FERMENTATION PROCESS

Abstract

Some embodiments relate to a method for producing a product of interest with a microbial host using an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid molecule is controlled.

Claims

1. Method for producing a product of interest with a microbial host, said method comprising the steps of: a) Providing the microbial host comprising an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, wherein the genetic activity of said first nucleic acid sequence is controlled; b) Optionally said auto-replicative extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence that is involved in the production of said product of interest, wherein the genetic activity of said second nucleic acid sequence is controlled independently from the one of the first sequence; c) Culturing said transformed microbial host under conditions allowing said transformed microbial host to express the first nucleic acid sequence to a given level to maintain the auto-replicative extra-chromosomal molecule into the growing microbial population and simultaneously genetically controlling the second sequence coding for said product of interest.

2. The method according to claim 1, further comprising transforming the microbial host with said auto-replicative extra-chromosomal nucleic acid molecule prior to or during step a), wherein the auto-replicative extra-chromosomal nucleic acid molecule optionally comprises the second nucleic acid sequence of step b), thereby providing the microbial host comprising the auto-replicative extra-chromosomal nucleic acid molecule.

3. A method according to claim 1 or 2, wherein at least in part of step c) conditions are such that the first nucleic acid sequence does not exhibit said genetic activity.

4. A method according to any one of claims 1 to 3, wherein the product of interest is purified at the end of the culturing step c).

5. A method according to any one of claims 1 to 4, wherein the product of interest is a microbial biomass, the auto-replicative extra-chromosomal nucleic acid molecule, the transcript of said second nucleic sequence, a polypeptide encoded by said second sequence or a metabolite produced directly or indirectly by said polypeptide.

6. A method according to any one of claims 1 to 5, wherein the microbial host is a bacterium, yeast, filamentous fungus or an algae

7. A method according to any one of claims 1 to 6, wherein the first nucleic acid sequence is operably linked to an inducible promoter.

8. A method according to any one of claims 1 to 7, wherein the first nucleic acid sequence comprises a sequence coding for an immunity gene whose expression confers to its host a resistance to the presence of a specific bacteriocin in the medium.

9. A method according to claim 8, wherein the sequence encoded by the first nucleic acid sequence confers to its host a resistance to the presence of at least two distinct bacteriocins in the medium.

10. A method according to claim 9, wherein the bacteriocin is B17, C7 or ColV and the immunity conferring resistance to a B17 is McbG, to C7 is either MccE or C-terminal MccE and to a ColV is Cvi.

11. A method according to any one of claims 1 to 10, wherein the auto-replicative extra-chromosomal nucleic acid molecule is a plasmid.

12. An auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to a microbial host wherein the genetic activity of said first nucleic acid sequence is controlled, and optionally comprising a second nucleic acid sequence that is directly or indirectly involved in the production of a product of interest.

13. An auto-replicative extra-chromosomal nucleic acid molecule according to claim 12, wherein the first nucleic acid sequence is operably linked to an inducible promoter.

14. An auto-replicative extra-chromosomal nucleic acid molecule according to claim 12 or 13 which is a plasmid.

15. An auto-replicative extra-chromosomal nucleic acid molecule according to any one of claims 12 to 14, wherein the first nucleic acid sequence comprises a sequence coding for an immunity gene whose expression confers to its host a resistance to the presence of a specific bacteriocin in the medium.

16. An auto-replicative extra-chromosomal nucleic acid molecule according to any one of claim 15, wherein the sequence encoded by the first nucleic acid sequence confers to its host a resistance to the presence of at least two distinct bacteriocins in the medium.

17. An auto-replicative extra-chromosomal nucleic acid molecule according to claim 16, wherein the bacteriocin is B17, C7 or ColV and the immunity modulator conferring resistance to a B17 is McbG, to C7 is either MccE or C-terminal MccE) and to ColV is Cvi.

18. A microbial host comprising the auto-replicative extra-chromosomal nucleic acid molecule of any one of claims 11 to 16, optionally wherein the microbial cell is a bacterium, yeast, filamentous fungus or an algae,

Description

DESCRIPTION OF THE FIGURES

[0098] FIG. 1: Construction: pSyn2-McbE/F: containing the gene McbE and F under Ptac.

[0099] FIG. 2: Construction: pSyn2-McbG: containing the gene McbG under Ptac.

[0100] FIG. 3: Construction: pMcbG 1.1: containing the gene McbG under P24.

[0101] FIG. 4: Construction: pMcbG 1.0: containing the gene McbG under P24 LacO.

[0102] FIG. 5: pBACT5.0 vector

[0103] FIG. 6: pBACT2.0 vector

[0104] FIG. 7: pBACT5.0-mcherry vector

[0105] FIG. 8: Tuning promoter. In the absence of inductor (upper part), repressor can bind to operator and prevent expression of selection gene. In the presence of inductor (lower part), repressor cannot bind to operator allowing expression of selection gene.

[0106] FIG. 9: Comparison of overexpression of protein X in E. coli with KanR (pKan-pLac) and with 2 immunities against microcines C7 and ColV (pBACT6.0-pLac). 5 mg of total extract was analysed in SDS-PAGE.

[0107] FIG. 10: Comparison of overexpression of iota-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE.

[0108] FIG. 11: Comparison of overexpression of lambda-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE.

EXAMPLES

Example 1: Use of Bacteriocin B17 and C7 as Selection Agent

1. Production of Bacteriocin B17, C7 and ColV

[0109] Strain used: C600: F.sup. tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 .sup.

[0110] Described in Appleyard Genetics 39 (1954), 440-452.

[0111] The vector used for producing Mic B17 is described in the table below.

TABLE-US-00015 TABLE X Vector used for producing Mic B17 Construct used pACYC184 containing the mccB17-producing genes pCID909 (mcbABCDEFG), chloramphenicol resistance

[0112] These constructs were described in detail in Rodriguez-Sainz, M. C., C. et al. 1990. Mol. Microbiol. 4:1921-1932.

[0113] The vector used for producing Mic C7 is Pp70. This vector is based on pBR322 and bears a 6000 bp DNA fragment with the mcc gene cluster (as described in Zukher I et al, Nucleic Acids Research, 2014, Vol. 42, No. 19 11891-11902).

[0114] The vector used for producing ColV is pUC-ColV (SEQ ID NO: 719). This vector is based on pUC57 and bear a 5000 bp DNA fragment with the ColV gene cluster. The strains harbouring these recombinant vectors were grown in LB medium at 37 C.

[0115] After an overnight culture the fermented medium was centrifuged and the supernatant flit red on a 0.2 micron filter.

[0116] The bacteriocin activity present in the supernatant was estimated by the size of the diffusion inhibition growth on a plate containing a sensitive strain.

2. Results

[0117] We demonstrated that we can use the supernatants that exhibit B17, C7 or ColV activities as classical antibiotics such as Amp, Kan or Chlo added in culture medium. Supernatant presenting such a bacteriocin activity were stored for several months (at least 12 months) at 20 C. and we did not observe a significant decrease of activity. Petri plates containing medium with such a bacteriocin activity were stored at +4 C. for several weeks (at least 4 weeks). We did not observe a decrease of activity. Therefore we demonstrated that such B17, C7 or ColV activities as present in culture medium are stable.

3. Conclusion

[0118] Bacteriocins B17, C7 and ColV produced by fermentation in laboratory are selection agents simple to produce, easy to use and stable in culture medium. These properties are similar to the ones of antibiotics used as classical selection agent.

Example 2: Identification of the Minimum Genetic Elements Necessary to Confer Resistance to C7 and B17

1. Construction of Needed Vectors

[0119] The literature has made it possible to determine the elements necessary for the production of the host against the production of its own bacteriocin, also in the case of B17 bacteriocin: McbG for B17, represented by SEQ ID NO: 699 and pumps (McbE and McbF for B17, represented by SEQ ID NO: 703). These genes are known to be necessary (or more precisely involved in protection against the action of bacteriocin B17). The literature for the B17 locus does not identify which is or is the sufficient element to give resistance.

[0120] We have separated genes from B17 immunity structures and cloned these into vectors behind an inducible promoter (Ptac).

[0121] Construction: pSyn2-McbG (FIG. 2, SEQ ID NO: 702): containing the gene McbG under Ptac

[0122] Construction: pSyn2-McbE/F (FIG. 1, SEQ ID NO: 703): containing the gene McbE and F under Ptac

[0123] We have separated the genes from B17 immunity structures and cloned them into vectors behind an inducible promoter (Ptac).

[0124] We have shown that low McbG expression (Ptac not induced) is sufficient to give the phenotype of resistance to the strain on the other hand the presence of McbE/F is toxic and did not allow to give a response As to the protection provided in relation to the presence of B17.

2. Results

[0125] Surprisingly it has been found that the C-terminal part of MccE which is represented by SEQ ID NO: 701 is sufficient to confer resistance to bacteriocin C7.

[0126] We have demonstrated that expression from a plasmid of the McbG and MccE genes (or truncated MccE, represented by SEQ ID NO: 701) are capable when cloned into a vector to give resistance to B17 and C7 respectively and that these proteins can be used as a vector selection marker in strains sensitive to these microcines/bacteriocins. The vector used is pBACT2.0 (FIG. 6, SEQ ID NO: 713).

[0127] SEQ ID NO: 710 represents the construct proC-McbG-CterMccE

[0128] We have demonstrated that expression from a plasmid of the Cvi and C-terminal part of MccE genes are capable when cloned into a vector to give resistance to ColV and C7 respectively and that these proteins can be used as a vector selection marker in strains sensitive to these microcines/bacteriocins. The vector used is pBACT5.0 (FIG. 5, SEQ ID NO: 712).

[0129] SEQ ID NO: 711 represents the construct proC-Cvi-CterMccE

3. Conclusion

[0130] It is therefore possible to use a single segment of small size represented by SEQ ID NO: 701 as a selection marker against C7.

Example 3: Can we Generate a Selectable Marker Using Little or No Energy from the Bacteria from McbG?

1. Vectors Constructed

[0131] To answer this question the McbG gene was cloned under a weak promoter P24 (SEQ ID NO: 707). The P24 promoter was described in Braatsch S et al, Biotechniques. 2008 September; 45(3):335-7.

[0132] We inserted the B17 McbG immunity structure gene and cloned the latter into vectors behind the weak constitutive promoter (P24). A second construct was generated with a P24 LacO hybrid promoter which is an inducible promoter repressed in the presence of lad and active in presence of IPTG.

[0133] Construction: pMcbG 1.0 (FIG. 4, SEQ ID NO: 704) containing the gene McbG under P24 LacO.

[0134] Construction: pMcbG 1.1 (FIG. 3, SEQ ID NO: 705) containing the gene McbG under P24.

[0135] The strains used are the following:

BL21(DE3): fhuA2 [lon] ompT gal ( DE3) [dcm] hsdS
DE3= sBamHIo EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 nin5

[0136] The BL21(DE3) strain was transformed with vector pMcbG1.0 or pMcbG1.1 (see FIG. 3 or 4). After transformation, transformants were selected on plates containing B17. Isolated colonies were re-grown and the plasmid they contained was analyzed by gel electrophoresis after treatment with relevant restriction enzymes.

2. Results

[0137] We showed that a weak transcription of McbG is sufficient to give the resistance to B17. In addition, we have shown that this selection marker is inducible via the P24 LacO promoter and that the vectors containing this gene gives the phenotype of resistance only in presence of IPTG.

3. Conclusions

[0138] It is possible to use the McbG gene as a selectable marker either constitutively or inducibly. Thus, it is shown that constitutive expression at a low level and inducible expression according to the need during the process, allows to reduce the energy burden for the producing cell, without loss of the plasmid from the producing cell.

Example 4: Production of m-Cherry Protein

[0139] SEQ ID NO: 714 represents the construct used for producing the m-cherry protein. This construct is depicted in FIG. 7.

[0140] The m-cherry protein was produced and visualised as the bacterial colony turns red on petri dish in the presence of IPTG.

Example 5: Tuning Promoter

[0141] We have prepared vectors with immunity selection that is tunable (see FIG. 8). Tuning the promoter allows us to switch the selection on or off. For the first time this allows to adapt the selective pressure to the need during the fermentation process, for example according to the loss by the host of the recombinant plasmid. This will provide an advantage by limiting the burden of energy for the host. Thus, such vectors improve the industrial outcome (recombinant product). Moreover, they are easy to use in any strain (no requirement for a special feature in the host genome) and require no antibiotics.

Example 6: Comparison of Antibiotic (Kanamycin) Selection with Immunity Selection

[0142] We have applied immunity selection on different recombinant proteins.

[0143] FIG. 9 shows the comparison of overexpression of protein X in E. coli with KanR (pKan-pLac) and with 2 immunities against microcines C7 and ColV (pBACT6.0-pLac). 5 mg of total extract was analysed in SDS-PAGE.

[0144] FIG. 10 shows the comparison of overexpression of iota-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE. The vector used is based on the pBACT5.0 vector (SEQ ID NO: 712).

[0145] FIG. 11 shows the comparison of overexpression of lambda-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE. The vector used is based on the pBACT5.0 vector (SEQ ID NO: 712).

[0146] The weak constitutive proC promoter used in this example allows to reduce the energy burden for the host.