Antioxidant-producing strain

Abstract

The present invention refers to a composition comprising Cutibacterium acnes (C. acnes) bacterial strains, wherein said strains are characterized by being modified to secrete a heterologous protein by insertion of an expression construct comprising at least one or more transcription promoter sequences, one or more ORFs (Open Reading Frames) encoding the heterologous protein, and one or more transcription termination sequences; wherein the heterologous protein is characterized by comprising at the N-terminus of the protein sequence, a secretion signal which is processed by C. acnes to secrete the protein, and wherein the heterologous protein is the superoxide dismutase enzyme.

Description

[0001]Antioxidant-producing strain Technical field The present invention relates to the field of medicine, in particular to an antioxidant-producing strain able to rescue keratinocytes from oxidative stress, preferably UV-induced oxidative stress. Background art The skin is the largest organ in the human body. Beyond its function as a barrier against pathogens or environmental factors, the skin holds relevant physiological functions such as heat regulation or immune surveillance. At the same time, it represents a crucial determinant of our physical appearance and social contact1. More than 3000 different conditions affect the skin, with almost one third of the world’s population having at least one2,3. These diseases have a significant impact on patients’ quality of life, ranging from life- threatening (malignant melanoma) to chronic and socially impairing conditions (psoriasis, atopic dermatitis (AD) or acne)3. Environmental factors also influence skin health. For instance, excessive UV exposure causes premature ageing and skin cancer4. Current treatments against the most frequent chronic skin conditions such as AD or psoriasis focus on symptom control and often cause side effects5,6. Novel treatments that target the underlying immune components of these diseases are effective, but they pose asignificant economic burden in national healthcare systems7 – e.g. dupilumab, a monoclonal antibody usedfor AD that blocks IL-4 and IL-13, has a yearly cost of more than 30,000$ per patient8. Engineered live biotherapeutic products (eLBPs) aim to combine the natural adaptation of human microbes to their host9with the synthetic design of sensors, circuits, and actuators that enhance the capabilities of those microbes. While some eLBPs have already shown encouraging results treating metabolic diseases10,11, infections12or cancer13in the gut or even the lung14,15, there are still few instances of skin eLBPs. The most promising examples focus on engineering lactic acid bacteria or Staphylococcus epidermidis for accelerated wound healing16or as cancer vaccines17and mosquito repellent18, with promising preliminary results in mice. These bacteria might produce transient benefits, but are suboptimal as a long term skin-resident eLBP. Lactic acid bacteria are not native skin residents, and their engraftmentis low. Despite being ubiquitous in the skin, S. epidermidis shows a lower relative abundance and a higherturnover compared to other skin microbes like C. acnes19,and might incur in horizontal gene transfer with related pathogens like S. aureus20.Cutibacterium acnes is the most abundant skin commensal21. This Gram-positive and facultative anaerobicbacterium predominantly colonizes the pilosebaceous units, where it metabolizes sebum lipids22. This specialized ecological niche constitutes a privileged location for eLBP deployment. The hair follicle is a physiological hotspot relevant for immune education, and the main route for topical absorption oftherapeutics23. The turnover of C. acnes in the skin is low19, and genomic studies report virtually monoclonalpopulations within pilosebaceous units24, with high genome stability across strains25; the low competition and low recombination events are relevant features regarding biosafety26. In addition, C. acnes can be transplanted from donor to recipient skin using topical applications, which has been shown to modulate therelative abundance of recipient C. acnes strains for up to several weeks27. Taken together, C. acnes is anideal chassis for skin eLBP development due to its location, high abundance, relevance in healthy skin, colonization capabilities and low turnover, as we demonstrated recently through a sebum modulation application28Brief description of the figuresFigure 1: Development of a toolbox for C. acnes genetic engineering. (A) List of all tools developed in thisstudy. (B) Schematics of C. acnes replicative plasmid optimizations (C) Average of population median mCherry fluorescent values of three independent replicates for nine different constitutive endogenous promoters, a synthetic consensus promoter (BBa_J23119) from E. coli and a control harbouring a no insert plasmid (Empty) for exponential and stationary growth phases. (D) Average of population median fluorescent values for C. acnes harbouring a fluorescent reporter (coloured bars) compared with a no insert plasmid control (grey bars). Values depicted above bars indicate average fold change of three independent replicates. (E) Pictures from C. acnes KPA171202 wild-type strain grown either as a lawn or as single colonies in a brucella media agar plate supplemented or not with X-gal. (F) Average of population median mCherry fluorescent values of three independent replicates for four different RBSs driving mCherry translation when expressed with the constitutive promoter PPA_RS07095 compared with an no insert plasmid control (Empty) for exponential and stationary growth phase. RBS_1 and RBS_2 correspond to strong E. coli RBSs with the conserved AGGAGG motif whereas RBS_3 and RBS_4 are de novo RBS designed with the RBS calculator29. (G) Average of population median mCherry (red) and sfGFP (green) fluorescent values of three independent replicates for five different transcriptional terminator sequences: the natural E. coli terminator ECK120033737, the synthetic terminator L3S2P21, and three endogenous terminator sequences from C. acnes. sfGFP and mCherry are located in the same transcriptional unit and the terminator is placed in-between. The whole transcriptional unit is expressed using the BBa_J23119 promoter and values are compared to a strain harbouring a no-insert plasmid control (Empty) and two control strains with spacer sequences (Spacer_1 or Spacer_2) but no terminator. The red dashed line indicates the mCherry fluorescent value for the no-insert plasmid control (Empty). (H) Average of population median mCherry fluorescent values of three independent replicates for four different C. acnes strains expressing mCherry controlled with the BBa_J23119 constitutive promoter (red) compared with the same strain harbouring a no insert plasmid (Empty, grey). The table below indicates the C. acnes Sequence Locus Sequence Typing (SLST)30or the recA-based phylotype31for each of the strains.Figure 2: CRISPR interference for quick prototyping of auxotrophies for biocontainment. (A) dCas9 andgRNA optimization against the genomic reporter lacZ (PPA_RS08600). Pictures show the absence or presence of blue colour in C. acnes lawn or single colonies grown in Brucella media agar plates supplemented with X-gal. (B) CRISPRi screen of C. acnes metabolic genes potentially leading to a single- gene knock-out amino acid auxotrophy based on homology. Optical density at 600nm (OD600) of C. acnes cultures grown for one day in the presence or absence of a specific amino acid compared to a control (dCas9) without gRNA cassette (i.e. without gRNA and its corresponding promoter). For instance, to evaluate His auxotrophy, the basal medium was supplemented with all amino acids except histidine and growth was measured both in the absence and presence of histidine for the auxotroph and dCas9 control. The dashed grey line indicates the starting culture OD600 at 0.1. Statistical significance was determined using a Student’s t-test to compare the strain when grown in the presence and absence of the studied amino acid. A p-value < 0.001 is denoted with three stars (***). (C) Agarose gel showing the PCR amplification of the lysA locus from a wild-type strain (WT), a strain with an erythromycin cassette insertion flanked by BxbI recombination sites (Insertion) and a third strain in which the erythromycin cassette has been removed through BxbI expression (Recombined). The diagram shows the process to obtain such as a strain and the expected size for each locus amplification. (D) Growth of a ΔlysA, ΔproA and ΔhisB knock- out strains compared to wild-type (WT) C. acnes in the presence or absence of L-ysine, L-Proline or L- Histidine after four days of incubation. The dashed grey line indicates the starting culture OD600, 0.1. Statistical significance was determined using a Student’s t-test to compare the strain when grown in the presence and absence of the studied amino acid. A p-value < 0.001 is denoted with three stars (***). Figure 3: Engineered sensing and actuation in C. acnes. (A) Schematics of a synthetic BBa_J23119-based promoter including operator sequences from the TetR and PhlF transcriptional repressors. Fold-change mCherry population median fluorescence upon induction, for different promoter architectures, in the presence or absence of the corresponding transcription factor. (B) Adaptation of temperature-responsive C. acnes endogenous genes for temperature sensing. The graph on top displays the coverage of RNA-seq reads for a specific genome region containing the differentially expressed temperature-responsive operon. The diagrams depict the plasmid design of two temperature sensing plasmids, both carrying the temperature-sensitive HspR transcription factor regulated by two distinct constitutive promoters (PPA_RS09160 and PPA_RS06745, respectively), alongside the temperature-responsive promoter controlling the pFAST reporter. The graph on the bottom right displays the average mRNA fold change increase in these two temperature sensing strains upon 42 ºC heat shock treatment measured by RT- qPCR. (C) Top: Western blot detection of SodC-F1 constitutively produced and secreted to the supernatant (labelled as SN) by an engineered C. acnes strain, compared to the cellular (labelled as C) fraction of thesame strain. Bottom: Western blot detection of aTc-inducible SodC-F1 production. Shown are supernatant(SN) fractions of uninduced (-) and induced (+) cultures. (D) Superoxide dismutase activity measured in the supernatant of three different SodC-F1-producing strains with different constitutive promoter strengths (P(PPA_RS09745), P(PPA_RS09225) and P(PPA_RS07095) ) compared to a no insert plasmid control (Empty) (E) Reactive oxygen species (ROS) levels measured with CM-H2DCFDA fluorescent dye for N / TERT-2G keratinocytes cells exposed or not to UV-B light. Cells were treated with the supernatant of strains producing SodC-F1 constitutively (from P(PPA_RS09225) or P(PPA_RS07095) promoter) or with the supernatant of a no-insert plasmid control (Empty).Figure 4. Western blot analysis of synthetic C. acnes cells engineered to produce and secrete proteins withanti-ROS activity. Both the cellular fraction (C) and the supernatant fraction (SN) were assessed for the presence of the desired proteins: the E. coli KatG catalase (left panel), the C. acnes catalase (right panel), and the E. coli O157:H7 SodC-F1 (both panels). Despite the use of the same promoter, RBS and signal peptide sequences for all three proteins, only the SodC-F1 was secreted to the supernatant, unlike the two catalases. Figure 5. The graph displays the coverage of RNA-seq reads for a specific genome region containing the differentially expressed ROS-responsive operon. The scheme depicts de genomic organisation. Thesequence logo displays the conservation of the OxyR binding motif compared with the OxyR motif from E.coli. Figure 6. Western blot protein detection of SODs constitutively produced and secreted to the supernatant (Supernatant) by engineered C. acnes strains, compared to the cellular fraction (Cell Lysate) of the same strains. The labels indicate the type of SOD (SodC-F1, dwSOD and hsSOD_1) and whether they were expressed from a genome insertion or an episomal plasmid. Figure 7. Superoxide dismutase activity measured in the supernatant of different SOD-producing strains with different SOD proteins (dwSOD and hsSOD_1) compared to C. acnes strain with no plasmid (C. acnes WT). Figure 8. Western blot protein detection of SODs constitutively produced and secreted to the supernatant (Supernatant) by engineered C. acnes strains, compared to the cellular fraction (Cell Lysate) of the same strains. The labels indicate the type of secretion peptide used for their secretion (sp1, sp2 and sp3). They were all expressed from a genome insertion. Figure 9. Superoxide dismutase activity measured in the supernatant of different SOD-producing strains. The strains produced the SodC-F1 with different secretion peptides (sp0, sp1, sp2 or sp3) and were expressed from an episomal plasmid (plasmids) or from a genome insertion (genome). The activity is compared to a strain that has an episomal plasmid with SOD gene but with no promoter (No promoter SOD (plasmid)) and another control with a genome insertion of the resistance cassette only (No SOD (genome)). Description of the embodiments of the inventionIn the present invention, we establish an enabling toolbox for the traditionally intractable C. acnes topromote the development of microbiome-based skin therapies (Fig.1A). We demonstrate the use of these tools to discover auxotrophies and to create an antibiotic marker-free strain for safe deployment in the skin. Moreover, we design a set of natural and synthetic inducible systems for the exogenous or environmental activation of gene expression. As a proof of concept for potential therapeutic applications, we create an antioxidant-producing strain able to rescue keratinocytes from oxidative stress, preferably UV-induced oxidative stress. To construct this toolbox, we optimized a plasmid from Propionibacterium freudenreichii previously described to replicate in C. acnes28,32,33(Fig.1B). First, we removed all non-essential sequences as well as many restriction sites commonly used in classical or Golden Gate cloning, reducing the total vector size by more than 2 kb. Additionally, we adapted the plasmid for a modular cloning scheme based on isothermal (Gibson) assembly that allows us to standardize all our genetic parts with common suffix and prefix sequences for quick and efficient cloning34. We also standardized and codon optimized two antibioticresistance cassettes – based on the ermE gene from Saccharopolyspora erythraea and the cmx gene fromCorynebacterium glutamicum – and used one or the other as the C. acnes selection marker. The plasmidbackbone contains two origins of replication (one for E. coli, the other for C. acnes) and two resistance cassettes (one for each of the bacterial hosts). To facilitate the straightforward replacement of those components, we flanked them with unique restriction sites. We wanted to develop a collection of genetic parts enabling us and others to control and program the behaviour of C. acnes. To this goal, we first tested a set of 9 endogenous constitutive promoters that we previously identified by RNA-seq (see Methods) spanning a wide range of expression levels, with most functioning in both exponential and stationary growth phases (Fig.1C). We also tested a synthetic E. coliconsensus promoter (BBa_J23119) because of its similarity to the C. acnes -10 consensus box35.Interestingly, this promoter yielded the highest expression among those tested (Fig.1C), and was thus usedto validate the suitability of 7 fluorescent proteins as C. acnes reporters, including the anaerobic fluorescenttag pFAST36. Using flow cytometry, we observed an increase in fluorescent signal for all reporters when compared with an empty-plasmid control (Fig.1D), and we selected mCherry for subsequent experiments due to its reported suitability in anaerobic bacteria after 2h maturation in aerobic conditions37,38. We alsovalidated the use of the endogenous copy of lacZ – encoding beta-galactosidase – as a genomic reporterin the presence of X-gal (Fig.1E). We next tested the strength of 4 different ribosome binding sites (RBS): two of these (RBS_3 and RBS_4) were designed using the RBS calculator29, while the other two (RBS_1and RBS_2) were strong RBS sequences from E. coli – indeed, the rRNA stretch that recognizes the Shine-Dalgarno sequence is only one nucleotide different between C. acnes and E. coli. Interestingly, the E. coliRBS sequences yielded the highest levels of mCherry production (Fig.1F). We next measured the transcriptional termination of five sequences: two strong terminators reported to work in E. coli (a natural one and a synthetic one)39and three C. acnes endogenous sequences selected from RNA-seq. We used a dual reporter system where sfGFP and mCherry were transcribed together but are separated by the evaluated terminator sequence (Fig.1G). All five terminators yielded mCherry levels that were reduced with respect to non-terminator spacer controls and with terminator efficiencies ranging from 70% to 90% compared with the Spacer_1 control (Fig.1G). Adults have a unique mixture of C. acnes strains21. Ideally, the engineering of different C. acnes strains could enable strain-specific eLBPs tailored to the specific patient microbial profile, therefore increasing engraftment. For this reason, we wondered how suitable our optimised plasmid and molecular tools were for other C. acnes strains spanning across two phylotypes31and four different Sequence Locus Sequence Typing (SLST) types30. These strains were able to incorporate our optimized plasmid and to express mCherry controlled by the BBa_J23119 synthetic promoter in exponential phase (Fig.1H). We used these basic genetic parts to further develop more advanced tools. To interrogate the function of endogenous genes, we sought to validate the use of CRISPR interference (CRISPRi) in C. acnes. We hadobserved that C. acnes is naturally able to degrade the synthetic compound X-gal to yield blue-colouredcolonies (Fig. 1E), and we hypothesized that this β-galactosidase activity was due to a genomic lacZhomolog, PPA_RS08600. We designed single guide RNAs (sgRNAs) against the 5’ end of the lacZ codingsequence, and we combined them with different expression levels of dCas9. Designs featuring high expression levels of dCas9 yielded colourless colonies in the presence of X-gal regardless of the sgRNA used, indicating an efficient repression of endogenous lacZ (Fig.2A). Next, we focused on designing a biocontainment strategy for C. acnes that ensures the safe deployment of therapeutically relevant strains. Our goal was to generate an auxotrophic strain without any heterologous antibiotic resistance gene. Due to the low genome integration efficiency in C. acnes28, we initially turned toplasmid-borne CRISPRi to screen for genes that would render C. acnes auxotrophic upon knock-out. Wetargeted 8 different putative gene candidates whose individual disruption would lead to an amino acidauxotrophy (Fig. 2B), based on their homology to auxotrophic strains described in the E. coli KEIOcollection40. We then evaluated which of the CRISPRi knock-downs impaired bacterial growth when the amino acid was not supplied in a rich defined custom medium (See methods). Lysine and proline biosynthesis pathway repression showed the highest growth defect compared to a dCas9-only control (Fig. 2B). Histidine, leucine, and tryptophan pathway repression also showed growth defects, but growth was also reduced in the dCas9-only control, suggesting that the wild-type expression of the targeted genes cannot compensate for the absence of those amino acids in the medium (Fig.2B). Interestingly, we did not manage to knock down the cysteine pathway, likely due to the relevance of this amino acid for C. acnes41.In the light of these results, we generated C. acnes ΔlysA, ΔproA and ΔhisB strains by using a homologousrecombination cassette harbouring the erythromycin resistance gene ermE (Fig.2C). To enable the removalof the genome-integrated antibiotic resistance gene, we flanked ermE with the attP and attB recombinationsites specific for the BxbI serine integrase. Constitutive expression of the BxbI gene from a replicative plasmid selected with a different antibiotic resistance marker (cmx) resulted in the successful removal ofthe complete ermE gene for all tested colonies after transformation (Fig. 2C). After plasmid curation, weobtained three strains without any heterologous antibiotic resistance that preserved the corresponding gene deletions. The lack of lysA and hisB was enough to impair the growth of C. acnes after four days of incubation in the absence of lysine or histidine, respectively (Fig.2D). In the case of histidine absence, the wild type had reduced growth after 24 h, as observed previously in the CRISPRi screening, but achieved a similar growth level to the histidine-supplemented culture after four days of growth, in contrast to the ΔhisB strain (Fig. 2D). We observed a similar behaviour for lysine supplementation, where the wild-type experienced a slower growth but recovered within four days, while the ΔlysA strain did not show any growth during that time. Regarding the proline auxotroph, proA deletion did not fully suppress growth, but yielded a growth delay in the absence of proline that might impact its ability to compete with other skin-residentmicrobes and therefore be enough for biocontainment within the natural niche - the human skin (Fig. 2D).While the concentration of free Lys, His and Pro in corneocytes appears to be low on the skin surface42, a more biologically relevant and tissue realistic skin model is needed to further evaluate the performance of these auxotrophies as a real-world biocontainment strategy. One of the key benefits of eLBPs is their ability to detect host or environmental signals and to dynamically produce relevant molecules with therapeutic potential. To build such a dynamic “smart” probiotic, weengineered different transcriptional sensors in C. acnes following two main strategies: the transplantationof known transcription factors (TFs) from other organisms, and the de novo discovery of natural geneticresources from C. acnes. For the first approach, we decided to focus on two well-known repressors commonly used in model bacteria, TetR and PhlF. We designed different inducible promoters by placingone or more operator sequences (tetO or phlO) upstream, downstream or between the -35 and -10 boxesof BBa_ J23119, and we combined the resulting promoters with different expression levels of the corresponding TFs (Fig. 3A). In the presence of the inducer (aTc for TetR, DAPG for PhlF), some of the designs yielded inducible mCherry expression of up to ~7-fold compared to the non-induced control (Fig. 3A). While sensors in model bacteria can display large dynamic ranges (of up to three or four orders of magnitude in some cases) as a result of numerous optimizations throughout years of research, the fold- induction of TFs used in non-model bacteria is generally more modest, often in the range of one order ofmagnitude43 - e.g. ten-fold for GlnR in Pseudomonas putida44, seven-fold for FNR in Synechocystis sp.PCC 680345, or 5-fold for AceT in Rhodococcus opacus46. To discover and co-opt the natural transcription factors already present in the C. acnes genome, weperformed an RNA-seq on C. acnes upon temperature shock, which represents an environmental signalwith potential relevance in the skin context. Indeed, temperature change-detection has been previously used to trigger the self-destruction of bacteria leaving the body (i.e. kill switch) for biocontainment47, or as an activation mechanism induced by ultrasounds48. We exposed C. acnes to a 42ºC heat shock for 15 minutes and we looked for upregulated genes with RNA-seq. From Hidden Markov Models (HMM) models and operon structure conservation, we identified a conserved DnaK-GrpE-DnaJ-HspR chaperon operon well-known in other bacteria to regulate protein refolding in heat-shock stress (Fig.3B). Specifically, the HspR transcriptional regulator is involved in the regulation of the operon49,50; we thus cloned the regulatory sequence together with the constitutively expressed HspR in our replicative plasmid. We tested two plasmid constructs differing only in the strength of the constitutive promoter used to express HspR (Fig. 3B). We measured the pFAST reporter mRNA expression by RT-qPCR after a 42ºC heat-shock and observed a 4 and 6-fold increase at the mRNA levels, respectively (Fig. 3B). The higher fold-change observed higher fold-change observed when HspR is expressed from the P(PPA_RS06745) promoter is likely due to the greater abundance of the repressor HspR due to its stronger constitutive promoter. Typically, higher repressor concentrations decrease basal expression levels and result in a greater change in expression upon induction51. Ideally, eLBPs should have the dual ability not only to sense the environment but also to produce proteins or molecules with therapeutic relevance directly on the appropriate biological niche. We thereforeengineered an antioxidant-secreting C. acnes strain for reducing oxidative stress in the skin. Superoxidedismutases (SODs) are enzymes that neutralize the superoxide anion (O2–) and protect cells from oxidativedamage. Among these enzymes, the SodC-F1 from the enterohemorrhagic E. coli O157:H7 has shown tobe functional in the periplasm, stable against proteases and with high catalytic activity, representing one of the principal defence mechanisms of this bacterium against Reactive Oxygen Species(ROS)52. Any type of unstable molecule that contains oxygen and that easily reacts with other molecules in a cell eventually causing oxidative damage to key cellular elements like DNA or RNA is considered a ROS.We hypothesized that expressing this enzyme in C. acnes could reduce ROS levels. To prove this, we firstcoupled the enzyme with a secretion signal from a highly secreted endogenous protein, RoxP53,54. Indeed, the protein was correctly produced and secreted to the supernatant (Fig. 3C), and it maintained its superoxide dismutase activity in the supernatant (Fig. 3D). We also demonstrated the usefulness of our newly-developed transcriptional sensors by constructing a strain that produces and secretes SodC-F1 in an aTc-inducible manner (Fig. 3C). We next challenged the immortalized keratinocyte cell line N / TERT- 2G55,56with UV-B to induce the formation of ROS. Thereafter, we treated the cells with C. acnes supernatants and quantified ROS levels using the fluorescent dye CM-H2DCFDA. The SodC-F1 producedby C. acnes was able to significantly reduce UV-mediated ROS levels in keratinocytes (Fig. 3E). Thereduction correlated with the strength of the promoter regulating SodC-F1 expression, suggesting that we are able to adjust the antioxidant activity of the engineered strain to match the requirements of the final application. Importantly, the secretion of SodC-F1, which is key for delivering its antioxidant activity, seemsto correlate with its small molecular size, since larger antioxidant proteins like the E. coli KatG or the C.acnes catalase remain in the cellular fraction and are not secreted despite the use of the same promoter,RBS and signal peptide sequence as for SodC-F1 (Fig.4). Moreover, deployments of this antioxidant eLBP could incorporate inducible SOD expression, either through artificial inducers like aTc (Fig.3C), or in response to environmental triggers, such as UV-damagemarkers produced by keratinocytes. For instance, a bacterial ROS sensor (such as OxyR57) controlling theexpression of SOD depending on the ROS produced by keratinocytes upon UV exposure would result in an adaptative antioxidant eLBP. Such sensors have been previously reported for the gut environment within an E. coli bacterial chassis to detect oxidative stress in situ58. In C. acnes, a putative oxidative stress sensor is homologous to the OxyR transcription factor from E. coli. Homologous genes in the OxyR regulon arealso present in C. acnes and are induced in the presence of oxidative stress generated with the commontoxic pesticide paraquat (Fig.5). The regulatory sequences of these promoters could be taken together withthe OxyR transcription factor homolog to induce gene expression in C. acnes with ROS, as it was previouslydone with the original OxyR sensor from E. coli57.In conclusion, we have established C. acnes as an amenable chassis for eLBPs development with potentialapplications in the skin. We describe a versatile toolbox for its engineering, as well as an auxotrophic strain and antibiotic-free methodologies for genetic engineering towards its safe application in the skin. We demonstrate the detection of both artificial and environmental cues by engineered C. acnes, and we provide a proof-of-concept demonstration of oxidative stress reduction on keratinocytes. Taken together, our resultspave the way for the use of C. acnes for microbiome-based skin therapies.Therefore, the instant disclosure relates to compositions of C. acnes and methods of secretingtherapeutically active proteins, preferably superoxide dismutase enzyme, for the treatment of diseases ordisorders. The recombinant bacteria disclosed herein are capable of high yield production of functionally active superoxide dismutase enzymes, which are secreted as active polypeptides, preferably as therapeutically active polypeptides. Thus, a first aspect of the invention refers to a composition comprisingCutibacterium acnes (C. acnes) bacterial strains, wherein said strains are characterized by being modifiedto secrete a heterologous protein by insertion of at least one or more ORFs (Open Reading Frames)encoding the heterologous protein, preferably in the genome, or at least an expression construct comprising at least one or more transcription promoter sequences, one or more ORFs (Open Reading Frames) encoding the heterologous protein, one or more transcription termination sequences and preferably one or more RBS (Ribosome Binding Site); wherein the heterologous protein is preferably characterized by comprising at the N-terminus of the protein sequence, a secretion signal which is processed by C. acnes to secrete the protein, and wherein the heterologous protein is the superoxide dismutase enzyme.In some embodiments, the genetically engineered or modified C. acnes of the disclosure comprises a genethat is operably linked to a promoter that is not associated with said gene in nature. For example, in someembodiments, the recombinant bacteria disclosed herein comprises a gene that is operably linked to adirectly or indirectly inducible promoter that is not associated with said gene in nature. As used herein, the term “coding region or encoding” refers to a nucleotide sequence that codes for a specific amino acid sequence. A regulatory region “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence,regardless of the distance between the regulatory element and the coding sequence. More specifically,operably linked refers to a nucleic acid sequence, e.g., a gene encoding an effector molecule, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the effector molecule described herein. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be “directly linked” to a regulatory sequence in a manner which allows expressionof the gene. In another embodiment, a gene may be “indirectly linked” to a regulatory sequence in a mannerwhich allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes. A regulatory region or sequence is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5 ’and 3 ’untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences and introns. A “promoter” as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5’ of the sequence that they regulate. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from promoters found in nature, and / or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene inresponse to a particular stimulus, e.g., in a cell- or tissue-specific manner, in response to differentenvironmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. A “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control. Some non-limiting examples of promoters useful in the present invention are HspR C. acnes (PPA_RS10230), HspR Operonpromoter temperature sensor from C. acnes (P(PPA_RS10245), OxyR C. acnes promoter ROS Sensing 1(P(PPA_RS10005)), OxyR C. acnes promoter ROS sensing 2 (P(PPA_RS10010)), or OxyR C. acnes transcription factor (PPA_RS10015): HspR C. acnes (PPA_RS10230) (SEQ ID NO 18): ATGGTGAGGCGCAGCAGGAATCTATCCGACGCTGTTGATCTTGCGGTGATCGACAAGGACGCTGCCATCTTCG CCATCTCAGTGGCGGCAGAGCTCGCCGGAATGCACCCTCAGACCCTGCGCACTTACGATCGGCTCGGGCTTGT AGTGCCCGAACGCACTCGGGGGCGGGGGCGTCGCTACTCGATGCGTGACGTTGCTGCGTTGCGGATGGTCCA GCATCTGTCCCAGGAAGAGGGGATTAACCTCAACGGCATCCGAAGGATCCTTCAGATGGAACGTAGGATCGAAC AGTTGTCGGAGCAGGTGGACGAGCTCACCGACACCGTGCGTGGGATGCAAGTGGCGCGTTACCAGGGCAGTG AAGATCCGAGAGTCTTTACTGCTGAGTCGACCGGCCGGGTGCATATGGGTCGGACGGTCGTTCATGCCCAGTT GGCACTGCCTTCTCATCGTGGTTGAHspR Operon promoter Temperature sensor from C. acnes (P(PPA_RS10245) (SEQ ID NO 19):CGTGAAGTGCGGAAGCTGCCGGACGGTTCGCCGTCCGGCATTTTTCATGGTCGGAAGGGGTCGTCTGAGGATG TCTTGACGAGACTTTCGGTGGGTGGGAATACTCCCGCCGGATCGTCGGTTGAGTGTAGCGAACTCAACTTCATG AAAAATCAGCCAACGTCAACTTGAGTGCTCGTGACTCAGGGTCTAGAGTTGAGTCGAAGTCGCTCAAGGGGATG TAGTCCCAGAACATTCGTCCATCAGGAGG OxyR C. acnes promoter ROS Sensing 1 (P(PPA_RS10005)) (SEQ ID NO 20): GGTTAAGGCCGCCCAATACATCTACACCCACCCAGGCGAGGTGTGCCCCGCCAAGTGGGAAGAAGGCGATGAG ACCCTCGCCCCGTCGATCGACCTCGTCGGCAAGATCTGA OxyR C. acnes promoter ROS sensing 2 (P(PPA_RS10010)) (SEQ ID NO 21): GACCATGGGGGCATCATGCCACGGGTGCGGGCCCTGGCGACACAGAGAGTCCCGAGTTCATGATCTTTATAGC TATTGACTATAGATGCTGGTTTAGTGATAGCCTTGATCTATCACATCGTCGACGAGAAAGGACACCC OxyR C. acnes transcription factor (PPA_RS10015) (SEQ ID NO 22): GTGAACCTTCATGATCTGCGGTATTTCGTCGCCCTGGCCGAAGAACACCACTTCGGCCGGGCTGCGGCGTCAT GCGGTGTCAGCCAGCCCACCCTCTCAACTCAAATCCACCGGCTCGAGAACGAGCTCGGCGTGGTGCTGGCCGT GAGGGTGGGACGACGCATCGAGATCACCCCAGTGGGGGAACGCATCGCCCAAGCTGCCCGTGACATGTTGGT GCTATCCGACGACATCCGGACCATGGCGCGGGAGGAAGCCGACCGGATGACCCTCCAGCTGAGGGTCGGGGT ATTCCCCACCTTAGGGCCGTTCGTGTTGCCTCAGGTGATCGCCCGATGTGACCAGCACGAGCCTGGCGTGAAC ATCCATATCGTCGAGAAGCGCACGGCAGAGCTGTTGGAGGCAATCCGCAACGGACAGTTAGACGTCGCCGCAG TGGCAGCCCCCGTTAATGATGACTCTTTGGTGACTATTCCCGTTTTTCGGGAGGAGTTCTTGCTCGTGACGGGG GCTGACGATGAGTTGGCCCGTGAGGAACGACCAATCAATCCGCACGACGTTCCCACCGAAGGGCTCATCCTCA TGTCGGAGGGACACTGTCTAAGAGACCAGGTGCTTGAGGTGTGCAGCCCGTCTCATCCGCTGCCTCCCGACACCCTCACGGCGGGATCCCTCGAGACGCTCCGCGAACTCGTATCGGTGGGCGCCGGTCAAACCCTCATGCCGCGCAGTGCGATCTCGGCACCACTTCATGCGGACCCGCGCATCGTGGTGCGGGAGTTCACCCACCCCAGACCCCAC CGCGACATCGTGGTGGCTGGCAGACCGGCCACAATGCACCGTCCGGCGGTGCAGACCCTGACCACCATCCTG CGGCAGCTACCAGGTGACGTCGTCGAGCCTCTCGGCGTTGCCGGAGCATCGTTGTGCTCTCACGGGATGTGA “Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, Ptac promoter, BBa_J23100, a constitutive Escherichia coli σS promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli o32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis oA promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis oB promoter (e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)). An “inducible promoter” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. An “inducible promoter” refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An “indirectly inducible promoter” refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.” As used herein, the term “sequence identity” as used herein is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%. The term “expression construct” as used herein, means the vehicle, e.g. vectors or plasmids, by which a DNA sequence is introduced into a host cell so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. “Expression construct” as used herein includes both, autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences. The terms "vector”, “DNA vector” and "expression vector” mean the vehicle by which a DNA sequence e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. “Vector” as used herein includes both, autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences, such as artificial chromosomes. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell. Specifically, the term “vector” or “plasmid” refers to a vehicle bywhich a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transformthe host and promote expression (e.g. transcription and translation) of the introduced sequence. As used herein, the term “transform” or “transformation” refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically-stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” organisms. The term “ROS reduction” or “reduction of ROS” refers to a decrease of any intracellular or extracellular ROS levels to levels similar (± 20%) to those of cells not treated with an oxidising agent, including (but not limited to) UV or paraquat. In some embodiments of the first aspect of the invention, the secretion signal is of SEQ ID No. 1 or a functional variant thereof that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the full / length sequence of SEQ ID NO 1 with the proviso that said functionalvariant can be processed by C. acnes cells to secrete the heterologous protein.PPA_RS09745signal peptide (nt) (SEQ ID NO 4): atgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgcc PPA_RS09745 signal peptide (aa) (SEQ ID NO 1): MFVQIAASLAAASSIALGIPGAA In some embodiments of the first aspect of the invention, the secretion signal can be selected from any oneof the following proteins (sp0, sp1, sp2 or sp3) that are known to be secreted in C. acnes or any functionalvariant thereof that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with the full / length sequence of sp0, sp1, sp2 or sp3, as shown below, with the provisothat said functional variant can be processed by C. acnes cells to secrete the heterologous protein.:● sp0: PPA_RS09745 Signal Peptide (RoxP):MFVQIAASLAAASSIALGIPGAA ●sp1: PPA_RS09975 Signal Peptide: MKVLRTSVLGLACATALVSSVGVSPAQA● sp2: PPA_RS03515 Signal Peptide (CAMP2 factor): MKKTHLVAPLLVGAMLVPAALSAPSAHA● sp3: PPA_RS10575 Signal Peptide (TAG Lipase): MKINARFAVMAASVAVLMAAAPIAQAIn some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the superoxide dismutase enzyme is theSodC-F1 from the enterohemorrhagic E. coli O157:H7 of SEQ ID NO 2 or any sequence that has at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of of SEQ ID No.2 that when secreted in one or multiple copies by using the secretion signal of SEQ ID NO 1 is capable of reducing ROS levels. SodC-F1 from E. coli O157:H7 (codon-optimized for C acnes) SodC-F1 (nt) (SEQ ID NO 9): (ATG)GAGCAGGAAGTGCCGATGAACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATC CAGGAGACCCCATACGGTCTGTTGTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGT CCACGAGAAGGGCAACTGCGCCCCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCA CTTTGACCCGAAGAATACTGGCAAGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCC TGTTCGTCACCCATGACGGCAAAGCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAA GGGCCGCTCcCTcATGCTcCACGCtGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtG CCCGCATGGCCTGCGGCATTATCCAGCATCATCACCATCACCACTGAtaa SodC-F1 (aa) (SEQ ID NO 2): (M)EQEVPMNLVSADGKEVSIGKITIQETPYGLLFTPALHSLSEGIHGFHVHEKGNCAPALKDGKPVAALSAGGHFDPK NTGKHLGPWSPDGHLGDLPALFVTHDGKANYPVLAPRLNSLKEIKGRSLMLHAGGDNHHDHPEPLGGGGARMACGI IQ It is noted that the SodC-F1 does not have a start codon (ATG) because it is fused to a N-terminal signal peptide, and the signal peptide does have the start codon. In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the superoxide dismutase enzyme is selected from anyone of the following alternative SODs for C. acnes expression: 1. Engineered superoxide dismutase from Deinococcus wulumuqiensis R12 from (Meng et al. 2025),which is herein denominated as dwSOD a. Sequence:MFVQIAASLAAASSIALGIPGAAAYTLPQLPYAYDALEPHIDARTMEIHHTKHHQTYVDNANKALEG TEWADLPVEELIQKLDQLPADKKGALRNNAGGHANHSLFWQVMGQGKGGQPSGELMDAIGSAF HSFDAFKQKFEDAAKTRFGSGWAWLVVRDGKLDVVSTANQDNPLMGEAVAGVSGTPILGVDVW EHAYYLNYQNRRPDYLAAFWNVVNWDEVAKRYAAAKHHHHHH 2. Human superoxide dismutase 1, which is here denominated as hsSOD-1:a. Sequence:MFVQIAASLAAASSIALGIPGAAWTGEDSAEPNSDSAEWIRDMYAKVTEIWQEVMQRRDDDGALH AACQVQPSATLDAAQPRVTGVVLFRQLAPRAKLDAFFALEGFPTEPNSSSRAIHVHQFGDLSQGC ESTGPHYNPLAVPHPQHPGDFGNFAVRDGSLWRYRAGLAASLAGPHSIVGRAVVVHAGEDDLGR GGNQASVENGNAGRRLACCVVGVCGPGLWERQAREHSERKKRRRESECKAAHHHHHH or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of any of the above, that when secreted in one or multiple copies by using any one of the secretion signals of the invention, in particular SEQ ID NO 1, is capable of reducing ROS levels. In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the superoxide dismutase enzyme is theSodC-F1 from the enterohemorrhagic E. coli O157:H7 of SEQ ID NO 2 and the secretion signal is of SEQID No.1. In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the one or more transcription promoter sequences is the promoter of SEQ ID NO 3, 56, or 15 or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID No.3, 5,6, or 15 that when operably linked to the heterologous protein is capable of expressing the said protein. Insome embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the one or more transcription promoter sequences is selected from the list consisting of: HspR C. acnes (PPA_RS10230), HspR Operonpromoter temperature sensor from C. acnes (P(PPA_RS10245), OxyR C. acnes promoter ROS Sensing 1(P(PPA_RS10005)), OxyR C. acnes promoter ROS sensing 2 (P(PPA_RS10010)), or OxyR C. acnestranscription factor (PPA_RS10015), or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of any of these promoters.pEND-100 Promoter (called “P (PPA_RS09745)”) of SEQ ID NO 3: acaccgacgatctgcacgggcgagtctaggggttatcggctacatgcgcgtcgccgcatcaggttcacgaaaccgaaaggcagacccaccccct accgggcataccgttgccgccgacacttcgatgacactatgcagtgctacacttcaactgaaaatctatatgattttcgtcaattc pEND-273 Promoter (called “P (PPA_RS09225)”) of SEQ ID NO 5: ggttgatctgtagtgctgccggggccgtgggttccggcagcactgtggactacgggcgggacgcgtgccctctgtgtgagttactgccggtgcccttta gctcctttcaatttggggagacgacgttgagaaggtatcgttaactgctggcttcgtgtgtgcggagccacaaccacgcccgcagggggtgtggg pEND-274 Promoter (called “P (PPA_RS07095)”) of SEQ ID NO 6: CCTGTCCTGATCATGATCCGTCCGGGGGCCGCTAGTTGGGCGGTCCCCGGACGATTTTGTGTACCC GCAGATGCGTGCCAGACAAGCGCCTCGGCTGTTTTGTGGGTTTGTGTTGGTGGTGGGGGTGTGTGT AGTGTGTGTTGGGCCGTTGGGGGCTGCGTGTGTGGTTCTTGGTGGTGGCTGTGTTTGGGATG pEND-387 Promoter (called “BBa_J23119-TetO2”, i.e. the aTC-inducible promoter) of SEQ ID NO 15: CAGCCTGCGGTCCGGTTGACACCCTATCAGTGATAGAGTATAATGCTAGCTCGCTGGGACGCCCG In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the one or more transcription promotersequences is the synthetic E. coli consensus promoter of SEQ ID NO 3.In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the expression construct further comprisesa ribosome binding sites (RBS): preferably the RBS is the E. coli RBS sequences of SEQ ID No 7, or 8 orany sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID No.7 or 8. RBS (called “RBS_1”) of SEQ ID NO 7: TCCACTTATAAATATAGAGGAGGTTTGAT RBS (called “RBS_2”) of SEQ ID NO 8: TAATTTTGTTTAACTTTAAGAAGGAGATATACAT In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the expression construct further comprisesa ribosome binding sites (RBS): preferably the RBS is the E. coli RBS sequences SEQ ID No 7.In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the expression construct is of SEQ ID NO9, 10, 11, or 16 or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identity with the full / length sequence of SEQ ID No.9, 10, 11, or 16. pEND-100 of SEQ ID NO 9: CAGCCTGCGGTCCGGacaccgacgatctgcacgggcgagtctaggggttatcggctacatgcgcgtcgccgcatcaggttcacgaaaccgaaaggca gacccaccccctaccgggcataccgttgccgccgacacttcgatgacactatgcagtgctacacttcaactgaaaatctatatgattttcgtcaattcTCCACTTATAAATATAGAGGAGGTTTGATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAAGTGCCGATGAACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCC ATACGGTCTGTTGTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGG GCAACTGCGCCCCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGA AGAATACTGGCAAGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACC CATGACGGCAAAGCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcC TcATGCTcCACGCtGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCC TGCGGCATTATCCAGCATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCG pEND-273 of SEQ ID NO 10: CAGCCTGCGGTCCGGggttgatctgtagtgctgccggggccgtgggttccggcagcactgtggactacgggcgggacgcgtgccctctgtgtgagttactg ccggtgccctttagctcctttcaatttggggagacgacgttgagaaggtatcgttaactgctggcttcgtgtgtgcggagccacaaccacgcccgcagggggtgtgggT CGCTGGGACGCCCGTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTGTTTAACTT TAAGAAGGAGATATACATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAA GTGCCGATGAACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCAT ACGGTCTGTTGTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGG CAACTGCGCCCCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAA GAATACTGGCAAGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCC ATGACGGCAAAGCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCT cATGCTcCACGCtGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCT GCGGCATTATCCAGCATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCG pEND-274 of SEQ ID NO 11: CAGCCTGCGGTCCGGCCTGTCCTGATCATGATCCGTCCGGGGGCCGCTAGTTGGGCGGTCCCCGGACGATTTT GTGTACCCGCAGATGCGTGCCAGACAAGCGCCTCGGCTGTTTTGTGGGTTTGTGTTGGTGGTGGGGGTGTGTG TAGTGTGTGTTGGGCCGTTGGGGGCTGCGTGTGTGGTTCTTGGTGGTGGCTGTGTTTGGGATGTCGCTGGGAC GCCCGTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTGTTTAACTTTAAGAAGGA GATATACATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAAGTGCCGAT GAACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCATACGGTCTG TTGTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGGCAACTGCG CCCCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAAGAATACTGG CAAGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCCATGACGGCA AAGCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCTcATGCTcCAC GCtGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCTGCGGCATTAT CCAGCATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCG pEND-387 of SEQ ID NO 16: CAGCCTGCGGTCCGGTTGACACCCTATCAGTGATAGAGTATAATGCTAGCTCGCTGGGACGCCCGTAGAACTCT GTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTGTTTAACTTTAAGAAGGAGATATACATatgttcgtc caaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAAGTGCCGATGAACCTGGTCTCGG CTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCATACGGTCTGTTGTTCACCCCCGCC CTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGGCAACTGCGCCCCGGCGCTTAAAG ACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAAGAATACTGGCAAGCACCTGGGCC CGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCCATGACGGCAAAGCCAACTACCCG GTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCTcATGCTcCACGCtGGtGGCGACAAT CACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCTGCGGCATTATCCAGCATCATCACC ATCACCACTGAtaaTCGCTGGGACGCCCGCTCGAGCAATAAACAGTTGATAGGGCTTCTCCGTTACCATGGTTCA GCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGGCGGTTTTCTT TTCTCTTCTCAATTCTTCTGACCTGTAACGAATAATAGATAGTAAAGTAGTCTCCGATTGAGTTTTCTCTGCCGAG TCCCACCCAGTTCTGTGATTTCAGTAAGTTGGTAATTGATACACTGTTGCGAGAACTGgTGCCTGGTAGTAGATA GGTTGTTATTGAGTAAGAAGGTAAAGTGAACGAAATCCCTGAAACTGAGACTGTAGAAAATAAGCTTGTCCAGACTATTGGATCCAAGAGATTTCTACACGATTGAGCACTGTCTCCTGCAGGCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCTAATAGATAAAGGATAGGTCTGGTAGTGTTGTTCGTTCTCG CAGGTAAATCAATAATACTCtGCAGTTCCGTAGACTTTTCAGTGGGACAGGGTAGCGATAACAGATAGATTGTAAT AAGACACAGTAGGTGCTCGTAGTTGCGTGAAGAGAACCGCTCAGGAAATCCAGTCAGAAGTATTGGTAATCGTT GAAAACTCAGTCGACCAGCCTGCGGTCCGGacaccgacgatctgcacgggcgagtctaggggttatcggctacatgcgcgtcgccgcatcag gttcacgaaaccgaaaggcagacccaccccctaccgggcataccgttgccgccgacacttcgatgacactatgcagtgctacacttcaactgaaaatctatatgattt tcgtcaattcTCGCTGGGACGCCCGTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTG TTTAACTTTAAGAAGGAGATATACATATGTCAAGACTAGATAAGAGCAAGGTCATTAACAGCGCACTGGAGTTACT AAACGAAGTTGGAATAGAGGGATTGACTACACGAAAATTAGCACAGAAATTAGGAGTAGAACAACCGACACTTTA TTGGCACGTTAAAAATAAAAGAGCTTTGCTGGATGCTTTAGCTATAGAAATGCTAGACCGACACCACACACATTTTTGCCCCTTAGAAGGAGAAAGTTGGCAAGATTTCCTACGTAACAACGCCAAATCATTTAGATGCGCGCTATTATCACACCGCGACGGCGCCAAAGTTCACCTCGGGACAAGACCTACGGAGAAGCAATACGAAACCCTAGAGAATCAATT AGCCTTTCTATGTCAACAAGGATTCAGTTTGGAAAACGCCCTCTACGCCCTATCAGCCGTTGGTCATTTTACATTG GGATGCGTTTTAGAGGATCAGGAACATCAAGTAGCAAAAGAAGAAAGAGAGACACCCACCACCGACAGCATGCC ACCATTATTACGTCAAGCTATTGAACTATTCGATCATCAGGGAGCGGAACCGGCATTTTTATTTGGCCTCGAATTA ATCATATGTGGTTTAGAAAAACAATTAAAGTGTGAATCTGGATCCCATCATCACCATCACCACTGAtaaTCGCTGG GACGCCCG In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the expression construct is comprised in aplasmid, preferably a plasmid capable of replicating in C. acnes and comprising an origin of replication forC. acnes. In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the strains are mutant live auxotrophicCutibacterium acnes (C. acnes) bacterial strains, preferably for Lysine, Histidine or Proline, with the provisothat said mutant live auxotrophic C. acnes bacterial strains do not comprise any heterologous antibioticresistance genes. In some embodiments of the first aspect of the invention, optionally in combination with any of the previous or subsequent embodiments of the first aspect of the invention, the expression construct is comprised in a plasmid and the plasmid comprising the said expression construct is selected from any one of SEQ ID NO 12, 13, 14, or 17 or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID No.12, 1314, or 17. pEND-100 of SEQ ID NO 12: GTGCGTGCGGGCGTGAGCGTTTCTACGCTGCGGCGCAGGAAATCAGAGCTTGAGGCTGCCGGAGCGACGGTA GACCCGTCCGGTTGGGTGGTGCCACTGCGTGCACTCAAGGTCGTTTTTGGGGTGTCAGATGAGACCTCGAATG CGAATACCAGTAAATCCTGACCTGTGAGTTGTGAATTCATAGGATAGATTCTGGAAACTTTACCGTCCGAGCTCC AGCCTGCGGTCCGGacaccgacgatctgcacgggcgagtctaggggttatcggctacatgcgcgtcgccgcatcaggttcacgaaaccgaaaggcaga cccaccccctaccgggcataccgttgccgccgacacttcgatgacactatgcagtgctacacttcaactgaaaatctatatgattttcgtcaattcTCCACTTATAA ATATAGAGGAGGTTTGATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAA GTGCCGATGAACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCAT ACGGTCTGTTGTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGG CAACTGCGCCCCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAA GAATACTGGCAAGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCC ATGACGGCAAAGCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCT cATGCTcCACGCtGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCT GCGGCATTATCCAGCATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCGCTCGAGCAATAAACAGTTGATA GGGCTTCTCCGTTACCATGGTTCAGCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCG GTGGACAGGATCGGCGGTTTTCTTTTCTCTTCTCAAGCTAGCGAAATCCCTGAAACTGAGACTGTAGAAAATAAG CTTCCGAAACTGAACCTGTTATTTTACGCTACGGAcgCCCGGTCATGACGCTGAGTTAGTGGCGCAGCTGCGCTC TGAGAACGAGTTTTTACGGCGTCAGGTCGAGCAGCAGGCGCGCACGATCGAACGGCAGGCTGAGGCACACGC GGTGGTCTCAGCGCAGCTCACACGGGTTGGCCAGCTTGAGGCCGGCGACGCAGCAGCACCGACACTGGCACC CGTTGAAAGGCCGGCTCCGCGACGGCGGTGGTGGCAGCGTCGGTAGCGGTCAGGATCGCTCTGGCGTGACGA GTGTGTCTGGCAGTGCGAACAGTTGCTCGACCAGTGGCAGCAGAAGCGAGATCGCTGCGTGGTGCTGTTCCTC GGTCAGTTCGTCGAGGACTGGCGGGTCTTGCTGCGTCCAGCCGATCGCCTCGGCGGCCAAGGTCAGTTCCAAG CTGTGCCAACGCACACGCCCCTCGGCTGACAGCTGAGTCTCGAACTGTGCAACTGGACCGGCCGGAAGATGCA CGTTGCCGAGGTCGTGAGTGGCCAAGCGCACGTCAAAGAGTGCTGCTTCGTAGCCGCGCAGAAATGGCAGTGC TCGGTCGATTCGGATCGGCCTGCCCAGGTACATTCCGGGCCGCTTGATGAACGCCTCCGCGTAGAAGCGCACC GTTCTCGGCCCGGCCTCGTGATCTGTCACTGTGCACGCTCCTCTCGATGGTTCTCGACGCTACCGGAGACCACC GACGTTCATGCCCAGCGCAGCGACCTGAAAGGACCAAGCCGAGTTAGCCGTGCTAACCGTATAGCTTGCTCCG TCGCCTCTGAGGGCAACCACCTGCGCAGCAGGTGGGCGGCAGCCCGCGCGCAAGCGCCTACCGGGTTTGGGCACAGCCCATAAATCAACGCCTCCGGTGTTGAAGCGATCGTGTGTCACGATTGCTATGCTTGCTACCCCTTCAGGGTTTTCGTATACACAAATCAAGTTTTTTCGTATACGCTAATGCCATGAGTGAGCATCTACTGCACGGCAAGCCCG TCACCAACGAGCAGATTCAGGCATGGGCAGACGAGGCCGAGGCCGGATACGACCTGCCCAAACTCCCCAAGCC ACGGCGCGGACGCCCGCCCGTAGGAGACGGTCCGGGCACCGTCGTACCCGTGCGTCTCGACGCGGCCACCG TTGCCGCTCTCACAGAACGAGCAACAGCCGAGGGCATCACGAACCGTTCAGACGCGATCCGAGCCGCAGTCCA CGAGTGGACACGGGTTGCCTGACCTCCACGACTCAGCACGCAAGCACTACCAACGAGACCGGCTCGACGACAC GGCCGTGCTCTACGCGGCCACCCACGTTCTCAACTCCCGGCCACTCGACGACGAAGACGACCCGCGCCGCTG GCTCATGATCGGAACCGACCCAGCAGGCCGCCTACTCGAACTCGTCGCACTGATCTACGACGACGGCTACGAA CTGATCATCCACGCAATGAAAGCCCGCACCCAATACCTCGACCAGCTCTAACCAAGAAAGGAACCTGATGAGCG ACCAGCTAGACAGCGACCGCAACTACGACCCGATGATCTTCGACGTGATGCGCGAGACCGCGAACCGCGTCGTCGCCACGTACGTTGCATGGGAAGATGAAGCCGCTGATCCCCGCGAGGCTGCGCACTGGCAGGCCGAGCGATTCCGCACCCGGCACGAGGTGCGCGCCGTCGACCCTGACAGCCGGCGAGCAGTTCAGGCGAAGATCGCACAGCT GCGCGAGGAACTAGCCGCAATGCCCGAACACGCCCCAGCCATCCCTTGGAGCAGGTGGCAGCGTCAGGGGAG TCGGGGGATGTTTGGCAGGGGATGTGGAAAGAGAGTTCGCTTTGCTCACATGGCTCAACCGGGTAACTAACTGA TATGGGGTCTTCGTCGCCCACTTTGAACACGCCGAGGAATGGACCACGCTGAACGTGACTCGCATGCTTCACTG CATGTATGGATTCGTTCGAGACGTTGTTCCCTGAGAGCTGGCTGCCACGCAAGCCGCTGGCGTCAGCCGAGAAGTCTGGGGCGTACCGGCACGTGACTCGGCAGAGGGCGCTGGAGCTGCCTTACATCGAAGCGAACCCGTTGGTCATGCAGTCCTTGGTCATCACCGATCGAGATGCTTCGGATGCTGACTGGGCCGCAGACCTCGCTGGGCTGCCT TCACCGTCCTACGTGTCCATGAACCGTGTCACGACCACCGGACACATCGTCTATGCCTTGAAGAACCCTGTGTG TCTGACCGATGCCGCGCGGCGACGGCCTATCAACCTGCTCGCCCGCGTCGAGCAGGGCCTATGCGACGTTCTC GGCGGCGATGCATCCTACGGGCACCGGATCACAAAGAACCCGCTCAGCACCGCCCATGCGACCCTCTGGGGC CCCGCAGACGCGCTCTACGAGCTGCGCGCCCTCGCACACACCCTCGACGAGATCCACGCACTGCCGGAGGCA GGGAACCCGCGTCGCAACGTCACCCGATCAACGGTCGGCCGCAACGTCACCCTGTTCGACACCACCCGCATGT GGGCATACCGGGCCGTCCGGCACTCCTGGGGCGGCCCGGTCGCCGAATGGGAGCACACCGTATTCGAGCACA TCCACCTACTGAACGAGACGATCATCGCCGACGAATTCGCCACAGGCCCCCTCGGCTTGAACGAACTTAAGCAC TTATCTCGATCCATTTCCCGATGGGTCTGGCGCAACTTCACCCCCGAAACCTTCCGCGCACGCCAGAAAGCGATCAGCCTCCGTGGAGCATCCAAAGGCGGCAAAGAAGGCGGCCACAAAGGCGGCATTGCCAGTGGCGCATCACGGCGCGCCCATACCCGTCAACAGTTCTTGGAGGGTCTCTCATGACCACACGTGAACGTCTCCCCCGCAACGGCT ACAGCATCGCCGCTGCTGCGAAAAAGCTCGGTGTCTCCGAGTCCACCGTCAAGCGGTGGACTTCCGAGCCACG CGAGGAGTTCGTGGCCCGCGTTGCCGCACGCCACGCGCGGATTCGTGAGCTgCGCTCGGAGGGTCAGAGCAT GCGTGCGATTGCTGCCGAGGTCGGGGTTTCCGTGGGCACCGTGCACTACGCGCTGAACAAGAATCGAACTGAC GCATGACCGTAACGCCGCACGATGAGCATTTTCTTGATCGTGCACCGCTTGGCACTACGTTCGCGTGCGGTTGC ACAGTGCGCGCCACGTTCTTATCCTGCGGCCATTGTGGCTACAGCCAATGGGGGGCATCAGCAACGGACGTTG AACCCGGTGGGCAAGTGTTACTCAGGGGGACATGCCCAGTCTGCGGCGCTCGGATTGACGGTATGGCAGTCGT GCATGCGGCCCCACCGTCAAACTCATTCAGGTATCAGTGAGAACCCTCATGGCACCCCCTCGTGACACGTTCTC GTTGCGATCAGCTGCGGCCAAGCTTGATCTTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTG TTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGC TGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCC CGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGT ATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCA GTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAA ACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACC GCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGC TTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTT CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTAT CGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTG TTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTT TCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCAA GCTTGTCGACGTTAACCTGCAGGCATGCGGATCCGGTACCAGCTGTCTCTTATACACATCTCTGCAGTTAACGCT GACCCGGACCACCACGACGACCCGGACGACCACCGGTACGACCACCACCAGACGGACCGCGGCGACCACCCT GGTCACCCTGTTCACGTTCACCGGTACGACCGTCGTCACGGTTACGACCAGAAGAACGACGGTCACCGTGACCAGACGCTTGCCGGTCTCTGCGATCTTGGCCCCCGGTACGTCTGTCACCACGGTCCTGACCACCGGTACGACGGTCGCCCCTATCCTGTCCACCCGTCCTACGACCACGCTGCGGCTGACCACCACGCGGTTCGTTTTTCTGGTCCAG ACGTTCGAACAGACGCAGCCACTGTTCCGGACGAACGTAAGCAACAACAGCACCACCACCAACACCAGCGTGG TCCAGAGCAGCTTCAACACGACGACGCGGGTATTTACGCAGCAGAGAAGCCTGGATGTTACCACCAACACCGG TGAAGCACAGTTCAACCATAGATTCGTAACGTTCCAGAGCAGCACCTTCCAGCAGCGGTTCAGCACGACGACGC AGACGCATGATAGCAGAGTCAACTTTCGGAACCGGTTTGAACAGACGACGATCGACTTTTTCAACGAATTCCCAT TCGAACAGCGGCCAGGTCATAACGGTCAGACGAGACCAACGACCGTAGTCACCGGTACGTTTACGAGCGAATT CCAGCTGGGTAACCATGGTAGCGGTTTCGATGGTCGGAGCTTCCAGGCACCAGTCAACGATAGCAGAGGTGAT ACCGTACGGGATAGCACCAACGAAAGCGAACGGTTCCGGCGGCGGTTCAGCGGTCAGGAAGTCAGCGTTAACA ACTTCGATGTTCGGGTGACCAGACAGTTTTTCACGCAGAGATTTAGCCAGACGCGGGTCGATTTCGTAAGAGGTAACCTGACGAGCACGGTCAGCCAGTTCACGGGTCAGCAGACCTTCACCCGGACCAGCTTCCAGAACCGGCAGGTCCGGACGCAGTTCAGCGGTTTCAGCGATACGAGCGATGGTTTTACGGTCACGCAGGAAGTTCTGGCCGAATTG GCGGCGGTTACGGTCACGCTCGGTACGGCCGAGGACGGGGCGATTCTGGTTGGGGTGCTGGCGGTCTTGGTT GCGGCGGCGGGGGCGGGGCTGTTCATCGGAGGAGGACATGACGATCTCCCGATGTGTCGTACGTATGGGAAT TGACGAAAATCATATAGATTTTCAGTTGAAGTGTAGCACTGCATAGTGTCATCGAAGTGTCGGCGGCAACGGTAT GCCCGGTAGGGGGTGGGTCTGCCTTTCGGTTTCGTGAACCTGATGCGGCGACGCGCATGTAGCCGATAACCCC TAGACTCGCCCGTGCAGATCGTCGGTGTGGATCCAGATGTGTATAAGAGACAGCTGGTACCGGCCGCTGCT pEND-273 of SEQ ID NO 13: GTGCGTGCGGGCGTGAGCGTTTCTACGCTGCGGCGCAGGAAATCAGAGCTTGAGGCTGCCGGAGCGACGGTA GACCCGTCCGGTTGGGTGGTGCCACTGCGTGCACTCAAGGTCGTTTTTGGGGTGTCAGATGAGACCTCGAATG CGAATACCAGTAAATCCTGACCTGTGAGTTGTGAATTCATAGGATAGATTCTGGAAACTTTACCGTCCGAGCTCC AGCCTGCGGTCCGGggttgatctgtagtgctgccggggccgtgggttccggcagcactgtggactacgggcgggacgcgtgccctctgtgtgagttactgcc ggtgccctttagctcctttcaatttggggagacgacgttgagaaggtatcgttaactgctggcttcgtgtgtgcggagccacaaccacgcccgcagggggtgtgggTC GCTGGGACGCCCGTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTGTTTAACTTT AAGAAGGAGATATACATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAA GTGCCGATGAACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCAT ACGGTCTGTTGTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGG CAACTGCGCCCCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAA GAATACTGGCAAGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCC ATGACGGCAAAGCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCT cATGCTcCACGCtGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCT GCGGCATTATCCAGCATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCGCTCGAGCAATAAACAGTTGATA GGGCTTCTCCGTTACCATGGTTCAGCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCG GTGGACAGGATCGGCGGTTTTCTTTTCTCTTCTCAAGCTAGCGAAATCCCTGAAACTGAGACTGTAGAAAATAAG CTTCCGAAACTGAACCTGTTATTTTACGCTACGGAcgCCCGGTCATGACGCTGAGTTAGTGGCGCAGCTGCGCTC TGAGAACGAGTTTTTACGGCGTCAGGTCGAGCAGCAGGCGCGCACGATCGAACGGCAGGCTGAGGCACACGC GGTGGTCTCAGCGCAGCTCACACGGGTTGGCCAGCTTGAGGCCGGCGACGCAGCAGCACCGACACTGGCACC CGTTGAAAGGCCGGCTCCGCGACGGCGGTGGTGGCAGCGTCGGTAGCGGTCAGGATCGCTCTGGCGTGACGA GTGTGTCTGGCAGTGCGAACAGTTGCTCGACCAGTGGCAGCAGAAGCGAGATCGCTGCGTGGTGCTGTTCCTC GGTCAGTTCGTCGAGGACTGGCGGGTCTTGCTGCGTCCAGCCGATCGCCTCGGCGGCCAAGGTCAGTTCCAAG CTGTGCCAACGCACACGCCCCTCGGCTGACAGCTGAGTCTCGAACTGTGCAACTGGACCGGCCGGAAGATGCA CGTTGCCGAGGTCGTGAGTGGCCAAGCGCACGTCAAAGAGTGCTGCTTCGTAGCCGCGCAGAAATGGCAGTGC TCGGTCGATTCGGATCGGCCTGCCCAGGTACATTCCGGGCCGCTTGATGAACGCCTCCGCGTAGAAGCGCACC GTTCTCGGCCCGGCCTCGTGATCTGTCACTGTGCACGCTCCTCTCGATGGTTCTCGACGCTACCGGAGACCACC GACGTTCATGCCCAGCGCAGCGACCTGAAAGGACCAAGCCGAGTTAGCCGTGCTAACCGTATAGCTTGCTCCGTCGCCTCTGAGGGCAACCACCTGCGCAGCAGGTGGGCGGCAGCCCGCGCGCAAGCGCCTACCGGGTTTGGGCACAGCCCATAAATCAACGCCTCCGGTGTTGAAGCGATCGTGTGTCACGATTGCTATGCTTGCTACCCCTTCAGG GTTTTCGTATACACAAATCAAGTTTTTTCGTATACGCTAATGCCATGAGTGAGCATCTACTGCACGGCAAGCCCG TCACCAACGAGCAGATTCAGGCATGGGCAGACGAGGCCGAGGCCGGATACGACCTGCCCAAACTCCCCAAGCC ACGGCGCGGACGCCCGCCCGTAGGAGACGGTCCGGGCACCGTCGTACCCGTGCGTCTCGACGCGGCCACCG TTGCCGCTCTCACAGAACGAGCAACAGCCGAGGGCATCACGAACCGTTCAGACGCGATCCGAGCCGCAGTCCA CGAGTGGACACGGGTTGCCTGACCTCCACGACTCAGCACGCAAGCACTACCAACGAGACCGGCTCGACGACAC GGCCGTGCTCTACGCGGCCACCCACGTTCTCAACTCCCGGCCACTCGACGACGAAGACGACCCGCGCCGCTG GCTCATGATCGGAACCGACCCAGCAGGCCGCCTACTCGAACTCGTCGCACTGATCTACGACGACGGCTACGAA CTGATCATCCACGCAATGAAAGCCCGCACCCAATACCTCGACCAGCTCTAACCAAGAAAGGAACCTGATGAGCGACCAGCTAGACAGCGACCGCAACTACGACCCGATGATCTTCGACGTGATGCGCGAGACCGCGAACCGCGTCGTCGCCACGTACGTTGCATGGGAAGATGAAGCCGCTGATCCCCGCGAGGCTGCGCACTGGCAGGCCGAGCGATT CCGCACCCGGCACGAGGTGCGCGCCGTCGACCCTGACAGCCGGCGAGCAGTTCAGGCGAAGATCGCACAGCT GCGCGAGGAACTAGCCGCAATGCCCGAACACGCCCCAGCCATCCCTTGGAGCAGGTGGCAGCGTCAGGGGAG TCGGGGGATGTTTGGCAGGGGATGTGGAAAGAGAGTTCGCTTTGCTCACATGGCTCAACCGGGTAACTAACTGA TATGGGGTCTTCGTCGCCCACTTTGAACACGCCGAGGAATGGACCACGCTGAACGTGACTCGCATGCTTCACTGCATGTATGGATTCGTTCGAGACGTTGTTCCCTGAGAGCTGGCTGCCACGCAAGCCGCTGGCGTCAGCCGAGAAGTCTGGGGCGTACCGGCACGTGACTCGGCAGAGGGCGCTGGAGCTGCCTTACATCGAAGCGAACCCGTTGGT CATGCAGTCCTTGGTCATCACCGATCGAGATGCTTCGGATGCTGACTGGGCCGCAGACCTCGCTGGGCTGCCT TCACCGTCCTACGTGTCCATGAACCGTGTCACGACCACCGGACACATCGTCTATGCCTTGAAGAACCCTGTGTG TCTGACCGATGCCGCGCGGCGACGGCCTATCAACCTGCTCGCCCGCGTCGAGCAGGGCCTATGCGACGTTCTC GGCGGCGATGCATCCTACGGGCACCGGATCACAAAGAACCCGCTCAGCACCGCCCATGCGACCCTCTGGGGC CCCGCAGACGCGCTCTACGAGCTGCGCGCCCTCGCACACACCCTCGACGAGATCCACGCACTGCCGGAGGCA GGGAACCCGCGTCGCAACGTCACCCGATCAACGGTCGGCCGCAACGTCACCCTGTTCGACACCACCCGCATGT GGGCATACCGGGCCGTCCGGCACTCCTGGGGCGGCCCGGTCGCCGAATGGGAGCACACCGTATTCGAGCACA TCCACCTACTGAACGAGACGATCATCGCCGACGAATTCGCCACAGGCCCCCTCGGCTTGAACGAACTTAAGCACTTATCTCGATCCATTTCCCGATGGGTCTGGCGCAACTTCACCCCCGAAACCTTCCGCGCACGCCAGAAAGCGATCAGCCTCCGTGGAGCATCCAAAGGCGGCAAAGAAGGCGGCCACAAAGGCGGCATTGCCAGTGGCGCATCACG GCGCGCCCATACCCGTCAACAGTTCTTGGAGGGTCTCTCATGACCACACGTGAACGTCTCCCCCGCAACGGCT ACAGCATCGCCGCTGCTGCGAAAAAGCTCGGTGTCTCCGAGTCCACCGTCAAGCGGTGGACTTCCGAGCCACG CGAGGAGTTCGTGGCCCGCGTTGCCGCACGCCACGCGCGGATTCGTGAGCTgCGCTCGGAGGGTCAGAGCAT GCGTGCGATTGCTGCCGAGGTCGGGGTTTCCGTGGGCACCGTGCACTACGCGCTGAACAAGAATCGAACTGAC GCATGACCGTAACGCCGCACGATGAGCATTTTCTTGATCGTGCACCGCTTGGCACTACGTTCGCGTGCGGTTGC ACAGTGCGCGCCACGTTCTTATCCTGCGGCCATTGTGGCTACAGCCAATGGGGGGCATCAGCAACGGACGTTG AACCCGGTGGGCAAGTGTTACTCAGGGGGACATGCCCAGTCTGCGGCGCTCGGATTGACGGTATGGCAGTCGT GCATGCGGCCCCACCGTCAAACTCATTCAGGTATCAGTGAGAACCCTCATGGCACCCCCTCGTGACACGTTCTCGTTGCGATCAGCTGCGGCCAAGCTTGATCTTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGA GCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTG TTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGC TGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCC CGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGT ATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCA GTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAA ACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACC GCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGT ACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTT CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTAT CGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGG GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTT TCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCAA GCTTGTCGACGTTAACCTGCAGGCATGCGGATCCGGTACCAGCTGTCTCTTATACACATCTCTGCAGTTAACGCT GACCCGGACCACCACGACGACCCGGACGACCACCGGTACGACCACCACCAGACGGACCGCGGCGACCACCCTGGTCACCCTGTTCACGTTCACCGGTACGACCGTCGTCACGGTTACGACCAGAAGAACGACGGTCACCGTGACCAGACGCTTGCCGGTCTCTGCGATCTTGGCCCCCGGTACGTCTGTCACCACGGTCCTGACCACCGGTACGACGG TCGCCCCTATCCTGTCCACCCGTCCTACGACCACGCTGCGGCTGACCACCACGCGGTTCGTTTTTCTGGTCCAG ACGTTCGAACAGACGCAGCCACTGTTCCGGACGAACGTAAGCAACAACAGCACCACCACCAACACCAGCGTGG TCCAGAGCAGCTTCAACACGACGACGCGGGTATTTACGCAGCAGAGAAGCCTGGATGTTACCACCAACACCGG TGAAGCACAGTTCAACCATAGATTCGTAACGTTCCAGAGCAGCACCTTCCAGCAGCGGTTCAGCACGACGACGC AGACGCATGATAGCAGAGTCAACTTTCGGAACCGGTTTGAACAGACGACGATCGACTTTTTCAACGAATTCCCAT TCGAACAGCGGCCAGGTCATAACGGTCAGACGAGACCAACGACCGTAGTCACCGGTACGTTTACGAGCGAATT CCAGCTGGGTAACCATGGTAGCGGTTTCGATGGTCGGAGCTTCCAGGCACCAGTCAACGATAGCAGAGGTGAT ACCGTACGGGATAGCACCAACGAAAGCGAACGGTTCCGGCGGCGGTTCAGCGGTCAGGAAGTCAGCGTTAACAACTTCGATGTTCGGGTGACCAGACAGTTTTTCACGCAGAGATTTAGCCAGACGCGGGTCGATTTCGTAAGAGGTAACCTGACGAGCACGGTCAGCCAGTTCACGGGTCAGCAGACCTTCACCCGGACCAGCTTCCAGAACCGGCAGG TCCGGACGCAGTTCAGCGGTTTCAGCGATACGAGCGATGGTTTTACGGTCACGCAGGAAGTTCTGGCCGAATTG GCGGCGGTTACGGTCACGCTCGGTACGGCCGAGGACGGGGCGATTCTGGTTGGGGTGCTGGCGGTCTTGGTT GCGGCGGCGGGGGCGGGGCTGTTCATCGGAGGAGGACATGACGATCTCCCGATGTGTCGTACGTATGGGAAT TGACGAAAATCATATAGATTTTCAGTTGAAGTGTAGCACTGCATAGTGTCATCGAAGTGTCGGCGGCAACGGTAT GCCCGGTAGGGGGTGGGTCTGCCTTTCGGTTTCGTGAACCTGATGCGGCGACGCGCATGTAGCCGATAACCCC TAGACTCGCCCGTGCAGATCGTCGGTGTGGATCCAGATGTGTATAAGAGACAGCTGGTACCGGCCGCTGCT pEND-274 of SEQ ID NO 14: GTGCGTGCGGGCGTGAGCGTTTCTACGCTGCGGCGCAGGAAATCAGAGCTTGAGGCTGCCGGAGCGACGGTA GACCCGTCCGGTTGGGTGGTGCCACTGCGTGCACTCAAGGTCGTTTTTGGGGTGTCAGATGAGACCTCGAATG CGAATACCAGTAAATCCTGACCTGTGAGTTGTGAATTCATAGGATAGATTCTGGAAACTTTACCGTCCGAGCTCC AGCCTGCGGTCCGGCCTGTCCTGATCATGATCCGTCCGGGGGCCGCTAGTTGGGCGGTCCCCGGACGATTTTG TGTACCCGCAGATGCGTGCCAGACAAGCGCCTCGGCTGTTTTGTGGGTTTGTGTTGGTGGTGGGGGTGTGTGT AGTGTGTGTTGGGCCGTTGGGGGCTGCGTGTGTGGTTCTTGGTGGTGGCTGTGTTTGGGATGTCGCTGGGACG CCCGTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTGTTTAACTTTAAGAAGGAG ATATACATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAAGTGCCGATGA ACCTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCATACGGTCTGTT GTTCACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGGCAACTGCGCC CCGGCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAAGAATACTGGCA AGCACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCCATGACGGCAAA GCCAACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCTcATGCTcCACGC tGGtGGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCTGCGGCATTATCC AGCATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCGCTCGAGCAATAAACAGTTGATAGGGCTTCTCCGT TACCATGGTTCAGCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATC GGCGGTTTTCTTTTCTCTTCTCAAGCTAGCGAAATCCCTGAAACTGAGACTGTAGAAAATAAGCTTCCGAAACTG AACCTGTTATTTTACGCTACGGAcgCCCGGTCATGACGCTGAGTTAGTGGCGCAGCTGCGCTCTGAGAACGAGT TTTTACGGCGTCAGGTCGAGCAGCAGGCGCGCACGATCGAACGGCAGGCTGAGGCACACGCGGTGGTCTCAG CGCAGCTCACACGGGTTGGCCAGCTTGAGGCCGGCGACGCAGCAGCACCGACACTGGCACCCGTTGAAAGGC CGGCTCCGCGACGGCGGTGGTGGCAGCGTCGGTAGCGGTCAGGATCGCTCTGGCGTGACGAGTGTGTCTGGC AGTGCGAACAGTTGCTCGACCAGTGGCAGCAGAAGCGAGATCGCTGCGTGGTGCTGTTCCTCGGTCAGTTCGT CGAGGACTGGCGGGTCTTGCTGCGTCCAGCCGATCGCCTCGGCGGCCAAGGTCAGTTCCAAGCTGTGCCAAC GCACACGCCCCTCGGCTGACAGCTGAGTCTCGAACTGTGCAACTGGACCGGCCGGAAGATGCACGTTGCCGAG GTCGTGAGTGGCCAAGCGCACGTCAAAGAGTGCTGCTTCGTAGCCGCGCAGAAATGGCAGTGCTCGGTCGATT CGGATCGGCCTGCCCAGGTACATTCCGGGCCGCTTGATGAACGCCTCCGCGTAGAAGCGCACCGTTCTCGGCCCGGCCTCGTGATCTGTCACTGTGCACGCTCCTCTCGATGGTTCTCGACGCTACCGGAGACCACCGACGTTCATGCCCAGCGCAGCGACCTGAAAGGACCAAGCCGAGTTAGCCGTGCTAACCGTATAGCTTGCTCCGTCGCCTCTGA GGGCAACCACCTGCGCAGCAGGTGGGCGGCAGCCCGCGCGCAAGCGCCTACCGGGTTTGGGCACAGCCCATA AATCAACGCCTCCGGTGTTGAAGCGATCGTGTGTCACGATTGCTATGCTTGCTACCCCTTCAGGGTTTTCGTATA CACAAATCAAGTTTTTTCGTATACGCTAATGCCATGAGTGAGCATCTACTGCACGGCAAGCCCGTCACCAACGAG CAGATTCAGGCATGGGCAGACGAGGCCGAGGCCGGATACGACCTGCCCAAACTCCCCAAGCCACGGCGCGGA CGCCCGCCCGTAGGAGACGGTCCGGGCACCGTCGTACCCGTGCGTCTCGACGCGGCCACCGTTGCCGCTCTC ACAGAACGAGCAACAGCCGAGGGCATCACGAACCGTTCAGACGCGATCCGAGCCGCAGTCCACGAGTGGACA CGGGTTGCCTGACCTCCACGACTCAGCACGCAAGCACTACCAACGAGACCGGCTCGACGACACGGCCGTGCTC TACGCGGCCACCCACGTTCTCAACTCCCGGCCACTCGACGACGAAGACGACCCGCGCCGCTGGCTCATGATCGGAACCGACCCAGCAGGCCGCCTACTCGAACTCGTCGCACTGATCTACGACGACGGCTACGAACTGATCATCCACGCAATGAAAGCCCGCACCCAATACCTCGACCAGCTCTAACCAAGAAAGGAACCTGATGAGCGACCAGCTAGAC AGCGACCGCAACTACGACCCGATGATCTTCGACGTGATGCGCGAGACCGCGAACCGCGTCGTCGCCACGTACG TTGCATGGGAAGATGAAGCCGCTGATCCCCGCGAGGCTGCGCACTGGCAGGCCGAGCGATTCCGCACCCGGC ACGAGGTGCGCGCCGTCGACCCTGACAGCCGGCGAGCAGTTCAGGCGAAGATCGCACAGCTGCGCGAGGAAC TAGCCGCAATGCCCGAACACGCCCCAGCCATCCCTTGGAGCAGGTGGCAGCGTCAGGGGAGTCGGGGGATGTTTGGCAGGGGATGTGGAAAGAGAGTTCGCTTTGCTCACATGGCTCAACCGGGTAACTAACTGATATGGGGTCTTCGTCGCCCACTTTGAACACGCCGAGGAATGGACCACGCTGAACGTGACTCGCATGCTTCACTGCATGTATGGAT TCGTTCGAGACGTTGTTCCCTGAGAGCTGGCTGCCACGCAAGCCGCTGGCGTCAGCCGAGAAGTCTGGGGCGT ACCGGCACGTGACTCGGCAGAGGGCGCTGGAGCTGCCTTACATCGAAGCGAACCCGTTGGTCATGCAGTCCTT GGTCATCACCGATCGAGATGCTTCGGATGCTGACTGGGCCGCAGACCTCGCTGGGCTGCCTTCACCGTCCTAC GTGTCCATGAACCGTGTCACGACCACCGGACACATCGTCTATGCCTTGAAGAACCCTGTGTGTCTGACCGATGC CGCGCGGCGACGGCCTATCAACCTGCTCGCCCGCGTCGAGCAGGGCCTATGCGACGTTCTCGGCGGCGATGC ATCCTACGGGCACCGGATCACAAAGAACCCGCTCAGCACCGCCCATGCGACCCTCTGGGGCCCCGCAGACGC GCTCTACGAGCTGCGCGCCCTCGCACACACCCTCGACGAGATCCACGCACTGCCGGAGGCAGGGAACCCGCG TCGCAACGTCACCCGATCAACGGTCGGCCGCAACGTCACCCTGTTCGACACCACCCGCATGTGGGCATACCGGGCCGTCCGGCACTCCTGGGGCGGCCCGGTCGCCGAATGGGAGCACACCGTATTCGAGCACATCCACCTACTGAACGAGACGATCATCGCCGACGAATTCGCCACAGGCCCCCTCGGCTTGAACGAACTTAAGCACTTATCTCGATC CATTTCCCGATGGGTCTGGCGCAACTTCACCCCCGAAACCTTCCGCGCACGCCAGAAAGCGATCAGCCTCCGT GGAGCATCCAAAGGCGGCAAAGAAGGCGGCCACAAAGGCGGCATTGCCAGTGGCGCATCACGGCGCGCCCAT ACCCGTCAACAGTTCTTGGAGGGTCTCTCATGACCACACGTGAACGTCTCCCCCGCAACGGCTACAGCATCGCC GCTGCTGCGAAAAAGCTCGGTGTCTCCGAGTCCACCGTCAAGCGGTGGACTTCCGAGCCACGCGAGGAGTTCG TGGCCCGCGTTGCCGCACGCCACGCGCGGATTCGTGAGCTgCGCTCGGAGGGTCAGAGCATGCGTGCGATTG CTGCCGAGGTCGGGGTTTCCGTGGGCACCGTGCACTACGCGCTGAACAAGAATCGAACTGACGCATGACCGTA ACGCCGCACGATGAGCATTTTCTTGATCGTGCACCGCTTGGCACTACGTTCGCGTGCGGTTGCACAGTGCGCG CCACGTTCTTATCCTGCGGCCATTGTGGCTACAGCCAATGGGGGGCATCAGCAACGGACGTTGAACCCGGTGGGCAAGTGTTACTCAGGGGGACATGCCCAGTCTGCGGCGCTCGGATTGACGGTATGGCAGTCGTGCATGCGGCCCCACCGTCAAACTCATTCAGGTATCAGTGAGAACCCTCATGGCACCCCCTCGTGACACGTTCTCGTTGCGATCA GCTGCGGCCAAGCTTGATCTTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTT GAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGA AGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAG TCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCT GTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCC ATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCA TCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAAC AGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAA GCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCAT GACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGT GCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCT CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAG TCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAG TTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAA TCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCG CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTAT TATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCAAGCTTGTCGACGTTAACCTGCAGGCATGCGGATCCGGTACCAGCTGTCTCTTATACACATCTCTGCAGTTAACGCTGACCCGGACCACCACGACGACCCGGACGACCACCGGTACGACCACCACCAGACGGACCGCGGCGACCACCCTGGTCACCCTGTT CACGTTCACCGGTACGACCGTCGTCACGGTTACGACCAGAAGAACGACGGTCACCGTGACCAGACGCTTGCCG GTCTCTGCGATCTTGGCCCCCGGTACGTCTGTCACCACGGTCCTGACCACCGGTACGACGGTCGCCCCTATCC TGTCCACCCGTCCTACGACCACGCTGCGGCTGACCACCACGCGGTTCGTTTTTCTGGTCCAGACGTTCGAACAG ACGCAGCCACTGTTCCGGACGAACGTAAGCAACAACAGCACCACCACCAACACCAGCGTGGTCCAGAGCAGCT TCAACACGACGACGCGGGTATTTACGCAGCAGAGAAGCCTGGATGTTACCACCAACACCGGTGAAGCACAGTTC AACCATAGATTCGTAACGTTCCAGAGCAGCACCTTCCAGCAGCGGTTCAGCACGACGACGCAGACGCATGATAG CAGAGTCAACTTTCGGAACCGGTTTGAACAGACGACGATCGACTTTTTCAACGAATTCCCATTCGAACAGCGGC CAGGTCATAACGGTCAGACGAGACCAACGACCGTAGTCACCGGTACGTTTACGAGCGAATTCCAGCTGGGTAACCATGGTAGCGGTTTCGATGGTCGGAGCTTCCAGGCACCAGTCAACGATAGCAGAGGTGATACCGTACGGGATAGCACCAACGAAAGCGAACGGTTCCGGCGGCGGTTCAGCGGTCAGGAAGTCAGCGTTAACAACTTCGATGTTCG GGTGACCAGACAGTTTTTCACGCAGAGATTTAGCCAGACGCGGGTCGATTTCGTAAGAGGTAACCTGACGAGCA CGGTCAGCCAGTTCACGGGTCAGCAGACCTTCACCCGGACCAGCTTCCAGAACCGGCAGGTCCGGACGCAGTT CAGCGGTTTCAGCGATACGAGCGATGGTTTTACGGTCACGCAGGAAGTTCTGGCCGAATTGGCGGCGGTTACG GTCACGCTCGGTACGGCCGAGGACGGGGCGATTCTGGTTGGGGTGCTGGCGGTCTTGGTTGCGGCGGCGGG GGCGGGGCTGTTCATCGGAGGAGGACATGACGATCTCCCGATGTGTCGTACGTATGGGAATTGACGAAAATCAT ATAGATTTTCAGTTGAAGTGTAGCACTGCATAGTGTCATCGAAGTGTCGGCGGCAACGGTATGCCCGGTAGGGG GTGGGTCTGCCTTTCGGTTTCGTGAACCTGATGCGGCGACGCGCATGTAGCCGATAACCCCTAGACTCGCCCG TGCAGATCGTCGGTGTGGATCCAGATGTGTATAAGAGACAGCTGGTACCGGCCGCTGCT pEND-387 of SEQ ID NO 17: tctagaGGAAATCAGAGCTTGAGGCTGCCGGAGCGACGGTAGACCCGTCCGGTTGGGTGGTGCCACTGCGTGCA CTCAAGGTCGTTTTTGGGGTGTCAGATGtGACCTCGAATGCGGAATTCATAGGATAGATTCTGGAAACTTTACCG TCCGAGCTCCAGCCTGCGGTCCGGTTGACACCCTATCAGTGATAGAGTATAATGCTAGCTCGCTGGGACGCCC GTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCGGTAATTTTGTTTAACTTTAAGAAGGAGATA TACATatgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgccGAGCAGGAAGTGCCGATGAAC CTGGTCTCGGCTGATGGCAAGGAGGTTTCCATCGGGAAGATCACCATCCAGGAGACCCCATACGGTCTGTTGTT CACCCCCGCCCTGCACTCGCTGAGCGAGGGTATTCACGGTTTCCACGTCCACGAGAAGGGCAACTGCGCCCCG GCGCTTAAAGACGGCAAGCCGGTCGCCGCGCTGTCCGCCGGTGGTCACTTTGACCCGAAGAATACTGGCAAGC ACCTGGGCCCGTGGTCCCCTGACGGCCACcTcGGtGACCTGCCGGCCCTGTTCGTCACCCATGACGGCAAAGCC AACTACCCGGTCCTGGCCCCGCGCCTCAACTCCCTGAAGGAGATCAAGGGCCGCTCcCTcATGCTcCACGCtGGt GGCGACAATCACCACGACCACCCAGAGCCCCTGGGCGGTGGCGGtGCCCGCATGGCCTGCGGCATTATCCAGC ATCATCACCATCACCACTGAtaaTCGCTGGGACGCCCGCTCGAGCAATAAACAGTTGATAGGGCTTCTCCGTTAC CATGGTTCAGCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGGC GGTTTTCTTTTCTCTTCTCAATTCTTCTGACCTGTAACGAATAATAGATAGTAAAGTAGTCTCCGATTGAGTTTTCT CTGCCGAGTCCCACCCAGTTCTGTGATTTCAGTAAGTTGGTAATTGATACACTGTTGCGAGAACTGgTGCCTGGT AGTAGATAGGTTGTTATTGAGTAAGAAGGTAAAGTGAACGAAATCCCTGAAACTGAGACTGTAGAAAATAAGCTT GTCCAGACTATTGGATCCAAGAGATTTCTACACGATTGAGCACTGTCTCCTGCAGGCTCGGTACCAAATTCCAGA AAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCTAATAGATAAAGGATAGGTCTGGTAGTGTTGTT CGTTCTCGCAGGTAAATCAATAATACTCtGCAGTTCCGTAGACTTTTCAGTGGGACAGGGTAGCGATAACAGATA GATTGTAATAAGACACAGTAGGTGCTCGTAGTTGCGTGAAGAGAACCGCTCAGGAAATCCAGTCAGAAGTATTG GTAATCGTTGAAAACTCAGTCGACCAGCCTGCGGTCCGGacaccgacgatctgcacgggcgagtctaggggttatcggctacatgcgcg tcgccgcatcaggttcacgaaaccgaaaggcagacccaccccctaccgggcataccgttgccgccgacacttcgatgacactatgcagtgctacacttcaactgaa aatctatatgattttcgtcaattcTCGCTGGGACGCCCGTAGAACTCTGTGAGGATAAAGTCTCCCTTACAGCCTGCGGTCCG GTAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCAAGACTAGATAAGAGCAAGGTCATTAACAGCGCACTGGAGTTACTAAACGAAGTTGGAATAGAGGGATTGACTACACGAAAATTAGCACAGAAATTAGGAGTAGAACAACCGACACTTTATTGGCACGTTAAAAATAAAAGAGCTTTGCTGGATGCTTTAGCTATAGAAATGCTAGACCGACACCA CACACATTTTTGCCCCTTAGAAGGAGAAAGTTGGCAAGATTTCCTACGTAACAACGCCAAATCATTTAGATGCGC GCTATTATCACACCGCGACGGCGCCAAAGTTCACCTCGGGACAAGACCTACGGAGAAGCAATACGAAACCCTAG AGAATCAATTAGCCTTTCTATGTCAACAAGGATTCAGTTTGGAAAACGCCCTCTACGCCCTATCAGCCGTTGGTC ATTTTACATTGGGATGCGTTTTAGAGGATCAGGAACATCAAGTAGCAAAAGAAGAAAGAGAGACACCCACCACC GACAGCATGCCACCATTATTACGTCAAGCTATTGAACTATTCGATCATCAGGGAGCGGAACCGGCATTTTTATTT GGCCTCGAATTAATCATATGTGGTTTAGAAAAACAATTAAAGTGTGAATCTGGATCCCATCATCACCATCACCACT GAtaaTCGCTGGGACGCCCGGACGTCCTATTACACTCGTCGTTGGAAACTGAAGATGCGGCCGCggaaacacagAAA AAAGCCCGCACCTGACAGTGCGGGCTTTTTTTTTcgaccaaaggTAGCGAACGACGAGTCACTGTTGAGGATAAATACTTTCTCTACTAGCTAGCTGTTACACAGGTCCTCAGCGGCGCGCCcgCCCGGTCATGACGCTGAGTTAGTGGCGCAGCTGCGCTCTGAGAACGAGTTTCGACCCTGACAGCCGGCGAGCtGTTCAGGCGAAGATCGCACAGCTGCGC GAGGAACTAGCCGCAATGCCCGAACACGCCCCAGCCATCCCTTGGAGCAGGTGGCAGCGTCAGGGGAGTCGG GGGATGTTTGGCAGGGGATGTGGAAAGAGAGTTCGCTTTGCTCACATGGCTCAACCGGGTAACTAACTGATATG GGcTCTTCGTCGCCCACTTTGAACACGCCGAGGAATGGACCACGCTGAACGTGACTCGCATGCTTCACTGCATG TATGGATTCGTTCGAGACtTTGTTCCCTGAGAGCTGGCTGCCACGCAAGCCGCTGGCGTCAGCCGAGAAGTCTGGGGCGTACCGGCACGTGACTCGGCAGAGGGCGCTGGAGCTGCCTTACATCGAAGCGAACCCGTTGGTCATGCAGTCCTTGGTCATCACCGATCGAGATGCTTCGGATGCTGACTGGGCCGCAGACCTCGCTGGGCTGCCTTCACC GTCCTACGTGTCCATGAACCGTGTCACGACCACCGGACACATCGTCTATGCCTTGAAGAACCCTGTGTGTCTGA CCGATGCCGCGCGGCGACGGCCTATCAACCTGCTCGCCCGCGTCGAGCAGGGCCTATGCGACGTTCTCGGCG GCGATGCATCCTACGGGCACCGGATCACAAAGAACCCGCTCAGCACCGCCCATGCGACCCTCTGGGGCCCCG CAGACGCGCTCTACGAGCTGCGCGCCCTCGCACACACCCTCGACGAGATCCACGCACTGCCGGAGGCAGGGA ACCCGCGTCGCAACGTCACCCGATCAACGGTCGGCCGCAACGTCACCCTGTTCGACACCACCCGCATGTGGGC ATACCGGGCCGTCCGGCACTCCTGGGGCGGCCCGGTCGCCGAATGGGAGCACACCGTATTCGAGCACATCCA CCTACTGAACGAaACGATCATCGCCGACGAgTTCGCCACAGGCCCCCTCGGCTTGAACGAACTTAAGCACTTAT CTCGATCCATTTCCCGATGGGTCTGGCGCAACTTCACCCCCGAAACCTTCCGCGCACGCCAGAAAGCGATCAGCCTCCGTGGAGCATCCAAAGGCGGCAAAGAAGGCGGCCACAAAGGCGGCATTGCCAGTGGCGCATCACGGCGtGCCCATACCCGTCAACAGTTCTTGGAGGGTCTgTCATGACCACACGTGAACGcCTCCCCCGCAACGGCTACAGC ATCGCCGCcGCTGCGAAAAAGCTCGGTGTCTCCGAGTCCACCGTCAAGCGGTGGACTTCCGAGCCACGCGAGG AGTTCGTGGCCCGCGTTGCCGCACGCCACGCGCGGATTCGTGAGCTgCGCTCGGAGGGTCAGAGCATGCGTG CGATTGCTGCCGAGGTCGGGGTTTCCGTGGGCACCGTGCACTACGCGCTGAACAAGAATCGAACTGACGCATG ACCGTAACGCCGCACGATGAGCATTTTCTTGATCGTGCACCGCTTGGCACTACGTTCGCGTGCGGTTGCACAGT GCGCGCCACGTTCTTATCCTGCGGCCATTGTGGCTACAGCCAATGGGGGGCATCAGCAACGGACGTTGAACCC GGTGGGCAAGTGTTACTCAGGGGGACATGCCCAGTCTGCGGCGCTCGGATTGACGGTATGGCAGTCGTGCATG CGGCCCCACCGTCAAACTCATTCAGGTATCAGTGAGAACCCTCATGGCACCCCCTCGTGACACGTTCTCGTTGC GATCAGCTGCGGCCAAGCTGATCTTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACtgatcaGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGgACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTT TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGGCGCCTCACCAaTGCTTGATgAGGGAGGCACCGATCTCGGCGATCTGACGGTTGCGCTCATCCATCGTGGCCTGGGAGCCGGTCGTGTAGATCACCACGATGCGGCTGGGCTTACCGTCgGGACCgAGAGCCGCGATGATGCCACGCGAGCCGCGCTCACC AGCACCCGACTTGTCCGCGATGAACCAACCGGCCGGGAGCGCGCTGCGCAACAGCGGGCCAGCGACCTTGTC GGCCTCCATCCAGTCGATCAGTTGCTGACGGGAGGCCAGAGTGAGGAGTTCACCGGTCAGGAGTTTACGGAGG GTGGTAGCCATAGCCGCGGGCATAGTCGTGTCGCGCTCGTCgTTcGGgATAGCCTCATTAAGTTCGGGTTCCCA ACGGTCCAGACGGGTCACGTGGTCGCCCATGTTGTGGAGAAAAGCCGTCAGCTCTTTCGGGCCACCGATGGTC GTCAACAGCAGATTGGCGGCAGTGTTGTCGGACATAGTGATGGCCGCGGAGCACAGCTCGCGCACGGTCATCC CATCGGTCAGATGTTTCTCCGTGACCGGGGAGTATTCGACCAGGTCGTTCTGGGAGTAATGAATGCGACGGCCC AGTTGCTCCTGCCCAGCATCCACCCGGCTAAGCACAGCGCCGCAGAGGAGGACCTTGAACGTGGACATCATGG GGAAACGCTCCTCAGGGCGGAAAGACTCCAGAATCTTACCGGAGTTCAGGTCCAACTCGATATAACCGACGCG CGCCCCCAGCTGATCCTCGGCGTCCTTGACCTTGACCAGGGTtTCCGGATGGGCAAAAACCGGCAAGCAAAAG GCCGCAAAAAATGGGATAAGGGCAACGCGGAAGTGCTGGATcGACATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGactagtTCCCCGAAAAGTGCCACCTGACGTTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTAT CACGAGGCCCTTTCGTCTaCAATCAGCGCTGACCCGGACCGCCACGACGGCCTGGACGACCGCCGGTACGACC ACCACCCGAAGGGCCACGACGACCACCCTGGTCGCCCTGCTCGCGCTCCCCGGTACGACCATCGTCGCGATTA CGGCCAGAGGACCGACGGTCACCGTGGCCGGAGGCCTGACGGTCGCGACGGTCCTGACCACCCGTCCGACG GTCGCCACGATCCTGGCCACCCGTACGGCGGTCACCACGATCCTGACCACCCGTACGACGGCCGCGCTGCGG TTGGCCACCGCGCGGCTCGTTCTTCTGGTCAAGGCGCTCGAAGAGCCGCAGCCATTGCTCCGGCCGGACGTAG GCCACGACAGCACCACCACCCACGCCGGCATGGTCCAGCGCAGCCTCCACGCGaCGaCGaGGGTACTTGCGCA GGAGGGAGGCTTGAATGTTGCCGCCGACACCAGTAAAACACAACTCCACCATGCTCTCGTAGCGCTCAAGGGC AGCACCCTCCAACAGCGGTTCAGCGCGGCGACGCAGACGCATGATGGCGGAATCGACCTTCGGGACCGGCTTGAACAAGCGGCGATCGACCTTCTCGACGAACTCCCACTCGAAGAGCGGCCAAGTCATGACCGTGAGGCGGGACCAGCGGCCATAATCACCGGTGCGCTTACGGGCAAATTCGAGTTGGGTAACCATCGTGGCGGTtTCGATCGTCGG AGCCTCCAGGCACCAATCGACGATCGCAGACGTGATGCCGTACGGGATCGCGCCGACGAAGGCAAACGGCTC GGGAGGGGGCTCGGCGGTCAGGAAGTCCGCGTTGACGACCTCGATATTCGGGTGGCCGGACAGCTTCTCACG CAGGGACTTCGCCAAGCGCGGGTCGATCTCGTAGCTCGTCACCTGGCGGGCCCGATCGGCAAGCTCACGGGT CAGCAGACCCTCGCCTGGGCCAGCCTCCAGGACCGGAAGGTCGGGGCGCAGTTCGGCCGTTTCCGCGATGCG GGCGATCGTCTTACGGTCACGAAGGAAATTCTGACCgAAtTGaCGGCGGTTGCGGTCCCGCTCAGTACGGCCCA GGACCGGGCGGTTCTGGTTCGGGTGCTGGCGGTCCTGGTTGCGGCGACGCGGGCGCGGTTGCTCGTCGGAC GACGACATGACGATCTCCCGATGTGTCGTACGTATGGGAATTGACGAAAATCATATAGATTTTCAGTTGAAGTGT AGCACTGCATAGTGTCATCGAAGTGTCGGCGGCAACGGTATGCCCGGTAGGGGGTGGGTCTGCCTTTCGGTTTCGTGAACCTGATGCGGCGACGCGCATGTAGCCGATAACCCCTAGACTCGCCCGTGCAGATCGTCGGTGTA second aspect of the invention refers to the composition as defined in the first aspect of the invention or in any of its preferred embodiments, for use in therapy. A third aspect of the invention refers to a pharmaceutical composition comprising the composition as defined in the first aspect of the invention or in any of its preferred embodiments, optionally further comprising pharmaceutically acceptable excipients and / or carriers. A fourth aspect of the invention refers to the composition as defined in the first or third aspect of the invention or in any of its preferred embodiments, for use in delivering the heterologous protein in a subject, preferably a human subject, in need thereof. Preferably for use as an antioxidant-producing strain able to rescue keratinocytes from oxidative stress in a subject, preferably a human subject, in need thereof. A fifth aspect of the invention refers to a non-therapeutic composition, such as a cosmetic composition, comprising the composition as defined in as defined in the first aspect of the invention or in any of its preferred embodiments. A sixth aspect of the invention refers to the use of the composition of the fifth aspect of the invention for delivering the heterologous protein in a subject, preferably a human subject, in need thereof. EXAMPLES Methods Strains and general growth conditions C. acnes KPA171202 (DSM16379) was obtained from the German Collection of Microorganisms and CellCultures (GmbH). All C. acnes strains were grown in Brucella agar plates (#1012, Condalab) at 37ºC inanaerobic conditions generated either with the GasPak EZ anaerobe pouch system (#BD260683, BD) or the AnaeroGen System (AN0025A, Thermo Scientific). For liquid cultures, a starting culture of 0.1 OD600 was inoculated in brain-heart infusion (BHI) media (#1400, Condalab) and grown at 37ºC 110 r.p.m. in anaerobic conditions. When appropriate, plates or liquid cultures were supplemented with either 5 µg mL-1chloramphenicol or 10 or 50 µg mL-1erythromycin. For measuring promoter and RBS strength in stationary phase, cultures were grown for 4 days. For the synthetic sensors, liquid cultures were incubated in the presence (or absence) of 100 ng mL-1 aTC or 5 µM DAPG for 3.5 or 5 days, respectively.For the experiments involving auxotrophies, C. acnes was grown using defined synthetic media based onDMEM F-12 medium lacking amino acids and glucose (D9807-02A, US Biological) supplemented with 3.151g / L glucose and 1.2g / L sodium bicarbonate. The pH was adjusted to 6 with HCl and the mediasterilized with a 0.2 µm filter. All amino acids were autoclaved and added unless indicated. C. acnes cultureswere inoculated as previously mentioned with a starting OD600 of 0.1. and grown in anaerobic conditions. Agarose 1.5% was added to the media when grown in solid in plates. To evaluate the auxotrophies, all aminoacids were added to the basal media except for the one being tested. For instance, to evaluate histidine auxotrophy, the basal medium was supplemented with all amino acids except histidine and growth was measured both in the absence and presence of histidine. For plasmid curation, cells were passaged twice by replating them in Brucella agar plates without the selecting antibiotic. Cells were then resuspended in BHI and serial dilutions were performed in order to obtain single colonies after plating. Plates with individual colonies were replica-plated in a Brucella agar plate with the appropriate antibiotic to identify colonies that had lost the plasmid. Plasmids and molecular biologyAll plasmids in this study were based on the Propionibacterium freudenreichii replicative vectorpBRESP36A32,33or on a pUC-19 based suicide vector for homologous recombination59. PCR fragments were usually amplified with either KAPA HiFi (KK2601, Roche) or Phanta Max (#001, Vazyme) and purified using QIAquick PCR purification or gel extraction kits (#28104 / #28704, Qiagen). Plasmids were built using a modular Gibson-based cloning method34. The method consists of two steps: step 1 relies on Gibson assembly of transcriptional units into individual intermediate plasmids; in step 2, these plasmids are digested, which generates flanking regions containing overlaps that drive a second Gibson assembly, thus yielding the final construct. For step 1, all DNA parts were flanked by the same Prefix (CAGCCTGCGGTCCGG) and Suffix (TCGCTGGGACGCCCG) sequences34. Basically, For and Rev primers annealing to Prefix and Suffix sequences, respectively, were used for PCRs that added unique linkers to the DNA parts (Table S2, Cloning primers). PCR amplifications were column-purified as specified above and assembled using a custom Gibson enzyme mix (Centre de Regulación Genomica CRG, 1 h 50 ºC) into backbones previously digested with the appropriate restriction enzymes (NEB, 1 h 37 °C) to generate the intermediate plasmids. In step 2, these intermediate plasmids were digested with enzyme combinations that produced overlapping ends, purified, and assembled as described above to yield the finalconstructs. Plasmids were transformed into chemically competent E. coli DH5α (MB00402, NZYtech) andplated in LB agar plates with the appropriate antibiotic (50 µg mL-1Ampicillin, 50 µg mL-1Kanamycin, 50 µg mL-1Spectinomycin or 25 µg mL-1Chloramphenicol). Liquid cultures were made in LB and incubated at37ºC 220 r.p.m. for plasmid extraction using the NZYMiniprep kit (MB01008, NZYtech). C. acnes plasmidswere first shuttled through a Δdam Δdcm ΔhsdMS E. coli strain harbouring the C. acnes IIIB methylase foravoiding R-M system degradation upon transformation. Plasmids shuttled through this methylation strainwere extracted either through the NZYMiniprep kit, the Miraprep protocol60 or the PureLink™ Maxiprep kit(K210017, Invitrogen) to obtain enough DNA mass for electroporation. When necessary, plasmids were washed with the Amicon®ultra 0.5mL 30K centrifugal filters (UFC503096, Millipore) according to manufacturer’s instructions to avoid arcing during the electroporation.C. acnes electrocompetent cell preparation and transformationC. acnes electrocompetent cells were prepared as previously described28. Briefly, C. acnes startinginoculum of 0.1 OD600 were grown for 24h until ~1 OD600. Then, cells were treated with 0.4M Sucrose and 10 µg mL-1penicillin G for 5h. Afterwards, cells were centrifuged at 2367 r.c.f. for 10 minutes at 4ºC and resuspended in electroporation buffer consisting of 272 mM sucrose, following a second centrifugation with the same conditions. Cells were resuspended in 1 mL and transferred to a 1.5 mL microcentrifuge tube where they were washed at 1657 r.c.f. for 10 minutes followed by 4 washes at 9391 r.c.f. for 1 minute each. After the last wash, cells were resuspended in electroporation buffer supplemented with 10% glycerol, washed once again at 9391 r.c.f. for 1 minute and resuspended in 50ul of electroporation buffer with 10% glycerol per 50 mL of starting culture volume. Finally, cells were frozen in liquid nitrogen and stored at - 75ºC for later use. For transformation, a cell aliquot was slowly thawed on ice, resuspended in 1 mL electroporation buffer and centrifuged at 9391 r.c.f. for 1 minute. After resuspension in the same initial volume with electroporation buffer, cells were diluted four times with electroporation buffer to a final volume of 40ul and either 1000ng of DNA were added for replicative plasmid transformation or 8000ng for the suicide vectors. For electroporation, the mixture was transferred to a precooled 1 mm electroporation cuvette (#1652089, Biorad) and electroporated at 1.5 kV, 25 µF and 400 Ω. Immediately after the pulse, cells were resuspended in 100 µL of BHI medium and plated on Brucella agar plates for 24h recovery at 37ºC in anaerobic conditions. Next day, cells were resuspended in 1 mL BHI, centrifuged at 1657 r.c.f. for 5 minutes, resuspended in 100 µL BHI and plated in 2 Brucella agar plates supplemented with the appropriate antibiotic for a 6-day incubation in anaerobic conditions at 37ºC. Flow cytometryC. acnes cells were analysed by flow cytometry on a LSR Fortessa 4L analyzer (BD) with a 488nm laserand 530 / 30 band pass filter for sfGFP, mCitrine and pFAST-Lime, a 405nm laser with a 450 / 50 band pass filter for Cerulean, and a 561nm laser with either a 610 / 20 band pass filter for EforCP and mCherry or a 585 / 15 for mKO2. Cells were centrifuged at 1657 r.c.f for 5 minutes and resuspended in PBS to ensure separation of cell events. FSC-H and SSC-H thresholds were set to exclude background events. Data wasanalysed using either FlowJo software (Treestar) or a custom R script using the flowCore package61. Thefluorescence median of each gated population was calculated and reported in this publication as the fluorescence value of a sample in arbitrary units (a.u.). Immunodetection of secreted SODLiquid cultures of recombinant C. acnes were grown in selective media for two days. Bacteria were collectedby centrifugation and the pellet (cell fraction, C) was resuspended in Bolt LDS Sample buffer (Thermo Fisher Scientific) containing 5% beta-mercaptoethanol. The supernatant (SN) containing the secreted SOD was subjected to trichloroacetic acid (TCA) precipitation: samples were precipitated in 10% trichloroacetic acid for 30 min on ice, and precipitates were pelleted by centrifugation at ~10,000g for 15 min at 4 °C. The pellets were washed twice with 500ul cold (−20 °C) acetone, air-dried, and resuspended in Bolt LDS Samplebuffer with 5% beta-mercaptoethanol. Cell and supernatant samples were boiled at 98ºC for 10 min, andequal amounts of C and SN fractions were analysed using SDS-PAGE and His-tag immunodetection.Briefly, a NuPAGE 4-12% gel (Thermo Fisher Scientific) was run at 120 V for 1 h 45 min, and proteins weretransferred onto a PVDF membrane (Immobilon-P Transfer Membrane, Merck Millipore) using a wet blotting apparatus running at 20 V for 1 h. Membranes were blocked overnight with 4% milk in TBST (Tris buffered saline (Bio-Rad 1706435) containing 0.05% Tween 80) and incubated for 1 h with mouse anti-His antibody (Bio-Rad MCA1396GA) diluted 1:800 in TBST-4% milk. Following three 10 min washes in TBST, membranes were incubated for 1 h in horseradish peroxidase-coupled anti-mouse antibody (SantaCruz sc516102) diluted 1:800 in TBST-4% milk, and washed again three times 10 min each in TBST. Membranes were developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and recorded using a ChemiDoc MP Imaging System (Bio-Rad). N / TERT-2G Cell culture, UV-exposure and AD monolayer model The immortalized keratinocyte cell line N / TERT-2G55,56was obtained from the J. Rheinwald laboratory (Harvard Medical School, Boston, USA). These cells were passaged in T25 or T75 flasks in keratinocyte- serum free EpiLife medium (#MEPI500CA, Gibco) with human keratinocyte culture supplement (HKGS) (#S0015, Gibco) based on previously published protocols62in a humidified 5% CO2 incubator. Alternatively, CnT-Prime (CNT-PR, CELLnTEC) media was also used. For UV-exposure, cells were seeded in 3.6 x 104cells mL-1in 24 well plates. When confluent, cells were exposed for 30 minutes to four UV-B lamps (302 nm) (Gel Dock XR+, Biorad). Then, cells were washed with Hanks’ balanced salt solution (HBSS) and dyed for 30 minutes at 37ºC with 20 µM 6-chloromethyl- 2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (C6827, Invitrogen). After washing again with HBSS, we applied the 0.2 µm filtered bacterial supernatant for 5h on the cells, and we measured the ROS levels using a M Nano Infitine 200 Pro plate reader (Tecan) at an excitation / emission of 485 / 535nm. SOD activity quantification 0.2 µm-filtered C. acnes supernatants were transferred to a 96-well plate to measure the SOD activity using an SOD activity assay kit (#CS0009, Sigma) following manufacturer’s instructions. Briefly, 20 µL from supernatants were mixed with 20 µL of the WST dye, and 160 µL of xanthine oxidase were added to startthe reaction. The SOD activity was calculated using a standard calibration curve. First, the absorbance at450nm from each sample was averaged across replicates. To obtain the linearized SOD rate, a blank control was subtracted from all samples and standards, and resulting values were divided by the absorbance of the control reaction lacking SOD. The standards were then used for a linear regression to obtain the calibration parameters as the slope and intercept. Then the following formula was applied. Where SampleLSR is the linearized SOD rate of the sample and the multiplication by 10 corresponds to the dilution factor. A calibration curve was only considered valid when its R2was higher than 0.95. RNA extraction Total RNA was isolated by E.Z.N.A. Total RNA kit (#R6834-02, Omega bio-tek). For bacterial RNA isolation, cultures were centrifuged at 8000 r.c.f. for 5 minutes and resuspended in 1 mL TRK Lysis buffer. Then, cells were lysed using Precellys®0.1 mm silica beads (432-3754, VWR) for 15 minutes in a Disruptor Genie (SI-D258, Scientific Industries) and the supernatant was collected after centrifugation for 4 minutes at 10.000 r.c.f. for further processing. For human RNA isolation, 350 µL of TRK lysis buffer was added directly to cells and taken for further processing according to manufacturer’s instructions. Briefly, either bacterial orhuman RNA in TRK buffer was mixed with an equal volume of fresh ethanol 70% and loaded in the RNA isolation columns by centrifugation at 13.000 r.c.f. for 1 minute. Then columns were washed with 500 µL of Wash Buffer I, 500 µL of Wash Buffer II twice and a last 2 minute centrifugation to let the column dry. The RNA was eluted with 40 µL RNase free water and concentration was measured with NanoDrop®One Spectrophotometer (ND-ONE-W, ThermoFisher) RNA-seq Isolated RNA was analyzed for purity and integrity using Bioanalyzer (Agilent Technologies GmbH, Germany) following library construction and RNA-sequencing by Macrogen Inc. (Seoul, South Korea) using the Truseq Stranded Total RNA and sequenced using an Illumina at 60M pair-reads depth. RNA-seq analysis was performed using the nf-core RNA-seq pipeline v3.063,64in Nextflow v20.12.0-edge65. Raw paired-end reads were trimmed using Trim Galore v0.6.6 and aligned to the C. acnes KPA171202 reference genome (Genbank AE017283) using STAR v2.6.1d66and SAMtools v1.1067. Quality control was performed using FastQC v0.11.9. Mapped reads were counted using mpileup from BCFtools in htslib v1.168. For temperature-sensitive promoters, differential gene expression analysis between three heat shock samples and three controls was performed on the normalized read counts using DeSeq269. Genes with a log fold- change greater than 3 were selected for further manual inspection of the read coverage across the genetic locus. We extracted either 200bp upstream the start codon or the whole intergenic region for those genes whose upregulation was consistent all over their ORF. Similarly, constitutive promoters were selected among the genes within the 15% lowest variation coefficient (CV) and after manual inspection of the read coverage for uniformity. Again, the regulatory sequence was identified as either the intergenic region or 200bp upstream from the start codon. For endogenous terminator sequences, mean change points were detected across the coverage pileup using the changepoint R package cpt.mean. Then, we calculated the segments between change points and selected those segments containing at least one stop codon thathad the greatest drop in expression compared with their neighbouring segments. After manual inspectionof those segments fulfilling these criteria, we selected as terminator sequences the intergenic regions within the selected segments. RT-qPCR 500ng of isolated total RNA were treated with DNaseI (18068015, Invitrogen) and used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis kit (K1622, ThermoFisher) according to the manufacturer’s protocols. Subsequent real-time quantitative PCR (RT-qPCR) was performed usingPowerUp™ SYBR™ Green Master Mix (A25742, Life Technologies). Target gene expression levels werenormalized using the housekeeping gene gyrase B (GyrB) from C. acnes. The ΔΔCT method was used to calculate relative mRNA expression levels70. Statistics and reproducibility Statistical analysis was performed using R and RStudio. Each experiment includes at least three independent replicates and graphs include the mean ± s.d. Unpaired one-way analysis of variance (ANOVA) was used for comparison between multiple groups followed by Tukey’s multiple comparison post hoc test. Results with p-value < 0.001 were considered highly statistically significant (***). Results with p- value < 0.05 were considered as significant (*). All blots and gels were repeated at least two times. Terminator efficiency was calculated with the following equation as previously described. Where Spacer_1 corresponds to the mCherry or the sfGFP fluorescence levels from the control harbouring only the Spacer_1 sequence and no terminator, and Terminator corresponds to the fluorescence levels of the terminator evaluated. Error bars were calculated with the following formula, propagating the error of three biological replicates: Experiments with alternative SODs to SodC-F1 and with different secretion peptidesWe have conducted experiments with alternative SODs to SodC-F1, which are also produced and secretedby C. acnes. In particular, the Applicant selected two alternative SODs for C. acnes expression: 1. Engineered superoxide dismutase from Deinococcus wulumuqiensis R12 from (Meng et al. 2025),which is herein denominated as dwSOD a. Sequence:MFVQIAASLAAASSIALGIPGAAAYTLPQLPYAYDALEPHIDARTMEIHHTKHHQTYVDNA NKALEGTEWADLPVEELIQKLDQLPADKKGALRNNAGGHANHSLFWQVMGQGKGGQP SGELMDAIGSAFHSFDAFKQKFEDAAKTRFGSGWAWLVVRDGKLDVVSTANQDNPLM GEAVAGVSGTPILGVDVWEHAYYLNYQNRRPDYLAAFWNVVNWDEVAKRYAAAKHHH HHH 2. Human superoxide dismutase 1, which is herein denominated as hsSOD-1:a. Sequence:MFVQIAASLAAASSIALGIPGAAWTGEDSAEPNSDSAEWIRDMYAKVTEIWQEVMQRRD DDGALHAACQVQPSATLDAAQPRVTGVVLFRQLAPRAKLDAFFALEGFPTEPNSSSRAI HVHQFGDLSQGCESTGPHYNPLAVPHPQHPGDFGNFAVRDGSLWRYRAGLAASLAGP HSIVGRAVVVHAGEDDLGRGGNQASVENGNAGRRLACCVVGVCGPGLWERQAREHS ERKKRRRESECKAAHHHHHHWe inserted these two sequences in the genome of C. acnes for a more stable expression as carried outwith the initial SodC-F1 from Escherichia coli. As shown in figure 6, the two alternative SODs were expressed properly and secreted. We also measured the activity of these two new SOD enzymes andobserved SOD activity compared to a wild type C. acnes strain KPA171202 (see figure 7). From thisexample, it is thus clear les that multiple SOD variants can be produced and expressed, and their culture supernatants display measurable superoxide dismutase activity relative to the wild-type, no-plasmid control,confirming secretion of functional enzyme in each case. This data thus demonstrates that the effect ofproducing and secreting SOD in C. acnes is reproducible across different SOD types, not an artifact of asingle construct,Furthermore, it is further note that we further selected three proteins that are known to be secreted in C.acnes, predicted their secretion signal peptide and fused it to the SodC-F1 from E. coli. We selected:● sp0: PPA_RS09745 Signal Peptide (RoxP):MFVQIAASLAAASSIALGIPGAA ●sp1: PPA_RS09975 Signal Peptide (probable acid phosphatase):MKVLRTSVLGLACATALVSSVGVSPAQA ●sp2: PPA_RS03515 Signal Peptide (CAMP2 factor): MKKTHLVAPLLVGAMLVPAALSAPSAHA● sp3: PPA_RS10575 Signal Peptide (TAG Lipase): MKINARFAVMAASVAVLMAAAPIAQAWe inserted them in the genome for more stable expression as well with the original signal peptide fromPPA_RS09745 (RoxP Protein). SodC-F1 was properly expressed as measured with western blot detectinga His tag in the protein (see figure 8). 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Claims

CLAIMS1. A composition comprising Cutibacterium acnes (C. acnes) bacterial strains, wherein said strains arecharacterized by being modified to secrete a heterologous protein by insertion of an expression construct comprising at least one or more transcription promoter sequences, one or more ORFs (Open Reading Frames) encoding the heterologous protein, and optionally one or more transcription termination sequences; wherein the heterologous protein is characterized by comprising at the N- terminus of the protein sequence, a secretion signal which is processed by C. acnes to secrete theprotein, and wherein the heterologous protein is the superoxide dismutase enzyme.

2. The composition according to claim 1, wherein the secretion signal is of SEQ ID No. 1 or a functionalvariant thereof that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the full / length sequence of SEQ ID NO 1 with the proviso that said functional variant can be processed by C. acnes cells to secrete the heterologous protein.

3. The composition according to any one of claims 1 or 2, wherein the superoxide dismutase enzyme isthe SodC-F1 from the enterohemorrhagic E. coli O157:H7 of SEQ ID NO 2 or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID No.2 that when secreted in one or multiple copies by using the secretion signal of SEQ ID NO 1 is capable of reducing ROS levels.

4. The composition according to any one of claims 1 to 3, wherein the superoxide dismutase enzyme isthe SodC-F1 from the enterohemorrhagic E. coli O157:H7 of SEQ ID NO 2 and the secretion signal is of SEQ ID No.1.

5. The composition according to any one of claims 1 to 4, wherein the one or more transcription promotersequences is the promoter of SEQ ID NO 3, 5 or 6, 15 or the inducible promoter of any one of SEQ ID NO 18 to 22, or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID No.3, 5 or 6, or 15 or SEQ ID NO.18 to 22, that when operably linked to the heterologous protein is capable of expressing the said protein.

6. The composition according to any one of claims 1 to 5, wherein the expression construct furthercomprises a ribosome binding sites (RBS): preferably the RBS is the E. coli RBS sequences of SEQID No 7 or 8 or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID No.7 or 8.

7. The composition according to any one of claims 1 or 2, wherein the expression construct is of SEQ IDNO 9, 10, 11, or 16, or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID NO 9, 10, 11, or 16.

8. The composition according to any one of claims 1 to 7, wherein the expression construct is comprisedin a plasmid, preferably a plasmid capable of replicating in C. acnes and comprising an origin ofreplication for C. acnes.

9. The composition according to any one of claims 1 to 8, wherein the strains are mutant live auxotrophicCutibacterium acnes (C. acnes) bacterial strains, preferably for Lysine, Histidine or Proline, with theproviso that said mutant live auxotrophic C. acnes bacterial strains do not comprise any heterologousantibiotic resistance genes.

10. The composition according to any one of claims 1 to 9, wherein the expression construct is comprisedin a plasmid and the plasmid comprising the said expression construct is selected from any one of SEQ ID NO 12, 13, 14, or 17 or any sequence that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full / length sequence of SEQ ID NO 12, 13, 14, or 17.

11. The composition according to any one of claims 1 to 10, for use in therapy.

12. A pharmaceutical composition comprising the composition as defined in any one of claims 1 to 10optionally further comprising pharmaceutically acceptable excipients and / or carriers.

13. The composition according to any of claims 1 to 10 or 12, for use in a method of treatment for deliveringthe heterologous protein in a subject, preferably a human subject, in need thereof, preferably for use as an antioxidant-producing strain in a method of treatment to rescue keratinocytes from oxidative stress in a subject, preferably a human subject, in need thereof.

14. A composition, such as a cosmetic composition, comprising the composition as defined in any one ofclaims 1 to 10.

15. A non-therapeutical cosmetic use of the composition according to claim 14 for delivering theheterologous protein in a subject, preferably a human subject, in need thereof.

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