CHAPTER 3 – Results
3.11. Increased MGGBQ in Recombinant E. coli Strains
3.11.1. Construction of E. coli CS8 and biosynthesis of MGGBQ in bioreactor
Chromosomal Integration of idi expression cassette in E. coli CS6 and analysis of protein expression on 2D Gel Electrophoresis
Plasmid pCAS10 was modified by ligation of HindIII / HindIII digested 1.1 kB FRT-cat-FRT fragment into 5.4 kB HindIII / HindIII digested pCAS10 fragment. Successful ligation produced a new plasmid pCAS10-FRT-cat-FRT. Clones obtained after transformation with ligation mixture were tested by digesting the plasmid with restriction enzyme HindIII which resulted in 1.1 & 5.4 kB. This corresponded to the expected size from restriction mapping.
pCAS10-FRT-cat-FRT was used as template for PCR amplification. Primers rbsD-integr and rbsK-integr (i.e. primer nr. 7 & 8 from table 6) were used for amplification of a fragment Ptac -idi-FRT-cat-FRT. This resulting fragment has a region homologous to some base pairs in rbsD and rbsK loci. E. coli CS6 strain carrying a plasmid pKD46 (expressing -recombinase induced by arabinose) was transformed by electroporation with the fragment Ptac-idi-FRT-cat-FRT. As a result, 6 transformants were obtained and were tested on MacConkey agar plate each supplemented with 4 different sugars (fucose, maltose, lactose and ribose). Each of these MacConkey agar plate were streaked with there respective control strain. All the 6 transformants were regarded as positive as none of them turned red on MacConkey agar plates with sugars. These 6 transformants were further tested with PCR using primers P1-Control-idi-rbs and P2-P1-Control-idi-rbs yielded PCR product of 1.8 kB size which corresponds with the expected size. The new strain was named as E. coli CS8-cat. Using plasmid pCP20, the catr cassette was removed from E. coli CS8-cat to obtain a new strain named E.
coli CS8 (shown in figure 3.64).
Figure 3.64: Scheme showing chromosomal integrated strains E. coli CS6 and E. coli CS8. The difference between the two is 4th expression cassette (idi) in ribose operon i.e. rbsDK locus in case of E. coli CS8 strain.
Overexpression level of Idi in the newly constructed E. coli CS8 strain was studied. E. coli CS6 and E. coli CS8 were cultivated in LB medium at 30 °C. Cultures were induced with 0.25 mM IPTG at OD 600nm of 0.8. Cell samples from both cultures i.e. 6 h after induction was E. coliCS6
hpt ptac RBS
(lacZYA)
crtE ptac RBS
(malEFG)
FRT FRT
hpd RBS
(fucIP)
FRT
E. coliCS8
hpt ptac RBS
(lacZYA)
crtE ptac RBS
(malEFG)
FRT FRT
hpd RBS
(fucIP)
FRT
idi RBS
(rbsDK)
FRT
E. coliCS6
hpt ptac RBS
(lacZYA)
crtE ptac RBS
(malEFG)
FRT FRT
hpd RBS
(fucIP)
FRT hpt
ptac RBS
(lacZYA)
crtE ptac RBS
(malEFG)
FRT FRT
hpd RBS
(fucIP)
FRT
E. coliCS8
hpt ptac RBS
(lacZYA)
crtE ptac RBS
(malEFG)
FRT FRT
hpd RBS
(fucIP)
FRT
idi RBS
(rbsDK)
FRT
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tested on 2D gel electrophoresis (see figure 3.65). Hpd and CrtE proteins were detected in both samples (overlapped and hence seen as dark spots marked at 40 kDa and 32 kDa respectively. Hpt-Syn being membrane associated was not detected on 2D gel. Idi protein has a calculated size of 20.5 kDa (ExPASy). An extra red spot at approx. 21.2 kDa was observed in E. coli CS8 (seen in red spots also marked by arrows).
Figure 3.65: 2 D Gel electrophoresis. In green is E. coli CS6 and in red is E. coli CS8. Hpd, and CrtE protein over lapped (both dark red coloured spots, shown by arrows) at approx. 40 and 32 kDa respectively. An additional red spot at approx. 20 kDa was seen in E. coli CS8 sample which corresponds to the expected size of Idi protein from E. coli.
To study the effect of idi expression cassette in the newly constructed strain E. coli CS8 on the MGGBQ (total) productivity it was cultivated in shaking flask in LB-Glycerol at 30 °C. As control E. coli CS6 was cultivated under same conditions. Cultures were induced with 0.25 mM IPTG at OD 600nm of 0.8. Results of this experiment can be seen in figure 3.66. Till 8.5 h both cultures had almost reached identical cell density (approx. OD600nm of 2.95). Between 8.5 and 24 h the cell growth in E. coli CS8 was affected and reached stationary phase at 36 h while the control E. coli CS6 grew further to reach a final OD600nm of 4.1. The total MGGBQ produced in E. coli CS8 after 24 h was more than 2.5 times higher (in terms of µg/g CDW) when compared to its control E. coli CS6 without idi expression cassette. Total MGGBQ yield after 48 h and 72 h in both cultures decreased but the decrease in E. coli CS8 was slower than in E. coli CS6 which suggests that MGGBQ was produced in E. coli CS8 but
E. coli CS8 E. coli CS6
50 kDa
37 kDa
25 kDa 75 kDa
20 kDa Idi
M
+
-pH 3 pH 10
E. coli CS8 E. coli CS6 E. coli CS8 E. coli CS6
50 kDa
37 kDa
25 kDa 75 kDa
20 kDa Idi
M
+
-pH 3 pH 10
Hpd
CrtE
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simultaneously oxidized and polymerized. After confirming the positive impact of expression of Idi in E. coli CS8 over E. coli CS6, E. coli CS8 was cultivated in bioreactor in minimal medium using glucose and/or glycerol as sole carbon and energy source to compare the respective carbon flux into total MGGBQ production.
Figure 3.66: Effect of chromosomally integrated idi expression cassette in E. coli CS8 on MGGBQ production. Shaking flask cultivation carried out at 30 °C in LB-Glycerol medium, induction with 0.25 mM at OD 600nm of 0.8. E. coli CS6 is used as a control for E. coli CS8. (A) Shows cell density in OD600nm as a function of time. (B) Shows the total MGGBQ production in µg/g CDW as a function of time. S.D calculated from 2 different samples taken during cultivation which were extracted and analyzed (HPLC) separately. Shaking flask cultivation of both strains were carried out thrice independently under identical conditions and medium. The S.D in all the three experiments w.r.t to the total MGGBQ production was less then 10 % (µg/l as well as µg/g CDW).
0 1 2 3 4 5
0 12 24 36 48 60 72 84
Time [h]
OD600 nm [-]
E. coli CS6 E. coli CS8
0 100 200 300 400 500 600
0 24 48 72
Time [h]
Total MGGBQ [µg/g CDW]
E. coli CS6 E. coli CS8
(A)
(B)
0 1 2 3 4 5
0 12 24 36 48 60 72 84
Time [h]
OD600 nm [-]
E. coli CS6 E. coli CS8
0 100 200 300 400 500 600
0 24 48 72
Time [h]
Total MGGBQ [µg/g CDW]
E. coli CS6 E. coli CS8
(A)
(B)
0 100 200 300 400 500 600
0 24 48 72
Time [h]
Total MGGBQ [µg/g CDW]
E. coli CS6 E. coli CS8
(A)
(B)
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Increased MGGBQ Production using E. coli CS8 Strain in Minimal Medium in Infors Bioreactor
E. coli CS8 was cultivated twice in minimal medium using glucose and twice in minimal medium using glycerol as sole carbon and energy source in bioreactor. Cultivation were carried out at 30 °C at constant pH of 7.0.
E. coli CS8 cultures in MM-Glucose and MM-Glycerol were induced with 0.25 mM IPTG at approx. OD600nm of 2.1 + 0.1 at 13.75 h and 16.0 h respectively (shown by (a) and (b) arrows in figure 3.67 A). E. coli CS8 in MM-Glucose medium (µmax of 0.24 h-1) grew better than in MM-Glycerol medium (µmax of 0.16 h-1). Cell growth in MM-Glucose after 30 h slowed down where the growth rates in both medium was almost identical (µ of 0.08 + 0.02 h-1). E. coli CS8 cultures in glycerol reached higher cell density (OD600nm of approx. 28.0) as compared to that achieved in E. coli CS8 in glucose.
No MGGBQ was produced before induction with IPTG either in Glucose or in MM-Glycerol (see figure 3.68). During batch process, the total MGGBQ produced in both medium was below 200 µg /g CDW. Fed batch process produced higher total MGGBQ levels in MM-Glycerol as compared to MM-Glucose. Acetic acid produced during the entire fed batch process in MM-Glycerol was relatively low (approx. 1.2 g/l) as compared to that produced in MM-glucose (approx. 2.1 g/l). This may be a possible reason for the slow growth in MM-Glucose after 30 h. As a result at the end of respective cultivations, total MGGBQ produced in E. coli CS8 in MM-Glycerol was at least 20 % higher (1285 + 117 µg/g CDW) than as that produced in E. coli CS8 in glucose (1076 + 105 µg/g CDW). Even though the difference in total MGGBQ was not significant, MM-Glucose produced almost 38 % of oxidized MGGBQ as compared to only 21 % in MM-Glycerol (figure 3.68).
MGGBQ total yield based on sugar, showed that since the start of MGGBQ production in MM-Glycerol (i.e. 16.5 h) the MGGBQ total resulted in higher yields as compared to that in MM-Glucose (figure 3.69). Highest MGGBQ total in MM-Glycerol reached 224 + 6 µg/g glycerol and that in MM-Glucose reached 187 + 4 µg/g glucose.
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Figure 3.67: Cultivation results of E. coli CS8 strain in Infors bioreactor in minimal medium using glucose and glycerol as carbon and energy source.
(A) Cell growth curve shown in OD600nm on left hand side y-axis, Glucose and Glycerol concentration in bioreactor vs. time shown on right hand side Y-axis. Vertical lines (a) and (b) indicate the time of induction with 0.25 mM IPTG in glucose and glycerol as carbon source respectively. Vertical lines (c) and (d), indicates the start of fed-batch cultivation by feeding glucose or glycerol stock solution respectively. (B) Feed rates of glucose and glycerol calculated used as sole carbon and energy source.
0 5 10 15 20 25 30
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glucose and Glycerol concentration [g/l]
OD (Glucose) OD (Glycerol) Glucose conc.
Glycerol conc.
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0 10 20 30 40 50 60 70
Time [h]
"F" Feed Rate [g/h]
Glucose Glycerol (A)
(B)
(c) (d)
(a) (b) 0
5 10 15 20 25 30
0 10 20 30 40 50 60 70
Time [h]
OD600nm [-]
0 1 2 3 4 5 6
Glucose and Glycerol concentration [g/l]
OD (Glucose) OD (Glycerol) Glucose conc.
Glycerol conc.
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0 10 20 30 40 50 60 70
Time [h]
"F" Feed Rate [g/h]
Glucose Glycerol (A)
(B)
(c) (d)
(a) (b)
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Figure 3.68: Reduced and Oxidized MGGBQ production in E. coli CS8 strain in bioreactor.
Cultivation was carried out in minimal medium with glucose/glycerol as sole carbon and energy source at 30°C using batch-fed batch cultivation strategy. Cultures induced with 0.25 mM IPTG.
(A) MGGBQ yield using glucose as carbon and energy source (B) MGGBQ yield using glycerol as carbon and energy source
0 200 400 600 800 1000 1200 1400
0,00 16,50
18,00 20,50
22,00 24,50
31,00 32,50
33,50 35,50
37,50 39,25
43,25 48,00 Time [h]
Reduced and Oxidized MGGBQ [µg/ g CDW]
Reduced MGGBQ Oxidized MGGBQ
(A)
0 200 400 600 800 1000 1200 1400
0,00 16,00
20,00 22,00
24,00 28,00
30,50 32,00
37,75 40,00
42,50 46,00
49,00 Time [h]
Reduced and Oxidized MGGBQ [µg/g CDW]
Reduced MGGBQ Oxidized MGGBQ
(B)
0 200 400 600 800 1000 1200 1400
0,00 16,50
18,00 20,50
22,00 24,50
31,00 32,50
33,50 35,50
37,50 39,25
43,25 48,00 Time [h]
Reduced and Oxidized MGGBQ [µg/ g CDW]
Reduced MGGBQ Oxidized MGGBQ
(A)
0 200 400 600 800 1000 1200 1400
0,00 16,00
20,00 22,00
24,00 28,00
30,50 32,00
37,75 40,00
42,50 46,00
49,00 Time [h]
Reduced and Oxidized MGGBQ [µg/g CDW]
Reduced MGGBQ Oxidized MGGBQ
(B)
Glucose
Glycerol
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Figure 3.69: Total MGGBQ yield calculated per gram of carbon source (glucose/glycerol) consumed during cultivation of E. coli CS8 strain in bioreactor in minimal medium at 30 °C.
0 50 100 150 200 250
0 10 20 30 40 50 60 70
Time [h]
Total MGGBQ [µg/g glucose consumed]
0 50 100 150 200 250
Total MGGBQ [µg/g glycerol consumed]
µg/g Glucose µg/g Glycerol
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Influence of co-expression of Dxs in E. coli CS6 and E. coli CS8 strains on MGGBQ production
After studying the positive effect of Idi co-expression in E. coli CS6 on the yield of MGGBQ
total, the influence of Dxs co-expression on total MGGBQ in E. coli CS6 and E. coli CS8 as host strains was tested.
dxs gene from Corynebacterium glutamicum was already cloned into pJF119HE (modified pJF119N) vector to obtain a new plasmid pJF119HE-dxs-C.glut (personal communication from Dr. Natalie Trachtmann), and was kindly provided for further expression and cultivation experiments. Protein over-expression was in E. coli was shown and enzyme was shown to be active during in-vitro tests (personal communication with Dr. N. Trachtmann). Before using the plasmid, protein over-expression was confirmed. SDS-PAGE and can be seen in figure 3.70. E. coli BW25113 / pJF119HE-dxs-C.glut was cultivated in LB-Amp100. As control E. coli BW25113 /pJF119HE was cultivated. Cultures were induced with 1 mM IPTG at approx. OD600nm of 0.8. Additional protein bands at approx. 68 kDa (Rf method) in lane 2 and 4 were observed after de-staining the acrylamide gel. These were absent in control strains (lane 1 and 3). Dxs has a calculated protein size of 69 kDa (ExPASy) which closely corresponded with the protein size obtained experimentally. This suggests that the Dxs from C. glutamicum was overexpressed in E. coli BW25113 strain with and without IPTG induction. Activity of the expressed Dxs protein was tested during in-vivo experiment, where the plasmid pJF119HE-dxs-C.glut was used for cultivation experiment for MGGBQ production, using E. coli CS6 and E. coli CS8 as host strains. Host strains with control vector (pJF119HE) were used as control strain.
Figure 3.70: SDS-PAGE for Dxs over-expression.
10 µg of protein sample (cell free crude extract) was loaded in each lane.
1: E. coli BW25113// pJF119HE-dxs-C.glut (3h) i.e.
before IPTG
2: E. coli BW25113/pJF119HE (3h) i.e. before IPTG M: molecular marker
3: E. coli BW25113/ pJF119HE-dxs-C.glut (8h) i.e. 5 h after IPTG
4: E. coli BW25113/pJF119HE (8h) i.e. 5 h after IPTG
Thick stained bands in lane 1 and 3 are marked with arrow representing the over-expressed Dxs protein (approx. 68 kDa) which are absent in control (lane 2 and 4 resp.)
37 kDa
25 kDa 50 kDa
75 kDa
20 kDa
1 2 M 3 4
37 kDa
25 kDa 50 kDa
75 kDa
20 kDa
1 2 M 3 4
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Shaking flask cultivation of E. coli CS6/ pJF119HE-dxs-C.glut and E. coli CS8/ pJF119HE-dxs-C.glut was carried out in LB-Glycerol-Amp100 at 30 °C to produce MGGBQ. As control, E. coli CS6/pJF119N and E. coli CS8/ pJF119N strains were cultivated under identical conditions. Cultures were induced with 0.25 mM IPTG at approx. OD600nm of 0.8. Till the time of induction all strains grew equally well. After induction E. coli CS6 and E. coli CS8 carrying pJF119HE-dxs-C.glut plasmid grew better than the strains carrying empty vector and reached two times higher cell mass after 72 h (see figure 3.71, graph A). E. coli CS8/dxs-C.glut strain reached higher cell density than E. coli CS6/ pJF119HE-dxs-C.glut.
Total MGGBQ production (figure 3.71, graph B) in strains carrying plasmid pJF119HE-dxs-C.glut was higher than strains carrying the empty vector. The increase in total MGGBQ in E.
coli CS6 /pJF119HE-dxs-C.glut (was 8.5 times as compared to E. coli CS6/ pJF119HE. The increase in MGGBQ in E. coli CS8 /pJF119HE-dxs-C.glut was 20 times as compared to E.
coli CS8/ pJF119HE. Over-expression of Dxs in E. coli CS6 and E. coli CS8 had a strong effect on total MGGBQ production without negatively affecting the cell growth. This experiment was repeated under same conditions to reproduce identical MGGBQ yields with standard deviations less then 10 % (in terms of µg/l and µg/g CDW). The reason for the increased total MGGBQ level in strains carrying pJF119HE-dxs-C.glut plasmid can be explained as the Dxs over-expressed increases the carbon flux via DXP pathway*. The difference in total MGGBQ produced in E. coli CS6/ pJF119HE-dxs-C.glut and E. coli CS8 pJF119HE-dxs-C.glut may be due to the Idi expressed in E. coli CS8 strain which perhaps increases the pool of FPP and IPP. Hence, it was decided to overexpress Dxs protein in E.
coli CS8 strain to increase the MGGBQ production level. Instead of using an extra copy of foreign dxs gene for chromosomal integration into another sugar operon a new chromosomal integration technique was used. (Albermann et. al. 2010). The native promoter of dxs in E.
coli CS8 strain was replaced by PT5 promoter to obtain a new construct. This is discussed in detail in next section.
* Independent attempts to study the effect of Dxs overexpression (using plasmid pJF119EH-dxs-C.glut) in another host strain and production system were unable to see any significant increase in product yield (Personal communication, Prof. Georg Sprenger), as seen in this study.
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Figure 3.71: Effect of co-expression of Dxs (in plasmid) in E. coli CS6 and E. coli CS8 on total MGGBQ production. Cultivations were carried out in shaking flask at 30 °C in LB-Glycerol-Amp100 medium. Cultures induced with 0.25 mM IPTG at approx. OD600nm of 0.8. (A) Line chart shows the cell density (OD600nm) calculated during cultivation. (B) Bar chart represents the total MGGBQ yield calculated in µg/g CDW. This experiment was repeated to confirm the results obtained. The S.D for OD and total MGGBQ yield was below 10 %.
0 400 800 1200 1600 2000 2400 2800 3200
0 18 42 50 72
Time [h]
Total MGGBQ [µg/g CDW]
E. coliCS8/pJF119HE-dxs-C.glut E. coliCS6/pJF119HE-dxs-C.glut E. coliCS8/pJF119HE
E. coliCS6/pJF119HE
0 2 4 6 8 10 12
0 12 24 36 48 60 72 84
Tim e [h]
OD600nm [-]
E. coliCS8/pJF119HE-dxs-C-glut E. coliCS6/pJF119HE-dxs-C-glut E. coliCS8/pJF119HE
E. coliCS6/pJF119HE
(B) (A)
0 400 800 1200 1600 2000 2400 2800 3200
0 18 42 50 72
Time [h]
Total MGGBQ [µg/g CDW]
E. coliCS8/pJF119HE-dxs-C.glut E. coliCS6/pJF119HE-dxs-C.glut E. coliCS8/pJF119HE
E. coliCS6/pJF119HE
0 2 4 6 8 10 12
0 12 24 36 48 60 72 84
Tim e [h]
OD600nm [-]
E. coliCS8/pJF119HE-dxs-C-glut E. coliCS6/pJF119HE-dxs-C-glut E. coliCS8/pJF119HE
E. coliCS6/pJF119HE
(B) (A)
175