taagagtcgt acaccacgaa agtccgcgta ctttttcact

9841 agtatcattg agaaaaatgg cttcttaacg aaaggtgcaa agcgtttgtt acctcctaac

9901 actgtaaatc ttccttcgaa ggtgcgcgcg tttacattcc ctagtgaggt tccgtcgaag

9961 gctaaggttg tctgggagaa attctcttcc caaccgaagt ttgagcggga tctgttttat

10021 catggaacgt ttcgcgatga ctttctgtgg ggtgtcagca gttccgcata ccagattgag

10081 ggtgcgtggg atgcagatgg taagggacct tcgatctggg ataactttac ccatacacca

10141 ggatctaatg tgaaggacaa cgcaacgggc gatattgctt gcgactccta ccatcagtta

10201 gatgccgatt taaatatgct gagggcattg aaggttaaag cgtaccgctt tagcattagc

10261 tggagtcgta tctttccgac tggtcgtaat agctcgatta attcgcatgg cgtggattat

10321 tacaaccgcc tgatcaatgg tctggttgct tcgaatatct ttcctatggt gacactgttt

10381 cattgggacc ttcctcaggc gttgcaggac atcgggggct gggaaaatcc tgcgcttatc 10441 gatctgtttg attcctatgc ggatttttgc tttcaaacct ttggggatcg ggtgaagttt

10501 tggatgacct ttaacgagcc gatgtatctt gcttggctgg gctacggttc aggcgagttt

10561 ccacctggtg ttaaggatcc aggctgggcg ccttatcgga ttgcacatgc tgtgatcaaa

10621 gcacatgcgc gggtgtatca tacatatgat gaaaaatacc gtcaagaaca aaagggtgtc

10681 attagtcttt cgttgtcgac tcattgggcg gaaccgaagt cgccaggtgt tccgcgggat

10741 gttgaggcgg cagatcggat gctgcaattt tcgttaggct ggtttgctca tccgatcttt

10801 aggaacggtg attacccgga taccatgaag tggaaggttg ggaaccggag cgagttgcaa

10861 catttggcga cttcacggtt gccatcgttt actgaggagg agaaacgctt tattcgcgca

10921 acggcggatg ttttttgtct gaacacgtat tattcacgca ttgtccagca taagaccccg

10981 cgccttaatc cgccatcgta tgaagatgat caggaaatgg cggaagagga agatccatca

11041 tggccttcga ctgctatgaa tcgtgctgcg ccgtggggaa cgcggaggct tctgaattgg

11101 attaaggagg aatatggcga tattccgatt tacattaccg agaacggcgt cgggttgact

11161 aatccgaaca cggaagatac cgatcggatt ttttaccata aaacctatat taatgaagct

11221 cttaaagcat accgcctgga tggtattgat ttacgtggtt acgtcgcgtg gagcttgatg

11281 gataattttg agtggttaaa cgggtatacc gttaagtttg gactttacca tgtcgatttt

11341 aataatacta accgacctcg cactgctcgg gctagtgcac gttattacac tgaggtcatt

11401 actaacaacg gaatgccttt agcacgagag gatgaatttt tgtatggtcg ttttcctgag

11461 ggatttattt ggtcagcggc ttcggccgcc tatcagattg aaggagcatg gcgggcagat

11521 ggtaagggct tatccatttg ggatactttt tcgcataccc cgttacgcgt tgaaaatgat

11581 gctattggtg atgtcgcgtg tgatagttat cataagattg ctgaagatct ggtcaccctg

11641 caaaatttag gcgtttcaca ttatcgcttt agcattagct ggtcccgcat tttacctgat

11701 ggaacgactc ggtatattaa tgaagctgga ttaaattatt atgtccgctt gattgatacg

11761 ttactggccg caagcattca accgcaggtg acgatttatc attgggatct gccgcagacg

11821 ctgcaggatg ttgggggttg ggaaaatgaa acgattgtgc aacgctttaa ggaatatgct

11881 gatgtgttgt ttcaacgatt aggcgataag gtgaaatttt ggattaccct gaatgaacca

11941 tttgtgattg catatcaggg gtatggttat ggcaccgcgg caccgggtgt ttccaatcgt

12001 ccaggcaccg ctccgtatat tgtgggccat aatctgatta aagcccatgc tgaagcctgg

12061 catttatata atgatgttta tcgggctagt cagggcggcg tgattagtat tacgattagc

12121 tcagattggg cagaaccgcg tgatccgtca aatcaagaag atgtggaagc ggcccgtcga

12181 tatgtgcaat ttatgggtgg gtggtttgcc catccgattt ttaaaaatgg tgattataat

12241 gaagttatga aaacgagaat tcgtgatcgt tcattggcgg ccggtctgaa taaaagtcgg

12301 ttgccggaat ttacggaaag tgaaaaacga cgtattaatg gaacgtatga tttttttggc

12361 tttaatcatt atacgaccgt tttagcgtat aatttaaatt atgcgaccgc gatttcatca

12421 tttgatgccg atcgcggtgt tgcctcaatt gccgatcgga gttggccaga ttcaggctca

12481 ttttggctga aaatgacgcc gtttggtttt cgaaggattc tgaattggtt gaaagaagaa

12541 tataatgatc caccgattta tgttacggaa aatggtgtta gtcagcgcga agaaaccgat 12601 ctgaatgata ccgccagaat ttattatttg cgcacctata ttaatgaagc cttgaaagcc

12661 gttcaggata aagttgattt gcgtggatat accgtttgga gtgccatgga taattttgaa

12721 tgggccaccg gtttttcaga acgttttggt ttgcattttg ttaattatag tgatccgtca

12781 ttgccaagaa ttccgaaagc cagtgccaaa ttttatgcca gtgttgttcg atgtaatggg

12841 tttccggatc cggccaccgg accgcatgcc tgtttgcatc agccggatgc cggtccgacc

12901 attagtccgg ttcggcagga agaagttcag tttttggggt tgatgttggg taccaccgaa

12961 gcccagaccg ccttgtatgt tttgtttagt ttggttttgt tgggcgtttg tggcttggcc

13021 tttttgtcat ataaatattg taaacggtca aaacagggga aaacccagcg tagtcagcag

13081 gaattgtcac cggttagtag tttttaatta gactcgaggt ccttatgtgg aggtttctgg

13141 gcatgcgagc gcgtttcagc agagcgtgaa ttggcgcgtt gaataatcgg cgtgtattgg

13201 ccttgcctgt gatgaccggg ctttggtcaa tgcagtcaag gtccttacac gcaggtttcg

13261 gtgccaatga gcgcgtttaa aaaaaaaccc cgcccctgac agggcggggt tttttttgaa

13321 ttcccatggc aaaattatga aaaagtctgt caattttgtt tcggcgaatt gataatgtgt

13381 tatactcacg taataggacg tgcttcaggc gtgttgcctg tacgcatgct gattcttcag

13441 caagactact acctcatgag agttatagac tcatggatct tgctttgaag ggttttgtac

13501 attataggct cctatcacat gctgaaccta tggcctatta cattttttta tatttcaagg

13561 aggaaaagac catgaagaag tttaatttta agaccatgtt gttgttggtt ttggcgagtt

13621 gtgtttttgg cgttgttgtt aatgttacca ccagtttggg cccgcagacc gcgattaccg

13681 cgcaggcgag taagttggaa attagtagta cctgtgatgc gattgttaat agtaagcgca

13741 gtgaattgga taagaagatt agtattgcgg cgaaggaaat taagagtgcg aatgcggcgg

13801 aaattacccc gagtcgcagt agtaatgaag aattggaaaa ggaattgaat cgctatgcga

13861 aggcggttgg cagtttggaa accgcgtata agccgttttt ggcgagtagt gcgttggttc

13921 cgaccacccc gaccgcgttt cagaatgaat tgaagacctt tcgcgatagt ttgattagta

13981 gttgtaagaa gaagaatatt ttgattaccg ataccagtag ttggttgggc tttcaggttt

14041 atagtaccca ggcgccgagt gttcaggcgg cgagtacctt gggctttgaa ttgaaggcga

14101 ttaatagttt ggttaataag ttggcggaat gtggcttgag taagtttatt aaggtttatc

14161 gcccgcagtt gccgattgaa accccggcga ataatccgga agaaagtgat gaagcggatc

14221 aggcgccgtg gaccccgatg ccgttggaaa ttgcgtttca gggcgatcgc gaaagtgttt

14281 tgaaggcgat gaatgcgatt accggcatgc aggattattt gtttaccgtt aatagtattc

14341 gcattcgcaa tgaacgcatg atgccgccgc cgattgcgaa tccggcggcg gcgaagccgg

14401 cggcggcgca gccggcgacc ggcgcggcga gtttgacccc ggcggatgaa gcggcggcgc

14461 cggcggcgcc ggcgattcag caggttatta agccgtatat gggcaaggaa caggtttttg

14521 ttcaggttag tttgaatttg gttcatttta atcagccgaa ggcgcaggaa ccgagtgaag

14581 attaattaga ctcgaggtcc ttatgtggag gtttctgggc atgcgagcgc gtttcagcag

14641 agcgtgaatt ggcgcgttga ataatcggcg tgtattggcc ttgcctgtga tgaccgggct

14701 ttggtcaatg cagtcaaggt ccttacacgc aggtttcggt gccaatgagc gcgtttaaaa 14761 aaaaaccccg cccctgacag ggcggggttt tttttaactt atatgacaat tttggtacag

14821 gagtcttcaa aagtggcaca gaaccaaagt gatggaaaaa taagaaactg cttgctttac

14881 tttgcctatt aatgctataa tgaaaatgta gaaaagatgg acgtgaaacc agttcatcaa

14941 aaaaagtaaa ggagactgtt caaccgtaat aggacgtgct tcaggcgtgt tgcctgtacg

15001 catgctgatt cttcagcaag actactacct catgagagtt atagactcat ggatcttgct

15061 ttgaagggtt ttgtacatta taggctccta tcacatgctg aacctatggc ctattacatt

15121 tttttatatt tcaaggagga aaagaccatg aagaagttta attttaagac catgttgttg

15181 ttggttttgg cgagttgtgt ttttggcgtt gttgttaatg ttaccaccag tttgggcccg

15241 cagaccgcga ttaccgcgca ggcgagtaag ttggaaatta gtagtacctg tgatgcgcgc

15301 tatagtagtg cggcggcgtt gccgagtccg attttgaatc cggatattcc gtataatcag

15361 ttgtttatta ataatgaatg gcaggatgcg gttagtaaga agacctttcc gaccgttaat

15421 ccgaccaccg gcgaagttat tggccatgtt gcggaaggcg atcgcgcgga tgttgatcgc

15481 gcggttaagg cggcgcgcga agcgtttcgc ttgggcagtc cgtggcgccg catggatgcg

15541 agtgaacgcg gccgcttgtt gaatttgttg gcggatttgg ttgaacgcga tcgcgtttat

15601 ttggcgagtt tggaaacctt ggataatggc aagccgtttc aggaaagtta tgcgttggat

15661 ttggatgaag ttattaaggt ttatcgctat tttgcgggct gggcggataa gtggcatggc

15721 aagaccattc cgatggatgg ccagcatttt tgttttaccc gccatgaacc ggttggcgtt

15781 tgtggccaga ttattccgtg gaattttccg ttggttatgc agggctggaa gttggcgccg

15841 gcgttggcga ccggcaatac cgttgttatg aaggttgcgg aacagacccc gttgagtgcg

15901 ttgtatttgg cgagtttgat taaggaagcg ggctttccgc cgggcgttgt taatattatt

15961 accggctatg gcccgaccgc gggcgcggcg attgcgcagc atgttgatgt tgataaggtt

16021 gcgtttaccg gcagtaccga agttggccat ttgattcaga aggcggcggg cgatagtaat

16081 ttgaagcgcg ttaccttgga attgggcggc aagagtccga gtattgtttt ggcggatgcg

16141 gatatggaac atgcggttga acagtgtcat gaagcgttgt tttttaatat gggccagtgt

16201 tgttgtgcgg gcagtcgcac ctttgttgaa gaaagtattt ataatgaatt tttggaacgc

16261 accgttgaaa aggcgaagca gcgcaaggtt ggcaatccgt ttgaattgga tacccagcag

16321 ggcccgcagg ttgataagga acagtttgaa cgcgttttgg gctatattca gttgggccag

16381 aaggaaggcg cgaagttgtt gtgtggcggc gaacgctttg gcgaacgcgg cttttttatt

16441 aagccgaccg tttttggcgg cgttcaggat gatatgcgca ttgcgaagga agaaattttt

16501 ggcccggttc agccgttgtt taagtttaag aagattgaag aagttgttga acgcgcgaat

16561 aatacccgct atggcttggc ggcggcggtt tttacccgcg atttggataa ggcgatgtat

16621 tttacccagg cgttgcaggc gggcaccgtt tgggttaata cctataatat tgttacctgt

16681 cataccccgt ttggcggctt taaggaaagt ggcaatggcc gcgaattggg cgaagatggc

16741 ttgaaggcgt ataccgaagt taagaccgtt accattaagg ttccgcagaa gaatagttaa

16801 ttagactcga ggtccttatg tggaggtttc tgggcatgcg agcgcgtttc agcagagcgt

16861 gaattggcgc gttgaataat cggcgtgtat tggccttgcc tgtgatgacc gggctttggt 16921 caatgcagtc aaggtcctta cacgcaggtt tcggtgccaa tgagcgcgtt taaaaaaaaa 16981 ccccgcccct gacagggcgg ggtttttttt ttgacatttc ttgaagaggt tggtataata 17041 gtaataggac gtgcttcagg cgtgttgcct gtacgcatgc tgattcttca gcaagactac 17101 tacctcatga gagttataga ctcatggatc ttgctttgaa gggttttgta cattataggc 17161 tcctatcaca tgctgaacct atggcctatt acattttttt atatttcaag gaggaaaaga

17221 ccatgaagaa gtttaatttt aagaccatgt tgttgttggt tttggcgagt tgtgtttttg 17281 gcgttgttgt taatgttacc accagtttgg gcccgcagac cgcgattacc gcgcaggcga 17341 gtaagttgga aattagtagt acctgtgatg cgaccaccag tgcgagtagt acccagatta 17401 gtcagaccaa taccggcagt cagccgaata atgaaaccac cggcgaaacc gcgcagagta 17461 gtgttaatag taccgcgacc gcgagtagta gtagtgttgc ggatttgccg agtagtagtg

17521 atagtaagag tagtattggc agtaccatta gtcagccgac cgttgataag aaggaaacca 17581 gtaagagtga taccgcggat aatgatttga ccaagagtgt taccaccagt gatagtgata 17641 aggcgttgcc gaccagtaag accaccttgc cgaccagtaa tgaacaggtt cagagtagtg 17701 ttggccagag tcagaccgat cagccggcga gtagtgcgac cattgcgacc aatgcggtta 17761 ccagtgatgt tagtcagaat gatcagtata atgaaccgta tcgcaatcag tatcattata

17821 gtagtagtca gaattggatt aatgatccga atggcttgtt ttatgatagt aagaccggct 17881 tgtataattt gtattatcag tataatccgg aaggcaatca gtggggcaat atgagttggg 17941 gccatgcggt tagtaaggat ttgattaatt ggacccagga agatgttgcg attccgatgt 18001 tgcagaatca gggctgggaa gattttacct ataccaatac caccggcagt ttgaaggata 18061 agggcgaagt tcgctatgtt ggcgttccga ccaccaattg gggcgatgcg gatggcaaga 18121 aggcgatttt tagtggcagt attgttgttg ataccaataa tgttagtggc ttgggcaagg 18181 atgcgatttt ggcgttttat accgcggatt atcagattgc gacccgcaag aatgatggcg 18241 cggaagatgg ctggggcacc tggattggct tgaccgaaat tcaggaacag catttggcgt 18301 atagtttgga tggcggcaag acctttattc agtatagtaa ggatggcaat gcggcgaatc 18361 cgcaggcgat tattccgacc agtatgaatc agggcggcga tgcggcgaat tttcgcgatc

18421 cgagtgttgt ttatgatgcg gttaataagc agtattattt gaccgttgtt agtggccagc 18481 aggcgttgat ttataagagt agtaatttgt tggattggac ctatgcgagt aagattgaac 18541 gcgaaaatga tgttggcaat ggcgtttggg aatgtccgag tttggttccg atgaaggttg 18601 cgggcaccaa tgaaaccaag tgggtttttt gtattagtgt tcagcagggc gcgcatgcga 18661 ccggcagtgg catgcagtat tatgttggca atatgaccgc ggatggcacc tgggttccgg

18721 aaagtagtaa gaccttgcag aatccgatga ccatggatag tggcgaagat ttttatgcgg 18781 gcattccgtt tagtaatatg ccggatggcc gcaccgttat gttggcgtgg cagagtaatt 18841 ggagttatgt tgatgaagcg aagaccagtc cgtggagtgg caatatgacc ttgccgcgcg 18901 aattgagttt gaagaagaat gcggatacca ccgatggcta tttgttgacc aataccgttg 18961 ttaaggaaat tgcgaataat gaagaagcga atgttattaa taaggcggaa agtaatttta

19021 ccgttacccg cagtgatgaa caggttcagt atgaaggcaa gcagtataag attagtgcga 19081 cctttagttg ggatgaagcg gataagccga agagtgttgg ctttaagttg cgcgttagtg

19141 atgatcagaa gtatgatatg attgttggct atgatttgac caccggcttg ttgtatgttc

19201 agcgcttgaa taccggcgaa ccgaatatgg gcgcgccgcg cgataagatg aatgcgaccg

19261 ttaatgcgga tggcagtatt accattaccg tttatgttga tgaaaccagt attgaagcgt

19321 ttgcgaatga tggcgaaaag agtattaccc agaatttttt tatgcgcccg gaaaatattg

19381 gcgatcaggc gaccaccggc gtttatgttt atagtaatga tggcaccacc aagattagtg

19441 atttgaccat taatccgatt accagtattt ggaatagtac cggccagttg accgaaaagt

19501 ttgttgatga aaatggcaat accattgcga gtgataagat tcagaccggc cgcgttggcc

19561 agagttatac cagtgaaagt gcgaccattc cgggctatgt ttttgttaag gaaaataccg

19621 atcatattaa tagtaatcag ttgtatacca cccagaatca gaccattacc tatacctatc

19681 gcgcgagtca ggcgagtgtt gttaccaagg ataccacctt ggttgcgggc ccgagtgcgg

19741 cgtggaatgc ggcggataat ttggttggcg cgaccgatgc ggatggcaat gcgttggcgg

19801 ttagtgattt gaccgttacc ggcgcggttg atccgaagac cccgggcacc tataccgtta

19861 cctatagtta taccgatgcg accggcagta agattagtaa ggaagcgacc gttaccgtta

19921 ttgcgagtaa ggcggatatt gttaccaagg ataccgcgat ggttgcgggc ccgagtgcga

19981 cctggaatgc ggttgataat tttgttgaag cgaccggcgc ggatggcaat gcgttgacct

20041 tgagtgattt gaccgttaat ggcgcggttg atccgaagac cccgggcacc tataccgtta

20101 cctatagtta taccgatgcg accggcaata agattagtaa gaaggcgacc gttaccgtta

20161 ttgcgagtaa ggcggatatt gttaccaagg ataccaccat ggttgcgggc ccgagtgcga

20221 cctggaatgc ggcggataat ttggttattg cgaccgatgc gaagggcaat gcgttggcgt

20281 tgagtaattt gaccgttacc ggcagtgttg atagtaagac cccgggcacc tataccgtta

20341 cctatagtta taccgatgcg gcgggcaata agattagtaa ggaagcgacc gttaccgtta

20401 ttgcgagtaa ggcggatatt gttaccaagg ataccaccat ggttgcgggc ccgagtgcgg

20461 cgtggaatgc ggcgaataat ttggttagtg cgaccgatgc ggatggcaat gcgttggcga

20521 tgagtaattt gaccgttacc ggcaccgttg atttgaagac ccagggcacc tataccgtta

20581 cctataccta taccgatgtt gcgggcaata agattagtaa ggaagcgacc gttaccgttt

20641 tgaccgaaaa ggaaaccaat attgaagata ataccggcag tagtattagt aatgatcgcg

20701 aaaatccgcc ggcgagtatt accggcaagg gcggcgatga tattcatcag aatgcgaaga

20761 ccaccatgac caagaagaag accgaaacct tgccgcaggc gggcaatcat gttaatgaat

20821 tggcgattgt tttgggccag atgattttgg cgatttgtgt tggcggcatt ttgtggttga

20881 agcgccgcgt taagcgcgtt taattagact cgaggtcctt atgtggaggt ttctgggcat

20941 gcgagcgcgt ttcagcagag cgtgaattgg cgcgttgaat aatcggcgtg tattggcctt

21001 gcctgtgatg accgggcttt ggtcaatgca gtcaaggtcc ttacacgcag gtttcggtgc

21061 caatgagcgc gtttaaaaaa aaaccccgcc cctgacaggg cggggttttt ttttctagaa

21121 ctagtggatc caaaataaaa agcgcctacc ccaccgacca aagtgaatgg gtagacgcct

21181 aacaaatact cggagcaaca aggctctttg tatacacatt tttacacagg agggcaataa 21241 tatggcggta ttcaagcgag ctaaccgaaa aagtaagcct tggggattcc agtattcata

21301 caaagtggat ggcatctcca agcagaaaac atcattttac aaaacaagaa aagaagctaa

21361 ggctgctgag gcgaagtacc tcgcttctac tggcggatct gtaaaaatcg atccagtgat

21421 cactttcgca gattggtatg acaagtggtt gcatacctac aagatacgtt ctgtttccga

21481 actgacgatg accaagtatg caacttcggg tacaatcatc agaaactact tcaaagacct

21541 taaattaatt gacttaacgc gcatgattta tcaacagttt attaacaact atattgatga

21601 cggttacggc cacaagcacg caaggcaatc agtccagaag ctacattcac acgctcatca

21661 agcaattatg gccgcagcag acgaaggttt gattaggcgc gattatgccg ctcatgcaga

21721 actgggtggt accgcaggca gatcagaaga cacaaaattt cttgaagctg atcagttcga

21781 gaaactgcga gattatgttg atcaatttgc caacccgcaa cgaattgctc tcatgatggt

21841 tcaaacggcc atatactctg gcgctcggct tggagaaatt ggtggcttaa cgtgggaaga

21901 tattgatgag aagaagagca ccatcagtat cgacaagacc ttcaagtaca ggtttgtcat

21961 tcgtaacgcg gatggtagct ggccagaccg tgaaaaagtc ttcggtccga ccaagactcc

22021 ttcaagtgtt cgtactatca aagtaagccc agttcttatc gctagcctcc ataagctcat

22081 attggctgac agaataaaag cgattaacaa tccgtaccat ttactgtttc ttgggccgac

22141 cggcttgcca atatatagca atggtgtcaa caaggaactt cgccgcgctc tcaaacatct

22201 tggtattgag cgtcctgggt tcggtttcca cggattgcgg cacacgcatg gcagctactt

22261 gctttataaa ggccttgaca ttcagtatgt atcacatcgc ctcggacacg aaaacgttgg

22321 cattaccacc aagatctata cacatctgct ggatgcgatg acacagaagc aggacgagaa

22381 agcaatgaat gtgttgtgac taaaaatcga accagagaaa gcggctcaat gtcaactgcc

22441 acaaggttta cagcacacat tcaattttcg atcacgaacc attttcctaa aaaatcgcaa

22501 tttcaggcta tttggttcga tgtggttcga tggattatat tttttagggg tttttcggag

22561 ttcagataaa tgcaagaatg ccggtttaaa gccatttctg agcactaaaa aagaccctct

22621 agggggettt gataccggtg atcggggtat cacggaatgt atacgtactg atatgattgc

22681 atttatgaca aaaagtggtt cgatgtggtt cgatgcttca aacgacagcg accaacaaca

22741 catctctata taataggtag aaatagcttt taagagttca gaaatatggg cacacaaact

22801 agtcatggca gtatttttta ttaaaatgtt agta

SEQ ID NO: 34 - amino acid sequence of Integrase (Lactobacillus phage A2) encoded by CDS of the pRac-DAO-lactase-int-attP-Amuc-aldh-fruct plasmid

SEQ ID NO: 35 - nucleotide sequence encoding the C-terminal anchoring pro tein ttgccgaata ccaatgaaaa gcatgaatat ggcattgcgg cggttggctt ggcgttgttg 60 agtttgatgg gcttgggcag taccttgttg tttcgcaagg cgaagcgcca g 111 SEQ ID NO: 36 - Codon optimized sequence used for lactase: tctgactgggagtcggaccgtaactttatctcgactgcgggacctcttaccaatgac ctattgcataatctatcaggactgctaggcgaccaatcgtcgaacttcgtggccggg gataaagatatgtacgtgtgtcaccagcctctgcctacattcctacctgagtacttt tcgtccttgcatgcatctcagatcacgcactataaagtatttctatcgtgggcacag ttgctcccagcgggcagtacgcagaaccctgatgagaaaacagtacaatgttaccgt cgccttttaaaagcattgaagacagcacggctacaacccatggttatattgcaccat cagacacttccggcgtcgacactgcggcgaacagaagcattcgctgacttatttgcg gactatgccacgtttgcgtttcacagcttcggcgatttagttggcatctggttcact ttctctgatttggaagaagttattaaggagctgccccatcaagagagtcgagcatca caactacagactctctccgacgctcaccggaaggcctatgagatctaccacgagagt tacgcgtttcagggtggcaagctctctgttgttttacgagccgaagacatcccggaa ttactgttagagccgccgatatctgctcttgcacaagacacagtcgactttttgtcc ttagatctctcctatgaatgccaaaatgaagcgtcgttacgccaaaaactttctaaa ttacagactattgagccaaaagtaaaggtattcatcttcaatttaaaattaccagat tgccctagcacaatgaaaaatcccgcttctttgcttttttccctttttgaagcgatt aacaaggatcaggttttaaccattggattcgatatcaatgaattcctgagttgctca tcatctagtaagaagagtatgtcgtgttcactgaccggatccttggctctgcaacca gatcagcaacaggaccatgaaactactgatagttcgcccgcaagcgcctaccagcgg atatgggaggcattcgcaaaccaatctcgagctgagcgcgacgctttcttacaggac acgttcccagaaggtttcctatggggggcgtctacaggtgcgtttaatgttgagggg ggatgggcggaaggcgggagaggcgtatccatctgggatccgcgtcggccattgaat acgaccgagggccaagccactttagaggtagcctctgacagttatcacaaggtggct tctgacgtggctctactatgtggcctgagagctcaggtatataagttcagcatttca tggagccggatcttccccatggggcacggctctagtccatcgcttcctggcgtagcg tactataacaagttgatagatcgattgcaggatgctggaattgaaccaatggccaca ctcttccactgggacttgccacaggcattgcaagatcacggagggtggcaaaatgaa tcggtggtggatgcgttcctcgactacgcagcgttctgcttttcgacctttggggac cgcgtcaagttgtgggtaacgtttcacgaaccttgggtcatgtcctatgcaggctac gggactggccaacatcccccaggcatttctgacccgggcgtagcgtcttttaaagta gcccaccttgttctaaaagcacatgctcgcacgtggcatcattataacagccatcat cgcccccaacaacaaggccacgtcgggatcgttcttaactccgattgggcagagccg ctttctcctgagcggccggaggatttacgcgcttcagaacggttcctccatttcatg ctcgggtggttcgcccacccggtctttgtcgacggagattatccagcaacattgaga acacagatacaacaaatgaaccgccagtgcagtcatccagtcgctcaattaccggaa tttacagaagcagaaaagcagctccttaagggttcggctgattttcttggcctttca cactacacgagccggttaattagcaatgctccccaaaacacctgtatcccaagctat gacacgattggcggcttttcacaacacgtcaatcacgtatggccacaaacaagctct tcctggatccgcgttgtcccgtggggcataagacgattgctgcaatttgtgagcttg gaatacacccggggtaaggtgccgatttatttagcgggcaatggcatgcccataggt gaatcagaaaacttgttcgacgattccttacgtgtcgactattttaaccagtacatt aacgaggtattgaaggcaatcaaagaagacagtgtggacgtccgctcttatatcgcc cgctctcttattgatggtttcgaaggaccctccgggtattcgcaacgtttcgggctg caccacgttaactttagcgactcttctaagagtcgtacaccacgaaagtccgcgtac tttttcactagtatcattgagaaaaatggcttcttaacgaaaggtgcaaagcgtttg ttacctcctaacactgtaaatcttccttcgaaggtgcgcgcgtttacattccctagt gaggttccgtcgaaggctaaggttgtctgggagaaattctcttcccaaccgaagttt gagcgggatctgttttatcatggaacgtttcgcgatgactttctgtggggtgtcagc agttccgcataccagattgagggtgcgtgggatgcagatggtaagggaccttcgatc tgggataactttacccatacaccaggatctaatgtgaaggacaacgcaacgggcgat attgcttgcgactcctaccatcagttagatgccgatttaaatatgctgagggcattg aaggttaaagcgtaccgctttagcattagctggagtcgtatctttccgactggtcgt aatagctcgattaattcgcatggcgtggattattacaaccgcctgatcaatggtctg gttgcttcgaatatctttcctatggtgacactgtttcattgggaccttcctcaggcg ttgcaggacatcgggggctgggaaaatcctgcgcttatcgatctgtttgattcctat gcggatttttgctttcaaacctttggggatcgggtgaagttttggatgacctttaac gagccgatgtatcttgcttggctgggctacggttcaggcgagtttccacctggtgtt aaggatccaggctgggcgccttatcggattgcacatgctgtgatcaaagcacatgcg cgggtgtatcatacatatgatgaaaaataccgtcaagaacaaaagggtgtcattagt ctttcgttgtcgactcattgggcggaaccgaagtcgccaggtgttccgcgggatgtt gaggcggcagatcggatgctgcaattttcgttaggctggtttgctcatccgatcttt aggaacggtgattacccggataccatgaagtggaaggttgggaaccggagcgagttg caacatttggcgacttcacggttgccatcgtttactgaggaggagaaacgctttatt cgcgcaacggcggatgttttttgtctgaacacgtattattcacgcattgtccagcat aagaccccgcgccttaatccgccatcgtatgaagatgatcaggaaatggcggaagag gaagatccatcatggccttcgactgctatgaatcgtgctgcgccgtggggaacgcgg aggcttctgaattggattaaggaggaatatggcgatattccgatttacattaccgag aacggcgtcgggttgactaatccgaacacggaagataccgatcggattttttaccat aaaacctatattaatgaagctcttaaagcataccgcctggatggtattgatttacgt ggttacgtcgcgtggagcttgatggataattttgagtggttaaacgggtataccgtt aagtttggactttaccatgtcgattttaataatactaaccgacctcgcactgctcgg gctagtgcacgttattacactgaggtcattactaacaacggaatgcctttagcacga gaggatgaatttttgtatggtcgttttcctgagggatttatttggtcagcggcttcg gccgcctatcagattgaaggagcatggcgggcagatggtaagggcttatccatttgg gatactttttcgcataccccgttacgcgttgaaaatgatgctattggtgatgtcgcg tgtgatagttatcataagattgctgaagatctggtcaccctgcaaaatttaggcgtt tcacattatcgctttagcattagctggtcccgcattttacctgatggaacgactcgg tatattaatgaagctggattaaattattatgtccgcttgattgatacgttactggcc gcaagcattcaaccgcaggtgacgatttatcattgggatctgccgcagacgctgcag gatgttgggggttgggaaaatgaaacgattgtgcaacgctttaaggaatatgctgat gtgttgtttcaacgattaggcgataaggtgaaattttggattaccctgaatgaacca tttgtgattgcatatcaggggtatggttatggcaccgcggcaccgggtgtttccaat cgtccaggcaccgctccgtatattgtgggccataatctgattaaagcccatgctgaa gcctggcatttatataatgatgtttatcgggctagtcagggcggcgtgattagtatt acgattagctcagattgggcagaaccgcgtgatccgtcaaatcaagaagatgtggaa gcggcccgtcgatatgtgcaatttatgggtgggtggtttgcccatccgatttttaaa aatggtgattataatgaagttatgaaaacgagaattcgtgatcgttcattggcggcc ggtctgaataaaagtcggttgccggaatttacggaaagtgaaaaacgacgtattaat ggaacgtatgatttttttggctttaatcattatacgaccgttttagcgtataattta aattatgcgaccgcgatttcatcatttgatgccgatcgcggtgttgcctcaattgcc gatcggagttggccagattcaggctcattttggctgaaaatgacgccgtttggtttt cgaaggattctgaattggttgaaagaagaatataatgatccaccgatttatgttacg gaaaatggtgttagtcagcgcgaagaaaccgatctgaatgataccgccagaatttat tatttgcgcacctatattaatgaagccttgaaagccgttcaggataaagttgatttg cgtggatataccgtttggagtgccatggataattttgaatgggccaccggtttttca gaacgttttggtttgcattttgttaattatagtgatccgtcattgccaagaattccg aaagccagtgccaaattttatgccagtgttgttcgatgtaatgggtttccggatccg gccaccggaccgcatgcctgtttgcatcagccggatgccggtccgaccattagtccg gttcggcaggaagaagttcagtttttggggttgatgttgggtaccaccgaagcccag accgccttgtatgttttgtttagtttggttttgttgggcgtttgtggcttggccttt ttgtcatataaatattgtaaacggtcaaaacaggggaaaacccagcgtagtcagcag gaattgtcaccggttagtagtttt

SEQ ID NO: 37: Codon optimized sequence used for histamine oxidase (dao): gagccatcac cggggacatt gcctcgaaag gcaggtgttt ttagcgacct gtcaaaccaa 60 gaattgaaag cagttcactc gtttttgtgg tcgaaaaagg aattgagact ccaaccgtca 120 agtacgacga cgatggccaa gaacaccgta ttcctcatcg aaatgttact tccgaaaaaa 180 taccatgtcc tccggttttt ggacaaaggc gagcggcatc ccgtgaggga agcacgggcc 240 gtgatttttt ttggcgacca agaacacccc aacgtcaccg aatttgctgt cggcccctta 300 ccagggccat gttatatgcg tgctttatct ccccgccctg gataccaaag ctcttgggcc 360 tctcgaccca taagtaccgc ggaatacgcc cttctatacc acacgttaca agaggcgact 420 aaaccgctcc atcagttctt tctaaatacc acgggctttt catttcaaga ctgccacgat 480 cggtgtcttg ctttcacaga cgttgctccc agaggcgttg caagcggaca aaggcgtagc 540 tggttgatta ttcagcgata cgtcgaggga tattttttac acccgaccgg cttagaattg 600 ctagttgacc atggtagtac ggacgcgggc cactgggcag tggagcaagt atggtacaat 660 ggaaaatttt acggatctcc tgaggagctt gctcgaaagt atgccgacgg tgaggtcgat 720 gtagtcgtac tagaagaccc gttgccgggt ggaaagggac acgatagtac cgaagaacca 780 cctttatttt cttcacacaa acctcgtggc gattttccga gcccaattca tgtatctggc 840 ccacgcttgg tacagccgca cggacctcgt ttccggcttg agggcaatgc agtcttgtat 900 ggcggatggt catttgcgtt tcggctgcgc tcgtcctcgg gcttacaggt acttaacgtg 960 catttcggtg gggaacgcat cgcatacgag gtctctgtgc aagaggcggt ggcgctgtacl020 ggaggtcata cgccagctgg gatgcagact aaatatttgg atgttggttg gggtctgggcl080 agcgtcacac atgaattggc cccaggcatt gattgcccag aaactgccac cttcttagatll40 actttccact actatgatgc tgatgatcca gtgcattatc ctagagcatt gtgcttgttcl200 gagatgccaa ccggcgtgcc tttacgacgt cacttcaact ccaactttaa ggggggtttcl260 aatttttatg ccggtttgaa agggcaggtg cttgtcctgc gtacgacaag tacggtttatl320 aactatgatt atatttggga ttttattttc tatccaaatg gggttatgga ggcgaaaatgl380 cacgcgactg gctatgtgca tgcaacattt tatacaccgg aggggetgcg tcatgggacgl440 cgtttacata cacatcttat tggcaatatt catacccatt tggttcatta tcgcgtcgatl500 ttggatgtgg cgggcactaa gaattccttt cagacacttc aaatgaagct tgaaaatatcl560 actaacccat ggtcgcctcg tcatcgagtt gttcaaccaa cgttggagca aacccaatatl620 tcctgggaac ggcaggccgc ctttcggttt aaacgcaagt tgccgaagta tttactgtttl680 acgtcgcctc aagagaaccc atggggtcat aagcgcactt atcggttaca gattcattcal740 atggcggatc aggttttacc gcctggctgg caggaagaac aggctataac ctgggcccgcl800 tatcctttgg cggttaccaa gtatcgcgaa agtgaactgt gttcatcgag tatctatcatl860 cagaatgatc cttggcatcc gcctgttgtg tttgaacagt ttttgcataa taatgaaaatl920 atcgaaaatg aagatctggt tgcatgggtt actgttggtt ttctgcatat tccgcattcgl980 gaagatattc cgaatactgc taccccgggt aatagtgttg ggtttctgct gcgcccgttt2040 aatttttttc cggaagatcc gagtctggct tcccgggata ccgttattgt ttggccgcgc2100 gataatggtc cgaattatgt tcagcggtgg attccggaag atcgggattg ttccatgccg2160 ccgccgttta gttataatgg tacctatcgc ccggtt 2196

SEQ ID NO: 38: - Signalling peptide sequence (spl_p0373) used for lactase and histamine oxidase (DAO): atgtataccgaaaataccggcaagcatcatcgcaatggcttgccggtttggttgttg ccgttgttggttgttattagtttttggggcgttagtcagaatattatggttgttgat gcgagtagtgttgat Detailed description of the invention

The present invention provides a probiotic comprising a GRAS mi crobiological organism, which GRAS microbiological organism comprises a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a function ally equivalent fragment of said soluble form of Amuc_1100, wherein said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof. The term “probiotic” as used herein in the context of the present in vention is defined as live microorganism, which when administered in adequate amounts, confers health benefit on the host. The probiotic may be in the form of a fermented dairy food product, a fermented non-dairy product, or a probiotic food supplement. Examples of a fermented dairy food product comprise yoghurt, yoghurt drinks, kefir, buttermilk, sour cream, viili, fil, and creme fraiche. Often dairy products are fermented with lactic acid bacteria such as Lactococcus, Lac tobacillus and Leuconostoc. However, in particular cheese may comprise bac teria and molds from other genera. Examples of fermented non-dairy products comprise pickled vegetables, sauerkraut, kimchi, pao cai, soy products including miso, tempeh, and soy sauce. Probiotic food supplements may be in the form of capsules, microcapsules, tablets, powders, and sachets, and may optionally be formulated to deliver the probiotic bacteria through the acidic environment of the stomach.

Generally recognized as safe (GRAS) is a designation of the United States Food and Drug Administration (FDA)designating that a chemical or sub stance added to food is considered safe by experts, and so is exempted from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tole rance requirements. The term “GRAS microbiological organism” as used herein in the context of the present invention is intended to mean that the microorgan- ism is known or is found to be suitable for consumption by a host, in particular a human, without causing a state of disease. Indeed, any organism causing a state of disease, i.e. a deterioration in health, would also not be considered as a probiotic. For example, Escherichia coli is not a GRAS microbiological organ ism. Thus, the terms “GRAS microbiological organism” and “probiotic” are in- tended to complement each other. Microorganisms which are intended to fulfill both requirements of a “probiotic” and a “GRAS microbiological organism” are exemplified in the review article of Fijan, “Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature” Int. J. Environ. Res. Public Health 2014, 11, 4745-4765, the content of which is incorporated herein by reference. In embodiments, the GRAS microbiological organism may be selected from the group of organisms consisting of a gram-positive bacteria, a gram-negative bacteria, and a yeast. In embodiments, the GRAS microbiological organism is selected from the group consisting of organisms of the genus Lactobacillus, Bifidobacterium, Brevibacil- lus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, Bacteroides, Prevotella, Parabacteroides, Ruminococcacaeae, Corynebacterium, Neisseria, Planococcaceae, Rothia, Ruminococcus, Veilo- nella, Coprococcus, Alistsipes, Clostridium, Lachnospiraceae, Faecalibacte- rium, Rikenellaceae, Comamonas, Dialister, Blautia, Roseburia, Turicibacter, and Saccharomyces. In embodiments, the GRAS microbiological organism is selected from the group consisting of organisms of the species Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus brevies, Lacto bacillus johnsonii, Lactobacillus fermentum, Lactobacillus reuteri, Bifidobacte- hum infantis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Enterococcus durans, Enterocococcus faecium, Streptococcus thermophilus, Pediococcus acidilactici, Leuconostoc mesentoroides, Bacillus coagulans, Ba cillus subtilis, Bacillus cereus, Saccharomyces boulardi. Preferably, the GRAS microbiological organism is not of the genus Akkermansia, in particular not not Akkermansia muciniphila.

The invention is particularly advantageous for embodiments, wherein the GRAS microbiological organism is selected from the group of organisms consisting of a gram-positive bacteria and a gram-negative bacteria. This is be- cause it is expected that the beneficial effects reported for Amuc_1100, in par ticular its Toll-like receptor 2 (TLR-2) agonistic activity, will further improve the beneficial health effects which are ascribed to the induction of TLR-2 by PAMPs found in the membrane of these microorganisms. A particular high expression of Amuc_1100 has been found in embodiments, wherein the GRAS microbio- logical organism is a gram-positive bacteria belonging to the order of lactic acid bacteria. As noted above, said GRAS microbiological organism comprises a food-grade expression vector. Several food-grade expression vectors are de scribed in the art. Food-grade expression vectors are characterized by contai ning only the DNA from homologous hosts or generally considered as safe or- ganisms, and by not being dependent antibiotic markers. Conseqeuntly, said food-grade expression vector may carry a food-grade selection marker, which provides prototrophy to an otherwise auxotroph GRAS microbiological organ ism. Suitable vectors for lactic acid bacteria are reviewed by Landete, Critical Review in Biotechnology, 2017, 37(3): 296-308, the content of which is incorpo- rated herein by reference. These vectors can also be used for identifying build ing blocks, which can be combined.

The various components of the food-grade expression vector are comprised in the vector in functional linkage. The expression “in functional link age” as used herein, is intended to mean that the respective component of the food-grade expression vector is arranged within said vector, such that they can bring about their intended function. A marker gene is in functional linkage in case the gene is expressed such that its gene product provides the selection ad vantage. A replicon is in functional linkage in case the vector or plasmid is re produced and maintained in the host cell due to the effect of said replicon. In the context of the nucleic acid encoding Amuc_1100, or a fragment thereof, said nucleic acid encoding Amuc_1100 or a fragment thereof is in functional linkage in case its gene product is expressed, such that its translated gene product is secreted into the host cells supernatant.

The food grade selection marker may be, for example, a marker se- lected from the group of alanine racemase (air), thymidylate snynthase (thyA), lactose phosphotransferase (lacF), and phospho-p-galactosidase (lacG). In one particular embodiment, the marker is alanine racemase (air), such as the alanine racemase (air) marker encoded by SEQ ID NO: 8. The air marker, and a food- grade expression vector using same is described in further detail in Nguyen et al. , J. Agric. Food Chem. 2011, 59: 5617-5624; and Bron et al. Appl. Environ. Microbiol. 2002, 68(11): 5663-5670; each the content of which is incorporated herein by reference. In embodiments, the food-grade expression vector carries the SFI71rep replicon, which has a broad functionality. The SFI71rep replicon is further described by Karlskas et al., PLOS One 2014, 9(3): e91125, the content of which is incorporated herein by reference. Other suitable replicons may be employed as well. In case it is intended that the construct is incorporated into the chromosome of the GRAS microbiological organism, the vector may com prise an integrational att-region, such as an attP-region, in particular the phage A2 int-attP region described in Alvarez et al., Virology, 250: 185-193 (1998), the content of which is incorporated herein by reference, or as exemplified in the examples and in SEQ ID NO: 16 herein. Other suitable integrative att-regions may be employed as well. An additional 5'UTR 'AGGAGGT' (SEQ ID NO: 13) sequence may be optionally inserted directly upstream of the Amuc-protein se quence and 3'UTR sequence 'TACTTGAA (SEQ ID NO: 14) directly dowstream of the Amuc-protein sequence followed by a terminator, for example iGEM-part BBa_B1006 (SEQ ID NO: 12).

Signal sequences steering the gene of interest to the secretion path way are known to the skilled person. For example, Dieye et al. J. Bacteriol. 2001 , 183(14): 4157, the content of which is incorporated herein by reference, disclose the M6 preprotein and the Usp45 preprotein signal peptide sequence, which provides secretion when fused to the gene product of interest. Whether a gene product of interest has been expressed and secreted into the supernatant of the host cell can be tested for by assays generally known in the art, including SDS- PAGE followed by Coomassie Blue Staining, or any immunological method in cluding dot blots, ouchterlony assays, western blots, or ELISA techniques. In any case, the food-grade expression vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a fragment of said soluble form of Amuc_1100. In embodiments, the nucleic acid sequence in said food-grade expression vector encodes a soluble form of Amuc_1100 having an amino acid sequence with at least 80% identity to SEQ ID NO: 2 (Amuc_1100), such as with at least 82% identity to SEQ ID NO: 2, such as with at least 84% identity to SEQ ID NO: 2, such as with at least 86% identity to SEQ ID NO: 2, such as with at least 88% identity to SEQ ID NO: 2, such as with at least 90% identity to SEQ ID NO: 2, such as with at least 92% identity to SEQ ID NO: 2, such as with at least 94% identity to SEQ ID NO: 2, such as with at least 96% identity to SEQ ID NO: 2, such as with at least 98% identity to SEQ ID NO: 2, for example with at least 99% identity to SEQ ID NO: 2. For example, the Amuc_1100 encoded by the nucleic acid sequence comprised in functional linkage in said food-grade expression vector may comprise one or more con servative or semi-conservative substitutions, as generally known in the art, or it may be a homolog or an allelic variant to Amuc_1100 of SEQ Dl NO: 2. In one embodiment, the nucleic acid sequence in said food-grade ex pression vector encodes a soluble form of Amuc_1100 having an amino acid sequence as set out in SEQ ID NO: 2. A protein sequence comparison can be conducted using a sequence comparison and alignment tool, such as the pub- licly available program BLASTp, wherein sequence identity is intended to mean the identity of two amino acids at the same position, when both sequences are aligned, and over the total length of SEQ ID NO: 2 (287 amino acids).

In embodiments, said nucleic acid sequence may also encodes for a fragment of said soluble form of Amuc_1100, which has a length of at least 100 and up to 286 amino acids. These fragments may, for example, be N- or C- terminally truncated fragments. Alternatively, these fragments may arise from internal deletion(s). For example, said fragment may have a length of up to 285 amino acids, up to 284 amino acids, up to 283 amino acids, up to 282 amino acids, up to 281 amino acids, up to 280 amino acids, up to 275 amino acids, up to 270 amino acids, up to 265 amino acids, up to 260 amino acids, up to 255 amino acids, up to 250 amino acids, up to 240 amino acids, up to 230 amino acids, up to 220 amino acids, up to 210 amino acids, up to 200 amino acids; and/or at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids, at least 230 amino acids, at least 240 amino acids, at least 250 amino acids, at least 260 amino acids, at least 270 amino acids, or at least 280 amino acids. In any case, the soluble Amuc_1100 protein or the fragment thereof must be selected such as that it maintains at least in part the functional proper ties observed for Amuc_1100 of SEQ ID NO: 2. The term “functionally equiva lent” or “functional properties” as used herein is intended to mean that the can didate protein maintains at least in part the property to increase the transepithe- lial electrical resistance (TEER), and/or the TLR-2 agonistic activity, observed for Amuc_1100 of SEQ ID NO: 2.

TLR-2 agonistic activity of the full length Amuc_1100 of SEQ ID NO: 2. TLR-2 agonistic activity can be determined using methods as described in the prior art, for example as described in Ottman et al. PLOS One 12(3): e0173004. Briefly, HEK-Blue hTLR2 cells (Invivogen, CA, USA) are grown and subcultured up to 70-80% of confluency using DMEM supplemented with 4.5 g/l D-glucose, 50 U/ml penicillin, 50 pg/ml streptomycin, 100 pg/ml Normocin, 2 mM L-gluta- mine, and 10% (v/v) of heat-inactivated FBS. For the experiment, cells are seeded in 180 pi in flat bottom 96-well plates and stimulated by addition of Amuc_1100 (fragment) protein to a final concentration of 5 pg/ml. Pam3CSK4 (10 ng/ml) are used as positive control, and culture medium is used as negative control. The 96-well plates are incubated for 20-24 hours at 37 °C in a 5% C02 incubator. Stimulation of the hTLR2 receptor activates NF-KB and AP-1, which induces the production of secreted embryonic alkaline phosphatase (SEAP), the levels of which are measured spectrophotometrically. SEAP secretion is de- tected by measuring the OD600 at 1 hour after addition of 180 mI of QUANTI- Blue (Invivogen) to 20 pi of induced FIEK-Blue hTLR2 supernatant. Experiments are performed in triplicate. The candidate soluble Amuc_1100 or the fragment thereof are considered to have or maintain TLR-2 agonistic activity in case its TLR-2 signalling activity, as determined using the foregoing assay, is at least 50% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 60% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 70% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 75% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 80% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, for example at least 85% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2 as measured in the above-described assay.

In addition, or alternatively, the property to increase the development of transepithelial electrical resistance can be tested for using the transepithelial electrical resistance (TEER) assay, as described in Ottman et al. PLOS One 12(3): e0173004. Briefly, 5 x 10 ⁴ Caco-2 cells/insert are seeded in Millicell cul ture inserts with a 3 pm pore size (Merck Millipore) and grown for 8 days, whereas the growth conditions are as described in Kainulainen et al. BMC mi crobiology, 2015, 15(1): 4, incorporated herein by reference. Transepithelial re sistance is determined using a Millicell ERS-2 TEER meter (Merck Millipore) from Caco-2 cell cultures at Oh, and 24 h after addition of 0.5 pg/ml of Amuc_1100 protein. The candidate soluble Amuc_1100 or the fragment thereof are considered to have or maintain the property to increase the development of transepithelial electrical resistance (TEER) in case its increase in TEER com pared to medium control, as determined using the foregoing assay, is at least 50% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 60% of the increase in TEER observed for Amuc 1100 of SEQ ID NO: 2, such as at least 70% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 75% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 80% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, for example at least 85% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2 as measured in the above-described assay.

Due to the degeneration of the genetic code, one and the same amino acid sequence can be encoded by different nucleic acid sequences. Indeed, dif ferent microorganisms have different preferences for encoding a particular amino acid. Depending on the abundance of the respective tRNAs in said mi croorganisms, expression of a gene product can be further improved by optimiz ing the nucleic acid sequence to the codon usage of the respective host. Thus, in embodiments, said nucleic acid sequence encoding for Amuc_1100 or a frag ment thereof can be optimized for expression in a genus selected from the group of Bifidobacterium, Bacillus, Brevibacillus, Lactococcus and Saccharomyces. For example, said nucleic acid sequence may have a sequence selected from SEQ ID NO: 3 to SEQ ID NO: 7.

Within this context, said nucleic acid sequence encoding for Amuc_1100 or a fragment thereof has at least 70% sequence identity to SEQ ID NO: 1 (Amuc_1100), such as at least 72% sequence identity to SEQ ID NO: 1, such as at least 74% sequence identity to SEQ ID NO: 1, such as at least 76% sequence identity to SEQ ID NO: 1 , such as at least 78% sequence identity to SEQ ID NO: 1 , such as at least 80% sequence identity to SEQ ID NO: 1 , such as at least 82% sequence identity to SEQ ID NO: 1, such as at least 84% se- quence identity to SEQ ID NO: 1 , such as at least 86% sequence identity to SEQ ID NO: 1, such as at least 88% sequence identity to SEQ ID NO: 1, such as at least 90% sequence identity to SEQ ID NO: 1, such as at least 92% sequence identity to SEQ ID NO: 1 , such as at least 94% sequence identity to SEQ ID NO: 1, such as at least 96% sequence identity to SEQ ID NO: 1, such as at least 97% sequence identity to SEQ ID NO: 1 , such as at least 98% sequence identity to SEQ ID NO: 1 , or at least 99% sequence identity to SEQ ID NO: 1. A nucleic acid sequence comparison can be conducted using a sequence comparison and alignment tool, such as the publicly available program BLASTn, wherein se quence identity is intended to mean the identity of two nucleotides at the same position, when both sequences are aligned, and over the total length of SEQ ID NO: 1 (864 nucleotides). In embodiments of the present invention, said soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100 does not need to comprise such a purification tag, as it is not re quired nor intended to purify Amuc_1100. Moreover, while food-grade expression systems are disclosed for pri mary use in organisms of the genus Lactobacillus, in embodiments these ex pression systems are used in genera other than Lactobacillus, in which these food-grade expression vectors are also functional.

One useful example of said food-grade expression vector is p3050al- rAmud 100-sh71 (SEQ ID NO: 9) or p3050Alr_Amuc1100-sh71 with 5’UTR, 3’UTR and terminator (SEQ ID NO: 15). Many (shuttle) vectors for gram positive bacteria or for yeasts may be used, these particular vectors show however the highest yielding.

In a further optional embodiment, the food-grade expression vector further comprises in functional linkage a nucleic acid sequence encoding for an enzyme with aldehyde dehydrogenase activity (EC 1.2.1.3), wherein said GRAS microbiological organism is capable of expressing said enzyme with aldehyde dehydrogenase activity. Assays for testing a sample for aldehyde dehydrogen ase activity are known in the field and commercially available. In one embodi- ment, the vector has an additional ethanol inducible promoter AlcA followed by human aldehyde dehydrogenase 1 B1 (UniProt P30837; SEQ ID NO: 10). A cor responding food-grade expression vector is exemplified in SEQ ID NO: 11 and SEQ ID NO. 33. Said vector is able to additionally express aldehyde dehydro genase following the consumption of potable ethanol. Acetaldehyde, a metabo- lite of ethanol, is carcinogenic and the expression vector enables providing al dehyde dehydrogenase locally to colon, so to turn acetaldehyde into acetic acid. By lowering acetaldehyde levels in the gut followed by, for example, excessive consumption of potable alcohol will lead to a decreased incidence or risk for colorectal cancer, and a reduced endogenous formation of (alcohol) depen- dency/addiction causing compound salsolinol. At the same time, it is reported that aldehyde dehydrogenase 1 expression is significantly higher in lean mice than in obese mice (Singh et al., Biochem Biophys Res Commun. 2015; 463(4): 768-773; and Yasmeen et al., Diabetes 2013; 62: 124-136; each of which is incorporated herein by reference). In addition or alternatively, the food-grade expression vector further comprises in functional linkage a nucleic acid sequence encoding for an enzyme with lactase activity (EC 3.2.1.108), wherein said GRAS microbiological organ ism is capable of expressing said enzyme with lactase activity (EC 3.2.1.108). Assays for testing a sample for lactase activity are known in the art and com mercially available. Lactase assists lactose-intolerant subject’s to digest lactose sugar, as found in dairy products. Further disclosure and guidance is provided in the examples herein below.

In addition or alternatively, the food-grade expression vector further comprises in functional linkage a nucleic acid sequence encoding for an enzyme with fructan beta-fructanase activity (also referred to as fructan beta-fructo- sidase activity; EC 3.2.1.80), wherein said GRAS microbiological organism is capable of expressing said enzyme with fructan beta-fructanase activity (EC 3.2.1.80). Assays for testing a sample for fructan beta-fructanase activity are known in the art and commercially available. Preferably, said fructan beta-fruc tanase activity arises from an enzyme, which is endogenous to the GRAS mi- crobiological organism, such that said endogenous enzyme is merely over-ex- pressed in said microorganism. For example, the enzyme may be the lactoba- cilli’s own fructan beta fructosidase. In embodiments, the enzyme is secreted in soluble form. In other embodiments, the enzyme is anchored into the cell wall of the GRAS microbiological organism. For example, the enzyme can comprise a C-terminal anchoring protein, such as the protein encoded by ttgccgaata ccaatgaaaa gcatgaatat ggcattgcgg cggttggctt ggcgttgttg 60 agtttgatgg gcttgggcag taccttgttg tttcgcaagg cgaagcgcca g 111 (SEQ ID NO: 35). Further disclosure and guidance is provided in the examples herein below.

Similar to lactase enzyme in subjects suffering from lactose intoler ance, providing fructan beta-fructanase ameliorates digestional problems in subjects, who do not tolerate well fructan containing foods, e.g. subjects suffer- ing from irritable bowel syndrome (IBS). Delivering fructan beta-fructanase to these subjects enables these subjects to eat fructan containing foods such as onions and wheat without suffering from bloating, abdominal cramps, flatulence, etc.

In addition or alternatively, the food-grade expression vector further comprises in functional linkage a nucleic acid sequence encoding for an enzyme with histamine oxidase activity and/or diamino oxidase activity (EC 1.4.3.22), wherein said GRAS microbiological organism is capable of expressing said enzyme with histamine oxidase activity and/or diamino oxidase activity (EC 1.4.3.22). Assays for testing a sample for histamine oxidase activity and/or dia mino oxidase activity (EC 1.4.3.22) are known in the art and commercially avail able. Further disclosure and guidance is provided in the examples herein below. Histamine oxidase is reported to ameliorate problems with people suffering from irritable bowel syndrome (IBS); see Maintz L, Novak N. Histamine and histamine intolerance. The American Journal of Clinical Nutrition. 2007;85(5): 1185-1196; the content of which is incorporated herein by refer ence. The food-grade expression vector can be maintained in an extra- chromosomal manner in the GRAS microbiological organism. In other embodi ments, the food-grade expression vector can be maintained in the chromosome of the GRAS microbiological organism. In the latter case, said food-grade ex pression vector is an integrative expression vector. For example, said vector further comprises a nucleic acid sequence encoding for attP and/oran integrase, such as int-attP. Further disclosure and guidance is provided in the examples herein below.

Useful examples are, for example, pRac-int-attP-Amuc (SEQ ID NO: 16), or pRac-DAO-lactase-int-attP-Amuc-aldh-fruct (SEQ ID NO: 33). Further disclosed is a method of preparing a probiotic as disclosed herein above, wherein the method comprises the step of introducing a food- grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a fragment of said soluble form of Amuc_1100, into a GRAS microbiological organism, such that said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof.

Methods for introducing the vector into the GRAS microbiological or ganism are known in the art, and include, for example, electroporation tech niques, or heat-shock techniques. The link between gut microbiota and health is well-recognized and described, and biotherapeutic strategies evolved in the recent years, including fecal microbiota transplant (FMT), as also reviewed in Hage et al. Frontiers in Microbiology 2017, 8: article 1889, the content of which is incorporated by refer ence. Moreover, Plovier et al. (Nature Medicine 2016, doi: 10.1038/nm.4236, the content of which is incorporated herein by reference) and Ottman et al. (PLOS One 2017, 12(3): e0173004, the content of which is incorporated herein by reference) demonstrate that Akkermansia muciniphila or the pasteurized bac terium improve metabolism in obese and diabetic mice. It was furthermore shown that these beneficial health effects are due to a membrane protein, Amuc_1100. When added as a His-tagged purified protein in soluble form, the following beneficial health effects were observed: a reduction in body weight gain, a reduction in fat mass gain, a decrease in intestinal energy absorption, normalization of plasma LPS concentration, normalizing / reducing plasma cho lesterol (in particular HDL-levels), normalizing / reducing plasma triglyceride lev els, and normalizing / reducing plasma glucose levels, and improving the intes- tinal barrier function (as can be followed, for example, by an increase in the development of transepithelial electrical resistance).

In addition, it was demonstrated Ottman et al. (PLOS One 2017, 12(3): e0173004, the content of which is incorporated herein by reference) that the soluble, His-tagged Amuc_1100 purified protein has TLR-2 agonistic activity, and is thus considered to be involved with cross-talk with the host. In the intes tine, TLR-2 regulates the expression of CYP1A1, an enzyme which is key in detoxication of certain carcinogenic substances. Recently, it was found that TLR-2 is involved in the activation of regulatory T cells (Tregs), that act to sup press immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. TLR-2 is also ex pressed by intestinal epithelial cells and subsets of lamina propria mononuclear cells in the gastrointestinal tract. TLR-2 has been observed downregulated in human papillomavirus-positive neoplastic keratocytres derived from uterine cer- vical preneoplastic lesions. Thus, TLR-2 is assumed to be associated with tu- morigenesis.

Thus, in a further aspect, the above-described probiotic is for use in medicine for therapeutic purposes. Likewise, disclosed is the use of a probiotic as defined herein above for the manufacture of a medicament. Accordingly, also provided is a method of treatment of a patient, comprising the step of orally ad ministering a probiotic as defined herein above to said patient. The patient may be a mammal, in particular a dog, cat, rat, or mouse. Preferably, the patient is a human patient. Dosages (cfu) will vary based on the formulation, the indication, and the physical state of the patient (for example dependent on the age and/or weight), but are commonly in the range of 10 ⁹ to 10 ¹⁰ CFU/day. Suitable dos ages can be determined by a person skilled in the art. More specifically, the probiotic is for use in the treatment and/or pre vention of obesity, diabetes, and/or hypercholesterolemia. Hence, the probiotic may be used for the manufacture of a medicament for the treatment of obesity, diabetes, and/or hypercholesterolemia. In case the probiotic provides enzymes with fructan beta-fructanase activity (also referred to as fructan beta-fructosidase activity; EC 3.2.1.80) and/or histamine oxidase activity and/or diamino oxidase activity (EC 1.4.3.22), the pro biotic can be advantageously used in the treatment of irritable bowel syndrome (IBS). Provided is thus a method for treating obesity, diabetes, and/or hy percholesterolemia in a patient, such as a human patient, comprising the step of orally administering a probiotic as defined herein above to said patient. Simi larly, also provided is a method for (i) reducing body weight gain, (ii) reducing fat mass gain, (iii) decreasing intestinal energy absorption, (iv) normalizing plasma LPS concentration, (v) normalizing / reducing plasma cholesterol (in par ticular HDL-levels), (vi) normalizing / reducing plasma triglyceride levels, and (vii) normalizing / reducing plasma glucose levels, and (viii) improving the intes tinal barrier function in a patient, such as a human patient, comprising the step of orally administering a probiotic as defined herein above to said patient. As used herein, the term “or” has the meaning of both “and”' and “or”

(i.e. “and/or”). Furthermore, the meaning of a singular noun includes that of a plural noun and thus a singular term, unless otherwise specified, may also carry the meaning of its plural form. In other words, the term “a” or “an” may mean one or more. It is apparent to the skilled reader that, as technology develops, the basic idea of the invention can be accomplished in many different ways. The invention and its embodiments are therefore not confined to the examples de scribed above, but may vary in the framework of patent requirements and the below claims. Examples

If not otherwise stated, the following example uses routine methods of molecular biology, as also described in reference textbooks in the art, in particular with regard to techniques concerning molecular cloning, polymerase chain reaction, and gel electrophoresis. See, for example, ‘Molecular Cloning: A Laboratory Manual’ by Michael Green and Joseph Sambrook, 4th edition, Cold Spring Har bor Laboratory Press, Cold Spring Harbor, New York. Example 1

In order to construct a food-grade expression and secretion vector comprising a nucleic acid sequence encoding for a soluble form of Amuc_1100, the plasmid p3050sNucA-sh71 was selected as the starting point. The plasmid p3050sNucA-sh71 is based on pSIP411 , described in Sorvig et al. Microbiology 2005, 151(7): 2439-2449 (the disclosure of which is incorporated herewith by reference), which is also the source of the sh71 replicon. The plasmid p3050sNucA-sh71 and its construction is described in Mathiesen et al. BMC Genomics 2009, 10: 425; and Karlskas et al. PLoS One, 2014, 9(3): e91125, the respective disclosure of which is hereby incorporated by reference. The plasmid p3050sNucA-sh71 (see Figure 1 in Karlskas et al.) was first linearized by diges tion with 4 restriction enzymes (BamH I, Afl III, Sal I, Hind III) yielding following bands in an agarose gel: 2852 bp (Afllll-Sall), 1962 bp (Afll ll-Bam HI), 1100 bp (BamHI-Afllll), 307 bp (Sall-Hindlll), 178 bp (Hindlll-Hindlll), 17 bp (Hindlll- Af I III); (linear: 2727(Afllll-End), 1962(Afllll-BamHI), 110O(BamHI-Afllll), 307(Sall-Hindlll), 178(Hindlll-Hindlll), 125(Start-Sall), 17(Hindll l-Aflll I)).

The bands containing the erythromycin resistance marker gene at 1.1 kb and NucA fragments at 0.3kb and 0.2kb were discarded, and the DNA was cleaned.

The sh71 -replicon (2kb band) was ligated back to the backbone lea ving BamHI-Afllll and Sall-Hindlll restriction site pairs open to which alanine ra- cemase and Amuc_1100 inserts were then ligated.

The food-grade alanine racemase (air) marker gene and its isolation is described in Nguyen et al. J. Agric. Food Chem. 2011, 59, 5617-5624, the content of which is incorporated herein by reference. The following is the se quence of the air marker gene in 5’ to 3’-direction:

(alanine racemase (air) atgcaagcgg caactgttgt gattaaccgc cgcgctctgc gacacaacct gcaacgtctt 60 cgtgaactgg cccctgccag taaaatggtt gcggtggtga aagcgaacgc ttatggtcac 120 ggtcttcttg agaccgcgcg aacgctcccc gatgctgacg cctttggcgt agcccgtctc 180 gaagaagctc tgcgactgcg tgcgggggga atcaccaaac ctgtactgtt actcgaaggc 240 ttttttgatg ccagagatct gccgacgatt tctgcgcaac attttcatac cgccgtgcat 300 aacgaagaac agctggctgc gctggaagag gctagcctgg acgagccggt taccgtctgg 360 atgaaactcg ataccggtat gcaccgtctg ggcgtaaggc cggaacaggc tgaggcgttt 420 tatcatcgcc tgacccagtg caaaaacgtt cgtcagccgg tgaatatcgt cagccatttt 480 gcgcgcgcgg atgaaccaaa atgtggcgca accgagaaac aactcgctat ctttaatacc 540 ttttgcgaag gcaaacctgg tcaacgttcc attgccgcgt cgggtggcat tctgctgtgg 600 ccacagtcgc attttgactg ggtgcgcccg ggcatcattc tttatggcgt ctcgccgctg 660 gaagatcgct ccaccggtgc cgattttggc tgtcagccag tgatgtcact aacctccagc 720 ctgattgccg tgcgtgagca taaagccgga gagcctgttg gttatggtgg aacctgggta 780 agcgaacgtg atacccgtct tggcgtagtc gcgatgggct atggcgatgg ttatccgcgc 840 gccgcgccgt ccggtacgcc agtgctggtg aacggtcgcg aagtaccgat tgtcgggcgc 900 gtggcgatgg atatgatctg cgtagactta ggtccacagg cgcaggacaa agccggggat 960 ccggtcattt tatggggcga aggtttgccc gtagaacgta tcgctgaaat gacgaaagtal020 agcgcttacg aacttattac gcgcctgact tcaagggtcg cgatgaaata cgtggattaa!080

(SEQ ID NO: 8)

For introducing same into the backbone vector, the air selection mar ker was PCR-amplified with 5' BamHI and 3' Afllll restriction sites.

The complete nucleic acid sequence encoding for Amuc_1100 is pub- licly available from the KEGG GENOME Database under reference ID T00376. Isolation of Amuc_1100 from Akkermansia muciniphila is also described in Plov- ier et al. Nature Medicine, doi: 10.1038/nm.4236, the disclosure of which is in corporated herein by reference. The nucleic acid sequence encoding a soluble form of Amuc_1100 (i.e. an Amuc_1100 encoding gene insert lacking it's signal sequence in the N-terminal residues 1-30) was synthesized with 5' Sail and 3' Hindlll -sites, and cloned into the above-mentioned vector backbone.

The following is the nucleic acid sequence encoding the soluble form of Amuc_1100, which lacks the first 30 N-terminal residues (in 5’ to 3’ direction): atcgtcaatt ccaaacgcag tgaactggac aaaaaaatca gcatcgccgc caaggaaatc 60 aagtccgcca atgctgcgga aatcactccg agccgatcat ccaacgaaga gctggaaaaa 120 gaactgaacc gctatgccaa ggccgtgggc agcctggaaa cggcctacaa gcccttcctt 180 gcctcctccg cgctggtccc caccacgccc acggcattcc agaatgaact gaaaacattc 240 agggattccc tgatctcctc ctgcaagaaa aagaacattc tcataacgga cacatcctcc 300 tggctcggtt tccaggttta cagcacccag gctccctctg ttcaggcggc ctccacgctg 360 ggttttgaat tgaaagccat caacagcctg gtcaacaaac tggcggaatg cggcctgtcc 420 aaattcatca aggtgtaccg cccccagctc cccattgaaa ccccggcgaa caatccggaa 480 gaatcggacg aagccgacca ggccccatgg actcccatgc ctctggaaat agccttccag 540 ggcgaccggg aaagtgtatt gaaagccatg aacgccataa ccggcatgca ggactatctg 600 ttcacggtca actccatccg tatccgcaac gaacggatga tgccccctcc catcgccaat 660 ccggcagccg ccaaacctgc cgcggcccaa cccgccacgg gtgcggcttc cctgactccg 720 gcggatgagg cggctgcacc tgcagccccg gccatccagc aagtcatcaa gccttacatg 780 ggcaaggagc aggtctttgt ccaggtctcc ctgaatctgg tccacttcaa ccagcccaag 840 gctcaggaac cgtctgaaga ttaa 864

(SEQ ID NO: 1)

The construct, p3050Alr_Amuc1100_sh71 (SEQ ID NO: 9), was then verified by DNA-sequencing and electrotransformed into the following compe- tent probiotic strains:

Genus -> species Lactobacillus Enterococcus L rhamnosus E. durans L acidophilus E. faecium L plantarum L casei Streptococcus L delbrueckii subsp. Bulgaricus S. thermophilus

L brevis L johnsonii Pediococcus L fermentum P. acidilactici L reuteri

Leuconostoc

Bifidobacterium L mesentoroides B. infantis

B. animalis subsp. Lactis Bacillus B. bifidum B. coagulans B. longum B. subtilis

B. breve B. cereus

Brevibacillus brevis Saccharomyces S. boulardii

Lactococcus L. lactis subsp. Lactis

Every recombinant strain secreted the protein Amuc_1100, when running the supernatant on a SDS-PAGE, and stained with Coomassie Blue. Example 2

In order to construct an integrative food-grade expression and secre tion vector comprising a nucleic acid sequence encoding for a soluble form of Amuc_1100, the inventors have constructed a shuttle vector pRac shown in SEQ ID NO: 18 comprising origin of replication (bps 1893-2481) for E. coli and sh71 -replicon (bps 5203-1715) for Lactobacillus.

Additionally, the plasmid pRac contains alanine racemase (bps 2862- 3998) as the selection marker for alanine auxotrophs under string constitutive promoter (bps 2482-2679) and 5'UTR leader sequence from Lactobacillus aci dophilus ATCC4356 S-layer protein gene (bps 2680-2861 ) and 3'UTR 3084746- 3084917 from Lactobacillus paracasei IJH-SONE68 followed by BioBrick termi nator BBa_B1006. To simplify the selection while manipulating the plasmid in E.coli, a beta-lactamase cassette was introduced as disposable selection mar ker in between of EcoRI and Xbal of the MCS comprising EcoRI, Xbal and Spel. This plasmid was transformed into E.coli HB101 grown on ampicillin and mi- niprepped out for the next step.

Next, integrase part containing phage attachment site (int-attP) was cloned out using PCR from pEM40-integration plasmid (Alvarez et al., Virology, 250: 185-193 (1998), the content of which is incorporated herein by reference; SEQ ID NO: 20) using primers fwd 5'-

AAACTAGTGGATCCAAAATAAAAAGCGCCT-3' (SEQ ID NO: 22), rev 5'- AAACTAGTTTGTGTGCCCATATTTCTGAACTCT-3' (SEQ ID NO: 23), creating Spel RE-sites in both sides of the int-attP -fragment. Agarose gel was run on the PCR product and the single band of the amplicon at ~1700bp was cut out and purified with GeneJET Gel Extraction Kit after which the amplicon and the circu lar plasmid were digested separately with Bcul (isoschizomer of Spel), the back bone vector dephosphorylated, phenol-chloroform extracted and the fragments were ligated together with T4 DNA Ligase.

The resulting plasmid was then transformed into E.coli HB101 grown on ampicillin and positive clone were screened by PCR. Following primers were used: Fwd 5'-cgggtacaatcatcagaaac-3' (in the int-attP region, SEQ ID NO: 24), rev 5'-ccttggtattcaacaaccaa-3' (at the plasmid backbone, SEQ ID NO: 25) yiel- ding a correct 2135 bp sized band of the amplicon on agarose gel. The plasmid pRac-int-attP (SEQ ID NO: 26) was then miniprepped out for the next step and additional 50% glycerol stock was made for further projects.

A 7802bp long linear fragment (SEQ ID NO: 29) containing codon optimized fragments of amuc_1100, acetaldehyde dehydrogenase X and fruc- tan beta-fructanase, each with LEISSCDA-propeptides (SEQ ID NO: 30) with EcoRI and Xbal RE-sites was created and simultaneously with the plasmid pRac-int-attP (SEQ ID NO: 26) from the previous step. In this step, the undesired beta-lactamase cassette from the plasmid backbone was also disposed of. Re sulted fragments were ligated together as previously described.

The construct was transformed into E. coli HB101 and positive clone was screened with PCR. Following primers were used for colony PCR: fwd 5'- cccagaatcagaccattacc-3' (in the fructan beta-fructanase part of the insert; SEQ ID NO: 31), rev 5'-ccttggtattcaacaaccaa-3' (at the plasmid backbone; SEQ ID NO: 32) which yielded 3992 bp band of the amplicon in the agarose gel. The construct pRac-int-attP-Amuc-aldh-fruct (SEQ ID NO: 16) was then miniprepped out for electroporation to L rhamnosus GG and additional aliquots of 50% glyce- rol stocks of E. coli HB101 containing the desired constructed plasmid were pre pared and placed into freezer at -80'C.

The electroporation of the previous construct pRac-int-attP-Amuc- aldh-fruct (SEQ ID NO: 16) into lethal alanine auxotroph of L rhamnosus GG (made before by employing CRISPR/Cas9) was performed as previously de- scribed (De Keersmaecker et al. Applied and Environmental Microbiology, 72(7): 4923-4930 (2006), the content of which is incorporated herein by reference) using the alanine racemase as the selection marker. After successful transfor mation, aliquots of 50% glycerol stocks were made from bacteria in MRS-liquid broth. The plasmid was then excreted by using ethidium bromide as growing the bacteria at 42 °C did not satisfactorily yield the shedding of the plasmid.

SDS-PAGE (stained with Coomassie blue) was performed on the cul ture broth to verify each of the desired proteins were indeed excreted into the growing medium and additional PCR reactions confirmed the integration was succesful before the novel strain was sequenced.

Example 3

The inventors created another fragment containing nucleic acid se quences encoding for histamine oxidase enzyme and lactase enzyme contai ning EcoRI restriction sites on both ends of the fragment. The fragment was ligated into the EcoRI-linearized construct pRac-int-attP-Amuc-aldh-fruct (SEQ ID NO: 16), resulting in the integrative food-grade expression and secretion vec tor pRac-DAO-lactase-int-attP-Amuc-aldh-fruct (SEQ ID NO: 33). A probiotic comprising a GRAS microbiological organism, which GRAS microbiological organism comprises pRac-DAO-lactase-int-attP-Amuc- aldh-fruct (SEQ ID NO: 33) is considered to be suitable in ameliorating problems in subjects suffering from irritable bowel disease (IBS) due to the histamine oxi- dase. See Maintz L, Novak N. Histamine and histamine intolerance. The Ameri can Journal of Clinical Nutrition. 2007;85(5): 1185-1196; the content of which is incorporated herein by reference.