3.1 Cell growth and segregation in recombinant bioprocesses
3.1.1 Cell growth in recombinant E. coli fed-batch cultivations
3 Results
In this thesis, a yeast α-glucosidase produced in E. coli was used as model system in order to obtain a more comprehensive knowledge about cell physiology in response to overexpression of heterologous genes during glucose limited fed-batch cultures. A limited number of studies was also performed with two other heterologous products, CRIMI (creatinine imino hydrolase) and ZZ protein, which were investigated in frame of an industry cooperation and a European network project. The combination of the experiences obtained from these different systems and the evaluation of the current literature on this subject, allows to make some broader conclusions about the cell behavior under strong recombinant gene induction. However, I specifically want to adress that the aim of this work was not to indicate the whole range of validity of the findings for the huge number of expressed proteins, but to describe thoroughly one model system. On this basis ongoing studies in our laboratory and of other groups will show which effect is caused by the specific properties of the protein and/or the expression system.
The experiments were generally performed under process-like conditions, namely by induction during the glucose limited growth phase of a fed-batch fermentation. Whereas the study is focusing in the first part on cellular responses and the maintenance of cell viability, certain specific questions in connection to the production scale are investigated in the second part of this work.
3.1 Cell growth and segregation in recombinant bioprocesses
plasmid for coexpression of the rare arginyl tRNA (argU). As the gene for α-glucosidase contains 19 aga/agg codons, corresponding to 3.23 % of the total α-glucosidase codon usage, which is ten times more than the average use of these codons in E. coli, the formation of the recombinant product is limited by argU tRNA as shown by Brinkmann et al. (1989). An approximately fourfold increase of the cellular product concentration was obtained by cotransformation of the pUBS520 plasmid (Fig. 3.1b,f).
c
f
Time [h]
0 5 10 15 20 25
Glucose [g L-1]
0.00 0.02 0.04 0.06 2.00 4.00 DCW [g L-1]
0 5 10 15 20 25
αα-glucosidase [U mL-1]
0 3 6 9 12
a
b
d
0 5 10 15 20 25
e
αα-glucosidase [mg g-1 DCW]
0 20 40
qp [mg g-1h-1]
0 20 40
g
h
St. 1.3 3 4 5 6 7 8 20 21 22 h i )
Figure 3.1: Growth characteristics of glucose limited fed-batch fermentations with E. coli RB791 pKK177glucC (a-d) and E. coli RB791 pKK177glucC pUBS520 (e-h) in dependence on the addition of IPTG for induction of the α-glucosidase (open symbol: no induction, filled symbol: induction by addition of IPTG). (a, e) biomass; (b, f) α-glucosidase concentration per dry cell weight after induction and specific product formation rate (---); (c, g) activity of soluble α-glucosidase; and (d, h) glucose concentration. The interrupted line represents the time of feeding start at a cell density of approximately 2 g L-1. The dotted line indicates the addition of IPTG (1 mM final concentration). (i) SDS-PAGE analysis of glucose limited fed-batch fermentations of E. coli RB791 pKK177glucC pUBS520 with induction of 1 mM IPTG.
Zero time indicates the point of feeding start.
Glucose limited fed-batch fermentations with a constant feed were performed with and without addition of IPTG as the inducing agent of the recombinant α-glucosidase. During the batch phase with 5 g L-1 glucose the strains grew with a maximum growth rate according to OD500 of 0.68 ± 0.08 h-1 (without pUBS520) and 0.79 ± 0.05 h-1 (with pUBS520). Glucose feeding was started at a defined cell density at the end of the batch phase causing a further exponential growth of the cells to the point of glucose exhaustion. From the point of glucose limitation the glucose concentration was
detected to be below 15 mg L-1 according to the low Ks value for glucose of E. coli and stayed at this low level up to the end of those cultivations without induction or with induction but without pUBS520. In the culture with pUBS520, the glucose concentration increased to about 1.5 g L-1 after addition of IPTG possibly due to a decrease of the glucose uptake capacity of the cells following induction. The specific growth rate µ decreased continuously due to the constant feed rate and the cell dry weight steadily increased to a value of about 25 g L-1. Acetate accumulated during the batch phase to a maximum concentration of about 0.6 g L-1 and was reconsumed within three hours after the onset of glucose limitation. α-glucosidase was very low in the fermentations without induction and could only be detected by activity analysis for the soluble protein (Fig. 3.1c,g).
When the inductor IPTG was added three hours after feeding start, α-glucosidase formation proceeded at a higher rate for approximately 3 hours (Fig. 3.1b,f), whereby the specific rate of α-glucosidase formation was much higher in the strain containing the pUBS520-plasmid than in the strain without the plasmid. In both systems, the specific product formation stops about 3 hours after induction which is connected to growth inhibition in the strain RB791 pKK177glucC pUBS520 (Fig.
3.1e), but not in the strain without pUBS520, in which growth was blocked only from seven hours after induction (Fig. 3.1a). The product appears to a dominant part in inclusion bodies and is about 4 % of the total cellular protein in the strain without pUBS520 but about 16 % of the total cellular protein in the strain containing pUBS520. In the case of these cultivations only less than 5 % of the product was soluble and showed activity, whereby the activity was lower in the system with coexpression of the argU tRNA (Fig. 3.1c,g). In principle, the soluble part of the α-glucosidase could be increased by changing cultivation conditions such as pH and temperature, as well as by lowering the amount of IPTG or induction by lactose in a lacY mutant (Kopetzki et al., 1989a,b).
Although optimization of the α-glucosidase activity was not the aim of this thesis, the activity (Fig. 3.1c,g) was measured and found that it increased after induction.
Effect of CRIMI production on cell growth. The plasmid pDSCrimi for overproduction of CRIMI was transformed into the same host strain E. coli RB791 and the cultivation was exactly performed by the same scheme as for α-glucosidase production in order to compare the two systems. Time profile of cell growth, product formation, substrate consumption and the corresponding specific rates are presented in Fig. 3.2. During the initial batch phase the cells grew exponentially with a maximum specific growth rate µmax of 0.80 h-1. The maximum specific glucose uptake rate qsmax in the batch phase was determined to be 1.2 g g-1 h-1. The biomass yield on glucose
was 0.64 g g-1. From the point of glucose limitation the glucose concentration was detected to be below 40 mg L-1 and stayed at this low level up to the end of the cultivation. In contrast to the α -glucosidase process, the growth proceeded also after induction of the CRIMI and the final cell dry weight was 22 g L-1.
a
DCW [g L-1 ]
0 5 10 15 20 25
µ [h-1 ]
0.0 0.3 0.6 0.9
c
Time [h]
0 10 20 30
Glucose [g l-1 ]
0.00 0.02 0.04 1.00 2.00 3.00 4.00 5.00
qs [g g-1 h-1 ]
0.0 0.5 1.0 1.5 b
Product [g L-1]
0 2 4 6
spe. product [mg g-1 DCW]
0 100 200 300
qp [mg g-1 DCW]
0 20 40 60
Figure 3.2: Growth characteristics of glucose limited fed-batch fermentation with E. coli RB791 pDSCrimi.
(a) cell dry weight (¡) and specific growth rate (µ,----), (b) creatinine imino hydrolase concentration (¡-g L-1, ∆-mg g-1 DCW) and specific product formation rate (qp,---), (c) glucose concentration (¡) and glucose uptake rate (qs, ----).
The interrupted line represents the time of feeding start at an cell density of approximately 2 g L-1. The dotted line indicates the addition of IPTG (1mM final concentration).
After induction the concentration of CRIMI increased with a rate of 45 mg g-1 h-1 and reached to a level of 160 mg g-1 DCW about four hours after induction, corresponding to about 30 % of the total cell protein. The total concentration of CRIMI at this time was about 1.7 g L-1 (Fig. 3.2c). During further cultivation the product concentration increased approximately linearly to about 5 g L-1 to the end of the cultivation. About two third of the product accumulated in the insoluble fraction and one third was soluble and active.
In difference to the strong inhibition effect of α-glucosidase production (Table 3.1), the synthesis of CRIMI had only minor effect on the growth. The inhibition of cellular growth following induction in the α-glucosidase system was further investigated by analyzing the cell populations to see what kind of cell segregation occurs following induction.
Table 3.1: Comparison of cultivation parameters of glucose limited fed-batch processes for production of α-glucosidase and CRIMI in E. coli RB791.
RB791 pKK177glucC RB791 pKK177glucC pUBS520 RB791 pDSCrimi
µmax [h-1] 0.68 0.79 0.82
qsmax [g g-1 h-1] 1.1 1.34 1.21
Yx/glucose 0.29 0.46 0.64
qp [mg g-1 h-1] 3.2 (initial qp) 15.5 (initial qp) 45
P [mg g-1] 15 50 160
Ratio of IB/soluble 99:1 99:1 2:1
DCW/OD500 0.22 a / 0.27 b 0.26 a / 0.21 b 0.29 a / 0.18 b
k d cfu [h-1] 0.35 0.74 c / 0.27 c 0.10
a : before induction, b: after induction, c: two phase process, more details see Fig.3.3.