Method for producing metabolites, peptides and recombinant proteins

Abstract

The present invention relates to a method for producing a molecule of interest in bacteria which is based on reversible growth arrest of the bacteria at cellular growth global control system level, thus allowing an improved yield of production of said molecule of interest.

Claims

1. A method for producing at least one metabolite, peptide, or recombinant protein of interest, said method comprising the steps consisting in: a) culturing bacteria comprising: (i) a gene encoding said recombinant protein or at least one gene encoding an enzyme involved in the production of said peptide or metabolite, wherein said gene is operably linked to a promoter, and (ii) genes encoding the ββ′ subunits of a bacterial RNA polymerase operably linked to an inducible promoter, wherein the promoter operably linked to the genes in (i) and (ii) are not the same, in a first culture medium comprising an inducer of the inducible promoter operably linked to the genes of (ii) and thereby inducing the expression of the genes encoding said ββ′ subunits, thereby inducing bacterial growth; b) culturing said bacteria in a second culture medium lacking the inducer and thereby inhibiting the expression of the genes encoding said ββ′ subunits and inhibiting bacterial growth while the gene encoding said recombinant protein or at least one gene encoding an enzyme involved in the production of said peptide or metabolite continues to be expressed, thereby producing said metabolite, peptide or recombinant protein; c) optionally iterating steps a) and b) successively; and d) optionally recovering said metabolite, peptide or recombinant protein produced by said bacteria.

2. The method according to claim 1, wherein said inducible promoter is an IPTG-dependent promoter.

3. The method according to claim 1, wherein the first culture medium of step a) comprises IPTG and the second culture medium of step b) is free of IPTG.

4. The method according claim 1, wherein the gene encoding said recombinant protein or said at least one gene encoding an enzyme involved in the production of said peptide or metabolite is transcribed by a second RNA polymerase having a catalytic subunit or catalytic subunits that are different from the sp′ subunits of the RNA polymerase operably linked to the inducible promoter.

5. The method according to claim 4, where said second RNA polymerase is the bacteriophage T7 polymerase.

6. The method according to claim 1, wherein said bacteria comprise at least two copies of the lacI gene.

7. The method according to claim 1, wherein said bacteria are Escherichia coli bacteria.

8. The method according to claim 1, wherein said genes encoding the ββ′ subunits of RNA polymerase are the rpoBC genes of the rpoBC operon.

9. The method according to claim 1, wherein the bacteria are cultured in step a) until a population density comprised from 0.1 to 100 OD.sub.600 is reached.

10. The method according to claim 1, further comprising a step a′) between steps a) and b) consisting in harvesting and optionally washing the bacteria cultured in step a) and transferring them into the second culture medium of step b).

11. The method according to claim 1, further comprising measuring the metabolic activity of the cultured bacteria during step b).

12. The method according to claim 11, wherein step c) is carried out when the metabolic activity measured during step b) decreases.

13. The method according to claim 1, wherein said first culture medium of step a) comprises M9 minimal medium supplemented with 0.4% glucose and 0.5 mM IPTG and said second culture medium of step b) comprises M9 minimal medium supplemented with 0.4% glucose and is free of IPTG.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: rpoBC operon in E. coli and cloning cassette.

(2) A. The rpoBC operon comprises a first transcriptional unit comprising the rplK and rplA genes, a second transcriptional unit comprising the rplJ and rplL genes, and a third unit comprising the rpoB and rpoC genes encoding the β and β′ subunits, respectively. A terminator (T) is present downstream of the rpoC gene. A transcription attenuator (A) is present between the rplL gene and the rpoB gene. The homologous sequences of the chromosome and the cassette are also shown (dotted).

(3) B. The cloning cassette of sequence SEQ ID NO: 4 or sequence SEQ ID NO: 7 comprises two homologous sequences (dotted) flanking the rrnBt1 terminator sequence (T), the spcR selection cassette, two lac operator sequences and the T5 inducible promoter.

(4) FIG. 2: Modifications of the chromosome of an E. coli bacterium according to the invention, such as IJ40 strain bacterium.

(5) The chromosome was modified by integration of an inducible promoter operably linked to the rpoBC operon and the replacement of genes galK and intS by two copies of the lacI gene.

EXAMPLES

Material and Methods

(6) Bacteria

(7) Escherichia coli strain IJ40 is derived from E. coli K12 BW25113 strain.

(8) Strain IJ40 was obtained from E. coli K12 BW25113 by integration into the chromosome of the T5 IPTG dependent-promoter in front of the rpoBC operon and two copies of the lacI gene (cf. FIG. 2). The cloning cassette comprising the T5 IPTG dependent-promoter is shown in sequence SEQ ID NO: 7. The two copies of the lacI gene have been integrated in replacement of the genes galK and intS (as shown in sequences SEQ ID NO: 5 and 6, respectively); they provide a large amount of the Lac repressor and prevent the appearance of fixed mutation.

(9) Plasmids

(10) pSB-crp-lux plasmid comprising a transcriptional fusion of the promoter of the gene encoding the Crp transcription regulator and the luxCDABE operon is used for assessing the cellular metabolism activity after growth arrest.

(11) pSKG plasmid, comprising two enzymes, the glycerol-3-P dehydrogenase (encoded by the gpd1 gene) and the glycerol-3-P phosphatase (encoded by the gpp2 gene) from yeast is used for the production of glycerol (Liang et al. (2011), Appl. Microbiol. BiotechnoL, 89:57-62).

(12) pUA66 plasmid carrying a transcriptional fusion of the pRM promoter of phage λ, which is constitutive in non-infected E. coli cells (Berthoumieux et al. (2013), Mol. Syst. Biol. 9:634), and the gene coding for the fast-folding, stable fluorescent protein GFPmut2 (Zaslaver et al. (2006), Nat. Methods 3:623-628) is used to transform Escherichia coli IJ40 strain for the production of a recombinant fluorescent protein.

(13) Medium

(14) M9 minimal medium has a standard composition, as for example described in Cold Spring Harbor Protocols (doi:10.1101/pdb.rec12295, 2010).

(15) The composition of the M9 minimal medium used in the examples is as follows: disodium phosphate: 6.8 g/l, potassium phosphate: 3 g/l, sodium chloride: 0.5 g/l, ammonium chloride: 2 g/l, magnesium sulphate: 0.24 g/l, calcium chloride: 0.011 g/l.

(16) The medium used for the bacterial growth is said M9 minimal medium supplemented with 0.4% glucose and 0.5 mM IPTG.

(17) The medium used for inhibiting bacterial growth is said M9 minimal medium supplemented with 0.4% glucose.

(18) Production of the Metabolite, Peptide or Recombinant Protein

(19) The method of production of the metabolite, peptide or recombinant protein of interest comprises the following steps.

(20) A pre-culture of strain IJ40 cells transformed with the plasmid of interest is carried out for 18 hours à 37° C. in a M9 medium supplemented with 0.4% of glucose containing 0.5 mM IPTG. At time zero, IPTG is removed from the overnight culture by centrifugation for 5 min at 4,000 g and the cells are washed with fresh M9 medium without IPTG. This operation is repeated twice and the inoculum size is adjusted to give equal optical density to all cultures. The washed cultures of equal optical density are diluted 100 fold into fresh medium supplemented with 0.4% of glucose and containing 0.5 mM IPTG to reach an initial OD.sub.600 of 0.01.

(21) This first step of culture is carried out at 37° C. until the density of population of 0.5 OD.sub.600 is reached. The cells of the first step are then harvested and washed with fresh M9 medium without IPTG, before being transferred into a fresh M9 medium supplemented with 0.4% of glucose without IPTG. This second step of culture is carried out at 37° C. until after about 10 h. The cultures of the first and second steps are shaken and the absorbance at 600 nm is read every 5 minutes by an automated plate reader.

(22) The cells of the second step may then be harvested and washed with fresh M9 medium without IPTG, before being transferred into a fresh M9 medium supplemented with 0.4% of glucose and containing 0.5 mM IPTG. The above first step and second step may then be repeated once or several times.

(23) Results

(24) (i) Assessment of the Cellular Metabolism Activity after Growth Arrest

(25) Escherichia coli IJ40 strain is transformed with the pSB-crp-iux plasmid comprising a transcriptional fusion of the promoter of the gene encoding the Crp transcription regulator, whose expression is known to vary little across growth phases (Kuhlman et al. (2007), Proc. Natl Acad. Sci. USA 104: 6043-6048), and the luxCDABE operon, which encodes the enzymes necessary for the production of bacterial luciferase as well as the production of the aldehyde substrate of bacterial luciferase.

(26) The production of the transformed strain is carried out with a first step of culture in the medium with IPTG and a second step of culture in the same medium free of IPTG as described in the Material and Methods.

(27) The metabolic activity after growth arrest of this strain manifests itself through a sustained glucose influx, leading to the maintenance of a high ATP/ADP ratio. This latter ratio can be measured using the luminescent reporter encoded by the pSB-crp-iux plasmid, since ATP is a co-factor required for luminescence production (Meighen et al. (1999), Microbiol. Rev. 55:123-142). In particular, a non-decreasing luminescence level per cell after growth arrest is an indicator of the maintenance of the ATP/ADP ratio. This is experimentally verified when the luminescence emitted by a population of E. coli cells is measured over time and divided by the optical density of the culture.

(28) Cellular metabolism is thus active after growth arrest and the above-mentioned method can be used for the production of metabolites, peptides or recombinant protein.

(29) (ii) Production of Glycerol in Bacteria with Improved Yield

(30) Escherichia coli IJ40 strain is transformed with the pSKG plasmid (or an equivalent plasmid expressing the same functional enzymes), allowing the expression of two enzymes, glycerol-3-P dehydrogenase (encoded by the gpd1 gene) and glycerol-3-P phosphatase (encoded by the gpp2 gene) from yeast (Liang et al. (2011), Appl. Microbiol. Biotechnol., 89:57-62). These enzymes convert the glycolysis intermediate dihydroxyacetone-phosphate into glycerol, thus turning the recombinant IJ40 strain into a glycerol production strain.

(31) The production of glycerol is carried out with a first step of culture in the medium with IPTG and a second step of culture in the same medium free of IPTG, as described in the Material and Methods.

(32) Glucose and glycerol concentrations are measured in the medium over time, by means of commercially-available kits and the yield is computed.

(33) The transformed IJ40 strain is capable of glycerol production during growth on glucose and, more importantly, during growth arrest. Moreover, the yield of glycerol in the growth-arrested state is higher than during growth on glucose.

(34) (iii) Production of a Recombinant Fluorescent Protein in Bacteria with Improved Yield

(35) Escherichia coli IJ40 strain is transformed with a pUA66 plasmid carrying a transcriptional fusion of the pRM promoter of phage λ, which is constitutive in non-infected E. coli cells (Berthoumieux et al. (2013), Mol. Syst. Biol. 9:634), and the gene coding for the fast-folding, stable fluorescent protein GFPmut2 (Zaslaver et al. (2006), Nat. Methods 3:623-628). The measured fluorescence is proportional to the total quantity of GFP in the cell population. The production of the recombinant fluorescent protein is carried out with a first step of culture in the medium with IPTG and a second step of culture in the same medium free of IPTG, as described in the Material and Methods.

(36) The fluorescence emitted by the culture and the optical density in a microplate reader are dynamically monitored.

(37) Cells continue to emit fluorescence after growth arrest and the GFP concentration per cell, obtained by dividing the fluorescence intensity by the optical density, is larger for the growth-arrested cells than for those growing on glucose.

CONCLUSIONS

(38) The method of the invention based on the possibly repeated arrest and restart of the GEM allows improving the production of molecules for biotechnological applications. Cellular metabolism is active after growth arrest and the method according to the invention allows improving the yield of metabolites, peptides, and recombinant proteins.