Bich Engincrng Jounal 164 202) 107782 Ca Biochemical Engineering Journal journal homepage: wwy.lsevier.com/locatefbe) Ethanol fed-batch bioreactor operation to enhance therapeutic protein ‘wate production in Pichia pastoris under hybrid-architectured ADH2 promoter Omar Wehbe, Oguz Ulag Yaman'*, Pinar Gabik* Deere of Chel nse Bich Retin Bgierig Lalas, Md at Teal Unies, 0500 Askar, Trey Depa’ of Bisel, ntl tly a eae raring Labora Grau Sea of Natural an Ale Ses, Ml Ea Tee Unter, 0580 Ankara. Trey ARTICLE INFO apsTRact ower hal fic recon Phin pstrs Races pa) Ibestaeiected ADHZ promote spree tes We presente ethanol fed batch bioreacior operations (FBBOS) designed for enhanced recombinant human growth bonnone (chGH) production in Pichia pastors eoustucted with novel hyd atchltetused ADH pro totes Pariacasss: The parameses in che fed batch fenmeatation of ethanol weie investigated, and the boundaries ofthe proton domain inter of the desig patameses wete detemined, Two FRBO design tethods were ed, and a platform aiming toast clini metabo forthe generation of constant speci srw ate (x) and substate concentration (Cpiog) Was established. The highese overall productivity (pi) Was ‘senerated with mode base continuous fee steam (CFS) desig with Hep ~ 0.035 i UPC yge <9 88 py 30mg"! and Gace ~34 mg The ethanol stat FBBO at Crear 0.5 8 allowed increased product, ter th high prostivty and he highest Cj) ~O.1 ig" at fan ~ 24h. The effer of Choy om specie rates for cell reaetions was epresented by the Haldane model in which the cell generation and dhGH production wee simulated with low mea Llativ ers of 65 and 16%, respectively The result indest heed fora Uiyarided CFS design model with kinetic expresions involving subst inition, We clade tht woe hybrid fe-baten design strategy tobe stated (O with the Initial petiod of the model-based FBBO, in eu, proceeded i) with the contol feeding with ethanol star FEBO enables gradual approach tothe hypeshetca erformanee of FBO. 1. INTRODUCTION presence of well-established fermentation methods for increased heter- logous protein production is its attribute [11 giteeted promoters are key components in the cll Factory design that enable precise ad enfianced expression of genes. Since now-toxie carbon source containing media may simplify downstream purification and regulatory documentation, using (D naturally occurring promoters such a lyceraldetyde-3-phosphate delydrogenase (GAP) promoter (Paap) Pichia pastoris isthe widely used nd ereatively engineered yeast and ‘compared with the fist model yeast Saccharonycescereisiae (0) Is ‘atively less studied and is lacking extensive functional annotation ‘studies [2]. Crabtree-negative P pastors can reach to high-cell densities (HCD) in fermentation processes due to low ethanol production rate under aerobic conditions where the respiratory mode of growth is ‘etivated [3,4]. P. pastoris can reach high prodiet titers owing to the tightly regulated methsnol-inducible naturally occurring alcohol exidase 1 4OXI) promoter (Pyox2) [5], and the engineered promoters [6,7] and the deregulated hybrid -architectured Psoxy-variants (8). Therefore itis ‘perfect model organism for directed evolution of yeast and sls0 (0 study the peroxisome biology and secretory pathway [5-10]. P- pastors ‘can be easily genetically modified to produce recombinant proreins ( proteins) and ean pesform pos ranslational eukaryotic modifiations such as glycosylation and disulfide bond formation. Besides, the n the glycolysis pathway [12,15] and engineered promoter based fernientation processes in glncose-based media (6, ad (engineered promoter variants (EPVs) in the alternative earbon source utlisetion pathways such as ethanol utilisation pathetay (EUT) [7,8], ate advan tageous for the production of biopharmaceuticals, compared to Paar bbsed fermentations using methanol. Promoter strength an regulation fre cumslaive effects of short and distinee DNA mots that facilitate binding of the transcriptional machinery elements recruited by the regulatory proteins [11]. To enhance promoter strength whereby the twanscription and expression through the generation of fine-tuned * Conesponding author at Department of Chemical Engineesing, Middle East Technical University, 06800 Ankara, Tukey Email addres pesll@meni.et (. Gab) spa /do.org/10.1016 be 2020.107782 Received 17 Februaty 2020, Received in vse forms 14 August 2020, Accepted 29 August 2020 Availabe online 2 Seprember 2020 1369 7034/6 2020 Elsevier BL All ights seve‘regulatory cenit, synthetic promoters have been constructed in the yeasts P. pastoris and 8. cerevisiae [68,14 17]. The state-of the art ‘studies reported by Ergiin et al. (7,8] have revealed that engineering based on comparative analysis of ranseripion factors in coordination ‘ith eleaeting DNA elements in S cerevisiae and P. pastors enables directed evolution ofthe gene-regulatory network forthe generation of novel regulatory cteuit(s). P. pastors alcohol delyrogenase 2 (ADH2) ‘promoter (Papya) in the EUT pathway is an ethanol inducible promoter {18}, and its finetional analysis (PAS chr2-1.0472) revealed that it isthe ‘only gene responsible for ethanol tilisation in P. pastorts (71. Upregulation of canscription and enhanced r-protein expression correlate with te sequence of synthetic mot and thelr Iocaton in the hybrid promoter architectures of EPVs in coordination with transcrip: tion faetors whieh are the design parameters in dhe engineering of binding sites (71. Ta inetense r protein expression through the gener {don of novel tegulatory circuits in P. pastoris over the EUT partway, the EPVs of Papi were designed by engineering of transcription factor binding sites (TFBS) determined by in silico analyses and mana ‘uration systematically by single handed replacement of specified TFBSS ‘with synthetic motifs for Marl, Cat and Aca binding. Compared with Pa at t= 20 h of batc-fermtentations, the hyrid-arhitectured EPV Pannacusi2 allowed the highest increase in enhanced protein ‘expression as 4.8fold on ethanol and 3.8-fold on methanol (7). In the forthcoming study [19], double-promoter expression systents (DPESS) 0} were designed with the EPVs Panwa usi2 (71 and Pmaoxt (3, respectively, to enhance and upregulate- deregulated gene expressions in P. pastoris on ethasol; and, compared the DPESs with te constituent Single promoter expression systems (SPESS). Reporter red uotescent protein and enhanced green forescent protein were expressed under Paowacnaia ad Pmaons. Tespectively, enabling determination of the transcription period and strength ofeach constituent inthe DPESS. Sw prior DPES performances were tested in extracellular human grovel hormone produetion and compared with its eonstirent SPES eon: streted with Paowa-casia in the barch-cultivations on ethanol [19] Human growth hormone (hGH), « non-glycosylated protein known as ‘sontatotropin, has been used to cree hypopicutary dwarfism, injuries, bone fractures, bleeding ulcers, and burs. It appears to be of consid ‘erable benefit to girls with Tumer's syndrome, ehildten with chronic renal failure and adults with growth homione deficieney or human immunodeficiency virus syndrome (21) [A majority of industrial bioreactors operate in the fed-batch mode whieh was invented in 1917 10 be able to feed & concentrated sugar solution to make baker's yeast at low and constant sugar concentration inthe bioreactor [22] Fed batch bioreactor operations (FREOS) are used ln industial microbial processes fo increase yield by preventing sub- ‘rate inhibition and carbon catabolite repression [23,24]. FBBOs are ‘superior to bateh operations, espectally when changing the concentra ‘on of nutriont(s) affees the yield or productivity, sie FBBOs allow ‘control and optimisation for » given praduction criterion by changing the continuous feed rate [25], During fed-batch bioreactor operation, i is essential to design @ continuous feed-stream (CFS) to prevent over feeding or underfeeding of substrates) to the bioresetor. For the design ‘of FBBOS, without feedback control () substrate feeding by CFS (261, Gi) nybeid fed-batch operation which starts by fed-batch in turn ‘switching to batch and chen proceeds in suceessivecyeles (271, (D) pulses, (iy) oscillations; and, to avoid dynamic changes in the concen tration ofa targeted substrate in bocenetor, with feedback control (») cconscanteoncenteation (substrate stat) based controled feeding, ane used. P. pastors fermentations are designed as ewo- to four phase HCD ‘ultvations on the eatbon sources methanol, glucose, glycetol, and for the fist ne on ethauo in this work, Theoretical analysis aad modelling ‘of fed-batch bioreactor lead to the design ofthe continuous feet-steant to be initiate afer a barch-operation phase (26,271. IF there i carbon source changeover, a transition phase on the next carbon source is needed to activate pathways that reconfigute the network atchiteture. At the onset of the depletion of the carbon source in the transition Bock ngmerg Jura 66 (2020) 10782 period, FBBO is started, which is marked ast =0 in the production pase indicating the cultivation tine to switch to fed atch operation [26,271 . pastors fermentation processes, exponential feeding of () meth nol der Paors (28,291, GH methanol and eo-substrate sorbicol under Po £90.31], and GHD glucose under Pegp £52, and, with feedback control, methanol-stat operation under Pay [235], were reporte. ‘The aim of this work is the investigation of the ethanol fed baceh bioreactor operation to euhance extracellular rprotein production with Panis casi2 In P. pastoris. We studied recombinant hGH (rhGH) produetion in P. pastors constrveted with Papyace12 in the ferment ‘don of ethanol as an alternative to the hGH production in P. pastors luider Por on methanol. The design strategy should be that the limiting substrate ethanol is ed with an appropriate and increased flow. rate to ads celular metabolism over erosstak through the integration of external and intemal stimull [11] for the generation of @ constant substrate concentration and a constant specific growth rate. For the design of FBBOs, the boundaries ofthe production domain in terms of the parameters of the methods are estimated besides the estimation set Of the parameters that gives good At and precisely designed FBBOG). Firs, we investigated the influences of ethanol concentration (Cox) of the growth of de cells in aicftered shake-bioreactors, AS eto it hibition isthe expected phenomenon, ethanol fed-batch bioreactor op erations were designed using two methods. The fis isthe model-based Daranuetrc design of continvous feed stream flow-sates (26,271 at three pre-determined specific growth rates (Hep = 0.020, 0.035, and 0.050 1), Considering (9 catabolite repression after switching t0 FBBO that ‘canbe caused by the low ethanol concentrations duting the low course ofthe intial CFS flow rates, and Gi ethanol inhibicion at high ethane) concentrations during the high-course of substrate feedings to maintain ‘onstant-ethanol concentration in the bioreactor, substrate-stat based controled feeding of ethanol (ethanol stat FBBOs) was investigated at three coustant Cro values (Cerogiee = 0.9, 1.0, and 1.5 gL). Un. strnenired Haldane kinesie equation was used for the mathematical description of the substrate inhibition at high ethanol concentrations (Cox) on the cell growth (y), ethanol uptake (qf) and r-protein produetion (gp) eates, besides the monotonically increasing unsteve tured Monod equation [25]. The substrateuptake (qeio#) and praduet formation (qp) rates were also modelled using Pir’ mainte nance energy and Luedeking.Piret equations, respectively. To describe the relationship of, qgions and gp With Ceo We ted the Haldane model together with mass conservation equations for the components ‘ethanol, cell, and r-protein of the fed-batch fermentation; and, the cel generation and ehGH prodietion were simulate. 2. Material and Methods 2.1. Microorganism and Pre-Culevation Conditions P. posoris carrying single-copy pADH2-Cat8-L2:KGH was co. structed using Paonia cas 2 #8 reported elsewhere (19) and was used as the extracellular hGH producer. Recombinant P. pastoris strain, stored imcryogenie tubes at 80 C, were streaked onto yeast peptone dextrose (1PD) agar plates containing 20 1" agat and YPD mesum consisting of 10 gL yerstextact, 20.8" peptone, 20g." dextrose and 100 yg mi? INTC (Nouseothiein), and were incubated for 48 hat 30°C. The grown cells were inoculated into 250-ntaiflered shake-bioreetors co: taining 50 mi buffered glycerol complex medium (BMGY) consisting of 10g yeast extract, 20 8 L" peptone, 10 gL geet, 3.4 gL? yeast nitrogen base without amino acids, 10g € ammonia sulphate, po ‘assum buffer (11.8 gL" KHzPOs and 3 gL" KeHPO« adjusted to pH 5.0 using 5 M KOM, 0.4 mg L: lter-seriized biotin and 34 met? fiker-sterlied chloramphenicol. The niesfitered.shake-bioreactors were incubated at 30 °C and 200 rpm: for approximately 24 hours until ft OD son of 4.6 was reached. The cells were then harvested by cent and resuspended fugation at 4500 g and 4 °C for 5 ‘medium All the shake bioreactor experi production 0py 200 min, in Vy =90 mi cultures 55, and N 2.2, Shake Bioreactor Experiments Following pre-cultivation, the cells were inoculated in air fitered shake-bioreactors in Vp =50 ml medium consisting of potassium buffer, 14.9 g 1° MgS047H:0, 1.2 1" G80,2H0, 15.3 gL? ‘ammonium sulphate, 34 mg L? ehoramphenieol, 44 mL? of filter: scerilzed trace salts medium (PTMD), containing: 6 g L* CuS0y, 80 mig L" KI,3 gL MnS0q, 0.2.8? NagMo0.,20 nig L? HaBOs, 0.5 g L? CoC 20 gL" ZnCl, 65 gL" FeS04-7H,0, 0.2 gL biotin and § mL? ‘of 9896 (W/W) H,804. The cells were inoculated Into exe air filtered shke-biorenctor at t< 0 h with an initial cell concentration of Cyo = (0.12 g L? by calibrating the initial ODso9 10 ea. 0.50, and Datel fermentation of ethatiol was started by ethanol addition at t= 0 h by ‘adjusting intial ethanol concentrations 0 Cros = 1 2.3, 4,5, 7, 8 ‘and 16 gL". The shake-bioreaetors were incubated at T =80 °C and N = 200 rpm, and the variations in ODjp were determined by sampling ‘every 2 ows until he stationary phase in each bioreactor was observed between 20 < t < 62h. All the experiments were performed in éupli- ‘ates, and the mean values of two techni replicates were presented as tmean = standard deviaion, 2.3. Three Phase Fed Batch Operation Conditions and Fermentation Medium After precultivation, the eels were inoculated into 2 1 basal sat medium (BSM) consisting of 12 g L" CaS042H,0, 149 g L” Mg804.7H,0, 4.1 g1." KOH, 18.28 L* KsS0., 40g glycerol, 53-4 mL HPO, (85%), 10 nL PTMA, 0.4 mig 1 biotin, 34 mg L* ehoran phenicol and 2 ml. antifoam (10% v/) and introduced into a 5. BIO- STAT Cplus bioreactor (Sartorius AG, Gottingen, Germany), The bioreactor cultvations consisted of three phases known as the glycerol bates phase (GBP), ethanol transition phase (ETP), and ethanol induc ‘ion phase (ETP). tn all the phases, the ells were cultivated at T =30 °C, 700 rpm, and Cyo = 15% dissolved oxygen (DO) maintained using & built-in PID controller adjusting the air and, flow-rate ratios through & ‘eascade system while keping the gas inlet -flow rate at 2 wan. Fed: bath, ‘operations were caried out under controlled pH operation condition at He =5.50 using a base solution containing 25% (v/v) NHOH [36]. Ia "he OBP, the only carbon source was glycerol and solely «sed fr the cell ‘growth to reach an HCD . The end of the GBP is marked with the ‘occurrence ofa instantateous DO. peak indicating that the glycerol I depleted, tha is, dhe first transition time ro site (0 the next pase, ETP. The ETP commenced by pumping ethanol in 18 min intervals into the bioreactor by a positive-dsplacentent pump (Watson Marlow 120, “ThermoFisher Scientific, MA, USA) ata flow-rate of ea. 0.4 ml. mint t0 ‘climate the eels to the nex earbon souree ethanol, whieh lasts 4. In tom, the EIP was initiated (marked as ¢ = 0) and lasted for 24 with ‘ech fed-batch operation condition under each strategy implemented as @ FoBOs with paramestie-designed CFS (PD) flow-rares calculated With peo Values, and (i) ethanol stat FBBOs at constant ethanol cox: centrations, Crorixe During the fed-batch operations, 100% pure ethanol was supplemented with 5 mL? PIM and 2 mL L biotin (0.0286 w/), and was fed by pumping into the cultivation medium, 2.8.1. FBBOs with parunetric-desigud continuous fordstreum (PD) low-res To desig the CPS flow-rate profiles in fed-batch bioreactor opera tions, based on the biochemical reaction engineering principles, the model-based PD" method (knovn as the exponential feeding) was used (26). The CFS containing ethanol was fed to the bioreactor to maintain the specific growth rate atthe predetermined values of Ypep = 0.020, (0035 oF 0.050 1, sg Eq. (1) oF 1D) (26,271 Bock ngmerg Jura 66 (2020) 10782 aw Seo 7 00!) aa) ry = Pesto) an) where Q() isthe dynamic ethanol volumetric feed fove-rate [Lh], F() Is the dynamic ethanol mass feed flow-rate (1), pen Is the pre determined specific growal rate [J intial fermentation volume was Vo = 21, Gx isthe initial cell concentration {g Lat the beginning of the EI indicated as ty = 0, ecanol inlet feed concentration was pre determined as Cp = 790 g 1%, the cell yield on the substrate was pre-determined a8 Yar 0.67 geellg?, and tis dhe cultivation tine (hy as explained in Supplementary Method S1 23.2. Bihanol-stat FOS Ethanol concentration in the cultivation medium was maintained constant a ether 0.5, 1.0 oF 1.5 g L* by an ethanol control unit (Met ador, Enzim Biyoteknoloi Ld, Ankara, Turkey) that indirectly measured the concentration of ethanol in the medium by analysing the compos ton ofthe exhanst gas fom the bioreactor. Consequently, the Row rate of ethanol was adjusted by the Pl algorithm of the contol unit and delivered by a positive-replacement pump. The ethanol feed stock solution was placed on a digital balance to moniter its utilisation seavimetrcal 24, Analyses 24.1. Bihanol and organic acid concentration measurements Ethanol concentrations were determined by gas chomatography (Agilent GC 6850, Wilmington, USA) equipped with a 30:m x 530 ym x 40 um Agilent 19095P-Q04E column and operated at a constant oven temperature of 170°C. Helm delivered ata constant presse of 19.62 si was used s the carrier gas with a 10:1 split ratio and 120 mL min splitflow. The TCD detector operated at 200 °C and 5 Hz with 12 mL iin" reference flow. 1 yl of sample was injeted snd run for'3 min. The excreted acetate and acetaldelyde concentrations were measured with fan HPLC (Waters Alliance 2695, Milford, MA, USA) as reported else where [971 242. Coll concentration measurements ‘The cell concentrations (Cx gow LD are presented based on the dy: cellweight (DCW), tn the airfiltered shake bioreactor experiments, Cy was determined by measuring the optical desty at = 600 nm with & UV-Vis spectrophorometer (Spectroquant Phato 300, Merck, Germany) tnd calculated using Eq 2): . = (ODuy\ Bitton Factor (028) @ Inthe FBBOs, was determined by cenrtuging 1 mLofthe samples 4500 g and 4°C for 10-min, discarding te supernatant and drying the pellets at 105 °C for 6b, The difference becween the empty and dried tmjcro-centrifge tubes represented the DCW. We performed tipicate salyses forall the samples, and the average values are presented in the Figures 24.3. rhGH concentration measurements hGH concentrations Were measited using an ELISA hGH quandif. cation kit in 96-well microplates according to the manufacturer's manna (Roche Diaghosties GmbH, Mannhelm, Germany). hGH stan dard supplied with the kit was prepared in duplicates in 1:2 serial dilution steps starting from 400 pg mi: until 12.5 pg mL. Absorbance ofthe samples was measured at = 405 nm using a microplate (ELISA) reader (Multiskan Sky Microplate spectrophotometer, Thermo Seient- fic, USA), We performed triplicate analyses forall the samples, ad the average values are presented in the Figures.3. Theory and Mathematical Models 3.1. Caleulaton of Specific Rates ‘The specific growth (Eq. 3, ethanol uptake. (Eq. 4), and product, formation- (Eq. 5) rates are derived from the mass conservation equa. ons (26,271, follows ax 0% a ® Eo aoe con = ~& (eet Set yy 2 csp) ® 1 (A, i, o£ (Gea) here piste specific growth rate ["], Cx the cell concentration [1 4}, ris the cultivation ime (h},V is the volume of the cultivation me- «lium [L, QC) is the dynanic volumetric iler steam Row-rate (1), ‘anon isthe specific ethanol uptake rate (g gh], Cpoi is the com FESEEE 2. Seheniaie desig ofthe expevinental sup.» i i i Bock ngmerg Jura 66 (2020) 10782 relocate Ceo 3") Fig. 2. Alcflkeved shake bioeactor experiments forthe determination ofthe effects of ethanol on the growth of P. pastoris a) Growth curves of P. pasts constructed with Paoyacuce on ifleent intial concentatios of ethanol, b) Vaiatons in ethan! couentatons withthe eultvation tine Blue ctl with dashed ines represeat the 1.0 g L® cultue, orange squares with dase ines represeat the 2 g L® culture, pple tangles with dashed lines represent the 3 gL" cular, green diamonds with sl lines represent the 4g culture, black diamonds with dashed ines represent the 5 gL? culre, magenta tuangles with sold Ue epresent he 7 cule, ick ed aquates with sll lines present the gL cule, yellow ereles wih soli ies epresent the 16 = ‘Leute ) Haldane model forthe growth of. pastrustisn constructed with Poy. n diferent inl conceeations of ethanol, Soli lack dots cepresent ‘experimental specific rates ad the red line represeats the Haldane move. Ero: ba fepreseat the saad deviation, ‘envelope of the family of curves of the lower ethanol concentration, ‘curves. ln the fermentation of ethanol, the course of ethanol concen: tration profiles reveals thatthe lower ethanol concentration decreases the pressure on the metabolism for the generation of higher cll eon: ‘centrations. The evidence shows dat, up t= 40, the envelope isthe Caio = 8 8 L! curve: and up to t = 60 h, is the Ceyyo = 16 gL (Ci Je), demonstrating that increasing the ethanol concentration in ‘creases the cell concentration atthe cost of prolonged cultivation tine (ig. Sb). The stationary phase started after t= 16,22, 24,26, 32, 36,38, ‘nd ate 5h forthe inital ethanol concentrations of Crime = 1,2, 3, 4,5, 7,8, and 16 21%, respectively. The onset ofthe stationary phase ‘was concomitant with the depletion of ethanol from the cultivation ‘mediuan (Vig. 3b). Ethanol concentrations dereased by more than 50% 1 = Slr and resulted inthe formation of the byproduct acetate. With the Increase In Casa the Mtl Cx nereased, that I a€Carrea = 16 87 reached Cy =11.5 gL" which is L.8.,1.9-,2.7-,3:1,,39, 46-,and 68 fold higher than that obtained at Cyosio 8,7, 5,4, 2,2, and 1 gL}, respectively, 4.2, Growth inition The cells grown on higher Czomo generated progressively slower sroveth rates and prolonged the cultivation tines (Fis. a), indicating the inhibitory role of ethanol on the growl. tn recombinant ?- pastors constructed with Panp.cos2 Producing eGED, Ergin et al. [7] ported ‘25 fold higher growth inhibition when the Gaoxo Was inerease from 41040 gL. Asean beseen in ig. e, the inhibition pater followed the Haldane model rather shan the Monod (Supplementary TableSt),wihere increasing ethanol concentrations increased the specific growth rates reaching @ maximum of yor = O14 hat 3 g L. Nevertheless, the ‘odel predicted a critical ethanol concentration of Cragson =1.97 gL? snd a theoreteal maximum specif growth tate Of pgas = 0.194 I! forthe P. pastoris strain constructed with Panyacnis (Supplementary ‘Table 81), The experimentally determined and predicted by the model, Ceeicn and Hae ae the frst reported values for a P. pastoris strain ‘grown on ethanol. For the yeast 8. cerevisiae [15], however, several studies were conducted on the growth on ethanol, Wher yay Wa te ported as 0.19 [40], which isin proximity to our experimental value Of fg = 0.14 4.3. Fed Batch Bioreactor Operations Based on the shake-bioreactor experiment rests, fod batch bone scior experiments wore designed using the alternative to methods for ‘continion feeding ofthe substrate ethanol forthe production of FAH ‘expressed in P pastors under Paonia cau They are (D the parametric continuous feedstream desigh abbreviated PO, and (i) the ‘controlled substrate concentration so called ethanol stt F8BOs, For the first group of the FBBOS, the PD dynanie volumetric Now rates were caleulated sing the pee deternsned speci growth rte en = 0.020, 10.038, and 0.050 hi, which determined to be <73% Haldane model's tas 0 avoid ethanol accanlation inthe bioreactor. The nex group of fexperinen’s was designed to keep the ethanol concentration in the catvation medium constant at pop = 05, 1.0 and 1.5 g13, which ‘were determined tbe 255 lower thn the Cz oy = 1.97 gL to avoid ‘overburdening the eel under the concentrations close 0 Gaon that represses the cellular metab 49.1. Ethanol Concentration Profiles 1 the FABOs designed with PD, the QQ profiles calculated with Veco = 0.020 and 0.038 resulted in depleted and undetectable ethanol concentrations thoughout the fed-batch eultvations in the biorenctr. The variations in the ethane] eoncentatons A xe = 0.020 10 operation indicate tha the QC designed with yy = 00020 8? Is @ limiting Pow ent profile forthe cell grout Section #2) and prdct fomtion (Section 43.3) whieh is wot scent forthe demand of the cells in the fermentation of ethanol. Tae Hex, at yep ~ 0.035 ‘operation, the CFS supplied ethanol eaablng a liear and prolonged cell {romth period with a Tow slope rg. da, where de product formation ‘ects with the highest rate betceen 6 << 9h and then slowdown (Cie Ab). The ress at pep — 0.035 b? indent tat by the clive tin de ¢> 9s etal supply allows higher ethanol uptake rats that, inhibit ch eal! grow and product formation. tthe FBBO designed ‘with pep ~ 0.080 8, ethanol concentration inthe bioreactor (Cron) increased up tot 6 hand reehed# gL, and then decreased 02 8? beteeen 9 <¢-< 12 Beyond > 12, the QC) designed with pay — (0.050 I i «high profile and resulted in enol aeeunlaion that reached 15 13 at = 211 The variations in Coo a ao = 2050 indicate thatthe CFS designed with iy = 0.050 1" provides high «thanol eoncentations forthe el growth (Section 4.3.2) which eased ‘overfeding of ethanol that inhibited tae product formation beyond t ‘Sh eSeeion 4.5) ‘The ean concentrations in ethanol star FBBOs were Kept constant Rhroughon the fed batch culations with menn vals of O64, 0.96, ‘and 1.0 gL? with a mean relative enor loser that 13% ofthese original Set pits of 05, 10, and 1.5 gL, respectively. 4.3.2. Gell Concentration Profiles In the FBBOs designed with PD flow-rates, the cell concenteations {fen = 0.020, 0.035, and 0.050 i" operation conditions were cose ‘and silly increased linearly up to = 6 hn the FBBO designed with Hero = 0.050 1, tne cell concentration was almost doubled over & breakthrough in chee hours (Cx = 65 ¢1°) and the growth proceeded up to t= 24 hand reached Gx = 87 8 1L'n the FBBOs with PD fow rates designed with pen = 0.020 and 0.035 i, after ¢= 6, the eo = 0.035 Ii" curve diverged and the cell generation in both operations proceeded linearly and reached the values of 54 nad 76 g L3, respec tively. Among the FBBOs designed with PD, the cell eoncentation at Bock ngmerg Jura 66 (2020) 10782 $e Coneatration (C,.9 1" Time ae teeter Tine Fig. 4. Vaultons ia the a) cll eoncenatloas, and b) s8GH eoneennaions withthe clvation tine in the FBBOs. Enpy purple etl eptesent Hoo — 1.020 i, lle green squares repese® nas ~ 0035 empty blue wines reptesetfoen 0080 filed te ices represent Coan 08 led lack damonds reptesent Conse = 1.0 gy led orange wangles present Cone = 1.5L" Bios bas Teptesnt the stat devition Hexeo = 0.050 i produced the maxinnam cell concentration (87 ¢ 1%) Which i 1.2- nnd 1.7-old higher than the cell concentration obrained With pep = 0.035 and 0.020 hi, respectvely ig. 4a). Ts, with peg = 0.020 hi, the eell concentration is the lowest as Gx = 52.1 gL? indicating Uh pyen = 0.020 bi is not sufficient to achieve w HCD fermentation proces. In the etianol-stat FBO, the Tocus of che cell growth curves with Cori 05, 1.0, and 1.58 were linear and similar between 6 15 hy where the srowth increses through a characteristic profile with a decelerating slope until t= 24 h and generates te highest ell concentration as © 135 g L*, Related the phenomena that eaused the two exceptions, the fis indicates the pressure on the cell growth created by te high ethanol‘concentration at Coiier = 15 gL. The next indicates the smoothly ‘operating central eazbon metabolism at Ceotsr = 1.0 8L? on ethanol which prolonged the cell growth untilt =24hand generated the highest «ell concentration tha isso ee, 15-fld higher than that with the FBBO designed with PDE flow-rate at Yao = 0.050 1 1m the literature, in P. pastors under Por, Using FBBOs with imethanol-sat operation 2.5 and 2 fold higher cll concentrations were ‘obtained compared to FBBOs with parametric designed CFS flo rates, for te production of rhGH on methanol (30,51) and endostarin £471 under Paor,tespectvey. 4.3.3. rhGH Concentration Profiles ‘nthe FBBO with the PDS, the ethanol low-rate calculated with Hyco = 0.020 h demonstrated limiting Q{0 profile which resulted in instantaneous consumption of ethanol, Cuou(®) = 0, that generated & ‘very slight cell formation but without prodvet formation (Fie. 44, )- The ext, pen ~ 0.035, the CFS supplied more ethanol that drives the «ells othe prov foriaton period at t= 6h which proceeded withthe highest rate among all the FBBOs up to € = 91, AtC> 9h, the product formation was alsostinterruped for sb hours until t = 15 hand then proceeded with a slight increase during 15 < t < 21 b producing the highest hGH (Cp ~ 43.6 mg L") among the FBBOs designed with the PDE (ig. Ab). This result at yes = 0.085 fn Indicates that the ‘alelate ethanol fow-rae profile isthe precise design of the CFS with, the predetermined parameters (Ys, Co. and Hep) t0 <9 that drives the cells to the production period berween 6 << 8 h with the ‘highest yield and productivity (ig. 4b). Neverteles, by the cultivation time > 9h, the ethanol supply according co the exponential structure of| the ethanol flow-rate profile was increased and allowed higher ethanol "uptee rates that inhibited the cell growth and product formation inthe cells The next isthe PD that is designed with « high acceleration ethanol flow-rate, calculated with yep ~ 0.050 h. The PD with Heo = 0.050 drives the cells tothe produet formation period also att ‘oh that proceeded until ¢ <9 h but producing almost rwofold less {hGH as Co = 24 mg L compared withthe FEBO at peep = 0.035 hi With feo ~ 0.050), itiseear that the product formation between 6 1 < 9] occurs under the pressure of ethanol inhibition. However, by the cultivation time t > 9h, according tothe exponential increase in the Q (© the inhibition effet of ethanol increased and intereupted the product formation in the ell ig, 4b). ‘The ethanol stat FBBOs produced significantly higher Cp values with the highest rhGH concentration stained at Cuopge = 0-98 LAS Coe 191 nig L which is 2.1-old higher dhan that of pen = 0.035 followed by Crone = 1.0 and 15 gL as Cp = 80.1 and 78.8 mg L?, respectively (Fi. ab). The hGH produecon with the FBBO controlled at (Coots = 05 gL" generated high rhGH concentrations with high proetion rates but interrupted rice uring the periods of 3 << 6 h and 15 << 18h, and reached Cpnge—91 mig Lat =24 h, When the ‘Cacia ms increased twofold withthe FBBO contolled at Cacti = 1.0 tne rhGH secretion was started with @ breakthrough atc > 6 h ‘and proceeded with high rates up to ¢ = 15 bv where with a second breakthrough reached Co~80.1 mg? att =21 h, Overall dite loner ‘ethanol inhibition efeec on the eels, the ethanolstat FBBO results, ‘reveal thatthe lowest ethanol concentration, thats Cooter =0.5 81, isthe favourable operation with the highest-eell and product formations (Cig. ab). tw the literature, likewise, in FBBOs on methanol, compared with the FBBOs esigned with PD", n sevenfold higher rhGH produc tion £50,311, and ewofold higher lipase activity [34] Were reported with the methanol stat FBBOSs. ‘The rests of the two design approaches forthe ethanol FBOs indicate thatthe continuows feding of ethanol is essential forthe cells for keeping the ethanol concentration low and within «narrow interval, to reach HICDs and high rprorein concentrations in P. pastors eon: structed with the ethanol inducible Papi-casta- The decease in rhGH ‘concentration for Crosse = 1.08" And pen — 0.085? after t=21 is caused by accumulated extracellular proteases that degraded the Bock ngmerg Jura 66 (2020) 10782 a Tine () » eal rn on (arenes a Tine (n) 8 (aah) Time th) Fig. 5. Vavlatons in the a) spel growth rates, b) specie ethana uptake tates, ane) spec rhGH plodveion rates ith the eulvaton tine fn the FBBOs. Empty puple circles represent jo ~ 0.020 by filled een squares represent jpen ~ 0.035, empty be tangles represent pep ~ 0.050 I, Bleue eitles represent Cane 05 § L fled Mack anonds epresent Gesotae = 1.0 & filed orange bangles teprsent Conan = 1-5 §‘Table 1 Bock ngmerg Jura 66 (2020) 10782 ‘Compavson of process variables, yes and productiities in the the FBBOs designed withthe pre-determined values and ethanol seat FBBOS FRO th PO at nw haga st FBO et Cassa oe EL) a 7287 or oa 7a Yoamang 2?) aaa oss ass ee as rtp) 200 120 355 ‘os aus pear) 017 5 0.025 mis .00035 «047 1.00095 053. 0.0083 a.n6B 0.009586 1.0080 Sewn (6e'® OUR Focons ost Faatad =a FaooHS © LoD onl: bone 12 000s rang #9) ane oat 021 S0012 ox: 0001s 0047 0.0050 oes + 0.0020 FBO: Fed-bach bloweactor operation, Coorie! Set ethanol coneeatiaton, Cpa: Maximus HAGH concentation, Yp¢1NGH 0 Bomass ye, Fw: Overall pro ‘ut nt Mean specific awh ae, quo Mean spec esol ULAR Te, deme: Mea INGH prodvetion Ite secreted hGH, is indeed noteworthy [43,48]. 4.3.4. Specific Growth Rates Im the FBBOs with PDS at ppep ~ 0.020 and 0.085 h, the exper imental specific growth sates (y)fhctuate around their mesa values oF Ynwor = 0.017 and 0.035 hi? with the eultvation time, respectively Cig: Sa, The proximity of the press Valves to theit respective eo ‘values indicates that the theoretically determined Yyvs and experimen: tally measured Cyo valieswsed in the calelaton of QO in Eq, (1) are the mpproprate values. The loc ofthe variations inthe specific growth rate with theculivation ine nthe FBBOs with PDS at yep 0.035 ih tower values) an eo = 0.050 1 with higher values) were similar up to = 124, with a mininum at =6 anda maxims at tn whee the fst generated the second minimum. Stil, the latter pro: ceeded with a decreasing slope and gave the second minimum between 15 <€< 18 h-coineiing with the onset of ethanol accumulation. The ‘thanol-sat FBBOs generated higher speifc growth rave values with (0.07

15 h (i. 5. 43.5. Specie Ethanol Uptake Rates The specific ethanol uptakerates (Geox) were higher during the 50s designed with the ethanol-stat operations (Fig. 5b). Among the FBBOs with the PDO, at pen = 0.050 HN, the qpons Vales were the highest bur lower than tha ofthe edhanol-sat FBBOs, except in shor, period between 7 << 11 hexhibited «breakthrough and thea a sharp taxinuum (Qaou = 0.13 g ¢" 1) at = 9b, which dectersed toa lower value (ca. deion = 007 gh by giving a platean between 15 15 h, except Chrotaer= 19 &L a he Gyo = 0:8. L! and 1.0 g LY FBBOS the specific rates of ethanol consumption (qrox) decreased (ig. Sb) ‘oncomicant with their ¥s € curves (Pig. Sa). The Yarton values of Coxe = 0.5, 1.0, bed 1.5 812 was determined ws 0:72, 071, and O71 8 £7, respectively. In the iterate, under the conttol of Paoxr it . pastoris on methanol relatively higher as values for substrate stat FBBOs were reported for the production of hGH [30,511 and lipase ba 43.6. Specific rhGH Production Rates ‘The specific hGH produetion rates (inthe FBBOs ofthe Pi Heo = 0.020 and 0.035 and at Crpae= 15 81" exhibited maxima of 0169, 0.102, and 0.0817 mg g*h att =O h (Pig. Se, respectively, coinciding withthe cultivation time that these FBBOs exhibited a sharp increase in the hGH concentration (Fig. 4b). Beyond t > 21 b, the decrease in for Gerona = 1.081 and peo = 0.035 h" isthe rest of the degradation of the secreted shGH, catalysed by the byproduct extracellular proteases) in the fermentation mediuin (29,44). tn the literature, under Paoxs om methanol, the substrate-stat method was re Ported as the supeior for the FBBOs forthe rhGH [30,31) and lipase DS at 44, Comparison of Fed: Batch Bloreactor Operations ‘The optimum cultivation ttt (gc) for fermentation of ethanol was determined fyax = 9 in the FBBOs designed with the PD fow- tates with Ypeod = 0.099 I ad gap = 0.050 since prolonged operation beyond fax decreases the productivity. The highest overall ‘volumetric productivity (po) ws produced in the FBBO with the PDT te = 0.035 in the period tp t0 Engr <9 h8 Fy =3.0 mg? Wt With a hGH concentration of 34.mg L that Is 80% higher than the sthanol-stat FBBO at Ceoitnr = 05 & Lat C= 9 he The ethanol stat FBBOS at Cron = 0.5 gL! and at Coon ~ 1.0 g L? allowed increased Droit ters with high produetivities, with the highest rhGH concen tration of90.1 mgt attyoe=241 (oy =3.79 mL") and BO mE? Eger = 21 (pp = BA gL"), respectively (Table ‘The #hGH yield on cll Cp) of piyen ~ 01035 is 2.5404 higher {han that ofthe yep = 0.050. I the ethanol stat FBBOS, the highest Yorx was generated with Ceoitax™= 1.5L at0.97 mg 3, which 1.2 tnd 14-fld higher than that of Crone = 0.5 and 1.0 1% respectively, tnd 12-fold lower than that of eo 0.0357. nthe fiternte, under Porson methanol, likewise, 1.5-f14 higher shGH to eel yields (pa) was reported for FBBOs designed with the PO? using pre-deternined ‘ales [30,51 The high volumetric productive coupled with high GH ters, makes the ethanol-starFBBOs advantageous. Overall, Crom, 5 gL isthe best FBO due tothe production ofthe highest rhGH ‘oncentraton slong with «high fpr Volumetric productivity and the protein to eell yield, Ypac (Table 1. 48, Kinetic Modelling 4.5.1. Ethanol tlabiion Models High ethanol concentrations exhibited inhibition on the growth, edhanol uptake, and ¢hGH production rates of P. pastors ws presented in Fig. 6. The specifi rates dereased after certain critical ethanol con centratlons (Cor) wile are detenmined a8 Cougions = 1.48 8 LyEthanol Concentration (Cae 3") » 008 anal Concentaton Coy, 3") ° ee anal Coneaaton Cyyye 9") Fig. 6. Haldane and Monod models fr the tations benween the mea Specific ates, drow ad) versus mean eidual ethanol eoncentiations: a) ‘specific growth sates versus ethanol concentations ) specific ethanol uptake tates esis ethanol eoneentatioas,e) specific rhGH production rates Yess ‘exianol concetations Solid bck dots represent the expedimental mea ‘specific rates, slid re Une the Haldane models, and dosed black Ines the ‘Monod models Bock ngmerg Jura 66 (2020) 10782 ‘Table 2 Model parametes forthe conelation between mesa specific rates nnd mean residual ethanol concentrations == ence ee = oe = mes ee Sey - = os a # i S Pere) ee ee station constant, Ky inion constant, X: Cal, P protl, 5: subsate ” ° om oor “oh om One » gpimaah) ney Fig. 7 Linear eatonships berween the specific rates a) Pit model forthe speci ethanol uptake rate vesus spel iow tat, andb} Luceking Piet odel forthe specific zhGH production tate veisus specific growth rate. Solid black dos represent the experimental mean data and solid lines present the linear tegieson meetsCosmony = 1.28 gL, and Coesonte = 0.75 gL for anon Hand ap, respectively. The ethanol inhibition obeys the Haldane model (able 2). ‘The high cooficent of determination values (R? > 0.90) and low mean. relative errors (RF maen< 1198) are the good indiestors ofthe adequacy of| the Haldane mode! for the specific rates vs Geox (Supplementary “Table $2). These results are congruent with the results reported for protein production in P, pastors under Paoxs where the relationship between the specific rates (qs, and qp) and the methanol concentration ‘was determined by the Haldane’s equation [39]. The inhibition exerted ‘by methanol on the growth of P, pastors under the contra of Pax Was also determined by using the Haldane's equation (25), 45.2. Kinetic Models for Specific Rates For P. pastoris constructed with Paonia casa the relatonship be tween gous nd (Eq. 13) is presented in ig. 7a. The yield coeficent Yes Was predicted a8 Yerion = 098 8 81 (Veoivx = 1.02 & &) which is 1.6-fold and 14-fol higher than the experimental yield values feo = 0.035 N? a Gaonse = 0-5 & L, espetively. Wheres, the naintenance energy demand was predicted a mpoy = 0.023 g "i . pastoris under Paoxs the maintenance coefictent on methanol was reported a8 muon = 0.013 g ¢*h” [40] and 0.016 g g*h F). ‘The relationship between gp and w was not linear and hence did not follow the Luedeking-Piret equation (Ps. 7b), whieh was further cortuborate by alow coefficient of determination (R? = 0:52) and high Eqaer = 0.22. The linear LnedekingPiret equation can predict acct rately, in theory, within a limited range of values in the produetion dma of the fermentation process with an impli assumption that specific growth rates are proportional with che faneton gp (221. Inthe FDBOs designed with the PD" at @ tao = 0.020", ethanol supply ‘with the Qt) profile indicates the lower bound of the production domain that is limited forthe cel growth and not sufficient for the product formation; and (2 uyen = 0.050 hb”, allowed the cell growth and protein profuetion up to ¢= 12 hand $ 9h, respectively, indicates the upper bound ofthe production domain. Therefore, among the FBBOS designed with the POS, only the data at jen ~ 0.035 h can be wed for the analysis using the Luedeking-Ptet equation (Pi, 7b). Related with the ethamol-stat FBBOs, the FBBO st Crone = 15 g L that is ‘qin to the Gatsoais (= 1.48 1, is a the upper bound ofthe pro ‘duction domains thetefore, the data belong tthe FBBOS at Crore = 05 gL! and 10 gL! can be used in the analysis using the Luedeking Pret equation, ‘The Luedeking-Ptet model was also rested ro predict he relationship between gy and or methanol-based fermentations under Paoxs {39442 49], Due tothe high-energy content of methanel, a it contains 50% ore electrons per carbon (6 elections per carbon higher product yield ‘on substrate values are expected on methanol than on ethanol and carbohydrates (4 eletrons per carbon). These extra electrons ca be potentially nse to reac ea, 40% higher product yes in the fermen tation of methaaol compared to ethanol and carbohydrates. Related for fan accurate explanation of the variation of gp with » with the Luedeking-Ptet equation, the requirement of two different submodels sch a5 qp-constant and linear was stated [99], 4521. Model Simulation and Validation ‘he Haldane models that relate p and p10 Caos Were sed co simulate the cell generation aud rhGH prodacion throughout the eu tivation. The cll generation with the eukivaton ine was well imu: Int with Tow REqea OF 0.065 (ig. Sa aed Table 3). The fed-batch ‘operation designed with the model-based PDS with yay — 0.020 sand 0.095 ae che ewo aostaceuraely simulated FBBOS. Whereas Uo = 0.050 having w higher RE of 0.087 because of te arp fveruatons ing. 5a) resulting from the ethanol aceumlaton inthe bioreactor: Concerning the ethanol stat FBBOS, Corie = 1.0 and 1.5 & LE" performed relatively beter than Caoiuer — 03 gL? (Eig. 8a, “Table 3). Overall, the cell growth simulations for the FBBOs designed with the model-based PDO fed flow rates were relatively better a Bock ngmerg Jura 66 (2020) 10782 ova sem Tene) Fig, Siulation of global state vasabes CV and GV over the ethanol fe bate perio for model validation. Sinlation of loa state vasiables CV and GN over the ethan fed bate pesiod for nee validation Viations i hea) cell genetation, CV, and b) AGH proueton CV, wih the ediation ein the FBBOs. Empey puple citles represent yn ~ 0020 I, filled green Squares represet Hyun = 0055, enpy bite tlanges fepreseat pen = 1.050 filed ted itcles represent Cane ~ 0.5 Lilet black atnonds represent Coen ~ LO g Us filled orange ingles epeseat Ccaea = 15 8 [EE Point data eprescnt the experimental vals whereas ted lines present the simulated results based on the proposed mde. ‘Tables Pesformiance ofthe proposed mode forthe mulation of the a) cel generation, ‘GW, and (b) HGH production, Cy, wit he eutvation time, nt fr ear Ren Rin Rinen Rls To WrScoment Ons! Gore omer 030 aa Gomn-loel! omy Ol aa bam tas, tal Gsc lst? Oome 021 amt alt om noes Sree" outa 813 mes mas tas ae BE: elaive eo, mr minionm, maxs anpredicting the growth behaviour In contrast, maintaining the ethanol ‘concentration constant doesnot allow a fixed growth rate inthe ethanol: stat FBBOs. In FBBOs producing lipase under the methanol induced Pox with the Haldane model, a low MRE = 5% was reported for the sinmation ofthe cell growah [59] ‘The shGH production in the fermentation of ethanol was better simulated forthe ethanol stat FBBOs (ig. 8b and Table 3). The best, simulated CV values belong to Cpoxan = 0.9 and 1.5 g L, whereas Garr = 1.0 gL” showed a relatively high REpusr = 0.21. The CV ‘values for the FBBOs designed with jy exhibited igh RE gay Vales ‘due tothe sharp qp peaks observed at t 9 h (Fig, Se). The proposed model does not take Into aecount me dependent qpvalues but rather the mean values, thus ie eannot account forthe fuetuations in gy nd, ‘consequently, the inscantaneons changes in CpV values [39]. This ex plains why the simulated mode! approached the experimental values of the FBBOS designed with yen towards the end ofthe cultivation time, 5. Conclusions For the fis time, we presented ethanol fed-batch bioreactor oper tions designed for enhanced extracellular protein. production in . pastoris constructed with the novel hybrid-archiveetured ADE2 pro: moter, Paizecao Using P pastoris cell actory constructed with Paz ‘cosa, we systematically investigated the state parameters in the fermentation of ethanol and determined boundaries of the production domiin in terms ofthe design parameters. Thereby, we demonstrated the consents affeting the strueture ofthe ethanol fed-batch biore ‘actor operation design for tailoring de now production strategies. As carbon catabolite repression is the expected phenomenon in the fermentation of ethanol, where he eels are exposed uot only to ethanol boutaso to muitple external simul simultaneously (111, determination ‘of the produetion domain in tems of ethanol concentration for each FBO design method is essential. The shake-bireactor experiments demonstrated that the lower ethanol concentrations ineseased the ‘sronth rates and shortened the cultivation tine. After 31" of ethanol, ‘decrease in the specific growth rates indicated the inhibitory role of ‘ethanol onthe growth, We demonstrated that the specif ates Gao bi ‘and qp) decreased after certain eiial ethanol concentrations (Coan) ‘which were determined ns Cnions = 14887, Grision = 1288, ‘and Geimotar = 0.75 8 U for den, vad ap, respectively. To adjust cellular mecaboisa for the generation of a constant spe ciffe growth rate and a constant substrate concentration in the biore ‘ctor, investigation of ethanol feeding with a progressively increasing flow-rate that controls cellular regulations on the transcription of pro moves (11 Is the focus ofthe design methods, Contextual, we used two FBBO design methods. tn the edhanol feeding with model-based parametric designed continuous feed-stream (PDS) flowrates, the ‘current mathematica model ignores the inhibition effect of the substrate in the kinetic expressions (26,271. the FBBOs designed with the PDS ‘tec ~ 0.020 * operation condition, eno eed flow rate profiles "he ining profile neaing the lower bound ofthe produetion domain forthe cll growth whichis nor sufficient forthe product formation. The "uppee bound ofthe production domain was determined by the FBO at peo = 0.050 Is! operation condition which allowed r-protein produc tion up 0 t= 8 h, Within the boundaries ofthe predution domain te FBO at Yeo = 0.035 h enabled linear and prolonged cell growth period with low slope where the product formation occurs with the highest rate between 6 << 9h AC pn = 0.085 I, the eulivation time tmec = 9 I indicates the optim fermentation time where te ‘ethanol fee lw ate determines the upper limita eo = 0.085 in Which the balance in the demanr-and-supply of ethanol favours the cel reactions forthe rprotein production. Considering. the results of the PDS in the ethanol stat FBBOs, ethanol concentrations were sett 0.5, 1.0, and 1.5 gL and kept almost constant throughout te fed-batch cultivations with mean values of 0.64, 0.96, and 1.6 # L?, respec tively. The ethano stat FBBOs produce significantly higher Gy values Bock ngmerg Jura 66 (2020) 10782 with the highest *hGH concentration attained at Cone = 0.5L ws Gomar=91 mig L att =24h which was 2.1-old higher than that of Ben (095 I, followed by Crotty = 1.0 and 1.5 Las Cp = 80.1 and 78.8 1g Li", respectively is 4. Based on our findings. we answer the long-standing question regarding the superior performance of the substratestat FBBOs (30,31, {41 than the substeate feeding with model based parametric designed continuous feed'stream, With the model-based PD substrate flow rate, using Heo values inthe production domain enabled appro priate cell growth and product formation profiles during a shorter cultivation peviod marked 48 far than the constantsubstate FBOS ith prolonged cultivation periods. Asthe present model (Eq 1) se for the PO Teed flow-rate ealeuations. ignores substrate inhibition, beyond pan the increase in substrate concentration dives the eas out af the production domain. We conchide that forthe continuovs substrate feed:Now rate design, an upgraded model with kinetic expressions involving substrate inhibition effet forthe rates of limiting substrate uptake Ct) ell fomaton (3 and product formation (fp) s require. Feeding the limiting substrate with an upgraded-model based ealela tion of the feed-low profile can adjust cellular metabolism for the seneration of a constant specific growth rate and a constant substrate concentration in the production domain, i being studied. ur inference is that an intermediate design strategy using the current model sup porte withthe results of precisely designed experimental programme tables an approach to the hypothetical performance of FABOs. Thus, our conetusionis that using a two-step hybrid fed-batch design strategy to be started (i with the initial-peiod ofthe model-based CFS, in turn, proceeded with the best performed ethan! st FBBO. Following the transition phase, the ist step ofthe FBBO should be designed with C the model-based (PD) ealelated feed flow-rates at Heep = 0.035 hi? Up 10 face $ h where the highest overall volumetric productivity (an) is produced; and, in tum (i) switching to the ethanolstat FBBO controled # Ceoitax = 05 81" Using directed evolution methods, engineering with synthetic Cats cis-acting DNA site upregulated the tanscription in ADH promoter in coordination with the master regulator transcription factor Cat8 and euhanced the expression on ethanol {7,11]. The framework of the regulation mechanism in. pastoris has been setup but needs in dept-nvestigation to clarify Snfl signalling pathway architetur, its subunits and catalytic domain, aetivation by phosphorylation and inactivation by dephosphorylation catalysed by a phospiatase with the regulatory protein involved in both processes (11). Overall, based on tur findings, we came to the conclusion that ethanol controls Sf sg naling. pathway [11,19] and crosstalk herween pathways enables fine-tuned transcription and eahanced expression for the rprotein production in . pastors with the hybrd-architectured ADH2 promote. Extracellular rprotein production in B. pastoris constructed with Paonia cu 21 ethanol enables the generation of promising r protein production platform, and the results ofthe FBBOs designed with the ro methods and our inferences contributes to the design of FBBOs and opens up new aventes for researc in the yeas. CcRediT authorship contribution statement ‘Omar Wehbe: Investigation, Bioreactor and analyeal experiments, Data curation, Modelling, Calculations, Results discussion, Visual Isation, Writing fst manuscript. Ou Ulas Yama: Modelling. Pinar GGalik: Conceptualization, Methodology, Resources, Supervision, Data curation, Results interpretation and discussion, Revision and writing final manuscript Declaration of Competing Interest ‘The authors report no declarations of interest.