PRODUCTION OF BACTERIOCINS

Abstract

The present invention relates to the production of bacteriocins, and in particular the production of bacteriocins of class I or II by recombinant expression in coryneform bacteria as the bacterial cell host. Also provided are modified coryneform bacteria for use as production hosts for production of bacteriocins.

Claims

1. A method of producing a Class I or Class II bacteriocin, said method comprising: (a) providing a modified bacterial strain of coryneform bacteria into which has been introduced a heterologous nucleic acid molecule encoding a Class I or Class II bacteriocin polypeptide; (b) culturing said modified strain under conditions suitable for expression of said bacteriocin polypeptide; and (c) optionally, harvesting said Class I or Class II bacteriocin polypeptide produced in step (b), wherein the bacteriocin polypeptide is an inactive precursor, and/or said strain is not susceptible to said bacteriocin and/or said bacteriocin polypeptide is a component polypeptide of a multi-peptide bacteriocin and said modified strain does not produce all other component polypeptides required to make a functional bacteriocin, wherein optionally said bacteriocin is harvested, where preferably the bacteriocin is isolated, purified or processed into a product.

2. (canceled)

3. The method of claim 1, wherein (a) when said bacteriocin is a Class I bacteriocin, the bacteriocin polypeptide is an inactive precursor, and when said bacteriocin is a class II bacteriocin, said strain is not susceptible to said bacteriocin and/or said bacteriocin polypeptide is a single polypeptide of a multi-peptide bacteriocin; (b) an expression vector comprising said nucleic acid molecule has been introduced into said modified strain, wherein said expression vector is capable of expressing said bacteriocin polypeptide in said strain; and/or (c) said nucleic acid molecule comprises a synthetic operon comprising: (i) a promoter controlling the expression of the following genes; (ii) a structural gene encoding the bacteriocin polypeptide; (iii) optionally, one or more genes encoding processing and/or transport proteins for production of said bacteriocin polypeptide; and/or (iv) optionally, one or more genes which provide the bacterial strain with immunity to the bacteriocin.

4-5. (canceled)

6. The method of claim 35, wherein (a) said synthetic operon comprises one or more genes encoding processing and/or transport proteins for production of said bacteriocin polypeptide; (b) said synthetic operon comprises (i) a promoter controlling the expression of the following genes; (ii) a structural gene encoding the bacteriocin polypeptide which is a chimeric bacteriocin polypeptide which comprises a heterologous leader sequence which is a leader sequence of a second bacteriocin; (iii) one or more genes encoding processing and/or transport proteins for production of said bacteriocin polypeptide wherein said genes are processing and/or transport proteins for processing and/or transporting said second bacteriocin; and (iv) optionally, one or more genes which provide the bacterial strain with immunity to the bacteriocin; or (c) said synthetic operon comprises (i) a promoter controlling the expression of the following genes; (ii) a structural gene encoding the bacteriocin polypeptide which is a chimeric bacteriocin polypeptide which comprises a heterologous leader sequence which is a Sec-dependent leader sequence; and (iii) optionally, one or more genes which provide the bacterial strain with immunity to the bacteriocin.

7-8. (canceled)

9. The method of claim 3, wherein said nucleic acid molecule comprises a synthetic operon, and wherein said genes are codon-optimised for expression in coryneform bacteria.

10. The method of claim 1, wherein said nucleic acid molecule is a self-replicating plasmid or a plasmid which has been integrated into the genome of the strain.

11. The method of claim 3, wherein said nucleic acid molecule comprises a synthetic operon, and wherein said promoter is an inducible promoter.

12. The method of claim 1, wherein (a) the strain does not express a protein capable of acting as a receptor for the class I or II bacteriocin to be expressed; and/or (b) said modified bacterial strain (i) does not contain a gene which provides the bacterial strain with immunity to the bacteriocin or (ii) contains a constitutively expressed gene which provides the bacterial strain with immunity to the bacteriocin.

13. (canceled)

14. The method of claim 1, wherein the Class I or Class II bacteriocin is a Class II bacteriocin.

15. The method of claim 14, wherein (a) said strain does not express a Group I mannose-specific phosphotransferase (PTS.sup.Man); (b) the leader sequence of said Class II bacteriocin comprises a double glycine motif, and/or (c) the Class II bacteriocin is a Class IIA, Class IIB or Class BD bacteriocin.

16-17. (canceled)

18. The method of claim 14, wherein the bacteriocin is selected from the group consisting of pediocin, lactococcin G, plantaricin EF, plantaricin JK, plantaricin NC08, lactococcin A, lactococcin B and garvicin Q.

19. The method of claim 14, wherein the nucleic acid molecule comprises (a) a synthetic operon comprising: (i) a promoter; (ii) a structural gene encoding the bacteriocin; and (iii) pedC and pedD genes, wherein preferably the structural gene encodes pediocin, or a chimeric bacteriocin polypeptide which comprises the leader sequence of pediocin; (b) a synthetic operon comprising: (i) a promoter; (ii) a structural gene encoding the bacteriocin; (iii) garC and garD genes; and (iv) optionally, one or more genes which provide the bacterial strain with immunity to the bacteriocin, wherein preferably the structural gene encodes garvicin Q, or a chimeric bacteriocin polypeptide which comprises the leader sequence of garvicin Q; or (c) a synthetic operon comprising: (i) a promoter; (ii) a structural gene encoding the bacteriocin polypeptide which is a chimeric bacteriocin polypeptide which comprises a heterologous leader sequence which is a Sec-dependent leader sequence; and (iii) optionally, one or more genes which provide the bacterial strain with immunity to the bacteriocin, wherein preferably the structural gene encodes garvicin Q.

20-21. (canceled)

22. The method of claim 14, wherein the Class II bacteriocin is a multi-peptide bacteriocin comprising 2 or more bacteriocin polypeptides, and the method comprises separately expressing each bacteriocin polypeptide in the bacterial strain, harvesting each bacteriocin polypeptide, and combining the bacteriocin polypeptides to prepare a bacteriocin complex.

23. The method of claim 1, wherein the Class I or Class II bacteriocin is a Class I bacteriocin, wherein optionally the method comprises harvesting the bacteriocin polypeptide and a further step (d) of cleaving the inactive precursor to remove the leader sequence; and/or wherein the Class I bacteriocin is a lantibiotic.

24-25. (canceled)

26. The method of claim 23, wherein the lantibiotic is selected from the group consisting of nisin, bisin, lacticin, subtilin, epicidin, epidermin, epilancin, salvaricin, sublancin, carnocin, variacin, cypemycin, gallidermin, mersacidin, actagardine, cinnamycin, duramycin, ancovenin, actagardine, cytolysin, staphylococcin and mutacin,

27. The method of claim 23, wherein the nucleic acid molecule comprises (a) a synthetic operon comprising: (i) a promoter; (ii) a structural gene encoding the bacteriocin; and (iii) nisB, nisC and nisT genes, wherein preferably the bacteriocin is nisin; or (b) a synthetic operon comprising: (i) a promoter; (ii) a structural gene encoding the bacteriocin polypeptide which is a chimeric bacteriocin polypeptide which comprises the leader sequence of nisin; (iii) nisB, nisC and nisT genes, wherein preferably the flavulin.

28. (canceled)

29. A product obtainable by a method as claimed in claim 1.

30-31. (canceled)

32. A strain of coryneform bacteria which has been modified to express a Class I or Class II bacteriocin polypeptide, wherein the bacteriocin polypeptide is an inactive precursor, and/or said strain is not susceptible to said bacteriocin and/or said bacteriocin polypeptide is a component polypeptide of a multi-peptide bacteriocin and said modified strain does not produce all other component polypeptides required to make a functional bacteriocin, wherein preferably said modified bacterial strain a) does not contain a gene which provides the bacterial strain with immunity to the bacteriocin or b) contains a constitutively expressed gene which provides the bacterial strain with immunity to the bacteriocin.

33. The strain of claim 32, wherein said strain comprises an expression vector comprising a nucleic acid molecule comprising a synthetic operon comprising: (i) a promoter controlling the expression of the following genes; (ii) a structural gene encoding the bacteriocin polypeptide; (iii) optionally, one or more genes encoding processing and/or transport proteins for production of said bacteriocin polypeptide; and/or (iv) optionally, one or more genes which provide the bacterial strain with immunity to the bacteriocin; wherein the nucleotide sequences of said genes are codon-optimised for expression in coryneform bacteria and wherein preferably the bacteriocin polypeptide is an inactive precursor.

34. The method of claim 1, wherein the bacterial strain is a species selected from Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium melassecola, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, Corynebacterium acetoacidophilum, Corynebacterium lilium, Corynebacterium casei, Corynebacterium stationis and Brevibacterium divaricatum.

35. The strain of claim 32, wherein the bacterial strain is a species selected from Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium melassecola, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, Corynebacterium acetoacidophilum, Corynebacterium lilium, Corynebacterium casei, Corynebacterium stationis and Brevibacterium divaricatum.

Description

BRIEF DESCRIPTION OF FIGURES

[0212] FIG. 1 shows (A) Microtiter plate growth inhibition assay using L. monocytogenes EGDe::pIMK2 (upper panel) or L. innocua LMG2785::pIMK2 (lower panel) to determine activity of a pediocin PA-1 standard solution at the indicated concentration in the assay. (B) Growth inhibition of L. monocytogenes EGDe::pIMK2 by supernatants of P. acidilactici 347 (Paci) collected in early stationary growth phase at the indicated dilution. Values are mean±standard deviation of at least n=3 independent experiments per condition. Dilution series sterile H.sub.2O (A) or MRS medium (B) were used as negative controls. Hatched bars indicate growth of the indicator strain without addition of diluted supernatant or pediocin, i.e. maximum growth. The bottom and top lines indicate OD.sub.600 of the positive (i.e. complete inhibition of growth) or negative (i.e. in the absence of an antimicrobial peptide), respectively. The broken middle line represents growth inhibition of 50%, i.e. the threshold to calculate bacteriocin units.

[0213] FIG. 2 shows (A) Growth (OD.sub.600) of C. glutamicum CR099 in 96-well microtiter plates in the presence of pediocin (black bars) nisin (grey), or bacitracin at the indicated concentrations. Values are mean±standard deviation of n=4-7 independent experiments. (B) Growth of C. glutamicum CR099 in standard batch culture in 2xTY medium with 2% Glc in the presence (+PA-1) or absence (−PA-1) of 2.0 μg/mL pediocin. Values are mean±standard deviation of n=4 independent experiments. Growth rates were calculated during exponential growth phase. (C) Growth inhibition of L. monocytogenes EGDe::pIMK2 by supernatants of C. glutamicum CR099 in 2xTY medium with 2% Glc plus 2 μg/mL pure pediocin (+CR099). Supernatants were collected during the experiment shown in (B) at the indicated time-points. As controls, pediocin was incubated under the same conditions in sterile medium (−CR099). Values are OD.sub.600 of the indicator strain L. monocytogenes EGDe in a growth assay at the indicated dilutions of the samples and are mean±standard deviation of n=3 independent experiments. In all graphs, the bottom and top lines indicate OD.sub.600 of the positive (i.e. complete inhibition of growth) or negative (i.e. in the absence of an antimicrobial peptide), respectively. The broken middle lines represent growth inhibition of 50%, i.e. the threshold to calculate bacteriocin units.

[0214] FIG. 3 shows (A) Genetic organization of the operon of P. acidilactici PAC1.0 for biosynthesis of pediocin PA-1. (B) Plasmid map of pEKEx2-pedACD introduced into C. glutamicum CR099. (C) Growth inhibition of L. monocytogenes EGDe::pIMK2 by supernatants of C. glutamicum CR099/pEKEx2-pedACD (pedACD) or the empty vector control strain C. glutamicum CR099/pEKEx2 (ped0). Bacteria were grown in 5 ml 2xTY medium in glass tubes and 2h after inoculation Glc (2 w/v) and IPTG (0.1 mM) were added to induce production of pediocin. Activity was measured in 2-fold serial dilutions of culture supernatants after o/N growth, i.e. early stationary growth phase, using the growth assay with L. monocytogenes EGDe::pIMK2 as indicator. Values are OD.sub.600 of the indicator strain and are mean±standard deviation of n=9 independent experiments. Hatched bars indicate growth of the indicator strain without addition of diluted supernatant or pediocin, i.e. maximum growth. The bottom and top lines indicate OD.sub.600 of the positive (i.e. complete inhibition of growth) or negative (i.e. in the absence of an antimicrobial peptide), respectively. The broken middle lines represent growth inhibition of 50%, i.e. the threshold to calculate bacteriocin units.

[0215] FIG. 4 shows (A) Growth of C. glutamicum CR099/pEKEx-ped0 (ped0) and C. glutamicum CR099/pEKEx-pedACD (pedACD) in 2xTY medium in baffled (+) and non-baffled (−) Erlenmeyer flasks. 2h after inoculation Glc (2% w/v) and IPTG (0.1 mM) were added to induce production of pediocin (B) Growth inhibition of L. innocua LMG2785::pIMK2 by different dilutions of supernatants of C. glutamicum CR099/pEKEx2-pedACD (pedACD) collected at the indicated timepoints during the growth experiment shown in (A). All values are mean±standard deviation of n=3 independent experiments. The bottom and top lines indicate OD.sub.600 of the positive (i.e. complete inhibition of growth) or negative (i.e. in the absence of an antimicrobial peptide), respectively. The broken middle lines represent growth inhibition of 50%, i.e. the threshold to calculate bacteriocin units.

[0216] FIG. 5 shows ion exchange chromatography of ammonium sulphate precipitated supernatants of P. acidilactici 347 (left panel) or C. glutamicum CR099/pEKEx-pedACD (right panel) following growth in non-baffled (−) Erlenmeyer flasks in 2xTY medium with glucose (2% w/v) and IPTG (0.1 mM). Black lines indicate absorbance at 280 nm in milli arbitrary units (mAU) and grey lines conductivity in mS/cm. Broken vertical lines indicate the boundaries of the peak fractions of the eluate collected for further analysis.

[0217] FIG. 6 shows (A) Growth inhibitory activity in different dilutions of fractions F8-10 of ion exchange chromatography of precipitated supernatant of C. glutamicum CR099/pEKEx-pedACD grow in 2xTY medium with 2% Glc in non-baffled Erlenmeyer flasks overnight. Samples were analyzed untreated or following incubation with proteinase K (+protK). All values are mean±standard deviation of n=3 independent experiments. The bottom and top lines indicate OD.sub.600 at baseline (i.e. complete inhibition of growth) or in the absence of antimicrobial peptides, respectively. The broken middle line represents growth inhibition of 50%, i.e. the threshold to calculate bacteriocin units. (B) SDS-PAGE of peak fractions F9 (IEX F9) of supernatants of C. glutamicum CR099/pEKEx-pedACD (CR099 pedACD) or P. acidilactici 347 (P. aci) analyzed by ion exchange chromatography. Pure pediocin (PA-1) was analyzed on the same gel as control. (C) Size exclusion chromatography of pooled ion exchange fractions F8-F10 from supernatants of P. acidilactici 347 (P. aci, middle panel) or C. glutamicum CR099/pEKEx-pedACD (Cg pedACD; lower panel) or pure pediocin (PA-1) as a control. The box indicates a signal in all samples with a size corresponding to pure pediocin. (D) Mass spectrometry of active fractions after reverse phase chromatography of pediocin PA-1 (upper panel) and pooled active cation-exchange fractions from supernatants C. glutamicum CR099/pEKEx-pedACD (lower panel) before applying to reverse phase chromatography. The boxes highlight peaks observed in both samples with almost identical mass/charge ratio (m/z) that match the calculated m/z of pediocin PA-1.

[0218] FIG. 7 shows growth inhibition of L. monocytogenes EGDe by different dilutions of supernatants of C. glutamicum CR099 strains harboring pEKEx2-derived constructs with ped genes in different combinations (A) or in different order and copy number (B). Bacteria were grown in 5 mL 2xTY medium in glass tubes and 2h after inoculation Glc (2% w/v) and IPTG (0.1 mM) were added. All values are mean OD.sub.600±standard deviation of n=3 independent experiments. The bottom and top lines indicate the OD.sub.600 at baseline (i.e. complete inhibition of growth) or in the absence of antimicrobial peptides, respectively. The broken middle lines represent growth inhibition of 50%, i.e. the threshold to calculate bacteriocin units.

[0219] FIG. 8 shows (A) Offline measurements of substrate, biomass and pediocin activity and (B) online measurements of critical process parameters during fermentation of C. glutamicum CR099/pEKEx-pedACD grow a bioreactor in CGXII medium supplemented with 2% Glc, 16 g/L tryptone and 10 g/L yeast extract under controlled fed-batch conditions (in A: biomass (DCW), bacteriocin acitivity (activity), glucose (Glc) and glutamate (Glu); in B: Dissolved oxygen (DO.sub.2), agitator speed (agitation) and aeration rate (aeration)). The timepoint of induction (t=7.5 h) is indicated by the vertical line.

[0220] FIG. 9 shows ion exchange chromatography of ammonium sulphate precipitated supernatants of C. glutamicum CR099/pEKEx-nisZBTC.sup.opt CR099/pPBEx-nisZBTC.sup.opt and CR099/pXMJ-nisZBTC.sup.opt grown in 2xTY medium with glucose (2% w/v) and IPTG (0.1 mM). Black lines indicate absorbance at 214 nm in milli arbitrary units (mAU) and grey lines conductivity in mS/cm. Broken lines indicate the boundaries of the peak fractions.

[0221] FIG. 10 shows (A) Growth inhibition of C. glutamicum CR099 by nisin standard solutions at the indicated concentrations in a spot-on-lawn assay. 10 μl of each nisin standard was spotted onto agar plates and, after drying, overlaid with a soft agar containing the indicator strain C. glutamicum CR099. (B) Spot-on-lawn assay with TCA-precipitated supernatant proteins of C. glutamicum CR099/pEKEx2 or C. glutamicum CR099/pEKEx-nisZBTC.sup.opt. 10 μl of the fractions were either spotted prior to or after treatment with trypsin. As a positive control, 10 μl of a 125 μg/ml nisin standard was spotted.

[0222] FIG. 11 shows (A) Plasmid map of pNZ-P.sub.nis-mCherry.sup.Lla introduced into L. lactis NZ9000. (B) A photo of the bacterial pellet taken after growth in GM17 medium containing 10 ng/ml nisin. The pellet already showed reddish color indicating efficient mCherry expression. (C) The recombinant strain L. lactis NZ9000/pNZ-P.sub.nis-mCherry.sup.Lla displays red fluorescence (fluorescence units per OD; FLU/OD) in the presence of nisin in a dose-dependent manner and with a limit of detection of less than 0.2-0.5 ng/ml of nisin.

[0223] FIG. 12 shows (A) Reverse phase chromatography of the peak fractions obtained by cation exchange chromatography of ammonium sulphate precipitated supernatant proteins of C. glutamicum CR099/pPBEx-nisZBTC.sup.opt (left panel) or CR099/pXMJ-nisZBTC.sup.opt (right panel) grown in 2xTY medium with glucose (2% w/v) and IPTG (0.1 mM). Black lines indicate absorbance at 214 nm in milli arbitrary units (mAU) and grey lines % of solution B in the elation buffer. Broken lines indicate the boundaries of the peak fractions collected for further analysis. (B) L. lactis NZ9000/pNZ-P.sub.nis-mCherry.sup.Lla was used to assay presence of nisin in different samples during purification of ammonium sulphate-precipitated supernatants of C. glutamicum CR099/pXMJ-nisZBTC.sup.opt (SN: plain culture supernatant; precip: ammonium sulphate precipitated supernatant proteins; IEX: peak fraction of ion exchange chromatography; RP peak fraction of reverse phase chromatography. pos: 10 ng/ml nisin; +/−: activation by trypsin).

[0224] FIG. 13 shows a graphical representation of the genetic constructs for the different approaches that are planned to achieve production of class IIB and IID bacteriocins using recombinant C. glutamicum strains. As an example, constructs for production of garvicin Q (A-D) or flavucin (E) are shown. (A) The gene for garvicin Q (garQ) will be cloned upstream of pedCD encoding the pediocin export apparatus. (B) A synthetic garQ gene encoding the pediocin leader peptide (also referred to here as the signal peptide, SP) fused to the garvicin Q coding sequence and replacing the garvicin Q leader peptide will be cloned upstream of pedCD. (C) The entire gar operon for garvicin Q and its native export apparatus will be cloned. (D) A synthetic gene for a hybrid pre-peptide consisting of any Sec-dependent SP will be cloned upstream of mature garvicin Q. Such pre-peptides will be secreted by the general Sec-dependent protein secretion system. (E) A synthetic gene for a hybrid pre-flavucin consisting of the SP of nisin fused to the sequence for the lantibiotic flavucin of Corynebacterium lipophiloflavum will be cloned upstream of nisBTC encoding the modification and transport proteins of nisin. All constructs will be cloned into pEKEx2, pXMJ-19 or pPBEx2 (not shown), or any other standard C. glutamicum expression vector, downstream of the IPTG-inducible P.sub.tac promoter of these plasmids.

[0225] FIG. 14 shows establishment of recombinant garvicin Q production by C. glutamicum. (A) Plasmid map of pPBEx2-garQICD.sup.Cgf introduced into C. glutamicum CR099 for recombinant production of garvicin Q. (B) Antimicrobial activity in C. glutamicum CR099/pPBEx2-garQICD.sub.Cgf and the empty vector control strain CR099/pPBEx2. Activity was analyzed using the fluorescent biosensor Listeria innocua LMG2785/pNZ-P.sub.help-pHluorin.sup.Lm assay and is indicated by a drop in the ratio of fluorescence intensities (emission at 510 nm) of the strain in LMB buffer after excitation at 400 and 470 nm (ratio RFU 400/470) following treatment with supernatants. Values are mean+/s standard deviation of supernatants obtained from n=3 independent cultures for each strain.

[0226] FIG. 15 shows establishment of Sec-dependent production of garvicin Q by C. glutamicum. (A) Plasmid map of pXMJ-SP.sub.ywaD-garQ.sup.Cgf introduced into C. glutamicum CR099 for recombinant production of garvicin Q via the Sec translocon. (B) Antimicrobial activity in supernatants of C. glutamicum CR099/pXMJ-SP.sub.ywaD-garQ.sup.Cgf (dark grey bars) and CR099/pPBEx2-garQICD.sup.Cgf (lighter grey bars). Activity was analyzed using the fluorescent biosensor Listeria innocua LMG2785/pNZ-P.sub.help-pHluorin.sup.Lm assay and is indicated by a drop in the ratio of fluorescence intensities (emission at 520 nm) of the strain in LMB buffer after excitation at 400 and 480 nm (ratio Em 520 Ex400/Ex480) following treatment with supernatants. Values are mean +/s standard deviation of supernatants obtained from n=3 independent cultures for each strain.

[0227] FIG. 16 shows establishment of flavucin production by C. glutamicum using the nisin biosynthetic machinery. (A) Plasmid map of pXMJ-SP.sub.nisflaA-nizBTC.sup.Cgf introduced into C. glutamicum CR099 for recombinant production of pre-flavucin. (B) Growth inhibition of C. glutamicum CR099/pXMJ19 by supernatants of strains CR099/pXMJ-SP.sub.nisflaA-nizBTC.sup.Cgf and CR099/pXMJ-nisZBTC.sup.Cgf. 10 μl of each supernatant were spotted onto agar plates and, after drying, overlaid with a soft agar containing the indicator strain. Where indicated supernatants were incubated with either trypsin or a soluble NisP protease. As a positive control 10 μl of a nisin standard (250 μg/ml) was spotted.

EXAMPLES

Example 1—Production of Pediocin in C. glutamicum

[0228] C. glutamicum is a Suitable Host for Recombinant Production of Pediocin PA-1

[0229] Pediocin PA-1 is a bacteriocin with potent antimicrobial activity against a range of Gram-positive microorganisms including important human pathogens such as Listeria monocytogenes. As a first step towards recombinant production of pediocin PA-1, a growth-dependent assay was established using L. monocytogenes EGDe::pIMK2 or L. innocua LMG2785::pIMK2 as indicator strains, and commercially available, purified pediocin (FIG. 1A). Using serial, 2-fold dilutions of a pediocin standard, 39 ng/ml of pediocin was determined as the minimal concentration to completely inhibit growth of the indicator strains under the conditions of the assay. This concentration was used in further experiments as a calibrator to estimate product concentrations in supernatants of producer strains.

[0230] To validate the assay, anti-microbial activity in supernatants of P. acidilactici 347, a natural producer of pediocin was measured following growth in MRS medium to early stationary growth phase (FIG. 1B). Complete inhibition of the indicator strain was achieved with a 1:128 dilution. Thus, supernatants of P. acidilactici 347 contain at least 5 μg mL.sup.−1 of active pediocin based on the calibration with the pediocin standard (FIG. 1A). According to a previously published method, activity is calculated as bacteriocin units per ml (BU mL.sup.−1) based on the highest dilution showing 50% inhibition of growth of the indicator. According to this definition, supernatants of P. acidilactici 347 contain 10240 BU mL.sup.−1.

[0231] The receptors for bacteriocins of the pediocin family on target organisms are the IIC and IID subunits of a sub-family of mannose-specific phosphotransferase systems (PTS.sup.Man; PMID: 19477899). In silico analysis of the genome of C. glutamicum CR099 and other strains of the species indicated that C. glutamicum does not encode a PTS.sup.Man, suggesting it may be resistant against pediocin. To corroborate this assumption, the resistance of C. glutamicum CR099 against different antimicrobial peptides was determined using an endpoint measurement of growth in 96-well microtiter plates (FIG. 2A).

[0232] Complete inhibition of growth was observed with bacitracin and nisin at concentrations of 390-781 ng mL.sup.−1. By contrast, C. glutamicum CR099 was able to grow in the presence of at least 12.5 μg mL.sup.−1 pediocin PA-1. Further experiments conducted in larger volumes under standard conditions of cultivation in baffled Erlenmeyer flask confirmed that 2 μg mL.sup.−1 of pediocin did not affect final optical density and growth rate (FIG. 2B). This indicates that production of pediocin by C. glutamicum CR099 at significant titers may be possible without adverse effects on growth.

[0233] Additionally, activity of pediocin was measured in the culture supernatant at select timepoints during the cultivation. Calculation of bacteriocin units revealed that, after a slight initial reduction from 2560 to 1280 BU mL.sup.−1, activity remained more or less stable for several hours in growing C. glutamicum CR099 cultures. After 24 hours, activities dropped to 160 BU mL.sup.−1 in the presence of C. glutamicum CR099 and the same reduction was observed in control incubation in 2xTY without bacteria. C. glutamicum does not show significant extracellular protease activity and pediocin activity is known to be sensitive to oxygen. The observed reduction in activity was therefore considered to be related to adsorption of the positively charged pediocin to the negative surface of bacteria and, at later timepoints, to oxidative inactivation. In summary, C. glutamicum CR099 was able to grow in the presence of high concentrations of pediocin and apparently did not degrade pediocin in significant amounts during active growth and was thus considered a suitable host for recombinant production.

Establishment of Pediocin Production in C. glutamicum CR099

[0234] To establish pediocin production in C. glutamicum CR099, the sequence of the biosynthetic operon of P. acidilactici PAC1.0 was retrieved from the NCBI GenBank database (accession no.: M83924.1; FIG. 3A). The operon comprises four genes located on a plasmid (pSRQ11) and consists of the structural gene pedA for the prepeptide of the bacteriocin, pedB for an immunity protein, and pedC and pedD encoding proteins required for processing, cleavage and export of the mature bacteriocin.

[0235] In P. acidilactici PAC1.0, all genes are transcribed from a single promoter upstream of pedA. The immunity protein confers resistance by a mechanism that depends on the receptor (PTS.sup.Man), which is absent in C. glutamicum CR099. Thus, pedB was considered dispensable. A synthetic pedACD operon for recombinant production of pediocin PA-1 was designed based on the protein sequences available on UniProt database (accession no.: P29430, P37249, and P36497). Gene sequences were codon-optimized for C. glutamicum, each equipped with a ribosome binding site, obtained as synthesized gene fragments, and cloned under the IPTG-inducible Ptac promoter into pEKEx2 by Gibson Assembly yielding pEKEx2-pedACD (FIG. 3B). The codon-optimised gene sequences for pedA, pedC and pedD respectively are shown in SEQ ID NOs 1, 5, and 7. This construct was successfully introduced into C. glutamicum CR099.

[0236] Supernatants of cultures of C. glutamicum CR099/pEKEx-pedACD grown to early stationary growth phase in 2xTY medium with 2% Glc and 0.1 mM IPTG in glass tubes contained up to 10240 BU mL.sup.−1 of antimicrobial activity against L. monocytogenes corresponding to a concentration of approx. 5 μg mL.sup.−1 of pure pediocin (FIG. 3C), i.e. levels comparable to those of the natural producer P. acidilactici 347 (FIG. 1B). By contrast, supernatants of the empty vector control strain C. glutamicum CR099/pEKEx-ped0 did not inhibit growth of L. monocytogenes EGDe::pIMK2 at all. Almost identical results were obtained with a second strain which contained the same operon and promoter in the pXMJ19 backbone.

[0237] In order to identify the compound responsible for inhibition of growth of the sensor strain and demonstrate that it is indeed pediocin, further experiments in larger culture volumes were conducted. Surprisingly, only very low activity was obtained when C. glutamicum CR099/pEKEx-pedACD was grown in baffled Erlenmeyer flasks for efficient oxygenation of the medium with a maximum of 640 BU mL.sup.−1 after 10 h of cultivation (FIG. 4).

[0238] However, growth inhibitory activity of C. glutamicum CR099/pEKEx-pedACD supernatants was dramatically increased when cultivated under the same conditions in non-baffled Erlenmeyer flasks. Under these conditions, a maximum of 5120 BU mL-1 (equivalent to approx. 2.5 μg mL.sup.−1 of pure pediocin) was observed at the end of the cultivation; FIG. 4B).

Identification of Pediocin in Supernatants of Recombinant C. glutamicum

[0239] For identification of pediocin, proteins in the supernatant of C. glutamicum CR099/pEKEx-pedACD grown over night in non-baffled Erlenmeyer flasks in 2xTY medium with 2% Glc and 0.1 mM IPTG were precipitated with 50% ammonium sulfate and further separated via cation exchange chromatography (CIEX). Similar to supernatants of P. acidilactici 347, a single peak was observed at 280 nm the onset of elution (FIG. 5). Peak fractions F8-F10 were collected and further analyzed. All peak fractions strongly inhibited growth of L. monocytogenes EGDe::pIMK2 with a maximum of 204,800 BU mL.sup.−1, which is equivalent to at least 25 μg mL.sup.−1 of active pediocin (FIG. 6A). In all fractions, activity was completely abolished by proteinase K treatment indicating that the active compound is a proteinaceous substance.

[0240] Additionally, SDS-PAGE revealed a single protein band in peak fractions of both P. acidilactici 347 and C. glutamicum CR099/pEKEx2-pedACD at around 5 kDa, which corresponds to the size of pediocin PA-1 (FIG. 6B). Size exclusion analysis of cation exchange chromatographic preparations of P. acidilactici 347 and C. glutamicum CR099/pEKEx2-pedACD supernatants revealed a single peak, which is identical to the elution volume of pure, commercially available pediocin (FIG. 6C).

[0241] Interestingly, both SDS-PAGE and size exclusion chromatography suggested that the preparations of C. glutamicum CR099/pEKEx2-pedACD contained pediocin as main product in high purity as indicated by a single band or peak. By contrast, samples prepared from P. acidilactici 347 supernatants contained several other peaks or signals indicative of further proteins/peptides possibly secreted by P. acidilactici 347 or present in MRS medium used for cultivation. In order to confirm the active compound, CIEX peak fractions were further analysed by reverse phase chromatography coupled to mass spectrometry (FIG. 6D). This identified a peptide in the supernatants of C. glutamicum CR099/pEKEx2-pedACD with an almost identical mass-to-charge ratio (m/z=4622.891) as pure pediocin PA-1 (m/z=4622.452).

Gene Complement and Order for Efficient Production of Pediocin by C. glutamicum

[0242] In order to assess the minimal operon for pediocin production by C. glutamicum, further synthetic constructs with ped genes in different combinations were cloned into pEKEx2 and corresponding plasmids were transformed into C. glutamicum CR099.

[0243] Measurements of activity in supernatants of strains with different combinations of the ped genes (pedA, pedAC, pedAD, pedCD, pedACD) using L. monocytogenes EGDe as sensor strain revealed that strains that lack any of the three genes did not contain pediocin activity in their supernatants (FIG. 7A).

[0244] Similarly, altering the gene order by moving the structural gene pedA to the end of the operon (pedCDA) or adding an additional copy of pedA (pedAACD) resulted in reduced activity in the supernatants of the respective strains (FIG. 7B).

Production of Pediocin in Bioreactors

[0245] In order to demonstrate feasibility of recombinant pediocin production on a larger scale, further experiments were performed with C. glutamicum CR099/pEKEx-pedACD in bioreactors under fed-batch conditions (FIG. 8). Media composition and culture conditions were similar to the shake flask experiments described above. During the growth phase (0-7.5 h) provided nutrients are consumed and biomass grew at an estimated growth rate of 0.5 h.sup.−1 (FIG. 8A), which was in good correspondence to the results of experiments in shake flasks (FIGS. 2 and 4). After 7.5 h, initial Glc was depleted and constant feed addition was started (50 mL h.sup.−1 and 1 L of feed) and production was induced (0.1 mM IPTG). During the induction phase, dissolved oxygen was reduced to 5% to prevent exhaustive oxidation of pediocin as indicated by baffled shake flask experiments (FIG. 4). Dissolved oxygen was tightly controlled by a split range control including stirrer speed (400-1200 rpm) and subsequent aeration rate adaption (18-80 L h.sup.−1; FIG. 8B). During induction, the fed glucose and glutamate were fully consumed and biomass was formed with a decreased growth rate (0.1 h.sup.−1 on average) before growth stopped at a biomass concentration of 46 g L.sup.−1 and after complete addition of nutrients (FIG. 8A). No significant amounts of other byproducts such as lactate or acetate were detected. Measurements of pediocin activity indicated active pediocin already 2.5 h after induction reaching a constant and high level of 20480 BU mL.sup.−1 at t=18.5-33.5 h of the experiment. This activity corresponds to approx. 10 μg mL.sup.−1 of pure pediocin. A slight decrease in activity (10240 BU mL.sup.−1) was observed at the end of the experiment (t=43.5 h).

[0246] Specific activity reached a maximum of 224.4 BU mL.sup.−1 OD.sup.−1 at t=18.5 h, i.e. 11 h after induction. This was in good agreement with the specific activities observed at the end (t=24 h) of the experiments in non-baffled Erlenmeyer flasks (200.9 BU mL.sup.−1 OD.sup.−1).

Example 2—Production of (Pre)Nisin in C. glutamicum

[0247] Preliminary experiments showed that growth of C. glutamicum CR099 is inhibited by nisin at concentrations above ˜100 ng mL.sup.−1 (FIG. 2). However, nisin is produced as prenisin, i.e. an inactive precursor of nisin. In the native producer, prenisin is activated after export by a dedicated protease NisP. However, prenisin can also be activated by treatment with the protease trypsin. A strategy for production of prenisin and subsequent activation by trypsin was adopted. To establish prenisin production in C. glutamicum CR099, the sequence of the nisin biosynthesis operon of L. lactis B1629 was obtained by genome sequencing. A synthetic operon encompassing the gene for the nisin Z precursor peptide (nisZ), a dehydratase for modification (nisB), a cyclase for formation of the thiolactone rings (nisC), and a dedicated transporter for the modified prepeptide (nisT) was designed based on the deduced protein sequences. Gene sequences were codon-optimized for C. glutamicum, each equipped with a ribosome binding site, obtained as synthesized gene fragments, and cloned under the IPTG-inducible P.sub.tac promoter into pEKEx2, pPBEx2 or pXMJ19 by Gibson Assembly yielding pEKEx-nisZBTC.sup.opt pPBEx-nisZBTC.sup.opt and pXMJ-nisZBTC.sup.opt. These constructs were successfully introduced into C. glutamicum CR099.

[0248] Supernatants of cultures of the recombinant strains C. glutamicum CR099/pEKEx-nisZBTC.sup.opt CR099/pPBEx-nisZBTC.sup.opt and CR099/pXMJ-nisZBTC.sup.opt were grown to early stationary growth phase in 2xTY medium with 2% glucose and 0.1 mM IPTG. Proteins were precipitated using ammonium sulphate and the precipitates were washed using ice cold acetone, resuspended in Tris/HCl buffer (pH 6.5) and analysed by ion exchange chromatography (FIG. 1). A single peak was observed in all samples at 214 nm the onset of elution.

[0249] Using a spot-on-lawn using C. glutamicum CR099 as an indicator strain (FIG. 10), no inhibition of growth was observed for proteins precipitated from the negative control, i.e. supernatants of the empty vector control strain C. glutamicum CR099/pEKEx2. However, the proteins precipitated from supernatants of C. glutamicum CR099/pEKEx-nisZBTC.sup.opt showed inhibitory activity of the indicator strain after treatment with trypsin (FIG. 10B) to a similar extent as a nisin standard at a concentration of 1-2 μg mL.sup.−1 (FIG. 10A). By contrast, the same protein preparation without further treatment did not inhibit growth of the indicator strain. This indicates that trypsin treatment was able to activate a compound present in the supernatants of C. glutamicum CR099/pEKEx-nisZBTC.sup.opt.

[0250] The sensitivity of the spot-on-lawn assay using C. glutamicum CR099 as an indicator strain is very low. In order to establish a sensor system with higher sensitivity and to demonstrate production of prenisin by C. glutamicum CR099/pEKEx-nisZBTC.sup.opt, a L. lactis sensor strain was generated. The strain L. lactis NZ9000/pNZ-P.sub.nis-mCherry.sup.Lla contains the NisRK two-component system and harbours a pNZ-derives plasmid for expression the red-fluorescent protein mCherry from the P.sub.nis promoter (FIG. 11A), which is activated by NisR in a strictly nisin-dependent manner. In this construct, the coding sequence of the mCherry protein is optimized for codon usage of L. lactis. The respective sensor strain results in reddish coloured bacteria after o/N growth in the presence of 10 ng mL.sup.−1 (FIG. 11B). In a 96-well microtiter plate assay, the limit of detection of this sensor strain was 0.25-0.5 ng mL.sup.−1 of active nisin (FIG. 11C).

[0251] The peak fraction of cation exchange chromatography of supernatant proteins precipitated by ammonium sulphate (FIG. 9) were further analysed by reverse phase chromatography. This revealed distinct peaks (FIG. 12A) that were absent in samples of the empty vector control strains. These peak fractions were collected and were clearly able to activate expression of mCherry in L. lactis NZ9000/pNZ-P.sub.nis-mCherry.sup.Lla following trypsin treatment (FIG. 12B). Similarly, peak fractions of cation exchange chromatography, precipitated supernatant proteins and plain supernatants treated with trypsin were able to induce mCherry expression in the sensor strain. By contrast, the sensor strain did not show any mCherry fluorescence when exposed to untreated plain culture supernatants.

[0252] Considering the signal intensity of the mCherry sensor, the dilution of samples in the assay and the signal intensity of the sensor treated with 10 ng mL.sup.−1 of nisin, all trypsin-activated samples contained at least 2 μg mL.sup.−1 of active nisin.

Example 3—Production of Further Bacteriocins in C. glutamicum

[0253] It has been demonstrated that it is possible to produce the class IIA bacteriocin pediocin PA-1 and the prepeptide of class I bacteriocin nisin using recombinant derivatives of C. glutamicum. Several approaches are possible to implement production of other bacteriocins depending on the class and nature of the peptide. For several class II bacteriocins, especially those that contain a specific double glycine (GG) motif in their leader sequence, it has been shown that the transport and modification machinery is promiscuous. For example, the GG-leader of lactococcin A (class IID) and its dedicated ABC transporter can be used to produce pediocin (class IIA). Fusion of the pediocin leader sequence to the coding sequence of colicin V allowed production of active colicin in a strain harbouring this construct and the genes required for pediocin secretion. Similarly, active divergicin A (class IIA) could be produced by fusion of the GG-leaders of leucocin A (class IIA), lactococcin A (class IID) or colicin V (unclassified). Thus, it is expected that class II bacteriocins that carry a GG motif in their leader sequence can be produced using the pediocin export apparatus (or the export apparatus of other bacteriocins with a GG-leader). Potential candidates amongst class II bacteriocins include pediocin, lactococcin G (class IIB), plantaricin EF (class IIB), plantaricin JK (class IIB), plantaricin NC08 (class IIB), lactococcin A (class IID), lactococcin B (class IID), and garvicin Q (class IID).

[0254] For all these bacteriocins, natural producers will be cultivated in standard media and supernatants will be tested for antimicrobial activity. These experiments will be carried out with bacteria that are shown to be sensitive to the bacteriocin to ensure that an active bacteriocin is present in the supernatants. Additionally, antimicrobial activity against C. glutamicum CR099 will be tested to assess toxicity of the product towards the anticipated production host. In a first round, only bacteriocins that do not inhibit growth of C. glutamicum CR099 will be taken into consideration for generation of recombinant producers. The coding sequences of these bacteriocins will be obtained as synthetic DNA constructs with sequences optimized for codon usage of C. glutamicum. In a first approach, these sequences will be cloned into the plasmids generated for pediocin production (pEKEx-pedACD, pXMJ-pedACD) replacing the pedA gene (FIG. 13A, shown for garvicin Q as the bacteriocin of interest by way of example). This illustrates expression of a bacteriocin which is processed and exported using the processing and transport genes of another bacteriocin. Alternative approaches for production of bacteriocins (not limited to class II bacteriocins) include:

[0255] a) the generation of chimeric bacteriocins consisting of the coding sequences of the leader sequence of one bacteriocin fused to another bacteriocin (FIG. 13B, shown for garvicin Q by way of example using a class II bacteriocin leader, e.g. the pediocin leader sequence/signal peptide);

[0256] b) cloning of the bacteriocin gene along with its native processing and export apparatus in a similar fashion as carried out for pediocin production strains (FIG. 13C, shown for garvicin Q by way of example);

[0257] c) expression of a hybrid pre-peptide consisting of any Sec-dependent signal peptide (SP) and mature bacteriocin. Such pre-peptides will be secreted by the general Sec-dependent protein secretion system and so not require specific transporters (FIG. 13D, shown for garvicin Q by way of example);

[0258] d) generation of a gene for a hybrid/chimeric bacteriocin consisting of the SP/leader sequence of a second bacteriocin (e.g. nisin) fused to the sequence for the bacteriocin of interest which is to be expressed. The corresponding synthetic gene of the bacteriocin of interest will be cloned upstream of the genes encoding the modification and transport proteins of the second bacteriocin (e.g. nisin, i.e. using NisBTC) allowing modification and secretion of the bacteriocin of interest with the biosynthetic machinery of the second bacteriocin. (FIG. 13E, shown for the lantibiotic flavucin of Corynebacterium lipophiloflavum, by way of example).

[0259] Lactococcin A and B and garvicin Q are single peptide bacteriocins. Thus, generation of the respective recombinant C. glutamicum strains is expected to be relatively straight forward with a plasmid containing the structural gene and genes for the export apparatus in one of the described setups resulting in a single producer. The class IIB bacteriocins lactococcin G and plantaricins EF, JK, and NC08 are two-peptide bacteriocins that require interaction of both peptides in specific molar ratios. For example, lactococcin G is fully active in complexes of α and β peptides in a molar ratio of 7:1 or 8:1 respectively 4. Here, separate producer strains will be generated for each of the peptides and bacteriocins will be produced separately, purified by downstream processing. The two peptides will then be combined to an optimized formula containing both peptides on molar ratios that ensures maximum activity.

Example 4—Production of Garvicin Q in C. glutamicum Using the GarCD Transporter

[0260] This Example illustrates the general methodology of cloning the bacteriocin gene along with its native export apparatus, using garvicin Q as the bacteriocin. The genetic construct used for this purpose is shown in FIGS. 13C and 14A.

[0261] GarQ is a class IID bacteriocin consisting of a single linear peptide that is produced by different strains of Lactococcus garvieae. The receptors of pediocin and GarQ are identical and were show to be IIC and IID subunits of group I mannose-family phosphotransferase system (PTS.sup.Man). C. glutamicum CR099 lacks a PTS.sup.Man and is therefore expected to be resistant to GarQ.

[0262] To establish GarQ production in C. glutamicum CR099, the sequence of the biosynthetic operon for GarQ was retrieved from the natural producer Lactococcus garvieae B1726. The operon comprises four genes consisting of the structural gene garQ for the prepeptide of the bacteriocin, garI for an immunity protein, and garC and garD encoding proteins that are probably required for processing, cleavage and export of the mature bacteriocin. A synthetic garQICD.sup.Cgl operon (.sup.Cgl: codon-optimized for C. glutamicum) for recombinant production of GarQ was designed with gene sequences codon-optimized for C. glutamicum, each equipped with a ribosome binding site, obtained as synthesized gene fragments, and cloned under the IPTG-inducible P.sub.tac promoter into pPBEx2 (Bakkes et al., 2020, supra) by Gibson Assembly yielding pPBEx-garQICD.sup.Cgl(FIG. 14). This construct was successfully introduced into C. glutamicum CR099. The codon optimized sequences for the genes Gar Q, I, C and D are shown in SEQ ID NOs. 75-78.

TABLE-US-00001 TABLE 1 Bacterial strains and plasmids used in this example. Relevant characteristics Source Strain Corynebacterium glutamicum CR099 C. glutamicum ATCC 13032, Baumgart et al. ΔCGP1, ΔCGP2, ΔCGP3, Appl. Environ. ΔISCg1, ΔISCg2 Microb. (2013):79(19):6006-15 Listeria innocua LMG2785 indicator strain unpublished Plasmid pNZ-P.sub.help- Reporter plasmid for pHluro Crauwels et al., pHluorin.sup.Lm assays to determine antimicrobial Front. Microbiol. activity in supernatants of (2018):9:3038 bacteriocin producers pPBEx2 E. coli/C. glutamicum shuttle Bakkes et al., vector; Ptacl; lacl.sup.q; oriC.g 2020, supra from pBL1.; oriE.c. ColE1 from pUC18; Kan.sup.r. pPBEx- pPBEx2 derivative for IPTG- C. Desiderato, garQICD.sup.Cgl inducible expression of the unpublished synthetic garicin operon results garQICD.sup.Cgl of Lactococcus garvieae B1726

[0263] Supernatants of this strain cultivated in a modified CGXII minimal medium containing 2% Glc and 0.2 mM IPTG contained antimicrobial activity against Listeria innocua LMG2785/pNZ-P.sub.help-pHluorin.sup.Lm, a recently described fluorescent biosensor for detection of baceriocins with membrane-damaging activity (Desiderato et al., Int. J. Mol. Sci. (2021): 22(16), 8615). This activity increased during cultivation of C. glutamicum CR099/pPBEx2-garQICD.sup.Cgf and was absent in supernatants of the empty vector control strain C. glutamicum CR099/pPBEx2 suggesting successful production of garvicin Q (FIG. 14B).

Example 5—Production of Garvicin Q in C. glutamicum Using Sec-Dependent Protein Secretion

[0264] This Example illustrates the general methodology of expression of a hybrid pre-peptide consisting of any Sec-dependent signal peptide (SP) and mature bacteriocin, using garvicin Q as the bacteriocin. The genetic construct used for this purpose is shown in FIGS. 13D and 15A.

[0265] In this example a synthetic gene consisting of coding sequences of the Sec-dependent secretion signal of aminopeptidase YwaD of Bacillus subtilis (peptide sequence shown in SEQ ID NO: 79) and mature garvicin Q was generated. This synthetic gene was cloned under the IPTG-inducible P.sub.tac promoter into pXMJ19 (Jakoby et al. Biotech. Tech. (1999):13(6):437-41) by Gibson Assembly yielding pXMJ-SP.sub.ywaD-garQ (FIG. 15A). This construct was successfully introduced into C. glutamicum CR099.

TABLE-US-00002 TABLE 2 Bacterial strains and plasmids used in this example.sup.1. Plasmid Relevant characteristics Source pXMJ19 E. coli/C. glutamicum shuttle vector; Jakoby, 1999, Ptacl; lacl.sup.q; oriC.g from pBL1.; supra oriE.c. ColE1 from pUC18; Cm.sup.r. pXMJ-SP.sub.ywaD- pXMJ19 derivative for IPTG- C. Desiderato, garQ.sup.Cgl inducible expression of the unpublished synthetic operon garQICD.sup.Cgl results for production of garvicin Q .sup.1strains CR099 and LMG2785 and plasmids pNZ-P.sub.help-pHluorin.sup.Lm and pPBEx-garQICD.sup.Cgl are as in Table 1.

[0266] Supernatants of C. glutamicum CR099/pXMJ-SP.sub.ywaD-garQ.sup.Cgl cultivated in a modified CGXII minimal medium containing 2% Glc and 0.2 mM IPTG contained antimicrobial activity against Listeria innocua LMG2785/pNZ-P.sub.help-pHluorin.sup.Lm, as used in Example 4. Activity was comparable to that measured in supernatants of C. glutamicum CR099/pPBEx2-garQICD.sup.Cgf, i.e. the strain producing garvicin Q using the garvicin-specific transporters GarCD (FIG. 15B). This demonstrates that active garvicin Q can be produced using a Sec-secretion signal of B. subtilis and the Sec translocon of C. glutamicum.

Example 6—Production of Flavucin Q in C. glutamicum Using Nisin Modification and Transport Machinery and Downstream Activation

[0267] This Example illustrates the general methodology of generation of a gene for a hybrid bacteriocin consisting of the SP of nisin fused to the sequence for the bacteriocin using flavucin as the bacteriocin. The genetic construct used for this purpose is shown in FIGS. 13E and 16A.

For production of a pre-peptide for a class I lanthipeptide using the biosynthetic machinery of nisin, a synthetic gene consisting of coding sequences of the leader peptide of nisin and the core peptide of flavucin, a class I lantibiotic produced by Corybebacterium lipophiloflavum (Van Heel et al. ACS Syn. Biol. (2016):5(1):1146-54), was generated. This synthetic gene was cloned under the IPTG-inducible P.sub.tac promoter into pXMJ19 (Jakoby et al., 1999, supra) by Gibson Assembly yielding pXMJ-SP.sub.nisflaA-nizBTC.sup.Cgl (FIG. 16A).

TABLE-US-00003 TABLE 3 Bacterial strains and plasmids used in this example. Plasmid Relevant characteristics Source pXMJ-nisZBTC.sup.Cgl pXMJ19 derivative for IPTG- Weixler et al. inducible expression of the Microb. Cell Fact. synthetic nisin operon (2022):21(1):11 nisZBTC.sup.Cgl for production of pre-nisin pXMJ-SP.sub.nis-flaA- pXMJ19 derivative for IPTG- D. Weixler, nisBTC.sup.Cgl inducible expression of the unpublished synthetic operon SP.sub.nisflaA- results nisBTC.sup.Cgl for production of pre-flavucin .sup.1 strain CR099 and plasmid pXMJ19 are as in Tables 1 and 2.
Strains CR099/pXMJ-SP.sub.nisflaA-nizBTC.sup.Cgl and CR099/pXMJ-nisZBTC.sup.opt were grown to early stationary growth phase in 2xTY medium with 2% glucose and IPTG (0.1 mM) and supernatants were collected for further analysis. Using a spot-on-lawn using C. glutamicum CR099/pXMJ19 as an indicator strain (FIG. 16B), no inhibition of growth was observed for untreated supernatants. However, inhibitory activity was observed for supernatants of both strains after treatment with trypsin or a soluble version of the NisP protease (sNisP; FIG. 16B). This suggests that both trypsin and sNisP are able to activate the pre-peptides of nisin or flavucin released into the supernatants by C. glutamicum CR099/pXMJ-nisZBTC.sup.opt or CR099/pXMJ-SP.sub.nisflaA-nizBTC.sup.Cgl, respectively. These results indicate successful production of fully modified pre-flavucin by C. glutamicum CR099/pXMJ-SP.sub.nisflaA-nizBTC.sup.opt and activation to mature flavucin by sNisP.

DESCRIPTION OF SEQUENCES—AS PROVIDED IN THE SEQUENCE LISTING

Pediocin

[0268] SEQ ID NO 1: pedA gene codon-optimised [0269] SEQ ID NO 2: PedA protein with leader (from UniProt P29430) [0270] SEQ ID NO 3: PedA protein without leader (from UniProt P29430) [0271] SEQ ID NO 4: PedA leader (from UniProt P29430) [0272] SEQ ID NO 5: pedC gene codon-optimised [0273] SEQ ID NO 6: PedC protein (from UniProt P37249) [0274] SEQ ID NO 7: pedD gene codon-optimised [0275] SEQ ID NO 8: PedD protein (from UniProt 36497)

Lactococcin G

[0276] SEQ ID NO: 9 Alpha with leader [0277] SEQ ID NO: 10 Alpha w/o leader [0278] SEQ ID NO: 11 Beta w leader [0279] SEQ ID NO: 12 Beta w/o leader

Plantaricin EF

[0280] SEQ ID NO: 13 E w leader [0281] SEQ ID NO: 14 E w/o leader [0282] SEQ ID NO: 15 F w leader [0283] SEQ ID NO: 16 F w/o leader

Plantaricin JK

[0284] SEQ ID NO: 17 J w leader [0285] SEQ ID NO: 18 J w/o leader [0286] SEQ ID NO: 19 K w leader [0287] SEQ ID NO: 20 K w/o leader

Plantaricin NC08

[0288] SEQ ID NO: 21 Alpha w leader [0289] SEQ ID NO: 22 Alpha w/o leader [0290] SEQ ID NO: 23 Beta w leader [0291] SEQ ID NO: 24 Beta w/o leader

Lactococcin A

[0292] SEQ ID NO: 25 w leader [0293] SEQ ID NO: 26 w/o leader

Lactococcin B

[0294] SEQ ID NO: 27 w leader [0295] SEQ ID NO: 28 w/o leader

Garvicin Q

[0296] SEQ ID NO: 29 w leader [0297] SEQ ID NO: 30 w/o leader

Nisin

[0298] SEQ ID NO: 31 Nisin Z with leader (from UniProt P29559) [0299] SEQ ID NO: 32 Nisin Z w/o leader (from UniProt P29559)

Lacticin

[0300] SEQ ID NO: 33 IctA with leader (from UniProt P36499) [0301] SEQ ID NO: 34 IctA w/o leader (from UniProt P36499)

Subtilin

[0302] SEQ ID NO: 35 spaS with leader (from UniProt P10946) [0303] SEQ ID NO: 36 spaS w/o leader (from UniProt P10946)

Epicidin

[0304] SEQ ID NO: 37 from UniProt 054220 [0305] SEQ ID NO: 38, sequence w/o leader

Epidermin

[0306] SEQ ID NO: 39 epiA with leader (from UniProt P08136) [0307] SEQ ID NO: 40 epiA w/o leader (from UniProt P08136)

Epilancin

[0308] SEQ ID NO: 41 elxA with leader (from UniProt 86047) [0309] SEQ ID NO: 42 elxA w/o leader (from UniProt 86047)

Sublancin

[0310] SEQ ID NO: 43 sunA with leader (from UniProt P68578) [0311] SEQ ID NO: 44 sunA w/o leader (from UniProt P68578)

Carnocin

[0312] SEQ ID NO: 45 cbnB2 with leader (from UniProt P38580) [0313] SEQ ID NO: 46 cbnB2 w/o leader

Variacin

[0314] SEQ ID NO: 47 from UniProt Q50848 [0315] SEQ ID NO: 48 sequence w/o leader

Cypemycin

[0316] SEQ ID NO: 49 cypA with leader (from UniProt E5K1B6) [0317] SEQ ID NO: 50 cypA w/o leader(from UniProt E5KIB6)

Gallidermin

[0318] SEQ ID NO: 51 gdmA with leader (from UniProt P21838) [0319] SEQ ID NO: 52 gdmA w/o leader (from UniProt P21838)

Mersacidin

[0320] SEQ ID NO: 53 mrsA with leader (from UniProt P43683) [0321] SEQ ID NO: 54 mrsA w/o leader(from UniProt P43683)

Actagardine

[0322] SEQ ID NO: 55 garA with leader (from UniProt P56650) [0323] SEQ ID NO: 56 garA w/o leader (from UniProt P56650)

Cinnamycin

[0324] SEQ ID NO: 57 cinA w leader (from UniProt P29827) [0325] SEQ ID NO: 58 cinA w/o leader (from UniProt P29827)

Duramycin

[0326] SEQ ID NO: 59 w leader [0327] SEQ ID NO: 60 w/o leader from UniProt P36504

Ancovenin

[0328] SEQ ID NO: 61 w/o leader from UniProt P38655

Enterococcal Cytolysin

[0329] SEQ ID NO: 62 mature ClyLl [0330] SEQ ID NO: 63 Cytolysin_ClyLl with precursor [0331] SEQ ID NO: 64 mature ClyLs [0332] SEQ ID NO: 65 Cytolysin_ClyLs with precursor:

Staphylococcin C55

[0333] SEQ ID NO: 66 Staphylococcins_C55b_SacbA [0334] SEQ ID NO: 67 Staphylococcins_C55a_SacaA

Mutacin

[0335] SEQ ID NO: 68 lanA w leader (from UniProt 68586) [0336] SEQ ID NO: 69 lanA w/o leader (from UniProt 68586) [0337] SEQ ID NO: 70 Consensus sequence for Class HA (wherein Xaa is any aa) [0338] SEQ ID NO: 71 nisZ gene codon optimised [0339] SEQ ID NO: 72 nisB gene codon optimised [0340] SEQ ID NO: 73 nisT gene codon optimised [0341] SEQ ID NO: 74 nisC gene codon optimised [0342] SEQ ID NO: 75 garvicin Q gene codon optimised (garQ.sup.Cgl) [0343] SEQ ID NO: 76 garvicin I gene codon optimised (garI.sup.Cgl) [0344] SEQ ID NO: 77 garvicin C gene codon optimised (garC.sup.Cgl) [0345] SEQ ID NO: 78 garvicin D gene codon optimised (garD.sup.Cgl) [0346] SEQ ID NO: 79 Sec-dependent secretion signal of aminopeptidase YwaD of Bacillus subtilis (peptide)