CHAPTER 3 – Results
3.3. Attempts for in-vivo biosynthesis of Phytylpyrophosphate (PPP) in recombinant E. coli
3.4.4. MGGBQ production in Infors bioreactor
In section 3.4, it was shown that the Hpd, CrtE and Hpt-Syn expressed in E. coli DH5 / pCAS29 strain were active as it resulted in MGGBQ production in LB-Glycerol-Amp100 medium in shaking flask. It was shown that HGA and GGPP was produced using MM-Glucose-Amp100 and MM-Glycerol-Amp100 in E. coli BW25113/pCAS2JF (shaking flasks) and in E. coli LJ110/pCAS30 (bioreactor) respectively, where either glucose or glycerol was supplemented as the sole carbon and energy source. To study the MGGBQ production and to study the robustness of E. coli BW25113 carrying a plasmid pCAS29 (a multi-copy vector), E. coli BW25113/pCAS29 was cultivated in bioreactor in Glucose-Amp100 and MM-Glycerol-Amp100.
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Figure 3.19: Scheme representing strain E. coli BW25113 / pCAS29. Ggh-Syn protein encoded by the ggh-Syn gene in plasmid pCAS29 has been shown in section 3.4.1 to be apparently inactive in-vivo (here marked with orange circle).
E. coli BW25113 / pCAS29 (shown in figure 3.19) was cultivated in a 3.6 L Infors bioreactor (Wald, Switzerland) in MM-Glucose-Amp100 and MM-Glycerol-Amp100. All fermentations carried out with E. coli BW25113/pCAS29 in bioreactor were operated initially in batch mode and later in fed batch mode (i.e. batch-fedbatch). During the fed-batch mode, glucose (in case of MM-Glucose-Amp100) or Glycerol (in case of MM-Glycerol-Amp100) was fed continuously using a feeding pump. Cultivation was done at 30 °C, at constant pH of 7.0 (maintained by addition of 5 N KOH) and pO2 concentration of 30 % oxygen saturation. Each fermentation was repeated, inorder to assess the reproducibility of the results. Results can be seen in Figure 3.20 to 3.22.
Cultures were induced with 0.25 mM IPTG (final conc.) at OD 600nm of approx. 2.0 in MM-Glucose-Amp100 (i.e. at 8 h) and in MM-Glycerol-Amp100 (i.e. at 18 h). At the end of batch process the cell density of 1.7 g/l CDW Glucose-Amp100) and 2.3 g/l CDW (MM-Glycerol-Amp100) was reached. Fed-batch cultivation was started by feeding glucose/glycerol at feed rates (g/h) shown in figure 3.21 such that there was no overflow of glucose or glycerol in the culture medium. Cultures in glycerol medium has a long lag phase compared to that in glucose medium. In glucose medium the cultures grew exponentially during batch process with maximum growth rate of 0.32 h-1 compared to 0.15 h-1 in glycerol medium. Cultures in glucose medium entered stationary phase at 46 h (final OD 600nm of 21.5 + 0.2) compared to 58 h in glycerol medium (final OD 600nm of 26.7 + 0.3).
E. coli BW25113 / pCAS29
ggh-Syn crtE hpt-Syn hpd
ptac RBS RBS RBS RBS
(Ggh-Syn expressed is most likely inactive)
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Figure 3.20: Batch-Fed batch fermentation of E. coli BW25113/pCAS29 in Infors bioreactor for MGGBQ production. Fermentation carried out at 30 °C, at constant pH of 7.0 and pO2 concentration of 30 % of saturation. (A) Cell growth curve and glucose concentration in bioreactor during fermentation in MM-Glucose-Amp100. (B) Cell growth curve and glycerol concentration in bioreactor during fermentation in MM-Glycerol-Amp100.
Cell density represented as OD600nm w.r.t time (scale on left hand side) and glucose or glycerol concentration in gram per liter of culture (scale on right hand side). Vertical lines (a) and (c) show the time of induction 0.25 mM IPTG (final conc.). Vertical lines (b) and (d) show the start of fed-batch process (glucose and glycerol feeding resp.).
0.1 1.0 10.0 100.0
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glucose [g/l]
OD Glucose
0.1 1.0 10.0 100.0
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glycerol [g/l]
OD Glycerol (a) (b)
(c) (d)
(B) (A)
Glucose
Glycerol 0.1
1.0 10.0 100.0
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glucose [g/l]
OD Glucose
0.1 1.0 10.0 100.0
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glycerol [g/l]
OD Glycerol (a) (b)
(c) (d) 0.1
1.0 10.0 100.0
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glucose [g/l]
OD Glucose
0.1 1.0 10.0 100.0
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glycerol [g/l]
OD Glycerol (a) (b)
(c) (d)
(B) (A)
Glucose
Glycerol
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Figure 3.21: Cell growth rate (µ) and Glucose/Glycerol Feed rate (F) during fed batch fermentation of E. coli BW25113/pCAS29. (A) Fermentation carried out in MM-Glucose-Amp100 (B) Fermentation carried out in MM-Glycerol-Amp100. Fermentation temperature of 30 °C, pH of 7.0, and pO2 concentration of 30 % saturation were maintained constant throughout the fermentation.
0.00 0.05 0.10 0.15 0.20 0.25 0.30
0 10 20 30 40 50 60 70
Time [h]
"µ" - Growth rate [h-1 ]
0.00 0.20 0.40 0.60 0.80 1.00 1.20
"F" - Glycerol Feedrate [gh-1 ]
µ F
0.00 0.05 0.10 0.15 0.20 0.25 0.30
0 10 20 30 40 50 60 70
Time [h]
Growth rate- µ [h-1 ]
0.00 0.20 0.40 0.60 0.80 1.00 1.20
Glucose Feedrate [gh-1 ]
µ F
(A)
(B)
0.00 0.05 0.10 0.15 0.20 0.25 0.30
0 10 20 30 40 50 60 70
Time [h]
"µ" - Growth rate [h-1 ]
0.00 0.20 0.40 0.60 0.80 1.00 1.20
"F" - Glycerol Feedrate [gh-1 ]
µ F
0.00 0.05 0.10 0.15 0.20 0.25 0.30
0 10 20 30 40 50 60 70
Time [h]
Growth rate- µ [h-1 ]
0.00 0.20 0.40 0.60 0.80 1.00 1.20
Glucose Feedrate [gh-1 ]
µ F
(A)
(B)
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Figure 3.22: MGGBQ production in E. coli BW25113 / pCAS29 in bioreactor in minimal medium in (A) glucose and (B) glycerol, as sole carbon and energy source. S.D values are calculated from the average values of 2 independent samples, which were separately extracted and analyzed by HPLC. In appendix, a graph showing MGGBQ concentration vs. time graph is shown as figure A.3-10.
The MGGBQ production shown here is the total MGGBQ. This is calculated as follows:
Total MGGBQ = [MGGBQ] reduced + [MGGBQ] oxidized in terms of reduced. [MGGBQ] oxidized in terms of reduced is calculated by conversion of oxidized form of MGGBQ into reduced form, in presence of ascorbic
acid. The following correlation was established and used to calculate the [MGGBQ] oxidized in terms of reduced. [MGGBQ] oxidized in terms of reduced = x/5.107, where [MGGBQ] oxidized in terms of reduced is in µg, and x is peak area of oxidized MGGBQ (at 253 nm) in mAU*min.
No MGGBQ was detected in any samples, before induction, in cells grown in MM-Glucose-Amp100 and MM-Glycerol-MM-Glucose-Amp100. This showed that the Ptac promoter is tightly control in minimal medium. Induction with 0.25 mM IPTG (final conc.) led to MGGBQ production reaching a level of 52 µg / g CDW and 49 µg / g CDW resp. (total MGGBQ) at the end of batch process (90 % of the total MGGBQ was in reduced form i.e. MGGBQ(Reduced)). Start of fed batch process resulted in linear increase in MGGBQ(Reduced) (MGGBQ(Oxidized) being constant) with time both in terms of product yield per gram dry biomass (µg/g CDW) or concentration (µg/l of culture) in case of MM-Glucose-Amp100 while in case of MM-Glycerol-Amp100 the increase in total MGGBQ was gradual till the end. Wherever the MGGBQ
(Reduced) product yield remained constant, the reduced MGGBQ concentration increased (can be seen for 28 and 31.5 h samples). The MGGBQ (Reduced) concentration increased continuously till 46 h, while the yield of product per gram dry biomass was constant since 38 h. After 40 h, the share of MGGBQ (Oxidized) started increasing and at the end of cultivation, MGGBQ (Oxidized) was approx. 40 % of the total MGGBQ. Oxygen concentration (pO2) in fermenter was always maintained constant at 30 % during fed-batch process.
At the end of cultivation, acetic acid accumulated in Glucose was 3.75 mM and in MM-Glycerol it was 2.45 mM. No HGA was detected in any of the samples before 34 h. Oxygen supply to the cells was maintained constant at 30 % of that saturated concentration. HGA
(A) Minimal medium with Glucose
0 100 200 300 400 500
0 10,5 15,8 20 22 25 28 31,5 34 38 40 44 46 48 50
Time [h]
Reduced and Oxidized MGGBQ [µg/g CDW]
Reduced Oxidized
(B) Minimal medium with Glycerol
0 100 200 300 400 500
0,0 15,0 20,3 22,3 25,5 26,5 32,0 34,5 41,0 44,5 47,0 51,0 59,0 63,0
Time [h]
Reduced and Oxidized MGGBQ [µg/g CDW]
Reduced Oxidized (A) Minimal medium with Glucose
0 100 200 300 400 500
0 10,5 15,8 20 22 25 28 31,5 34 38 40 44 46 48 50
Time [h]
Reduced and Oxidized MGGBQ [µg/g CDW]
Reduced Oxidized
(B) Minimal medium with Glycerol
0 100 200 300 400 500
0,0 15,0 20,3 22,3 25,5 26,5 32,0 34,5 41,0 44,5 47,0 51,0 59,0 63,0
Time [h]
Reduced and Oxidized MGGBQ [µg/g CDW]
Reduced Oxidized
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concentration in medium increased after 36 h and reached 1.62 mM (glucose medium) and 1.87 mM (glycerol medium) at the end of fermentation. During the end phase of cultivation where cell growth dropped down to below 0.05 h-1, the oxygen uptake dropped down and as a result the excess oxygen concentration may have influenced the oxidation of HGA and MGGBQ. Supernatant samples at the end of fermentations were brown in colour.
A test for plasmid segregational stability (described in section 2.2.1.4) was performed to find out how many recombinant E. coli cells lost the plasmid pCAS29, during bioreactor cultivation (MM-Glucose-Amp100 and MM-Glycerol-Amp100), over a period of 48 h. Results of the plasmid segregational stability test are shown in figure 3.23. In case of MM-Glucose-Amp100, after 40 h, approx. 50 % cells were plasmid carriers (based on ampicillin resistance). During the batch process (15 h in MM-Glucose-Amp100 and 21 h in MM.Glycerol-Amp100) approx. 85-90 % of the recombinant E. coli cells possessed the plasmid pCAS29. Most of the plasmid loss occurred during the fed-batch process. 40 h after induction approx. 50 % of E. coli cells still carried the plasmid (in glucose medium) compared to 60 % (in glycerol medium).
0 20 40 60 80 100 120
0 12 24 36 48 60 72
Time [h]
E. coli BW25113 cells still carrying AmpR plasmid pCAS29 [%]
Glucose Glycerol
Figure 3.23: Plasmid stability test for plasmid pCAS29 during fermentation of E. coli BW25113/pCAS29 strain in bioreactor. Blue colour indicates fermentation in MM-Glucose-Amp100 and pink colour indicates fermentation in MM-Glycerol-MM-Glucose-Amp100. S.D is calculated based on the average of two independent measurements.
MGGBQ yield was also calculated in terms of µg of total MGGBQ produced per gram of carbon source consumed. A comparison of MGGBQ yield achieved during cultivation of E.
coli BW25113/pCAS29 in MM-Glucose-Amp100 or MM-Glycerol-Amp100 can be seen in
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figure 3.24. The MGGBQ yield in terms of µg total MGGBQ produced per g of carbon source cannot be compared directly w.r.t time, as the glycerol cultures had long lag phase and hence was ontinued till 58 h (i.e. both cultures were cultivated for sample number of hours after induction with IPTG i.e. approx. 40 h). For most of the fermentation time, the MGGBQ yield (carbon) in glucose was higher, compared to that in glycerol.
Figure 3.24: Total MGGBQ yield shown in terms of µg product per g of glucose or per g of glycerol consumed during bioreactor cultivation of E. coli BW25113/pCAS29 in minimal medium with glucose/glycerol and Amp100; cultivated at 30 °C at constant pH 7.0.
Table 3.10: Overview showing the highest MGGBQtotal produced in E. coli BW25113/pCAS29 strain in Infors fermenter
µg/l µg/g CDW µg/ g Glucose µg/l µg/g CDW µg/ g Glycerol E. coli BW25113/pCAS29 2164 ± 199 325 ± 33 78 ± 5 4591 ± 236 554 ± 29 128 ± 11
Glucose Glycerol
Total MGGBQ Total MGGBQ
In this section, it was shown that except Ggh-Syn, all the other enzymatic reactions in the MGGBQ biosynthesis pathway were functional. Overall, fermentation in MM-glycerol was better with respect to MGGBQ concentrations, yields (MGGBQ per gram CDW), and yields (MGGBQ per g of sugar source if compared with identical hours after induction).
Segregational stability of plasmid pCAS29 was also better in medium with glycerol compared to glucose.
0 20 40 60 80 100 120 140
0 10 20 30 40 50 60 70
Time [h]
Total MGGBQ Yield [µg/g Glucose or µg/g Glycerol]
µg/g Glucose µg/g Glycerol
40 h after induction (Glucose) 40 h after
induction (Glycerol)
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