3 Results RESULTS Cell growth and segregation in recombinant bioprocesses

Transcription

1 RESULTS 3 3 Results In this thesis, yest α-glucosidse produced in E. coli ws used s model system in order to otin more comprehensive knowledge out cell physiology in response to overexpression of heterologous genes during glucose limited fed-tch cultures. A limited numer of studies ws lso performed with two other heterologous products, CRIMI (cretinine imino hydrolse) nd ZZ protein, which were investigted in frme of n industry coopertion nd Europen network project. The comintion of the experiences otined from these different systems nd the evlution of the current literture on this suject, llows to mke some roder conclusions out the cell ehvior under strong recominnt gene induction. However, I specificlly wnt to dress tht the im of this work ws not to indicte the whole rnge of vlidity of the findings for the huge numer of expressed proteins, ut to descrie thoroughly one model system. On this sis ongoing studies in our lortory nd of other groups will show which effect is cused y the specific properties of the protein nd/or the expression system. The experiments were generlly performed under process-like conditions, nmely y induction during the glucose limited growth phse of fed-tch fermenttion. Wheres the study is focusing in the first prt on cellulr responses nd the mintennce of cell viility, certin specific questions in connection to the production scle re investigted in the second prt of this work. 3.1 Cell growth nd segregtion in recominnt ioprocesses Cell growth in recominnt E. coli fed-tch cultivtions In most cses, the overproduction of recominnt proteins to high level of the cell protein results in n inhiition of the cellulr growth. Although the inhiition of growth is lso ovious in shke flsk cultures, I used simple glucose limited fed-tch fermenttion process with constnt feed rte, which is common method to rech higher cell densities nd therefore to increse volumetric yields. Although, in mny industril processes n exponentil feed is often used fter n initil tch phse for certin period, I used the constnt feed method due to its higher reproduciility, nd s the inducing signl is mostly given to the culture in industry when the feed ws switched to constnt rte. Growth inhiition fter induction of recominnt -glucosidse. Fed-tch cultivtions for α-glucosidse production were performed with the recominnt strin E. coli RB791 contining the plsmid pkk177glucc. This host-vector system ws prtilly used in connection with the pubs5

2 RESULTS 31 plsmid for coexpression of the rre rginyl trna (rgu). As the gene for α-glucosidse contins 19 g/gg codons, corresponding to 3.3 % of the totl α-glucosidse codon usge, which is ten times more thn the verge use of these codons in E. coli, the formtion of the recominnt product is limited y rgu trna s shown y Brinkmnn et l. (1989). An pproximtely fourfold increse of the cellulr product concentrtion ws otined y cotrnsformtion of the pubs5 plsmid (Fig. 3.1,f). Glucose [g L -1 ] DCW [g L -1 ] -glucosidse [mg g -1 DCW] -glucosidse [U ml -1 ] e 4 f c g d h i ) St h q p [mg g -1 h -1 ] Figure 3.1: Growth chrcteristics of glucose limited fed-tch fermenttions with E. coli RB791 pkk177glucc (-d) nd E. coli RB791 pkk177glucc pubs5 (e-h) in dependence on the ddition of IPTG for induction of the α-glucosidse (open symol: no induction, filled symol: induction y ddition of IPTG). (, e) iomss; (, f) α-glucosidse concentrtion per dry cell weight fter induction nd specific product formtion rte (---); (c, g) ctivity of solule α-glucosidse; nd (d, h) glucose concentrtion. The interrupted line represents the time of feeding strt t cell density of pproximtely g L -1. The dotted line indictes the ddition of IPTG (1 mm finl concentrtion). (i) SDS-PAGE nlysis of glucose limited fedtch fermenttions of E. coli RB791 pkk177glucc pubs5 with induction of 1 mm IPTG. Zero time indictes the point of feeding strt. Glucose limited fed-tch fermenttions with constnt feed were performed with nd without ddition of IPTG s the inducing gent of the recominnt α-glucosidse. During the tch phse with 5 g L -1 glucose the strins grew with mximum growth rte ccording to OD 5 of.68 ±.8 h -1 (without pubs5) nd.79 ±.5 h -1 (with pubs5). Glucose feeding ws strted t defined cell density t the end of the tch phse cusing further exponentil growth of the cells to the point of glucose exhustion. From the point of glucose limittion the glucose concentrtion ws

3 RESULTS 3 detected to e elow 15 mg L -1 ccording to the low K s vlue for glucose of E. coli nd styed t this low level up to the end of those cultivtions without induction or with induction ut without pubs5. In the culture with pubs5, the glucose concentrtion incresed to out 1.5 g L -1 fter ddition of IPTG possily due to decrese of the glucose uptke cpcity of the cells following induction. The specific growth rte µ decresed continuously due to the constnt feed rte nd the cell dry weight stedily incresed to vlue of out 5 g L -1. Acette ccumulted during the tch phse to mximum concentrtion of out.6 g L -1 nd ws reconsumed within three hours fter the onset of glucose limittion. α-glucosidse ws very low in the fermenttions without induction nd could only e detected y ctivity nlysis for the solule protein (Fig. 3.1c,g). When the inductor IPTG ws dded three hours fter feeding strt, α-glucosidse formtion proceeded t higher rte for pproximtely 3 hours (Fig. 3.1,f), wherey the specific rte of α-glucosidse formtion ws much higher in the strin contining the pubs5-plsmid thn in the strin without the plsmid. In oth systems, the specific product formtion stops out 3 hours fter induction which is connected to growth inhiition in the strin RB791 pkk177glucc pubs5 (Fig. 3.1e), ut not in the strin without pubs5, in which growth ws locked only from seven hours fter induction (Fig. 3.1). The product ppers to dominnt prt in inclusion odies nd is out 4 % of the totl cellulr protein in the strin without pubs5 ut out 16 % of the totl cellulr protein in the strin contining pubs5. In the cse of these cultivtions only less thn 5 % of the product ws solule nd showed ctivity, wherey the ctivity ws lower in the system with coexpression of the rgu trna (Fig. 3.1c,g). In principle, the solule prt of the α-glucosidse could e incresed y chnging cultivtion conditions such s ph nd temperture, s well s y lowering the mount of IPTG or induction y lctose in lcy mutnt (Kopetzki et l., 1989,). Although optimiztion of the α-glucosidse ctivity ws not the im of this thesis, the ctivity (Fig. 3.1c,g) ws mesured nd found tht it incresed fter induction. Effect of CRIMI production on cell growth. The plsmid pdscrimi for overproduction of CRIMI ws trnsformed into the sme host strin E. coli RB791 nd the cultivtion ws exctly performed y the sme scheme s for α-glucosidse production in order to compre the two systems. Time profile of cell growth, product formtion, sustrte consumption nd the corresponding specific rtes re presented in Fig. 3.. During the initil tch phse the cells grew exponentilly with mximum specific growth rte µ mx of.8 h -1. The mximum specific glucose uptke rte q smx in the tch phse ws determined to e 1. g g -1 h -1. The iomss yield on glucose

4 RESULTS 33 ws.64 g g -1. From the point of glucose limittion the glucose concentrtion ws detected to e elow 4 mg L -1 nd styed t this low level up to the end of the cultivtion. In contrst to the α- glucosidse process, the growth proceeded lso fter induction of the CRIMI nd the finl cell dry weight ws g L -1. Product [g L -1 ] DCW [g L -1 ] Glucose [g l -1 ] c µ [h -1 ] spe. product [mg g -1 DCW] qs [g g -1 h -1 ] 6 4 q p [mg g -1 DCW] Figure 3.: Growth chrcteristics of glucose limited fed-tch fermenttion with E. coli RB791 pdscrimi. () cell dry weight ( ) nd specific growth rte (µ,----), () cretinine imino hydrolse concentrtion ( -g L -1, -mg g -1 DCW) nd specific product formtion rte (q p,---), (c) glucose concentrtion ( ) nd glucose uptke rte (q s, ----). The interrupted line represents the time of feeding strt t n cell density of pproximtely g L -1. The dotted line indictes the ddition of IPTG (1mM finl concentrtion). After induction the concentrtion of CRIMI incresed with rte of 45 mg g -1 h -1 nd reched to level of 16 mg g -1 DCW out four hours fter induction, corresponding to out 3 % of the totl cell protein. The totl concentrtion of CRIMI t this time ws out 1.7 g L -1 (Fig. 3.c). During further cultivtion the product concentrtion incresed pproximtely linerly to out 5 g L -1 to the end of the cultivtion. Aout two third of the product ccumulted in the insolule frction nd one third ws solule nd ctive. In difference to the strong inhiition effect of α-glucosidse production (Tle 3.1), the synthesis of CRIMI hd only minor effect on the growth. The inhiition of cellulr growth following induction in the α-glucosidse system ws further investigted y nlyzing the cell popultions to see wht kind of cell segregtion occurs following induction.

5 RESULTS 34 Tle 3.1: Comprison of cultivtion prmeters of glucose limited fed-tch processes for production of α -glucosidse nd CRIMI in E. coli RB791. RB791 pkk177glucc RB791 pkk177glucc pubs5 RB791 pdscrimi µ mx [h -1 ] qs mx [g g -1 h -1 ] Y x/glucose q p [mg g -1 h -1 ] 3. (initil q p ) 15.5 (initil q p ) 45 P [mg g -1 ] Rtio of IB/solule 99:1 99:1 :1 DCW/OD 5. /.7.6 /.1.9 /.18 k d cfu [h -1 ] c /.7 c.1 : efore induction, : fter induction, c : two phse process, more detils see Fig Cell segregtion nd plsmid stility fter IPTG induction By replic plting it ws shown tht the growth inhiition fter induction of α-glucosidse production is connected to strong decrese in the ility of the cells to form colonies on gr pltes. In the cse of E. coli RB791 pkk177glucc, the ility to form colonies decresed y specific rte constnt of.35 h -1 from 1 % t 3 h fter induction to out 1 % within 15 hours (Fig. 3.3). In contrst to the control culture where no significnt cell segregtion ws found, more thn 8 % of the culture reched the stte of non-dividing cells within few hours, shortly fter ccumultion of α-glucosidse. Cell numer or cfu [ml -1 ] : totl cell numer : cfu of P + c: cfu of P - 1 k d1 =.74 k d =.7 k d =.35 1 c 1 3 Figure 3.3: Comprison of () totl cell numer, () colony forming units of plsmidcontining cells (P + ) nd (c) cfu of plsmid-free cells (P - ) during fed-tch cultivtions of E. coli RB791 pkk177glucc (, ) nd E. coli RB791 pkk177glucc pubs5 (, ) in dependence on the ddition of IPTG for induction of the α-glucosidse (open symol: no induction; filled symol: induction y ddition of 1 mm IPTG). The nlysis of glucose (see Fig. 3.1d) showed tht ll the cron which ws fed continuously to the culture ws tken up in the system without pubs5, which proved tht the culture, lthough nongrowing, exerted metolic ctivity. This ws further supported y nlysis of the respirtory ctivity. The specific consumption rtes of oxygen (q O ) nd cron dioxide (q CO ) were not significntly

6 RESULTS 35 lower thn the rtes of non-induced culture (Fig. 3.4). A fluorescence ssy for chrcteriztion of living from ded cells (BcLight, Moleculr Proes, USA) sed on the different permeility of the memrne for the two fluorescence mrkers showed tht ll cells were vile without induction with induction RQ 1..5 q CO [mmol g -1 h -1 ] Y CO/S [mol mol -1 ] q O [mmol g -1 h -1 ] c d Y O/S [mol mol -1 ] 6 4 e Figure 3.4: Fed-tch cultures of E. coli RB791 pkk177glucc without induction (--) or with induction ( ) y 1 mm IPTG. Comprison of the respirtory quotient (RQ, ), cron dioxide evlution rte (q CO, ), oxygen uptke rte (q O, d), yield of cron dioxide nd oxygen per glucose (Y CO/S, c; Y O/S, e). Figure 3.5: Colonies grown on pltes: () efore induction ll colonies were of the sme size; () 1 h fter induction culture segregtion into smll nd lrge colonies. The recovery of the cells on oth, NBII pltes with nd without the ntiiotic mpicilline, ws not ruptly inhiited, ut the cells recovered on the pltes with different time dely. In difference to non-induced cell which formed lrge colonies within 1 hours, fter induction the smll portion of cells which ws le to form colonies, showed time dely of up to 48 hours (Fig. 3.5). These smller colonies were counted s positive ccording to their colony forming ility. However, more thn 8 % of the cells from 6 h fter induction did not recover to growth. Concluding from these results, the popultion of cells which is shifting to the non-growing stte is vile ut incompetent for further division.

7 RESULTS with old medium + 1 g/l glucose OD 5. with new medium, without IPTG OD 5 3. with new medium. with IPTG OD 5 cfu on NB-plte cfu on Amp-plte cfu of plsmidfree-cell t f = h t f = 5 h t f = 9 h t lg = 8.5 h 4 t lg = ; µ =.75 [1/h] t lg = 11 h < t<4.5; µ= < t< ; µ~ t lg =? t lg = 11 h <t< 4; µ=.15 4< t<15; µ~ t lg =? t lg > 15 h t lg = 11 h colony forming ility [1 8 cfu/ml] t f = 15 h t lg > 1 h t lg > 1 h Figure 3.6: Reinocultion experiments into shke flsks with new minerl slt medium with cells from different phses during the fed-tch fermenttion of E. coli RB791 pkk177glucc. To exclude tht n inhiitory compound in the medium is cusing growth inhiition in the cse of induced cells, smples were inoculted to fresh or spent medium where only new glucose ws dded. In oth cses, smples from the fermenttion with lrge dividing-competent popultion strted to growth nerly immeditely (Fig. 3.6). However, long lg phse ws oserved in fresh s well in spent medium, if cells were tken s inocolum from the phse of the fermenttion where more thn 9 % of the cells were unle to recover to growth on pltes. The growth of these cultures ws contriuted to the overgrow of prt of the cells which were lso found to grow on pltes. The growth inhiition ws stronger if the strin with the plsmid pubs5 ws used. In this system, growth inhiition strted erlier nd the colony forming ility decresed y higher specific rte of.74 h -1 during the first 3 hours fter induction. After this first period the k d decresed to.7 h -1. Furthermore, s ovious in Fig. 3.3c, in ll cultures performed for α-glucosidse production, cells which hd lost the mpicilline resistnce could e detected y replic plting. The numer of these cells, which oviously do not contin the pkk177glucc plsmid, incresed to the end of the fermenttions. This cell popultion contriuted to out one third of the totl cell numer hours 1 ln (OD 5 )

8 RESULTS 37 fter IPTG ddition in E. coli RB791 pkk177glucc pubs5. No plsmid loss ws oserved in the control cultivtions without induction. In contrst to the plsmid pkk177glucc, the second plsmid pubs5 with the knmycine resistnce ws lwys stily mintined c Extrcellulr protein [g g -1 DCW] [g L -1 ] [g g -1 DCW] [g L -1 ] d Figure 3.7: Comprison of extrcellulr protein content during fed-tch fermenttions of E. coli RB791 pkk177glucc (, ) nd E. coli RB791 pkk177glucc pubs5 (, ) in dependence on induction of the α-glucosidse (open symol: no induction, filled symol: induction y ddition of IPTG). Extrcellulr protein ws mesured to investigte to which mount the non-dividing popultion is contriuting to cell lysis. The specific extrcellulr protein content reched up to level of. g g -1 DCW for the cells with pubs5 nd.8 g g -1 DCW for the cells without pubs5, which indicted tht the cells without pubs5 hd higher lysis rte (Fig. 3.7,d). Although the cell growth ws inhiited y the induction of α-glucosidse, in connection to cell lysis there ws no significnt difference etween the cultures with nd without induction. These results suggest tht the non-dividing cells were not lysing. The onset of incresing ccumultion rte of extrcellulr proteins correlted with the time when cultivtion ws shifted from tch phse to limited growth in oth systems. Surprisingly, in the culture with pubs5 the cells lysis ws lso inhiited fter induction for out 4 hours. Effect of CRIMI production on cell segregtion nd plsmid stility. The totl cell numer nd colony forming units during cultivtion of E. coli RB791 pdscrimi re represented in Fig No up-growth of plsmid-free cells ws oserved fter induction in this strin, ut prt of the cell popultion lost the ility to divide. The rte of the development of non-dividing popultion ws detected to e out.1 h -1 from 3 hours fter induction. Under this constnt rte the prt of the popultion, which hs lost its colony forming ility, ws out 9 % of the totl cells t the end of

9 RESULTS 38 the experiment. The nlysis of extrcellulr protein indicted tht the lysis rte is higher direct fter induction with mximum level of 1.7 mg g -1 h -1. cfu; cell numer [ml -1 ] Plsmid stility Extrcellulr protein content [g L -1 ] c totl cell numer cfu on NB-pltes cfu on Amp-pltes cfu/totl cell numer [%] [g g -1 DCW] Figure 3.8: Cell segregtion nd plsmid stility of fed-tch fermenttion with E. coli RB791 pdscrimi. ) Totl cell numer ( ) nd colony forming units (cfu; which re presented s totl cfu on NB pltes ( ) nd the prt of mpicilline resistnt cells ( )), ) Plsmid stility ( ) nd cell segregtion into vile ut no culturle cell ( ), c) extrcellulr protein content (g L -1, ; g g -1 DCW, ). Induction ws performed y ddition of IPTG to finl concentrtion of 1mM (dotted line) In summry, significnt differences were detected in connection to the influence of the recominnt production on the cellulr growth, cell survivl nd plsmid stility, etween the two recominnt processes, lthough oth products, α-glucosidse nd CRIMI, ccumulted to more thn 1 % of totl protein. Furthermore, in oth systems the cell segregtion into non-dividing cells ws oserved nd the popultion of cells which is shifting to the non-growing stte is vile ut incompetent for further division. This phenomenon ws erlier descried y Andersson et l. (1996,). The uthors showed tht the non-dividing cell popultion hs certin metolic ctivity, such s respirtion nd glucose uptke, nd ws therefore discussed to fulfill the requirements of the vile ut nonculturle (VBNC) sttus, which is known from the environmentl microiology. However, the VBNC sttus in recominnt cultures is neither well documented, nor relly proved. From the ville dt it is not cler, why this segregtion occurs nd wht is the moleculr ckground of the growth inhiition. Therefore, in the following chpter the sttus of the cells will e more thoroughly descried y investigting generl cellulr processes nd the energetic sitution of the cell in connection to induction of the recominnt product in glucose limited fed-tch cultivtions.

10 RESULTS Cellulr responses fter strong induction of recominnt -glucosidse 3..1 Activity of repliction, trnscription nd trnsltion Inhiition of cellulr rections s consequence of -glucosidse formtion. The growth inhiition in α-glucosidse expression systems, with nd without pubs5, is connected to the inhiition of cellulr rections, such s repliction, trnscription, nd trnsltion. The ctivity of repliction, trnscription, nd trnsltion were investigted y rdioctive incorportion experiments. Fig. 3.9 shows y incorportion of [ 14 C]-L-leucine nd [ 3 H]-thymidine tht trnsltion nd repliction re inhiited to more thn 8 % within three hours (trnsltion) or two hours (repliction) respectively in shke flsk culture of E. coli RB791 pkk177glucc. In contrst to this, trnscription ws rther stle. There ws reduction of [ 3 H]-uridine incorportion to 5 % within pproximtely 4 h fter induction. OD 5 1. A B Cultivtion time [h] Reltive Rte of Incorportion Figure 3.9: Inhiition of trnsltion () nd repliction () ( ), mesured y the reltive incorportion rte of [ 14 C]-leucine or [ 3 H]-thymidine respectively following induction of α-glucosidse gene expression in shke flsk cultures of E. coli RB791 pkk177glucc. The incorportion of the rdioctive molecules ws clculted on cell sis nd ws set to 1 % t the time of induction. Growth of the culture is shown y OD 5 ( ). Induction ws performed y ddition of 1 mm IPTG t time zero. However, ll three rections were very fst inhiited in the cells contining the pubs5 plsmid which re suspected to contin higher content of the rgu trna. This ws shown not only in shke flsk cultures (dt not shown), ut lso in glucose limited fed-tch fermenttion (Fig. 3.1). In the fermenttion, ll three processes, repliction, trnscription, nd trnsltion were inhiited much fster thn in control fermenttion without induction, where these processes were very much relted to the decrese of the specific growth rte. The trnsltionl ctivity ws decresed to out 5 % during the glucose limited growth (Fig. 3.1), wheres the trnscription ws rely influenced y the growth rte reduction. After induction of α-glucosidse production the incorportion of [ 14 C]-L-leucine ws further reduced to out 1 % of the rte which ws mesured

11 RESULTS 4 during logrithmic growth. A slight increse of the rte ws mesured from 5 hours fter induction, which is relted to the upcoming plsmid-free cell popultion. The level of trnscription ws even more reduced fter IPTG ddition to out 3 % of the level efore induction, ut recovered to out 3 % if the plsmid-free cells were growing up. The incorportion of [³H]-thymidine ws even fster inhiited thn trnsltion, which gin shows the high dependence of repliction not only on trnsltion, ut on the synthesis of limiting fctor which hs recently discussed s the DnA protein (Kogom, 1997). Leucin incorportion reltiv ctivity Uridine incorportion Thymidine incorportion c Figure 3.1: Comprison of the reltive incorportion rte of [ 14 C]-Lleucine (), [ 3 H]-uridine (), nd [ 3 H]-thymidine (c) s mesure of trnsltion, trnscription nd repliction ctivity in fed-tch cultivtions of E. coli RB791 pkk177glucc pubs5 without induction (s control, open circles) nd with induction y 1 mm IPTG (closed circles). The incorportion of the rdioctive molecules ws clculted on cell sis nd the vlues from the tch phse of growth were set to 1 %. Induction ws performed y ddition of 1mM (finl concentrtion) of IPTG (dotted line) Cultivtion time [h] In summry, it hs een shown y the incorportion experiments tht the overexpression of α-glucosidse strongly influenced the ctivity of repliction, trnscription nd trnsltion, ut to different level. Trnscription ws most strongly inhiited, wht would hve consequences for the induction of stress responses fter induction of the α-glucosidse. I ssume tht this inhiition is direct mintennce effect, tht mens n effect of the competition of the lrge mount of recominnt mrna for the ville riosomes nd therefore reduction of importnt proteins which re involved in these processes.

12 RESULTS Plsmid mplifiction fter induction In order to etter understnd the ppernce of plsmid-free cells following induction, nmely, whether plsmids re quickly degrded fter induction y process which could e similr to tht, pulished y Neuuer et l. (1996), or whether this plsmid-free popultion is cused y n upgrowth of plsmid-free cells y the growth dvntge which these cells would hve, if the growth of the producing cells is inhiited, we followed the plsmid content in glucose limited fed-tch cultivtions of E. coli RB791 pkk177glucc efore nd fter induction. Although there were no indictions in the literture for chnging plsmid copy numer fter induction of P tc promoter controlled expression systems, the plsmid content incresed on cellulr sis from out 1 copies per cell to out 4 copies fter induction corresponding to fctor of four (Fig. 3.11). In contrst to this oservtion, no increse of the plsmid copy numer per cell ws found in cultivtions without induction. The increse of the plsmid content ws prtly connected to the ccumultion of multimers of the plsmid pkk177glucc, which were visile on grose gels (Fig. 3.1). Plsmid content [mg g -1 DCW] Plsmid copy numer / Cell Figure 3.11: Comprison of plsmid content () nd plsmid copy numer sed on plsmidcontining cells () during fed-tch fermenttions of E. coli RB791 pkk177glucc (, ) nd E coli RB791 pkk177glucc pubs5 ( ) in dependence on the ddition of IPTG for induction of the α-glucosidse (open symol: no induction, filled symol: induction y ddition of IPTG). The feeding strted t zero hours (interrupted line) efore glucose ws exhusted. Plsmid concentrtions were clculted from spectrophotometricl nlysis nd were verge vlues of t lest three independent preprtions. Bsed on the repliction rte which decresed to pproximtely 8 % of the vlue mesured efore IPTG ddition, within two hours fter induction (Fig. 3.1) y mesuring the incorportion of [ 3 H]-

13 RESULTS 4 thymidine, the reltive incorportion rte of 8 % corresponds firly well to the clculted plsmid DNA content of n E. coli cell (out 1 copies of the plsmid pkk177glucc with size of kp), which is less thn 8 % of the totl DNA concentrtion of the cell (on ssumption of chromosome numer per cell of 1.85, chromosome size of 47 kp, clculted from dt given y Bremer nd Dennis, 1996). From these results, we conclude tht chromosoml repliction is nerly completely inhiited within two hours fter induction. Furthermore, it is ovious from Fig. 3.1 tht stle repliction (which includes plsmid repliction) pproximtely proceeds with constnt incorportion rte. Therefore, we ssume tht repliction proceeds only with the numer of plsmids which were replicting efore mplifiction strted. There seems to e no new formtion of repliction complexes, nd therefore the plsmid copy numer increses linerly, confirming the mesurements of Fig h h h 4 h 7 h 17 h 19 h 1 h St.. h 3 h 34 h 45 h 5 6 h 67 h 8 h h 131 h 411 h 81h St 1.3 h 3 h 4 h 5 h 6 h 7 h 8 h h h c. Figure 3.1: Plsmid preprtion of smples from the fed-tch cultivtions of E. coli RB791 pkk177glucc without induction () nd with induction () nd of E coli RB791 pkk177glucc pubs5 with induction (c). The smples were extrcted from the sme mount of cells ccording to OD 5 vlue y the Qigen method. The feeding ws strted t zero hours. The decrese of the plsmid content in this smple is due to the up-growth of plsmid-free cells. The different DNA nds re different isomers of the pkk177glucc plsmid, which cn e trnsferred to the liner plsmid form y HindIII digestion (St - DNA size stndrd, 1 k ldder). A trnsient plsmid mplifiction ws lso oserved y fctor of two in the system contining the oth pkk177glucc nd the pubs5 plsmids (Fig. 3.11). However, plsmid mplifiction ws only oserved for the pkk177glucc plsmid nd not for pubs5 which mintined t level of 1 copies per cell.

14 RESULTS 43 In contrst to the pkk177glucc plsmid, the plsmid pdscrimi ws stily mintined through the cultivtion, nd no significnt mplifiction occurred fter induction, just slight increse of the plsmid copy numer (Fig. 3.13). Plsmid content [mg g -1 DCW] Plsmid copy numer / Cell Figure 3.13: Plsmid content ( ) nd plsmid copy numer ( ) sed on plsmid-contining cells during fed-tch fermenttions of E. coli RB791 pdscrimi with induction y ddition of IPTG (dotted line). The feeding strted t zero hours (dshed line) efore glucose ws exhusted. Furthermore, ColE1-derived plsmids contining different recominnt genes which re controlled y the tc promoter were mplified following induction with IPTG, ut no mplifiction occurred if product formtion ws not induced. Plsmid mplifiction occurred in E. coli B strins s well s in K-1 strins with different plsmids (rop + nd rop - ) coding for vrious heterologous proteins. The mplifiction ws not cused y toxic effect of IPTG, ut ws relted to strong inhiition of trnsltion nd chromosoml repliction fter the induction of heterologous gene expression (for further results nd discussion see Teich et l., 1998) Influence of -glucosidse production on the chromosoml DNA supercoiling Inhiition of cellulr rections s consequence of α-glucosidse formtion nd the plsmid mplifiction due to the inhiition of chromosoml repliction were descried in the sections ove. Here I wnted to investigte the sttus of induced cells. Similr to Andersson et l. (1996), high product formtion ws connected to ppernce of non-culturle cells, which hd not lost ll metolic ctivities, nd even succeeded to mintin some glucose uptke nd respirtory ility. The development of non-dividing popultion with certin metolic ctivity fter overexpression of recominnt gene ws sometimes compred to the sttus of vile ut non-culturle cells (Andersson et l., 1996). However, the results from overproduction of α-glucosidse might lso imply tht the non-dividing cells perform the intermedite stte from growing to ded cell.

15 RESULTS 44 time relted to induction [h] -3.5 h h 4 h 6.5 h 16 h RB791 pkk177glucc pubs5 RB791 pkk177glucc -1 h 1 h 3 h 5 h 19 h Reltive er of nucleoid [%] Cultivtion time [h] Figure 3.14: Electron microscopic imges () of cells during different phses of glucose limited fed-tch fermenttions of E. coli RB791 pkk177glucc (upper pnel) nd of E. coli RB791 pkk177glucc pubs5 (lower pnel) with induction of α-glucosidse gene expression. The white re inside the cells corresponds to the chromosoml DNA which ecomes visile fter urnyl cette tretment. The verge reltive re of the nucleoid reltive to the totl cell re from the fermenttion of E. coli RB791 pkk177glucc ws clculted fter tretment of the photogrphs with n imge nlysis procedure, which is descried in the methods section nd is presented in (). The numers indicte the evluted cells for ech smple. The 5 % nd 75 % quntils re shown y the grey oxes nd rs respectively. Among different possiilities which would mke cell unle to recover on pltes, dmge of the chromosoml DNA would cuse the cells to e unle to replicte if this dmges would e not repired. Becuse of the high synthesis of the recominnt product, which ccording to rough estimte from the specific product formtion rte contriutes to out 85 % of the totl protein

16 RESULTS 45 synthesis shortly fter induction nd the strong decrese of trnsltion nd trnscription shortly fter induction, the induction of the SOS regulon, which is the cellulr response to DNA dmge, should e impired fter induction. However, single or doule strnd reks in DNA which would cuse n inility for repliction, were not detected y electrophoretic methods or y using poptosis test kits (dt not shown). Also chloroquine incorportion experiments, which cn e used to detect the degree of supercoiling of smll plsmids, filed to detect the decrese of supercoiling of the pkk177glucc plsmid, s it is too lrge for these investigtions. However, there ws indiction in the literture (Hecker et l., 1986) tht the cells, which re unle to induce the stringent response nd therefore dying within some dys if they re cultivted on pure phosphte uffer, show relxtion of the chromosome. Therefore, electron microscopy studies were crried out for checking the chnge in the chromosoml DNA during the recominnt fermenttions (Fig. 3.14). In electronmicroscopic imges the chromosome of vitl cell ppered condensed nd covered out 15 % of the cytoplsmic spce (see Fig. 3.14). In contrst, the chromosome ws extended to out 3 % of the cytoplsm in non-dividing or ded cell. The electronmicroscopic nlysis of smples from the fermenttions with induction indicted decrese in the pcking density of the chromosome, which ws ovious y the increse of the reltive chromosoml re. This could e sttisticlly proved y imge nlysis for E. coli RB791 pkk177glucc (Fig. 3.14). The nlysis ws impossile with the high production strin with coexpression of the rgu trna, s the lrge inclusion odies distur the imge nlysis. However, in this cse lot of cells, which hd decondensed chromosome, cn e oserved in the imges. From the results, the decrese in the density of the chromosome could e one importnt spect of the loss of cell division ility. This could e cused in principl y different mechnisms which will e discussed in the following section Anlysis of DNA inding protein (H-NS) nd LexA protein fter induction As ove mentioned, the loose of supercoiling could e cused y not repired strnd reks, y the reduced synthesis of DNA inding proteins fter induction, or y n uncontrolled ort of repliction. High product synthesis might lso influence the production of DNA inding proteins, ecuse the recominnt product is synthesized in competition to the cellulr proteins. Among them, H-NS seems to e the most importnt plyer connected to gene regultion. Spurio nd coworkers (199) showed tht the djustment of the H-NS concentrtion plyed n importnt role in cell

17 RESULTS 46 viility nd nucleoid condenstion. Therefore, the loss of DNA condenstion could e ssumed to e connected to decrese in the H-NS concentrtion. In contrst to the expecttion tht the mintennce of H-NS ws distured y the α-glucosidse production, the level of H-NS did not significntly decrese fter induction of the α-glucosidse within four hours fter induction (see Fig. 3.15). An interesting spect, which is not further discussed here, is tht the more thn twofold increse of the specific concentrtion of H-NS during the trnsient period from the tch phse to the fed-tch phse. This is interesting, s it confirms the oservtions of Dersch et l. (1993) nd Flconi et l. (1996), who hve oserved tht the level of H-NS increses in the erly sttionry growth phse, wherey the control mechnism seems to e sed on sensitive regultion y the concentrtions of Fis nd H-NS itself (Flconi et l., 1996). H-NS + H-NS - -1h 1h 3h 4h 6h 7h 8h H-NS reltive concentrtion [%] Cultivtion time [h] Figure 3.15: Anlysis of the H-NS protein during different phses of glucose limited fedtch fermenttion of E. coli RB791 pkk177glucc pubs5 with induction y 1 mm of IPTG (t three hours, interrupted line). Reltive levels of H-NS were determined y immunolot nlysis (). Sme cell concentrtions ccording to OD 5 were pplied to ech lne. Smples of MC41 hns + nd PD3 hns were pplied s reference. Bnds representing H-NS were quntified densitometriclly (), nd the dt otined were normlized for the vlue determined t the point of induction (smple 3). The feeding ws strted t zero hours (dotted line). Furthermore, the loss of DNA condenstion could e lso cused y DNA strnd reks or DNA dmge, which should e connected to the induction of the SOS regultory network. The expression of the genes which elong to this DNA dmge-inducile network re controlled y complex circuitry involving the RecA nd LexA proteins. LexA cts therey s the repressor of more thn genes, including its own lexa gene. As the SOS response is connected to clevge of the LexA protein y RecA, the typicl response fter SOS induction is connected to trnsient decrese of the LexA level. Indeed, rpid decrese of the LexA level ws found directly fter induction of the

18 RESULTS 47 α-glucosidse, which indictes DNA dmge (see Fig. 3.16). However, LexA is not coming up gin, s it does in non-induced cell, in which DNA dmge is induced y DNA dmging gents (see Sssnfr nd Roerts, 199). In contrst to our expecttion, the LexA protein ws found remining t the low level nd not coming up. The light increse in the lst smple (Fig. 3.16) is relted to the eginning up-growth of plsmid-free cell popultion, which, of course, hd the norml level of LexA nd contriutes to out 5 % of the totl cells in the culture t this time (compre to Fig. 3.3c) h 3 h 3h 3.5h 4h 5 h 6h 7h reltive vlue of lexa Cultivtion time [h] Figure 3.16: Anlysis of the LexA protein during glucose limited fed-tch fermenttion of E coli RB791 pkk177glucc pubs5 with induction y 1 mm of IPTG (t three hours, interrupted line). Reltive levels of LexA were determined y immunolot nlysis (). Sme cell concentrtions ccording to OD 5 were pplied to ech lne for demonstrtion. Bnds representing LexA were quntified densitometriclly (), nd the dt otined were normlized for the vlue determined t the point of induction. The feeding ws strted t zero hours The energy sitution following induction of -glucosidse The mintennce of the DNA superhelicity is n energy dependent process nd hs een shown to e connected to ATP/ADP rtio (vn Workum et l., 1996). Therefore, the ATP concentrtion nd the energy chrge (EC) in the cell were determined to check the question whether chnge in ATP nd EC fter induction of the α-glucosidse could cuse the relxtion of the DNA. Neither ATP, nor ADP nd AMP showed significnt chnge in their cellulr levels in control fermenttion of E. coli RB791 pkkglucc pubs5 without induction. The energy chrge (EC) vried in rnge etween.8 nd.95 nd the sum of the denosine phosphte concentrtion (AXP) did not chnge significntly (Fig f).

19 RESULTS 48 ATP [µmol g -1 ] g AXP [µmol g -1 ] d j 1 1 h 8 e k ADP [µmol g -1 ] ATP/ADP c i.8 f l AMP [µmol g -1 ] Energy chrge Cultivtion time [h] Cultivtion time [h] Figure 3.17: The level of denosine nucleotides nd energy chrge during fed-tch fermenttion of E. coli RB791 pubs5 in dependence on induction of the α-glucosidse (-f: no induction, g-l: induction y ddition of IPTG). The clcultion of ATP/ADP rtio efore induction ws sed on the men vlue of the two fermenttions (Fig. e, k). In the contrst to the control fermenttion, the denosine pool showed significnt chnges following induction of the α-glucosidse (Fig. 3.17g-l). First, the ATP level slightly incresed within one hour, which ws connected to n incresed respirtion (qo, see Fig. 3.18) nd decresed DOT (Fig. 3.49). However, t out 1.5 hours fter induction respirtion egn to decrese, nd from this point the ATP concentrtion lso decresed nd finlly reched very low level, which ws only out 3 % of the pre-induction level. This considerle decrese is connected to rpid increse of the ADP nd AMP levels, which in the sum were higher thn the sum of denosine phosphtes efore induction. The high increse of ADP nd AMP cnnot e explined only from the decrese of ATP, ut might e triggered y RNA degrdtion, s indicted y S8 immunolot nlysis (Fig. 3.19), which is similr to the system erlier descried y Dong et l. (1995). A sustntil mount of the riosomes ws lso degrded in this α-glucosidse production system fter induction. The rise of the lower phosphorylted nucleotides hd strong impct on the energy chrge, which decresed elow.3. Furthermore, the levels of AMP nd ADP do not only increse intrcellulrly, ut oth nucleotides re lso found in significnt mounts (out 5 %) in the extrcellulr medium. Even ATP ws detected in minor mounts in the cultivtion roth. In conclusion, it could e ssumed tht the decrese of the ATP level ut especilly the decrese of the energy chrge contriute to the loss of superhelicity.

20 RESULTS 49 RQ q O [mmol g -1 h -1 ] Y O/S [mol mol -1 ] c without induction with induction Figure 3.18: Comprison of the respirtory quotient (RQ, ), oxygen uptke rte (q O, ), nd yield of oxygen per glucose (Y O/S, c) etween fedtch cultures of E. coli RB791 pkk177glucc pubs5 without induction (----) nd with induction ( ) y 1 mm IPTG (dotted line). reltive mount of S8 [%] Cultivtion time [h] Figure 3.19: Concentrtion of S8 protein in glucose limited fed-tch fermenttions of E. coli RB791 pkk177glucc pubs5 with induction y 1 mm of IPTG t three hours fter feeding strt nd without induction (filled column, s control). The dt otined were normlized for the vlue determined t the point of induction. The feeding ws strted t zero hours Inhiition of glucose uptke rte fter overexpression of recominnt genes As discussed efore, it is only possile tht plsmid-free popultion grows up from very low level within few hours in glucose limited fed-tch if this popultion cn grow with pproximtely µ mx. Therefore, one should ssume tht the glucose uptke rte of the induced cells is significntly reduced in comprison to non induced culture, where never plsmid-free cells were coming up. This hypothesis ws checked y mesuring the mximum glucose uptke rte during different phses of the fed-tch fermenttions. An experiment ws designed to determine whether the mximum glucose uptke rte decrese fter overexpression of recominnt protein (Fig. 3.). In glucose limited fed-tch fermenttions q smx cn e esily determined y ddition of glucose pulse nd mesuring the consumption of glucose y rpid smpling. The glucose uptke ws connected to incresed respirtion, which ws ovious y declining DOT (Fig. 3.1). When the dded glucose mount ws so high to sturte the cellulr uptke system(s), the DOT pproched level tht corresponded to the q O t the mximum glucose

21 RESULTS 5 uptke rte or to the respirtory cpcity if this ws lower thn the mximum glucose inflow. Then the DOT kept t this vlue s long s the glucose uptke is constnt. Only if the glucose pproches level which i not sturting the uptke system(s) (tr), the DOT is rising. For E. coli this DOT rise is very rpid t cell concentrtions in the g cell dry weight per liter rnge due to the very low Ks vlue for glucose tht is in the order of few mg L -1 (Neuuer et l., 1995). RQ glucose, cette [g - L -1 ] q O, q qo, CO qco [mmol [mmol/gh] g -1 h -1 ] Respirtory Quotient c t P t R Acette DOT Glucose qco qo DOT [%] Figure 3.: Responses following glucose pulse during glucose limited fed-tch. (): Glucose ( ), cette ( ) nd DOT( ). The symols indicte mesured vlues, the lines correspond to simultion results. (): q O ( ) nd q CO ( ), (c): Respirtory quotient ( ). The glucose pulse is indicted y n rrow. From the DOT curve q smx cn e esily clculted y the time intervl etween the response of the DOT signl fter the glucose pulse nd tr, s expressed y the formul: qsmx= tr [Glc+ (F*Si)] tp V*X*(tR-tP) ( 4 ) The ppliction of this clcultion of the q smx only y the DOT signl ws restricted to the following conditions: (1) It cn e only pplied if the growth is limited y glucose, nd () if there is n respirtory cpcity which llows the decrese of DOT.

22 RESULTS ) E.coli RB791 pkk Glucose concentrtion [g L -1 ] DCW [g L -1 ] ) E.coli RB791 pdscrimi DOT [%] q smx [g g -1 h -1 ] Figure 3.1: Cell growth, glucose consumption, DOT nd q smx during different phses of fed-tch cultivtions of E. coli RB791 with pkk177glucc () or pdscrimi () with induction of 1mM IPTG (dotted line). Feeding strt t zero hour (interrupted line). The clculted q smx ws out 1.1 to 1.3 g g -1 h -1 during the tch phse. This cpcity for the uptke of glucose did not decrese ut even slightly incresed during the shift from glucose unlimited to limited growth. However, fter induction of the α-glucosidse, the q smx decresed to out 5 % of the q smx of the tch phse in the cultivtion of E. coli RB791 pkk177glucc (Fig. 3.1). q Smx ws even more reduced in the strin E. coli RB791 pkk177glucc pubs5 (Fig. 3.). This cpcity for uptke of glucose decresed to out 5 % of the mximum glucose uptke rte of the tch phse during control fermenttion without induction independent on the growth rte. After induction of the α-glucosidse rpid reduction in the mximum glucose uptke rte to out 4 % of the q smx of the tch phse ws determined. The nlysis of the residul glucose in the cultivtion medium resulted in q smx of.37 g g -1 h -1. Interestingly, q Smx ws lso inhiited in E. coli RB791 pdscrimi fed-tch cultivtions fter induction of CRIMI, ut lower rte thn in the α-glucosidse system (Fig. 3.1).

23 RESULTS 5 q smx [g g -1 h -1 ] without induction with induction upshift F979 DOT upshift glucose glucose Figure 3.: Comprison of q smx during different phses of fed-tch cultivtions of E. coli RB791 pkk177glucc pubs5 with induction ( ) nd without induction (----). Feeding strt (verticl interrupted line) nd induction with 1mM IPTG (verticl dotted line) re indicted. These results indicted tht the specific cpcities for glucose uptke (q scp ) nd oxygen consumption (q ocp ) were not constnt during the course of fed-tch fermenttion. Both could e significntly reduced y the production of recominnt proteins, which were importnt in considertion to the upgrowth of plsmid-free cells. The residul glucose in the medium cused n unlimited growth of the plsmid-free popultion tht cn rech considerle level of the totl popultion within few hours. The knowledge out the kinetics of the decrese of q smx nd q O ws used in model to simulte the up-growth of the plsmid-free popultion on the sis of monod kinetics. The mesured growth prmeters cn e correctly fitted y this model (lso see Fig. 4.3 nd Tle 4.3). 3.3 Stress responses fter induction of recominnt gene expression A decrese of the specific glucose uptke cpcity fter induction of α-glucosidse would result in severe cron nd energy strvtion if glucose is the only cron source. Furthermore, the inhiition of glucose uptke could e suggested to induce glucose strvtion specific cellulr responses, such s the camp/crp medited response, the generl stress response nd the stringent response. In this section, the effect of the overexpression of heterologous genes (α-glucosidse nd CRIMI) in E. coli on the level of different stress response regultors ws investigted Level of the stringent response regultor ppgpp It is well estlished tht the generl stress response nd the stringent response re importnt dpttion strtegies of E. coli in order to survive dverse environmentl conditions, such s prolonged strvtion (McCnn et l., 1991). Confirming the results of Andersson et l. (1996), Fig. 3.3 shows tht the level of the lrmone ppgpp ws highly elevted in the trnsient phse from unlimited growth to glucose limittion in fed-tch fermenttions, ut decresed lso fst nd remined t higher level during the glucose limited growth phse thn in the unlimited growth phse.

24 RESULTS 53 When smples were collected during the fermenttion nd exposed to glucose strvtion (see Fig. 3.4), ppgpp concentrtion rised within 6 sec to out.3 µmol g -1 DCW confirming the results of Neuuer nd coworkers (1995) tht chnges in the glucose vilility y chnges in the feed rte re immeditely responded y n pproprite chnge of the ppgpp level (lso see Andersson et l., 1996). ppgpp [µmol g -1 DCW] c d RB791 - pubs5 - IPTG - pubs5 + IPTG + pubs5 + IPTG DCW [g L -1 ] Figure 3.3: Cellulr concentrtion of ppgpp (, µmol g -1 DCW) nd the dry cell weight (, g L -1 ) in fedtch cultivtions of E. coli RB791 pkk177glucc with nd without pubs5. () E. coli RB791; () E. coli RB791 pkk177glucc without induction; (c) E. coli RB791 pkk177glucc with induction; (d) E. coli RB791 pkk177glucc pubs5 with induction. The time of induction is indicted y dotted line t 3 h fter feed strt. ppgpp is suggested to increse lso in () nd (c) within three hours fter feeding strt, however, no dt were nlyzed in this time frme for these two fermenttions ppgpp [µmol g -1 DCW] h 4h. 1 3 Time fter removl of smple from the culture [sec] Figure 3.4: ppgpp concentrtion fter removl of smple from the culture (t time zero). The two different smples were tken t the time 3 h nd time 4 h from the glucose limited fed-tch fermenttion of E. coli RB791 without plsmid. As descried in section 3..6, we found tht the cellulr glucose uptke cpcity ws strongly inhiited fter induction of the α-glucosidse, nd concluded tht the cells would run y this into glucose strvtion. Proly, this would lso result in ppgpp response, which hs een shown for humn SOD y the group of K. Byer (Cserjn-Puschmnn et l., ). Thus, similr to the

25 RESULTS 54 induction of het shock like response following overexpression of recominnt gene which hs een descried for mny proteins y mny uthors (Goff & Golderg, 1985, 1987; Kosinski & Biley, 1991; Kosinski et l., 199), the stringent response nd following, y the positive impct of ppgpp on rpos expression, lso the generl stress response might e induced fter strong induction of recominnt gene. In order to nswer the question whether the stringent response lrmone ppgpp plys ny role in the growth inhiition nd decrese of colony forming units fter induction of the tc-promoter directed overexpression of the α-glucosidse, the level of ppgpp fter induction ws nlyzed y HPLC nd is shown in Fig. 3.3c,d. Similr to the control cultivtion with the plsmid-less strin, there ws n increse of ppgpp in ll cultures, when they run into glucose limittion (shown only for RB791 pkk177glucc pubs5 in Fig. 3.3d). After the pek, the level of ppgpp incresed to out % of the vlue of the tch phse in the glucose limited growth phse. However, fter induction of the α-glucosidse expression, ppgpp ws found to drop elow the detection limit within two hours (Fig. 3.3c,d), independently, whether the strin contined the pubs5 plsmid or not. The results suggest tht no stringent response is induced fter overproduction of α-glucosidse in oth systems with nd without plsmid pubs The s S - relted generl stress response Level of s S fter induction of recominnt -glucosidse. The rpos encoded sigm fctor σ S (σ 38, RpoS, KfF) is mster regultor which is induced in response to vriety of environmentl stress conditions tht include strvtion for vrious nutrients, entry into sttionry phse, high osmolrity, cid shock, nd high or low temperture (Lnge & Hengge-Aronis, 1991; Hengge- Aronis, 1996,, 1999). Furthermore, it hs een proved tht rpos gene expression is positively regulted y ppgpp (Gentry et l., 1993; Lnge et l., 1995). Therefore, it is worth to see on the level of σ S in connection to the surprising result of the ppgpp decrese fter induction of α-glucosidse. As shown in Figure 3.5 nd c (see lso Fig. 3.6,c), the concentrtion of σ S incresed, ccordingly to the ppgpp level, in the control fed-tch cultures t the point when the culture run into glucose limittion independent, whether the strin contins plsmid or not. A 1.5 to two-fold higher level of σ S thn in the tch phse ws mintined up to the end of the cultivtion if no induction ws performed, lthough σ S slightly decresed to out 5 % of the mximum vlue in some cses.

26 RESULTS 55 reltive σ S concentrtion reltive σ S concentrtion c - pubs5 - IPTG + pubs5 - IPTG d - pubs5 + IPTG pubs5 + IPTG 5 1 Figure 3.5: σ S in fed-tch cultivtions of E. coli RB791 pkk177glucc with nd without pubs5. () E. coli RB791 pkk177glucc without induction; () E. coli RB791 pkk177glucc with induction; (c) E. coli RB791 pkk177glucc pubs5 without induction; (d) E. coli RB791 pkk177glucc pubs5 with induction. Reltive σ S levels (grey rs) were normlized for the vlues determined during exponentil growth fter immunolot nlysis nd densitometricl quntifiction. The time of induction is indicted y n interrupted line. Time zero corresponds to the strt of glucose limittion ccording to the DOT signl (not shown). ) ) h F h c) h d) h Figure 3.6: Immunolots of the cellulr levels of σ S in glucose limited fed-tch fermenttions of E. coli RB791 pkk177glucc with nd without pubs5. () E. coli RB791 pkk177glucc without induction; () E. coli RB791 pkk177glucc with induction; (c) E. coli RB791 pkk177glucc pubs5 without induction; (d) E. coli RB791 pkk177glucc pubs5 with induction. A time of zero hours is relted to the feed strt. IPTG ws dded t 3 h to the cultivtions of pnels () nd (d). After IPTG ws dded to cultures of E. coli RB791 pkk177glucc pubs5 the concentrtion of σ S significntly decresed to only % of the vlue t the induction point within one hour (Fig. 3.5d). The reduction of σ S ws lso mesured in cultures with E. coli RB791 pkk177glucc without pubs5 (Fig. 3.5). In ccordnce to these results, no increse of σ S level ws mesured

27 RESULTS 56 fter ddition of IPTG in shke flsk cultures of oth strins where the level of σ S ws closely to the detection limit t the point of induction (dt not shown). Effect of clpp muttion on the overexpression of the -glucosidse. Opposite to our expecttions, the nlysis of stress response regultors showed tht neither ppgpp nor σ S is induced in response to the growth inhiition cused y overproduction of the α-glucosidse. Rther, the concentrtions of σ S nd ppgpp decresed to the detection limit. According to the results of Schweder nd coworkers (1996) tht σ S is n unstle protein nd is degrded y the ClpXP protese, the effect of clpp-deficient muttion on the overproduction of recominnt α-glucosidse in Escherichi coli ws investigted, s one could suggest tht the decresed level of σ S could e responsile for the loss of cell viility fter induction of the α-glucosidse. In ccordnce to the results of Schweder nd coworkers (1996) high level of σ S ws found y immunolot nlysis in the clpp mutnt strin efore, nd lso fter induction with IPTG (Fig. 3.7c). The nlysis of the colony forming ility showed significnt higher viility of the clppdeficient strin in comprison to the wildtype (Fig. 3.7). By ssumption tht the colony forming ility is decresed y first order kinetics, this differences re relted to specific deth rte k D of.5±.8 h -1 for the clpp + wildtype strin nd.6 h -1 for the clpp mutnt. Generlly the clpp mutnt grew significntly more slowly (µ mx =.51 h -1 ) thn the wildtype (µ mx =.79±.5 h -1 ), possily due to higher level of σ S. This higher level of σ S in the clpp mutnt further incresed fter glucose limittion during the trnsient to glucose limited growth nd remined t high level during the fed-tch phse of growth. This high level ws not influenced y induction of the α-glucosidse in the clpp mutnt (Fig. 3.7c). From this result one could suggest tht the higher σ S level is responsile for the etter survivl. However, the high level of σ S nd the etter survivl in the clpp mutnt did not improve the yield of the α-glucosidse (Fig. 3.7). On the contrry, the α-glucosidse level in the clpp mutnt ws clerly lower thn in the wild type. The corresponding specific production rtes (q p in g per g cell dry weight nd hour) were 15 mg g -1 h -1 for the clpp + wildtype strin nd 4 mg g -1 h -1 for the clpp mutnt. Interestingly, the growth of the culture ws not inhiited fter induction in this mutnt, ut ws pproximtely the sme s in the culture without induction.

28 RESULTS 57 Colony forming units [ml -1 ] α-glucosidse [mg g -1 ] clpp rpos wt clpp wt wt clpp Figure 3.7: Colony forming ility () nd α-glucosidse production () of E. coli RB791 pkk177glucc pubs5 ( without induction; with induction) nd of the corresponding clpp ( ) nd clpp rpos ( ) mutnts in glucose limited fed-tch cultivtions. The feeding strt is indicted y dotted line nd the point of IPTG ddition is shown y the interrupted line. The dt origin from two independent fermenttions of ech strin. (c) Immunolot of the cellulr levels of σ S in the clpp mutnt E. coli RB791P pkk177glucc pubs5 with induction. 1 clpp rpos c -3 h -h h 1 h 3 h 4 h 5 h 6 h 7 h Furthermore, experiments with clpp rpos doule mutnt (RB791PS) were performed in order to nswer the question whether the high level of σ S competes with σ 7 for the ville RNA polymerse nd thus is responsile for the lower production of α-glucosidse in the clpp mutnt. In the clpp rpos doule mutnt µ mx ws very similr to the growth rte of the wild type. But in contrst to the wild type, the doule mutnt, similr to the clpp mutnt, showed clerly longer lg phse of growth (lso see Dmeru nd John, 1993). Following induction, the clpp rpos doule mutnt showed strong loss of viility (k d =.79 h -1 ), lthough surprisingly the yield of α-glucosidse ws not s high s in the cultivtion of the wildtype, nd only slightly higher thn in the clpp mutnt (Fig. 3.7). However, the nlysis of α-glucosidse mrna showed tht the induction of the recominnt gene ws the sme in the doule mutnt s in the wild type, ut lower in the clpp mutnt (see Fig. 3.9f). These results indicte tht σ S competes with σ 7 for the ville RNA polymerse in ccordnce to our suggestions, ut on the other hnd, tht σ S is lso importnt for the survivl of the cells fter stress nd recominnt gene induction. Effect of CRIMI overproduction on the generl stress response. In contrst to the cse of α-glucosidse overproduction where no increse of σ S ws determined fter ddition of IPTG, the induction of recominnt CRIMI cused strong increse of σ S (Fig. 3.8).

29 RESULTS 58 ) h 4 ) 3 reltive σ s Figure 3.8: Cellulr σ S response (: immunolot, : reltive σ S vlue) during glucose limited fed-tch culture of E. coli RB791pDSCrimi. A time of zero hours is relted to the feed strt (interrupted line). IPTG ws dded t 3 h to the cultivtion (dotted line). After the ddition of IPTG to the culture, the concentrtion of σ S incresed mrkedly within four hours, clerly demonstrting rpid generl stress response to the overproduction of the heterologous protein CRIMI. Furthermore, the level of σ S remined t higher level up to the end of the cultivtion. Although oth products, α-glucosidse nd CRIMI, were ccumulted to more thn 1 % of totl cell proteins nd to mjor prt s inclusion odies, significnt differences were detected in connection to the influence of the recominnt production on the cellulr growth nd cell survivl. The results suggest tht induction strength on the trnscriptionl level nd the strength of the riosome inding site, even the gene codon usge of recominnt gene influence the ehvior of stress signls. Moreover, n pproprite generl stress response my e importnt for mintennce of protein synthesis over longer time intervl in fed-tch fermenttions Comprison of mrna levels of genes controlled y different s fctors As descried ove, the oserved higher σ S level in the clpp mutnt ws suggested to compete with σ 7 for the ville RNA polymerse molecules, which consequently would lower the σ 7 -promoter directed expression of the α-glucosidse in the clpp mutnt. Therefore, the mrna levels of different σ S nd σ 7 dependent genes were nlyzed y investigting the mrna level of the σ 7 -dependent α-glucosidse gene, the rpos gene nd the σ S -dependent gene osmy. The σ 7 -dependent rpoa mrna coding for the α-suunit of the RNA polymerse ws used s control.

30 RESULTS 59 6 α-gluc f α-gluc 4 rpos g rpos 1 reltive concentrtion 1 6 c d osmy dnk h i osmy dnk 4 e rpoa k rpoa Figure 3.9: Concentrtion of different mrna s in glucose limited fed-tch cultures of E. coli RB791 pkk177glucc pubs5 (-e) with induction (lck rs) nd without induction (reference, striped rs) in comprison to corresponding cultivtions with the RB791P (gry rs) nd the RB791PS (white rs) strins (f-k). The concentrtions were clculted on the sis of the totl RNA nd normlized to the vlues determined short efore induction. The zero vlues in grph (c) of osmy mrna elong to the fermenttion with induction of α-glucosidse. No osmy mrna could e detected in the clpp rpos mutnt. The time of IPTG ddition is indicted y dotted line. Fig. 3.9 shows tht the level of the rpos mrna is drsticlly reduced following induction of the α-glucosidse production in the wild type. This differs from the control cultivtion without induction where the level ws higher during the fed-tch phse thn the exponentil growth phse. The decrese in the rpos mrna level fter induction of the tc-promoter is consistent with the decrese in the σ S level, nd the low ppgpp level. Furthermore, the low level of σ S, consequently negtively influences the expression of genes, which re positively controlled y σ S, such s osmy. After induction of the α-glucosidse, the mount of osmy mrna decresed elow the detection limit nd

31 RESULTS 6 did not rise to the end of the cultivtions (19 hours fter induction, dt not shown). In contrst, the osmy mrna level in the control cultivtion without induction incresed with dely of some hours fter σ S (Fig. 3.9c), possily ecuse it is negtively controlled y further fctors, such s camp nd Lrp (Lnge et l., 1993). The level of the osmy mrna ws very high in the clpp mutnt in greement with the high σ S level in this strin. It ppers tht osmy is exclusively trnscried y the σ S RNA polymerse holoenzyme. No osmy mrna could e detected in rpos mutnt (dt not shown). In order to prove whether the competition of σ S nd σ 7 for RNA polymerse lowers the induction of α-glucosidse t the mrna level in the clpp mutnt, the α-glucosidse mrna level in the clpp rpos doule mutnt ws investigted. In this doule mutnt, α-glucosidse mrna incresed within one hour fter induction to pproximtely the sme level s in the wild type, which would e consistent with the hypothesis. However, in contrst to the wild type, the mount of the α-glucosidse mrna decresed more quickly. Wheres the highest level of α-glucosidse mrna ws detected in the wildtype three hours fter induction, the vlue in the clpp rpos doule mutnt ws highest one hour fter induction. rpoa mrna ws mesured s control for host vegettive gene which is trnscried y σ 7 RNA polymerse holoenzyme. The mount of this mrna ws influenced only to minor extent y the induction of the α-glucosidse. However, within pproximtely two hour fter induction the concentrtion of rpoa mrna drops to 5 %, corresponding to high synthesis of dnk mrna (Fig. 3.9d) nd groel mrna (not shown here, see Jürgen et l., sumitted mnuscript) t this time. Thus, one could suggest competitive effect of σ 3 shortly fter induction, which ws lso supported y the D PAGE nlysis of the smples from the sme fermenttions where higher level of DnK nd GroEL ws detected fter induction of the tc-promoter directed overexpression of the α-glucosidse (Jürgen et l., sumitted mnuscript). This is in ccordnce with numer of erlier studies from other groups (Goff & Golderg, 1985, 1987; Kosinski & Biley, 1991; Kosinski et l., 199), which showed tht the het shock response is induced y recominnt protein production. Interestingly, n increse of the dnk mrna level ws only found in the wild type, ut not in the clpp nd clpp rpos mutnts, possily due to the low synthesis of α-glucosidse in oth mutnts, ecuse it is known tht the incorrectly folded product induces the het shock like response in recominnt production systems.

32 RESULTS Level of camp in fed-tch fermenttions of E. coli camp is further nucleotide with regultory role in the cellulr response to glucose limittion nd strvtion. Although the phenomenon of camp formtion y the denylte cyclse in connection to glucose vilility nd its influence in the trnscriptionl gene regultion in connection to CRP re intensively studied, the knowledge is low concerning the mechnisms of camp efflux nd reconsumption nd possile consequences for the cell physiology. However, it is well-known phenomenon tht the cellulr level of camp drmticlly increses fter glucose exhustion nd is kept t intermedite levels if glucose concentrtions re low (Sier, 1996). camp enles the camp receptor protein (CRP, CAP) to inding t crp regions triggering the stimultion of severl genes (Eright et l. 1989). Although camp is very fst recting molecule in the cell, it is chemiclly stle nd cn e esily determined in medium smples. According to Mtin (198), lmost ll (over 99.9%) of the camp mde y E. coli grown t severl dilution rtes in chemostt ws excreted into the medium. They suggested tht extrcellulr camp possily lso hs role in sensing cell density or other intercellulr communiction. Therefore, camp ws nlyzed in the fermenttion medium, to investigte whether differences cn e determined from vrious fermenttions nd to which extent the camp dt correlte to the other stress response regultors. Extrcellulr camp nlysis fter overproduction of -glucosidse. Figure 3.3 illustrted the kinetics of the medium camp concentrtion during fed-tch cultivtions of E. coli RB791 pkk177glucc with nd without pubs5. After totl consumption of initil glucose the level of camp in medium egn to increse in oth strins, lthough the ccumultion rte ws different nd rnged from.46 µmol L -1 h -1 to.3 µmol L -1 h -1 within the first 4 hours of the glucose limited phse. In the non-induced cultivtion of E. coli RB791 pkk177glucc pubs5 camp ws ccumulted in the cultivtion medium to concentrtion of 9 µm (Fig. 3.3). Similrly, no significnt difference ws oserved in the cultivtion of E. coli RB791 pkk177glucc fter induction (Fig. 3.3). The efflux of camp mintined constnt level nd the specific camp concentrtion in medium ws t n intermedite level of.8 ±.1 µmol g -1 DCW. Surprisingly, the medium camp concentrtion strted to decrese pproximtely one hour fter induction of the α-glucosidse in cultures with E. coli RB791 pkk177glucc pubs5 (Fig. 3.3c). The camp ws reconsumed with rte of.97 µmol L -1 h -1. At the sme time the increse of the glucose concentrtion in the medium (Fig. 3.1h) cn e detected. Furthermore, the volumetric concentrtion of camp incresed gin from 11 hours fter feed strt, where glucose ecme

33 RESULTS 6 limiting gin due to up-groeth of plsmid-free cells. However, from this time period only the upcoming plsmid-free cells were supposed to produce camp, s the concentrtion ws only incresing very slowly. This suggestion is sed on the clcultion of the camp ccumultion y ssuming the sme rte of camp efflux s fter the tch phse, when the culture ecme glucose limited for the first time. camp content [µmol L -1 ] c E. coli RB791 pkk177glucc + pubs5 - IPTG - pubs5 + IPTG + pubs5 + IPTG, 1,5 1,,5,, 1,5 1,,5,, 1,5 specific camp content [µmol g -1 DCW] Figure 3.3: Concentrtion of extrcellulr camp ( µmol L -1 ; µmol g -1 DCW) in glucose limited fed-tch fermenttions of E. coli RB791 pkk177glucc with nd without pubs5. () E. coli RB791 pkk177glucc pubs5 without induction; () E. coli RB791 pkk177glucc with induction; (c) E. coli RB791 pkk177glucc pubs5 with induction. The interrupted line represents the time of feeding strt nd the dotted line indictes ddition of IPTG. 5 1,,5, Influence of recominnt CRIMI product on the concentrtion of camp. Besides strong increse of σ S s descried in section 3.3., overproduction of CRIMI ws lso connected to continuous ccumultion of camp in the culture with rte of 1.77 µmol L -1 h -1 tht ws not chnged y the ddition of IPTG (Fig. 3.31). The specific concentrtion of camp reched level of 1.7 µmol ḡ 1 DCW. In contrst to the α-glucosidse, the efflux of camp ws not ffected y overproduction of CRIMI.

34 RESULTS 63 camp[µmol L -1 ] E.coli RB791 pdscrimi, 1,5 1,,5 specific camp content [µmol g -1 DCW], 1 3 Figure 3.31: Concentrtion of extrcellulr camp ( µmol L -1 ; µmol g -1 DCW) during glucose limited fed-tch fermenttion of E. coli RB791 pdscrimi. The interrupted line represents the time of feeding strt nd the dotted line indictes ddition of IPTG. Comprison of ppgpp, s S nd camp level in glucose limited fed-tch cultivtion of E. coli MC41 rela +. Furthermore, the level of ppgpp, σ S nd camp ws mesured in glucose limited fed-tch cultivtion of E. coli MC41 rela + (Fig. 3.3). The smples were otined from the cultivtions performed y the group of G. Lrsson (KTH, Stockholm) within EU-project (BIO4- CT98-167) to study cell physiology under fed-tch cultivtion conditions. The cultivtion ws performed to rech cellulr density of 7.5 g dry cell weight per liter in 1 L fermenter with such feed protocol tht n initil exponentil feed ws followed y constnt feed. In this nonrecominnt fed-tch cultivtions, the level of ppgpp reched n pproximtely constnt level during the exponentil feed phse nd incresed slightly fter the chnge to the constnt feed (Fig. 3.3). Fig. 3.3 illustrtes the levels of two different cellulr sigm fctors. The house keeping sigm fctor σ 7 showed no significnt chnges during the cultivtion. In contrst, the level of σ S incresed out twofold from the sl level of cells growing with the mximum growth rte (first smple in Fig. 3.3c). Interestingly, σ S lso tendentiously decresed during the lst hours of the fermenttion. The level of intrcellulr camp ws determined to e out.5 µmol g -1 DCW during the exponentil feed phse nd ccumulted to mximum of 1 µmol g -1 DCW during the constnt feed phse. Menwhile, the extrcellulr camp ccumulted exponentilly to 15 µm in the cultivtion medium (Fig. 3.3c). However, if the specific growth rte decresed elow.5 h -1 shortly fter the shift from the exponentil feeding to the constnt feeding, the efflux of camp ecme lower nd finlly the extrcellulr camp concentrtion declined. This chnge in the camp ccumultion ehvior seems firly well to correlte with the switch from phse two of growth (limittion) to phse three (severe strvtion) in the model of different growth phses presented y Chesro (199).

35 RESULTS 64 DCW [g L -1 ] 1 5 exponentil feed constnt feed ppgpp [µmol g -1 DCW] 3. 3 reltive σ S 1 1 reltive σ 7 camp [µmol g -1 DCW] c intrcellulr camp 3 1 camp [µmol l -1 ] 1 3 Figure 3.3: Cell growth nd the level of stress regultors during glucose limited fed-tch cultivtions of E. coli MC41 rela + in 1 L fermenter. ) Cell growth ( ) nd ppgpp concentrtion (r), ) reltive σ S (open r) nd reltive σ 7 (filled r), c) concentrtion of extrcellulr camp ( µmol L -1 ; µmol g -1 DCW) nd intrcellulr camp ( µmol g -1 DCW). The interrupted line mrks the time point of chnge from exponentil to constnt feed. Error rs indicte vritions of smples tken from two independent fed-tch cultivtions. 3.4 Cell segregtion nd stress responses in lrge-scle cultivtions One tsk of this thesis ws to study the cell growth kinetics nd physiology during lrge-scle fermenttions which were performed within EU network project sed on integrtion of microil physiology nd fluid dynmics, in order to develop generlized methodologies nd tools to identify criticl prmeters to e included in new scle-up methodologies for microil ioprocesses. Here I present the dt of lrge-scle fed-tch fermenttions of E. coli with nd without overproduction of recominnt protein ZZ (17.7 kd), modified B domin of stphylococcl protein A, in industril 1 m 3 nd 3 m 3 scle iorectors respectively. The mjor ojective of our group ws the nlysis of cell viility, plsmid stility, energy chrge nd stress response in lrge-scle processes Lrge-scle cultivtions of E. coli W311 In order to get comprehensive informtion out the cell growth nd sustrte consumption in industril scle ioprocesses, three fed-tch fermenttions of E. coli W311 with different stirrer configurtions nd feed strtegies were performed in the 3 m 3 stirred tnk rector with n initil

36 RESULTS 65 working volume of m 3 (see Tle 3.). The growth profiles nd on-line dt from one of the three similr cultivtions re shown in Fig nd Fig This fermenttion (ET1) ws performed with stndrd Rushton configurtion (see Fig. 3.37) nd ws fed with glucose to the top of the fermenter, just elow the liquid surfce. Ammoni for ph-control ws dded ove the liquid surfce. DCW [g L -1 ] Feed rte [L h -1 ],5,4,3, µ [h -1 ] 1 5,1 glucose [mg L -1 ] ,5 1,,5 q s [g g -1 h -1 ],, Figure 3.33: Fed-tch cultivtion of E. coli W311 in 3 m 3 iorector. ) cell dry weight ( ), specific cell growth rte (µ, ---) nd feed profiles ( ); ) mesured glucose concentrtion ( ) nd specific glucose uptke rte (---). (The glucose dt were provided y B. Xu). During the first phse with exponentil feeding the specific growth rte styed pproximtely constnt t.39 h -1. The second growth phse, fter switch from qusi exponentil feeding to constnt feeding t 8 hours, ws chrcterized y grdully declining specific growth rte (Fig. 3.33). Within the constnt feed phse, the overll iomss yield per glucose ws clculted to e.8 gg -1. The glucose concentrtion ws low ut showed slightly incresing tendency from 5 mg L -1 to 65 mg L -1 (Fig. 3.33). The mximl specific glucose uptke rte ws 1.3 g g -1 h -1 during the first phse of the cultivtion.

37 RESULTS m 1 m 1 9 DOT [%] 6 4 O (%) 18 6 CO (%) 15 3 Air flow [m 3 min -1 ] ph Tempertur [C ] d c m 1 m Stirrer [rpm] Addition of NH 3 [l] rq (mol CO /mol O ) Yo/s (mol O /mol Glucose) Yco/s (mol CO /mol Glucose) c d Time (h) q q O (mmol O g -1 h -1 CO (mmol CO g -1 h -1 ) ) Figure 3.34: On-line mesurement during fed-tch cultivtion of E. coli W311 in 3 m 3 iorector [ET1]. Figure 3.35: Respirtion dt during lrge-scle fermenttion with E. coli W311 [ET1]. The on-line mesurement during fed-tch cultivtion of E. coli W311 indicted tht DOT nd ph depend on the plce in the rector where the electrode re instlled, which suggests rector inhomogeneity (Fig. 3.34,c). In this fermenttion foming ws cused overtitrtion of NH 3 nd hence the ph incresed to 7.5 fter 5 h of fermenttion (Fig. 3.34c). Due to the limittion of power input, the stirrer speed only rnged from 5 rpm to 15 rpm (Fig. 3.34). The DOT ws regulted y rising the ir flow to the rector limit of.8 vvm. Therefore, n incresed CO concentrtion of 8 % in exhust gs ws oserved during the exponentil feed phse. After chnging to constnt feed, the concentrtion of exhust O nd CO remined pproximtely constnt nd the respirtion quotient RQ ws out 1.1 (Fig. 3.35,). This mens tht the mount of produced cron dioxide per consumed glucose (Y CO/S ) nd the mount of consumed oxygen per consumed glucose (Y O/S ) incresed with time. Theoreticlly, the quotient Y O/S is 6 for the complete oxidtion of glucose. The quotient Y O/S incresed from 1.7 to.5 during the cultivtion fter constnt feed strt nd indicted tht more nd more of the consumed glucose ws used for mintennce energy.

38 RESULTS 67 OD L Preculture Fermenter 3 m 3 Fermenter.8 15 c ET1 ET ET µ [h -1 ] Cell numer [ml -1 ] d Figure 3.36: Comprison of three similr lrge-scle cultivtions of E. coli W311 [ET1 ( ), ET ( ), ET3 ( )]. nd show the pre-cultivtions in the 15 L iorector, wheres c nd d show the fed-tch fermenttions in the 3 m 3 iorector., c) OD 5 (closed symols) nd specific growth rte µ (open symols);, d) colony forming units (open symols), nd totl cell numer (closed symols). () Rushton stirrer (rdil pumping) () Sc 6SRGT (rdil pumping) (c) Sc 4SHP (xil upwrd pumping) Figure 3.37: Stirrers used in the lrge-scle cultivtions (Vráel et l., 1999; for further informtion see Tle 3.). A comprison of cell growth from three similr lrge-scle cultivtions with different stirrer configurtion (Fig. 3.36) is presented in Figure The finl vlues of these fermenttions re summrized in Tle 3.. The dt from the precultures in the 15 liter scle indicted n exponentil growth phse y opticl density nd colony forming units with specific growth rte of.56 ±.5 h -1 (Fig. 3.36,). During preculture OD 5 of 1 corresponded to cell numer of ml -1. The growth in fermenttions ET1-3 ws very similr nd no cell segregtion into non-culturle cells ws oserved to the end of the cultivtions (Fig. 3.36d). The opticl cell density profile in the 3 m 3 scle indictes two distinct phses (Fig. 3.36c) ccording to exponentil feed nd constnt feed phse. Within the first 1 hours cells grew with constnt specific growth rte of