BACTERIAL STRAIN AND METHOD FOR HIGH THROUGHPUT OF SUGAR IN THE MICROBIAL CONVERSION INTO BIOSYNTHETIC PRODUCTS

Abstract

The present invention relates to recombinant Escherichia coli (E. coli) host cells comprising, in relation to wild-type cells, at least one mutation selected from the group consisting of deletion of the gene relA (relA); amino acid substitutions R290E and K292D in the protein guanosine-3,5-bis pyrophosphate 3-pyrophosphohydrolase (bifunctional (p)ppGpp synthetase II; SpoT) (spo T[R290E;K292D]); and amino acid substitution G267C in the protein pyruvate dehydrogenase subunit E1 (AceE) (aceE[G267C]). Said recombinant host cells are characterized by increased sugar uptake rates that lead to increased productivity when using said cells for the production of biosynthetic products. The present invention further relates to respective methods for the biosynthetic production of a product of interest using said host cells.

Claims

1. A recombinant Escherichia coli (E. coli) host cell, wherein said cell comprises the following mutations in relation to a wild-type cell: (i) deletion of the gene relA (relA); (ii) amino acid substitutions R290E and K292D in the protein guanosine-3, 5-bis pyrophosphate 3-pyrophosphohydrolase (bifunctional (p)ppGpp synthetase II; SpoT) (spoT[R290E;K292D]); and (iii) amino acid substitution G267C in the protein pyruvate dehydrogenase subunit E1 (AceE) (aceE[G26C]).

2. The recombinant host cell according to claim 1, wherein said cell is derived from E. coli strain E. coli K-12.

3. The recombinant host cell according to claim 2, wherein said cell is derived from E. coli strain E. coli K-12 substrain MG1655.

4. A method for the biosynthetic production of a product of interest (POI), wherein said POI is produced in a recombinant host cell according to claim 1.

5. The method according to claim 4, wherein said POI is a protein and said protein is expressed in said recombinant host cell.

6. The method according to claim 4, wherein said POI is an organic molecule and said organic molecule is produced in said recombinant host cell as a metabolite.

7. The method according to claim 6, wherein said organic molecule is selected from the group consisting of pyruvate, lactate, and acetate.

8. The method according to claim 6 or claim 7, wherein the starting material for the production of said metabolite is a sugar.

9. The method according to claim 8, wherein said sugar is selected from the group consisting of glucose, fructose, mannose, xylose, arabinose, and sucrose.

10. The method according to claim 4, wherein the metabolic activity of the recombinant host cells is reduced by limiting the amount of available nitrogen sources.

11. (canceled)

Description

[0035] The figures show:

[0036] FIG. 1:

[0037] Batch cultivation of the Escherichia coli MG1655 wild-type strain in a minimal medium supplemented with 18 g/L glucose as sole C-source and 40 mM NH.sub.4.sup.+ as sole N-source at starting conditions. After 6 hours glucose is still in excess and the nitrogen source is consumed to a minimum residual concentration. Exponential bacterial growth stops immediately. Data points and error bars derive from three parallel fermentations n=3.

[0038] FIG. 2:

[0039] Batch cultivation of the Escherichia coli K-12 MG1655 aceE[G267C] strain in a minimal medium supplemented with 30 g/L glucose as sole C-source and 40 mM NH.sub.4.sup.+ as sole N-source at starting conditions. After 16 hours glucose is still in excess and the nitrogen source is consumed to a minimum residual concentration. Exponential bacterial growth stops immediately. Data points and error bars derive from three parallel fermentations n=3.

[0040] FIG. 3:

[0041] Batch cultivation of the Escherichia coli K-12 MG1655 relA spoT[R290E;K292D] strain in a minimal medium supplemented with 28 g/L glucose as sole C-source and 40 mM NH.sub.4.sup.+ as sole N-source at starting conditions. After 5.4 hours glucose is still in excess and the nitrogen source is consumed to a minimum residual concentration. Exponential bacterial growth stops immediately. Data points and error bars derive from three parallel fermentations n=3.

[0042] FIG. 4:

[0043] Batch cultivation of the Escherichia coli K-12 MG1655 relA spoT[R290E;K292D] aceE[G267C] strain in a minimal medium supplemented with 28 g/L glucose as sole C-source and 40 mM NH.sub.4.sup.+ as sole N-source at starting conditions. After 15 hours glucose is still in excess and the nitrogen source is consumed to a minimum residual concentration. Exponential bacterial growth stops immediately. Data points and error bars derive from three parallel fermentations n=3.

[0044] The present invention will be further illustrated by the following examples without being limited thereto.

EXAMPLES

[0045] Material and Methods:

[0046] Media and SolutionsPreculture Minimal Medium

[0047] Solution A: (10Salts)

TABLE-US-00001 NaH.sub.2PO.sub.42 H.sub.2O 98.44 g/L K.sub.2HPO.sub.4 46.86 g/L (NH.sub.4).sub.2HPO.sub.4 13.21 g/L (NH.sub.4).sub.2SO.sub.4 26.80 g/L Na.sub.2SO.sub.4 8.80 g/L pH was adjusted to pH 7.0 with KOH

[0048] Solution B: (1000Ca.sup.2+)

TABLE-US-00002 CaCl.sub.22 H.sub.2O 14.70 g/L

[0049] Solution C: (1000Mg.sup.2+)

TABLE-US-00003 MgSO.sub.47 H.sub.2O 246.48 g/L

[0050] Solution D: (2000Trace Elements Solution=TES)

TABLE-US-00004 FeCl.sub.36 H.sub.2O 16.70 g/L Na.sub.2-EDTA 20.10 g/L ZnSO.sub.47 H.sub.2O 0.18 g/L MnSO.sub.4H.sub.2O 0.10 g/L CuSO.sub.45 H.sub.2O 0.16 g/L CoCl.sub.26 H.sub.2O 0.18 g/L

[0051] Solution E: (1000Vitamin)

TABLE-US-00005 Thiamine HCl 10.00 g/L

[0052] Solution F: (50% glucose w/v)

TABLE-US-00006 -D(+)-GlucoseH.sub.2O 500.00 g/L

[0053] Media and SolutionsBatch Minimal Medium

[0054] Solution A.2: (10Salts)

TABLE-US-00007 NaH.sub.2PO.sub.42 H.sub.2O 98.44 g/L K.sub.2HPO.sub.4 46.86 g/L (NH.sub.4).sub.2HPO.sub.4 13.21 g/L (NH.sub.4).sub.2SO.sub.4 12.68 g/L Na.sub.2SO.sub.4 8.80 g/L pH was adjusted to pH 7.0 with KOH

[0055] Solution B: (1000Ca.sup.2+)

TABLE-US-00008 CaCl.sub.22 H.sub.2O 14.70 g/L

[0056] Solution C: (1000Mg.sup.2+)

TABLE-US-00009 MgSO.sub.47 H.sub.2O 246.48 g/L

[0057] Solution D: (2000Trace Elements Solution=TES)

TABLE-US-00010 FeCl.sub.36 H.sub.2O 16.70 g/L Na.sub.2-EDTA 20.10 g/L ZnSO.sub.47 H.sub.2O 0.18 g/L MnSO.sub.4H.sub.2O 0.10 g/L CuSO.sub.45 H.sub.2O 0.16 g/L COCl.sub.26 H.sub.2O 0.18 g/L

[0058] Solution E: (1000Vitamin)

TABLE-US-00011 Thiamine HCl 10.00 g/L

[0059] Solution F: (50% glucose w/v)

TABLE-US-00012 -D(+)-GlucoseH.sub.2O 500.00 g/L

[0060] Preculture Shaking Flask Cultivation

[0061] To prepare the preculture minimal medium, solutions A, B and C, described above, were prepared separately and also separately sterilized at 120 C. for 20 min. Solutions D, E and F were separately prepared and sterile filtrated at 0.2 pm pore size. For 1 L of ready-to-use preculture medium 100 mL 10salts, 1 mL 1000Ca.sup.2+ stock solution, 1 mL 1000Mg.sup.2+ stock solution, 0.5 mL 2000TES, 1 mL 1000Vitamin stock solution, 10 mL 50% w/v glucose stock solution and 886.5 mL sterile water were mixed. Sterile 500 mL Erlenmeyer shaking flasks with baffles were filled with 60 mL of the preculture minimal medium. For each strain the preculture was carried out in parallel with three uniquely inoculated shaking flasks at 37 C. and constant agitation. After incubation, the bacterial cells were harvested by centrifugation (4500g, 10 min, 4 C.) and diluted to an Optical Density of about 8.0 in a volume of 5 mL. This cell suspension was used for inoculation of the bioreactors.

[0062] Batch Cultivation

[0063] To prepare the batch minimal medium, solutions B and C, described above, were prepared separately and also separately sterilized at 120 C. for 20 min. Solutions D, E and F were separately prepared and sterile filtrated at 0.2 m pore size.

[0064] All fermentation processes were carried out in a parallel cultivation system consisting of three identical HWS glass bioreactors with a working volume of 250 mL each. After assemblage of the cultivation system, every bioreactor was separately filled with 20 mL of 10salts (solution A.2) and 160 mL of water. This diluted salt solution was sterilized in every bioreactor at 120 C. for 20 min. After sterilization a total volume of 15 mL containing: 7.2 mL 50% w/v glucose stock solution (E. coli K-12 MG1655 wild-type) or 11.2 mL 50% w/v glucose stock solution (E. coli K-12 MG1655 relA spoT[R290E;K292D], E. coli K-12 MG1655 relA spoT[R290E;K292D] aceE[G267C]) or 12 mL 50% w/v glucose stock solution (E. coli K-12 MG1655 aceE[G267C]), 0.2 mL 1000Ca.sup.2+ stock solution, 0.2 mL 1000Mg.sup.2+ stock solution, 0.1 mL 2000TES, 0.2 mL 1000Vitamin stock solution and 7.1 mL water or 3.1 mL water or 2.3 mL water, respectively, was added sterile to every vessel. Each bioreactor was inoculated with 5 mL of a concentrated preculture giving a starting Optical Density of 0.2. Fermentations were performed at a constant temperature of 37 C., agitation and good oxygen supply. The process length varied for every E. coli K-12 MG1655 strain. Individual fermentation durations are mentioned for the corresponding strains in Examples 1 to 4, below.

[0065] Nitrogen-Limited Batch Cultivation Phase

[0066] Each and every batch cultivation process started with identical conditions, except of varying initial glucose concentrations for the processes of E. coli K-12 MG1655 wild-type/E. coil K-12 MG1655 relA spoT[R290E;K292D]) and E. coli K-12 MG1655 aceE[G267C]/E. coli K-12 MG1655 relA spoT[R290E;K292D] aceE[G267C]. Despite the actual amount of glucose, this sole C-source was always in vast excess at the beginning of every fermentation. This also extends to all additional nutrients in the batch minimal medium, as listed above. For the first couple of hours every E. coli K-12 MG1655 strain was growing exponentially at its very specific maximum growth rate and consumed glucose with its individual biomass-specific uptake rate under non-limited conditions. This state is termed as Exponential Growth in FIGS. 1, 2, 3 and 4. A fixed nitrogen concentration in the minimal medium enables the bacterial cells to form a certain total biomass before entering nitrogen-depleted nutritional conditions. The subsequent N-limited growth phase is further termed as Nitrogen-limited Growth in FIGS. 1, 2, 3 and 4. During this late stage of the fermentation process bacterial growth was highly limited due to nitrogen-depletion. However, the glucose concentration remained abundantly and the rates for biomass-specific glucose consumption under limited growth conditions could be calculated.

[0067] Example 1:

[0068] In this example Escherichia coli K-12 MG1655 wild-type was cultivated under preculture conditions in shaking flasks, as described above, for 12 hours with constant agitation. These bacterial cells were then transferred into the three bioreactors under sterile conditions to start the batch cultivation process. Escherichia coli K-12 MG1655 wild-type cells were cultivated for a total period of 9 hours with a starting concentration of glucose being 18 g/L. In the exponential growth phase the maximal biomass-specific growth rate was 0.7180.007 h.sup.1 and glucose was consumed at a biomass-specific rate of 1.7650.056 g.sub.Glc/g.sub.cdw.Math.h (cdw: cell dry weight). As can be seen in FIG. 1, these cells were growing exponentially during the first 6 hours of cultivation before all the NH.sub.4.sup.+ in the minimal medium was depleted and the nitrogen-limited growth phase was reached. During the following 3 hours of cultivation the bacterial cells showed a limited linear growth behavior and also a linear progression of glucose consumption. The biomass-specific glucose uptake rate in the nitrogen-limited cultivation phase from hour 6 to 9 averaged at a value of 0.2450.011 g.sub.Glc/g.sub.cdw.Math.h and the biomass-specific growth rate dropped to a value of 0.0430.004 h.sup.1.

[0069] Example 2:

[0070] In this example Escherichia coli K-12 MG1655 aceE[G267C] was cultivated under preculture conditions in shaking flasks, as described above, for 29.5 hours with constant agitation. These bacterial cells were then transferred into the three bioreactors under sterile conditions to start the batch cultivation process. Escherichia coli K-12 MG1655 aceE[G267C] cells were cultivated for a total period of 23.5 hours with a starting concentration of glucose being 30 g/L. In the exponential growth phase the maximal biomass-specific growth rate was 0.2010.004 h.sup.1 and glucose was consumed at a biomass-specific rate of 1.5120.022 g.sub.Glc/g.sub.cdw.Math.h. As can be seen in FIG. 2, these cells were growing exponentially during the first 16 hours of cultivation before all the NH.sub.4.sup.+ in the minimal medium was depleted and the nitrogen-limited growth phase was reached. During the following 7.5 hours of cultivation the bacterial cells showed a limited linear growth behavior and also a linear progression of glucose consumption. The biomass-specific glucose uptake rate in the nitrogen-limited cultivation phase from hour 16 to 23.5 averaged at a value of 0.3140.012 g.sub.Glc/g.sub.cdw.Math.h and the biomass-specific growth rate dropped to a value of 0.0080.004 h.sup.1.

[0071] Example 3:

[0072] In this example Escherichia coli K-12 MG1655 relA spoT[R290E;K292D] was cultivated under preculture conditions in shaking flasks, as described above, for 11 hours with constant agitation. These bacterial cells were then transferred into the three bioreactors under sterile conditions to start the batch cultivation process. Escherichia coli K-12 MG1655 relA spoT[R290E;K292D] cells were cultivated for a total period of 8.4 hours with a starting concentration of glucose being 28 g/L. In the exponential growth phase the maximal biomass-specific growth rate was 0.7150.003 h.sup.1 and glucose was consumed at a biomass-specific rate of 1.7700.059 g.sub.Glc/g.sub.cdw.Math.h. As can be seen in FIG. 3, these cells were growing exponentially during the first 5.4 hours of cultivation before all the NH.sub.4.sup.+ in the minimal medium was depleted and the nitrogen-limited growth phase was reached. During the following 3 hours of cultivation the bacterial cells showed a limited linear growth behavior and also a linear progression of glucose consumption. The biomass-specific glucose uptake rate in the nitrogen-limited cultivation phase from hour 5.4 to 8.4 averaged at a value of 0.3520.016 g.sub.Glc/g.sub.cdw.Math.h and the biomass-specific growth rate dropped to a value of 0.0140.002 h.sup.1.

[0073] Example 4:

[0074] In this example Escherichia coli K-12 MG1655 relA spoT[R290E;K292D] aceE[G267C] was cultivated under preculture conditions in shaking flasks, as described above, for 29 hours with constant agitation. These bacterial cells were then transferred into the three bioreactors under sterile conditions to start the batch cultivation process. Escherichia coli K-12 MG1655 relA spoT[R290E;K292D] aceE[G267C] cells were cultivated for a total period of 21.3 hours with a starting concentration of glucose being 28 g/L. In the exponential growth phase the maximal biomass-specific growth rate was 0.290 0.012 h.sup.1 and glucose was consumed at a biomass-specific rate of 1.7910.059 g.sub.Glc/g.sub.cdw.Math.h. As can be seen in FIG. 4, these cells were growing exponentially during the first 15 hours of cultivation before all the NH.sub.4.sup.+ in the minimal medium was depleted and the nitrogen-limited growth phase was reached. During the following 6.3 hours of cultivation the bacterial cells showed a limited linear growth behavior and also a linear progression of glucose consumption. The biomass-specific glucose uptake rate in the nitrogen-limited cultivation phase from hour 15 to 21.3 averaged at a value of 0.5960.023 g.sub.Glc/g.sub.cdw.Math.h and the biomass-specific growth rate dropped to a negative value of 0.0100.004 h.sup.1.

[0075] Example 5:

[0076] Specific glucose consumption as determined in Examples 1 to 4 above is summarized in the Tables below.

[0077] Table 1 shows the comparison of biomass-specific rates in different Escherichia coli K-12 MG1655 mutant strains and the wild-type during the nitrogen-limited batch cultivation phase. Values are calculated from at least three parallel fermentations n3. For further comparison, the literature value for ms.sup.true is given in the last row of Table 1. It designates the true maintenance coefficient for glucose for non-growing cells at carbon-limitation conditions.

TABLE-US-00013 TABLE 1 Glucose uptake Growth (N-limited) (N-limited) qs [g/g.sub.cdw .Math. h] [h.sup.1] Strain E. coli K-12 MG1655 wild-type 0.245 0.011 0.043 0.004 E. coli K-12 MG1655 aceE[G267C] 0.314 0.012 0.008 0.004 E. coli K-12 MG1655 relA 0.352 0.016 0.014 0.002 spoT[R290E; K292D] E. coli K-12 MG1655 relA 0.596 0.023 0.010 0.004 spoT[R290E; K292D] aceE[G267C] Escherichia coli ms.sup.true 0.057 0.000 0.000

[0078] Table 2 shows the comparison of biomass-specific rates in different Escherichia coli K-12 MG1655 mutant strains and the wild-type during the initial batch cultivation phase of exponential growth with all nutrients in excess. Values are calculated from at least three parallel fermentations n3.

TABLE-US-00014 TABLE 2 Glucose uptake Growth (Excess) (Excess) qs [g/g.sub.cdw .Math. h] [h.sup.1] Strain E. coli K-12 MG1655 wild-type 1.765 0.056 0.718 0.007 E. coli K-12 MG1655 aceE[G267C] 1.512 0.022 0.201 0.004 E. coli K-12 MG1655 relA 1.770 0.059 0.715 0.003 spoT[R290E; K292D] E. coli K-12 MG1655 relA 1.791 0.059 0.290 0.012 spoT[R290E; K292D] aceE[G267C]

[0079] Discussion:

[0080] According to the present invention, increased sugar uptake rates have been achieved in resting cells by specific targeted interventions into E. coli metabolism and metabolic regulation.

[0081] Concerning metabolic regulation, it is known that the stringent response in E. coli plays a central role under conditions of limited substrate availability. In this context, the alarmone (p)ppGpp (guanosine penta- or tetraphosphate) is an important signal for the induction and mediation of the regulatory response. Previous studies have shown that an increase in L-lysine production can be achieved by an increase of (p)ppGpp availability following over-expression of (p)ppGpp synthetase I, encoded by the E. coli gene relA. With respect to E. coli metabolism, it has been shown that introduction of an artificial ATPase activity into E. coli , leading to a reduction of the available amount of ATP, results in increased glucose uptake.

[0082] Thus, an increase in ppGpp synthesis, e.g. by over-expression of relA, should be advantageous for the intracellular availability of carbon sources. Further, an artificial reduction of the availability of ATP should result in E. coli sugar uptake rates. However, in the present invention, it has been surprisingly found that reduction of ppGpp synthesis by deletion of relA, optionally in combination with a reduction of remaining ppGpp synthetase activity of SpoT by introduction of the spoT[R290E;K292D] mutation, and optionally in further combination with introduction of the mutation aceE[G267C], results in the desired phenotype of increased sugar uptake rates in resting cells which is two- to three-fold higher as compared to wild-type cells.