Kinetic Statins Copia

Journal of Biotechnology 130 (2007) 422435

A macrokinetic modelling of the biosynthesis oflovastatin by Aspergillus terreusMarcin Bizukojc , Stanislaw Ledakowicz

Department of Bioprocess Engineering, Technical University of Lodz, ul. Wolczanska 213/215, 90-924 Lodz, PolandReceived 20 December 2006; received in revised form 20 April 2007; accepted 7 May 2007

Abstract

In this work a simple kinetic model to describe the biosynthesis of lovastatin by Aspergillus terreus ATCC 20542 was proposed. Several seriesof experiments were conducted at different media compositions. The concentrations of C- and N-sources were changed over a wide range and sowere the initial biomass concentrations. From these runs the relationships ruling the substrates uptake, biomass and product formation were learnt.Lovastatin biosynthesis appeared to be partly growth associated. The inhibitive effect of organic nitrogen on lovastatin biosynthesis was foundand lactose appeared to be an important limiting substrate in the formation of lovastatin. The parameters of the model were evaluated on the basisof the kinetic data obtained in the separate experiments made in triplicate at two chosen media compositions. Other results obtained at differentmedia compositions were independent of the ones mentioned above and used for the verification of the model. The validity of the model was alsoexamined for the lactose-fed fed-batch run. Finally, a sensitivity analysis of the model parameters was performed. The formulated model, althoughrelatively simplified, described the experimental data quite well and could be regarded as the background for further attempts to mathematicallydescribe the process of lovastatin biosynthesis. 2007 Elsevier B.V. All rights reserved.

Keywords: Lovastatin; Mevinolinic acid; Aspergillus terreus; Modelling; Kinetics

1. Introduction

Lovastatin, belonging to statins, is a widely used antihy-percholesterolemia drug. The main producers of statins arePenicillium citrinum, Monascus ruber and Aspergillus terreus(Manzoni and Rollini, 2002; Endo, 2004). Lovastatin is a natu-ral product originated from A. terreus (Monaghan et al., 1980).This secondary metabolite is extensively excreted from fungalcells into a medium in the form of !-hydroxy acidmevinolinicacid (Casas Lopez et al., 2003).

There are many papers concerning the biosynthesis of lovas-tatin. Some of them deal with the biochemical mechanism rulingthis process. From the biochemical point of view the biosynthe-sis of mevinolinic acid is performed at two stages. The firststage is catalysed by the nonaketide synthase (EC 2.3.1.161),which belongs to type I polyketide synthases and catalyses thenine-step formation of the polyketide (Shen, 2003). This PKS

Corresponding author. Tel.: +48 42 631 37 04; fax: +48 42 636 56 63.E-mail address: [email protected] (M. Bizukojc).

stage leads to the lovastatin precursor 4a,5-dihydromonacolineL (Sutherland et al., 2001):

AcCoA + 8malonyl-CoA + 11NADPH + 10H++ S-adenosyl-l-methionine

EC 2.3.1.1614a, 5-dihydromonacoline L + 9CoA + 8CO2+11NADP + S-adenosyl-l-homocysteine + 6H2O

In the post-PKS stage several oxidation steps with the participa-tion of molecular oxygen lead to mevinolinic acid (Sutherlandet al., 2001).

Other papers concern the influence of media composition onthe process of mevinolinic acid production. The optimum car-bon source was widely sought by many authors (Sitaram Kumaret al., 2000; Casas Lopez et al., 2003; Lai et al., 2003). Also avariety of nitrogen-sources were tested with regard to the opti-misation of mevinolinic acid biosynthesis (Hajjaj et al., 2001;Casas Lopez et al., 2003; Lai et al., 2003).

Casas Lopez et al. (2005) and Lai et al. (2005) investigatedthe influence of dissolved oxygen in the medium on the process

0168-1656/$ see front matter 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2007.05.007

M. Bizukojc, S. Ledakowicz / Journal of Biotechnology 130 (2007) 422435 423

NomenclatureNomenclaturecLAC lactose concentration (g l1)cLAC0 initial lactose concentration (g l1)cN organic nitrogen concentration (g l1)cMEV mevinolinic acid concentration (g l1)cX biomass concentration (g l1)cX0 initial biomass concentration (g l1)C/N carbon to nitrogen ratio(C/N)0 initial carbon to nitrogen ratiokMEV second order constant rate for mevinolinic acid

formation (g MEV l g LAC1 g X1 h1)KI,N organic nitrogen inhibition constant (g N l1)KMEVI,N organic nitrogen inhibition constant for mevino-

linic acid formation (g N l1)KLAC Contois type saturation constant of lactose

towards biomass (g LAC g X1)KMEVLAC Contois type saturation constant of lactose for

mevinolinic acid formation (g LAC g X1)KN Contois type saturation constant of organic nitro-

gen towards biomass (g LAC g X1)LAC lactoseMEV mevinolinic acid (lovastatin)max maximum specific biomass growth rate (h1)N organic nitrogen originated from yeast extractqMEVmax maximum specific formation rate of mevinolinic

acid (g MEV g X1 h1)rLAC lactose volumetric uptake rate (g LAC l1 h1)rMEV mevinolinic acid volumetric formation rate

(mg MEV l1 h1)rN organic nitrogen volumetric uptake rate

(g N l1 h1)rX biomass volumetric formation rate (g X l1 h1)t time of the process (h)X biomassYE0 initial yeast extract concentration (g l1)YX/LAC biomass to lactose yield coefficient

(g X g LAC1)YX/N biomass to nitrogen yield coefficient (g X g N1)

and tried to enrich the aeration gases with oxygen. Lai et al.(2005) proved that pH level is of little significance and should notbe definitely adjusted to any level during the process. Bizukojcet al. (2007) have recently revealed that the supplementationof the cultivation media with B-group vitamin, especially withnicotinamide, calcium d-pantothenate and pyridoxine, leads toan enhancement of the lovastatin production, which is connectedwith a high demand for reduced NAD and CoA at the PKS stageof mevinolinic acid formation. Also the influence of sporulationconditions (light exposure) and the age of spores on the lovastatinproduction was investigated (Rodrguez Porcel et al., 2006).

Nevertheless till now no attempt to formulate any kineticmodel of lovastatin biosynthesis has been made, although fila-mentous fungi have been the object of kinetic modelling both

structured and unstructured for many years. For example, vari-ous proposals for structured modelling of penicillin formation byPenicillium chrysogenum were published in the last decade (Pauland Thomas, 1996; Zangirolami et al., 1997; Paul et al., 1998).Also structured models were proposed for citric acid formationby Aspergillus niger (Alvares-Vasquez et al., 2000; Bizukojc andLedakowicz, 2003). The unstructured or simple kinetic modelsfor filamentous fungi were less frequently presented (Goudarand Strevett, 1998; Kelly et al., 2004; Lisowska et al., 2006;Bizukojc and Ledakowicz, 2006).

The aim of this work is to formulate a simple kinetic modelfor lovastatin formation by A. terreus on the basis of our ownexperimental data.

2. Materials and methods

2.1.1. Strain and media

The strain A. terreus ATCC 20542 was employed in thebiosynthesis of lovastatin (mevinolinic acid). All experimentswere carried out in a rotary shaker in flat bottomed flasks of150 ml working and 500 ml total volume at 30 C and 110 rpm.The inoculum was prepared from the spores grown on the maltextract slants. The spores were washed with 10 ml of sterileinoculation media up to the concentration about 107 spores/ml,suspended in the inoculation medium and precultivated for 24 h.The inoculation was made in the proportion 1:20, apart fromruns X1 and X3, in which the ratios of 1:75 and 1:10 wereused, respectively, in order to change the initial concentration ofbiomass.

The basic cultivation medium, which followed the suggestionof Casas Lopez et al. (2003) contained lactose: 20 g l1 (10 g l1in the preculture), yeast extract: 8 g l1 (0.762 0.005 g N l1),potassium dihydrophosphate KH2PO4: 1.51 g l1, magnesiumsulphate MgSO47H2O: 0.52 g l1, sodium chloride NaCl:0.4 g l1, zinc sulphate ZnSO47H2O: 1 mg l1, ferric nitrateFe(NO3)9H2O: 2 mg l1, biotin: 0.04 mg l1 and 1 ml solutionof trace elements per 1 l of medium. The solution of trace ele-ments contained sodium borate Na2B4O710H2O: 100 mg l1,manganese chloride MnCl2: 50 mg l1, sodium molybdateNa2MoO42H2O: 50 mg l1 and copper sulphate CuSO45H2O:250 mg l1.

The experimental scheme is presented in Table 1.Runs Z1 and Z2 were made in triplicate separately and inde-

pendently of the other runs. The averaged data calculated fromthese runs were used to evaluate the model parameters.

2.1.2. Analytical methods

Lactose was determined by HPLC (Waters, USA) in a Shodex1011 column. It was eluted with 0.01N H2SO4 at the flow rate1 ml min1 at 50 C and detected by a refractive index detector.Mevinolinic acid was determined by HPLC in a Waters Nova-pak C18 4"m column (3.9 mm 150 mm). The elution wasmade with CH3CN0.1% H3PO4 (60:40, v/v) at the flow rate1 ml min1, at 25 C. A photodiode array detector at = 238 nmwas used. The standard solution of mevinolinic acid was pre-

424 M. Bizukojc, S. Ledakowicz / Journal of Biotechnology 130 (2007) 422435

Table 1Experimental scheme for all runs

Run Initial biomass concentration,cX0 (g l1)

Initial yeast extractconcentration, YE0 (g l1)

Initial lactose concentration,cLAC0 (g l1)

Initial carbon tonitrogen ratioa (C/N)0

X1 0.031X2 0.104 8 20 14.5X3 0.21

N1 2 37.4N2 4 24.8N3 0.104 6 20 16.8N4 8 14.4N5 12 10.7

L1 5 9.7L2 10 14.7L3 0.104 4 20 21.6L4 40 45.5

Z1 0.104 4 20 24.0 0.3Z2 0.104 8 20 14.3 0.1FB1b 0.104 4 20 25.0

a The value given in this table is determined experimentally upon data concerning elemental composition of yeast extract: C = 41%, H = 6.22%, O = 28.22%,N = 10.9%, S = 0.6%, ash = 13.06%.

b Run performed in the fed-batch manner, for the feeding scheme details see further in the text.

pared according to Casas Lopez et al. (2003). The by-productspresent in the medium were separated with the use of a WatersSymmetry Shield RP18 5"m column (4.6 mm 250 mm) bythe following gradient elution: CH3CN0.1% H3PO4 (40:60,v/v) up to 7 min and CH3CN0.1% H3PO4 (60:40, v/v) from 8to 30 min.

Organic nitrogen was determined alkacymetrically, afterdigestion of the sample with concentrated sulphuric acid andhydrogen peroxide and subsequent water vapour distillation ofthe released ammonium ions (Buchi, Switzerland). Biomass wasassayed as dry weight.

The elemental analysis of the yeast extract (complex nitrogensource) was performed with the help of an Elemental AnalyzerNA2500 (CE Instruments, Italy).

2.1.3. Modelling tools

Microsoft Excel software was used as a tool for linearregression calculations. The non-linear regression calculations,the parameters estimation and the solution of the differen-tial equations were performed with the help of the followingnumerical procedures implemented in Easy-fit software (KlausSchittkowski 2001, University of Bayreuth). The modifiedquasi-Newton least squares optimisation was used to estimatethe parameters of the model. The implicit Radau method ofthe 5th order for stiff equations was a solver of the differentialequations.

3. Results

3.1. The run of mevinolinic acid formation at differentprocess conditions

The mode of association of mevinolinic acid production withA. terreus biomass growth was investigated from two points of

view: proportionality between the initial biomass concentrationand mevinolinic acid yield and the association of biomass growthwith product formation.

It was observed that the final lovastatin yield increased withthe amount of inoculum. The experimentally quantified meanyields of mevinolinic acid to biomass YMEV/X for run X1,X2 and X3 were equal to 1.119 0.127, 1.352 0.069 and1.795 0.178 mg MEV g X1, respectively. Nevertheless, fol-lowing the literature data and the question of aeration of themedium, other experiments were conducted at the initial biomassconcentration at the level about 0.1 g l1 (inoculation ratio equalto 1:20).

Mevinolinic acid was produced with the varying volumetricformation rate from the beginning to the end of the run and atfirst glance no strict correlation between lovastatin formationand biomass growth could be noticed. It was already excreted inthe trophophase: during exponential growth (up to 24 h) and laterduring linear growth. The exponential growth phase at A. terreuswas investigated earlier and presented elsewhere (Bizukojc andLedakowicz, 2005). After 100 h of the run the fungus turned intothe idiophase. In this phase lovastatin was still produced but withthe lower volumetric formation rate than earlier. In order to findthe correlation between product formation, biomass growth andsubstrate uptake, the method proposed by Luedeking and Piret(1959) was used.

In Fig. 1 the association of product formation with biomassgrowth was presented in the form of the time evolution of volu-metric rates of lactose and organic nitrogen uptake and biomassand mevinolinic acid formation. The data for these calculationscame from runs Z1 (at limited nitrogen content: YE0 = 4 g l1,Fig. 1a) and Z2 (for basic medium, Fig. 1b). Analysing thisgraph, it may be assumed that biosynthesis of mevinolinic acid ispartially growth associated because the maxima of rX and rMEVdid not coincide. The maximum of rMEV was delayed for about20 h for both cases. Additionally, some information concerning


Fig. 1. Changes of volumetric rates of substrates uptake rLAC and rN, product rMEV and biomass rX formation in time for the averaged runs Z1 (a) and Z2 (b).

the correlation between substrate uptake, biomass formation canbe deduced from Fig. 1. The maximum of rX went together withrN, which could be the evidence that the main limiting substratefor biomass formation was rather N-source than C-source. Fur-thermore, rMEV strictly correlated with rLAC, but not with rN.And last but not least, comparing Fig. 1a and b, it is well seenthat the maximum of rMEV was less delayed when more nitrogenwas present in the medium (about 10 h). Another evidence for thepartially growth-associated mevinolinic acid formation will bepresented while analysing the fed-batch run (FB1) in Section 7.

As lovastatin does not contain any nitrogen moieties in itsstructure (C24H36O5), its formation is connected with nitrogenutilisation only to the extent to which the amount of nitrogeninfluences biomass amount in the system. Therefore, lactosemay be the most important factor influencing the productionof mevinolinic acid. Fig. 2 shows the influence of initial lac-tose concentration on mevinolinic acid formation. It is evident

that when lactose is depleted, the biosynthesis of mevinolinicacid is ceased, which is especially well seen for the run withinitial lactose concentration equal to 5 g l1. In runs L2 and L3more lovastatin was obtained because more lactose was initiallyadded. Nevertheless, at the highest initial lactose concentra-tion in run L4 (40 g l1) despite lactose was still present in themedium, the production of mevinolinic acid slowed down signif-icantly and its final yield was comparable with the result obtainedin run L3 (Fig. 2).

As it has been mentioned above, organic nitrogen does notdirectly participate in the biosynthesis of mevinolinic acid. Whatis more, many authors claim that it inhibits lovastatin formation(Casas Lopez et al., 2003; Lai et al., 2003; Hajjaj et al., 2001).So the lower concentration of nitrogen was in the system, thebetter mevinolinic acid yield should be obtained. Fig. 2 showsalso the influence of initial concentration of yeast extract in therange from 2 to 12 g l1 on mevinolinic acid formation by A. ter-


Fig. 2. Influence of initial lactose (runs from L1 to L4) and yeast extract (runs from N1 to N5) concentration on lovastatin production; experimental points areconnected with the spline curves.

reus. The highest concentration of mevinolinic acid was obtainedin run N2, in which yeast extract was added at the amount of4 g l1. Although in run N1 nitrogen concentration was lower,the highest titre of lovastatin was not achieved, due to strongnitrogen limitation with regard to biomass. Its concentration inthe idiophase was about 7 g l1. It remains in agreement with theearlier revealed correlation between mevinolinic acid productionand biomass concentration in the system. In run N2 a moderatebiomass growth was observed (about 10 g l1 in the idiophase),while in runs N3 and N4 biomass concentration was the high-est (about 12 g l1 in the idiophase), as the sufficient amountof nitrogen was supplied. In runs from N3 to N5 the mevino-linic acid production decreased significantly with the increaseof nitrogen content. In run N5 only traces of lovastatin weredetected in the medium.

In the investigated system two main substrates, lactose asC-source and yeast extract as N-source, were present. As thecomplex nitrogen source used contains also carbon in the formof amino acids, it is important from the point of lactose utilisa-tion kinetics to know if these amino acids interfere with lactoseutilization, i.e. amino acids were used as a carbon source.

In runs from N2 to N4 volumetric uptake rates of lactosewere approximately the same and, between 48 and 140 h of therun, were equal to 0.16, 0.17 and 0.18 g LAC l1 h1, respec-tively, despite the fact that different amounts of amino acidswere available for the fungus, as initial yeast extract variedfrom 4 to 8 g l1. Only in run N1 this rate was slightly lower

(0.11 g LAC l1 h1) due to small biomass growth. Only in runN5 either lactose (0.044 g LAC l1 h1) or nitrogen were hardlyconsumed. It is probably due to the fact that the fungus wasdeprived of the carbon source because of strong repressioncaused by organic nitrogen.

Therefore, it can be claimed that in all runs lactose wasutilised as a sole carbon source and yeast extract as a sole nitro-gen source and amino acids seem not to play the role of a carbonsource.

4. Modelling

4.1. Verbal formulation of the modelOn the basis of the results presented above the following

assumptions for the model for lovastatin production by A. terreuscan be formulated.

Lactose is regarded as a sole carbon source in the investigatedsystem. Although there are amino acids in the medium whichare originated from yeast extract, they are not utilised as a car-bon source independently of the concentration of both lactoseand yeast extract.

As in the fungal system high amounts of biomass are usuallypresent, it is reasonable to use for the substrates utilisation ratethe Contois model which takes the amount of biomass intoaccount (Goudar and Strevett, 1998; Lisowska et al., 2006).


Yeast extract is regarded as a sole nitrogen source as no ammo-nium salts are added to the medium.

The excess of nitrogen exerts an inhibitive effect on lovastatinbiosynthesis (Fig. 2) and lactose uptake, which is also claimedby many authors (Hajjaj et al., 2001; Casas Lopez et al., 2003;Lai et al., 2003; Bizukojc and Ledakowicz, 2005).

As it was presented above, mevinolinic acid biosynthesis ispartially growth associated. This observation determines theform of mevinolinic acid balance consisting of the term takingthe product formation associated with biomass growth intoaccount and the term regarded the non-associated productformation which is, however, also dependent on biomass andlactose concentration.

Lactose is both utilised for biomass formation and biosynthe-sis of mevinolinic acid. Therefore, the total lactose volumetricuptake rate is the sum of uptake rates for biomass growth andmevinolinic acid formation.

Although Casas Lopez et al. (2004) claim that lovastatininhibits its own synthesis, the product inhibition term wasnot added. They observed a significant inhibition when thespiked lovastatin concentration exceeded 200 mg l1. In thepresent experiments such concentration was not achieved andfor the sake of simplicity this term was omitted.

4.2. Model equations

On the basis of the verbal formulation of the model the follow-ing equations were proposed. Four components were balanced:lactose as a carbon source, cLAC, nitrogen originated from yeastextract, cN, mevinolinic acid, cMEV and biomass, cX.

dcLACdt

= 1YX/LAC

max cLACcLAC+KLAC cX

cNcN +KN cX

KI,NKI,N + cN cX

1YMEV/LAC

qMEVmax

cLACcLAC +KMEVLAC cX

KMEVI,N

KMEVI,N + cN cX (1)

dcNdt

= 1YX/N

max cLACcLAC+KLACcX

cNcN +KN cX cX

(2)

dcMEVdt

= qMEVmax cLAC

cLAC +KMEVLAC cX K

MEVI,N

KMEVI,N + cN cX

+kMEV cLAC cX (3)dcXdt

= max cLACcLAC +KLAC cX

cNcN +KN cX cX (4)

4.3. Determination of model parameters

All model parameters were determined on the basis of theaveraged runs Z1 and Z2 (Table 2). Three of them were directlydetermined from the experimental data. These were maximumbiomass growth rate max, maximum specific formation rate ofmevinolinic acid qMEVmax and two yield coefficients YX/LAC andYX/N. The determination of maximum biomass specific growthrate was made from the relation ln(cX) = f(t). The maximumspecific formation rate of mevinolinic acid was directly foundfrom its time evolution curve. This curve was calculated by theapproximation of mevinolinic acid and biomass curves by meansof non-linear regression. In each case the specific formation rateachieved its maximum values in the middle trophophase about4050 h.

As nitrogen was only used for biomass growth and wasnot built into the lovastatin molecule, the linear correlation"cX = YX/N"cN led practically to the accurate determinationof this model parameter. The determination of yield coeffi-cient biomass on lactose was performed in the similar way("cX = YX/LAC"cLAC), although the participation of lactose inthe biosynthesis of mevinolinic acid of the partially growth-associated type might have led to a higher estimation error thanfor YX/N. As only two runs Z1 and Z2 were used to determine theyield coefficients, it might be doubtful whether these yield coef-ficients were really independent of the medium composition.Therefore, these values were compared to the ones determinedfor the other batch runs, excluding Z1 and Z2. The chosen results

Table 2Parameters of the model estimated on the basis of run Z1 and Z2

Type of the parameter Value Estimation method

Maximum specific biomass growth rate, max (h1) 0.120 0.010 Linear fit: ln(cX) = f(t)Maximum specific formation rate of mevinolinic acid, qMEVmax (g MEV g X1 h1) 1.9 104 0.3 104 Averaged from experimental

dataSecond order constant rate for mevinolinic acid formation, kMEV (g MEV l g LAC1 g X1 h1) 5.42 106a; 1.89 106b Non-linear optimisationBiomass to lactose yield coefficient, YX/LAC (g X g LAC1) 0.483 0.051 Linear fit: cX = f(cLAC)Biomass to nitrogen yield coefficient, YX/N (g X g N1) 20.06 2.11 Linear fit: cX = f(cN)Mevinolinic acid to lactose yield coefficient, YMEV/LAC (g MEV g LAC1) 7.06 104a; 2.2 104b Non-linear optimisationContois type saturation constant of lactose towards biomass, KLAC (g LAC g X1) 1.63 Non-linear optimisationContois type saturation constant of organic nitrogen towards biomass, KN (g LAC g X1) 8.84 102a; 0.278b Non-linear optimisationContois type saturation constant of lactose for mevinolinic acid formation, KMEVLAC (g LAC g X1) 13.23 Non-linear optimisationOrganic nitrogen inhibition constant, KI,N (g N l1) 0.158 Non-linear optimisationOrganic nitrogen inhibition constant for mevinolinic acid formation, KMEVI,N (g N l1) 9.65 102 Non-linear optimisation

a For run Z1.b For run Z2.


Fig. 3. Determination of yield coefficients YX/N and YX/LAC for runs from N1 to N5, from X1 to X3 and from L1 to L4.

are shown in Fig. 3. It occurred that at each run independentlyof (C/N)0, initial biomass concentration and mevinolinic acidconcentration both YX/LAC and YX/N do not change significantlyand the values determined from averaged runs Z1 and Z2 arereliable.

Other model parameters were determined with the use of thenon-linear optimisation procedure from the data obtained in Z1and Z2 runs. Fig. 4 shows a comparison of simulated curvesand experimental points. In Table 2 all parameters of the modelare collected. Good agreement of the simulated curves with theexperimental data was achieved. Only three parameters had tobe varied, depending on medium composition, in order to fit thecurves to the experimental data from run Z1 and Z2. They wereKN, YMEV/LAC and kMEV. The reasons for this will be presentedfurther in Section 7.

5. Verification of the model

The formulated model was then verified with the use of thedata from the independent experiments. All batch experiments,excluding Z1 and Z2, were used for this purpose. Although theobservations made in these experiments were helpful in the ver-bal and mathematical formulation of the model, none of thesedata were used for the evaluation of the model parameters.

The simulations were performed with the use of all param-eters gathered in Table 2, apart from one: the second orderconstant rate for mevinolinic acid formation kMEV. This rateconstant is connected with the non-growth-associated forma-

tion of mevinolinic acid and had to be tuned in order to fit theexperimental data, which eventually occurred to be acceptable.Its reasons will be discussed further. At runs, in which YE0 wasequal to 8 and 12 g l1, the parameters connected with nitrogenmetabolism were the same as in run Z2. For other runs theyfollowed the parameters for run Z1. Results of the simulationsand experimental data are shown in Fig. 5. The values of kMEVfor each run are collected in Table 3. Second order rate con-stant increased for the runs, in which more mevinolinic acidwas formed. It was independent of the initial carbon to nitro-gen ratio too. Usually kMEV was higher, if C/N was elevated bymeans of the decrease of N-source level, not by the increase oflactose content. More detailed analysis is presented in Section 7.

Table 3Comparison of kMEV values for the runs conducted at different conditions

Run (C/N)0 kMEV (106 g MEV l g LAC1 g X1 h1)X1

14.51.00

X2 1.80X3 1.90

N1 37.4 13.4N2 24.8 7.40N3 16.8 2.75N4 14.4 1.50N5 10.7 0.40

L1 9.7 25.0L2 14.7 15.2L3 21.6 8.70L4 45.5 2.42


Fig. 4. The simulated curves and averaged experimental points for runs Z1 (a and b) and Z2 (c and d).

6. The application of the model for the fed-batch run

Another verification of the validity of the model was made,when it was used to simulate the time course of lactose, nitro-gen, mevinolinic acid and biomass in the fed-batch run. For thepurpose of modelling of the fed-batch process the system ofdifferential equations was rearranged to the following form.

dcLACdt

= 1YX/LAC

max cLACcLAC+KLAC cX

cNcN +KN cX

KI,NKI,N + cN cX

1YMEV/LAC

qMEVmax

cLACcLAC +KMEVLAC cX

KMEVI,N

KMEVI,N + cN

cX + F (t)V

(cfeedLAC cLAC) (5)

dcNdt

= 1YX/N

max cLACcLAC +KLAC cX

cNcN +KN cX

cX F (t)V

cN (6)

dcMEVdt

= qMEVmax cLAC

cLAC +KMEVLAC cX K

MEVI,N

KMEVI,N + cN cX

+kMEV cLAC cX F (t)V

cMEV (7)dcXdt

= max cLACcLAC +KLAC cX

cNcN +KN cX cX

F (t)V

cX (8)dVdt

= F (t) (9)

where F(t) is the feeding profile. The feeding profile was asfollows: three times at t = 96, 144 and 196 h, 10 ml of the solutioncontaining lactose at the concentration cfeedLAC = 110 g l1 wasadded to the flasks. This way the volume of the culture increasedstep by step from 157 to 167 ml, then to 177 ml and finally to187 ml. Fig. 6 shows the simulated curves and experimental datafor the fed-batch run. Agreement of the simulated curves withthe experimental data seems to be acceptable.

Fig. 7 illustrates the results of parameter sensitivity analysis.Relative absolute sensitivity versus time curves were calculatedfor all parameters with regard to lactose, nitrogen, mevinolinicacid and biomass. A detailed discussion of the sensitivity ofmodel parameters will be presented in the next section.


7. Discussion

Analysing the influence of biomass amount on the lovas-tatin biosynthesis, it was found that the inoculation withmore biomass led to the better yield of the process (runsfrom X1 to X3). Nevertheless, the duplication of initialbiomass amount did not lead to the duplication of lovas-tatin yield (1.352 0.069 mg MEV g X1 for run X2 and1.795 0.178 mg MEV g X1 for run X3). That is why theinoculum ratio was sustained at 1:20 as for run X2 due to thereasons mentioned earlier in Section 3 and because of the factthat the other factors influenced the process to a higher extent.

Mevinolinic acid formation occurred to be partially growthassociated (Fig. 1) and similar results were obtained by CasasLopez et al. (2005). Although they did not try to evaluate thiscorrelation because their considerations were focused on thehydrodynamic aspects of lovastatin biosynthesis, even roughanalysis of the time curves for biomass and lovastatin presentedby them confirms this thesis.

The proper amount of C-source is crucial when the ele-vated amounts of lovastatin are to be obtained. Some authorseven suggested to use over 100 g l1 of glucose in the medium(Novak et al., 1997), glycerol at 70 g l1 (Manzoni et al., 1998),

sucrose at 50 g l1, lactose at 70 g l1 (Lai et al., 2005) or lactoseat 100 g l1 (Casas Lopez et al., 2005). Although different C-sources were previously used, Casas Lopez et al. (2003) claimthat slowly utilisable carbohydrates as lactose or glycerol arethe most useful. It was also confirmed in our previous works(Bizukojc and Ledakowicz, 2005; Bizukojc et al., 2007). SitaramKumar et al. (2000) observed the negative effects connectedwith the deficiency of C-source in the medium and they strug-gled against it using the fed-batch system. Nevertheless, theseauthors also claimed that the increase of lactose concentrationat the beginning of the process did not lead to the significantlybetter yield of lovastatin. Although many authors started theircultivations with very high initial C-source concentration, asmentioned above, in our investigations the significantly betteryield for elevated initial lactose was not confirmed (Fig. 2).Therefore, starting of the feeding with lactose in the early idio-phase appeared to be a better solution (Fig. 6). The fed-batchsystem for lovastatin biosynthesis is also suggested by Novak etal. (1997), Sitaram Kumar et al. (2000) and Casas Lopez et al.(2003).

With regard to the influence of nitrogen, it must be noted thatorganic nitrogen although necessary for biomass growth, is alsothe inhibitor of mevinolinic acid biosynthesis. The higher C to

Fig. 5. Comparison of simulated curves and experimental data for independent runs: run X1 (a), run X2 (b), run X3 (c), run N1 (d), run N2 (e), run N3 (f), run N4(g), run N5 (h), run L1 (i), run L2 (j), run L3 (k), run L4 (l).


Fig. 5. (Continued )

N ratio is set, the higher amount of lovastatin can be achieved(Casas Lopez et al., 2003; Lai et al., 2003; Hajjaj et al., 2001;Bizukojc and Ledakowicz, 2005). In the experiments performedat yeast extract concentration of 4 g l1 the highest amount ofmevinolinic acid was obtained (Fig. 2). On the one hand, itmeans that when more nitrogen was applied, more biomasscould be achieved. But at the same time it would not lead tothe better yield of mevinolinic acid. On the other hand, nitro-gen content must not be lowered in the system too much, as itresults in a significant decrease of biomass amount, as it wasobserved in run N1, and in consequence led to a decrease inthe amount of lovastatin. This decrease of the lovastatin titrewas expected, as the lovastatin concentration was also the func-tion of biomass content, as it was proved in experiments fromX1 to X3.

Concerning the formulated model, the values of the chosenmodel parameters should be thoroughly discussed. First of all,it could be expected that the same set of parameters were suit-able for any run independently of the conditions and mediumcomposition. It was not possible. The first problem was with thenitrogen saturation constant, which had to be different for theruns, in which initial yeast extract concentration was equal orhigher than 8 g l1 (KN = 0.278 g N l1) in comparison to runswith less nitrogen input in which it was equal to 0.0884 g N l1.

The reason was probably connected with the biosynthesis ofextracellular proteins which is organic nitrogen dependent. Dueto the fact that protein biosynthesis was not modelled in sucha simplified approach as presented in this work, the changeof the parameter had to be accepted. The values of the twoother parameters were tuned because of other reasons. Theseparameters are YMEV/LAC and kMEV. As A. terreus is a fungus ofrich secondary metabolism, it must not be treated as homofer-mentative towards lovastatin in any of the runs performed.By-products were always present in the medium. Many authorsclaim that such compounds (aromatic and non-aromatic) origi-nated from the polyketide metabolism as 6-methylsalicylic acid,pigment precursors, citrinin, sulochrin, asterric acid, (+)-geodinbutyrolactone-I are the by-products at the lovastatin biosynthesisand that their production is certainly nitrogen or carbon or evenC to N ratio dependent (Schimmel et al., 1998; Schimmel andParsons, 1999; Hutchinson et al., 2000; Couch and Gaucher,2004). In the present work it was observed that the mediumgot yellowish, especially in the runs in which nitrogen amountwas reduced and at highly elevated C to N ratio (late stages ofthe fed-batch run). Therefore, several samples were addition-ally investigated by the qualitative HPLC analysis. Fig. 8 showsa chromatogram (scans at 238 and 280 nm) in which the peaksof hypothetical by-products are depicted.


Fig. 5. (Continued )

The wavelength 238 nm is usually used for the detection ofmevinolinic acid and lovastatin due to characteristic decalinerings in the mevinolinic acid molecule, which is responsiblefor light absorption at this wavelength (Monaghan et al., 1980;Kysilka and Kren, 1993). Similar unidentified peaks, as in Fig. 8,at 238 nm for A. terreus broths were observed by Morovjan etal. (1997). As other substances detected in the chromatogramabsorb the light at this wavelength and at the same time they havedifferent retention times, they must have a different structure,apart from decaline-like rings, and are probably the metabolitesof polyketide pathway. The concentration of by-product 6 is thehighest and this is (+)-geodin. It had a maximum absorbance at280 nm and was recognised with the use of a standard kindlydonated by Prof. Isao Fujii (University of Tokyo). Its signifi-cant amounts (expressed in terms of the height of a peak) wereobserved at high C/N ratios in N1, N2, N3, L3 and L4 runs.Nevertheless, a more detailed investigation of by-products andthe methods to eliminate them is going to be the subject of thefurther experiments. Summing up, if only one metabolic prod-uct is taken in the model into account, lactose balance is notsatisfied, so YMEV/LAC does not express exactly the amount oflactose utilised for mevinolinic acid formation. Nevertheless, itmust be also mentioned here that in the chromatogram depictedin Fig. 8 only the polyketide-like products are probably detected,

and there is no certainty that these are all products of this typeproduced by A. terreus. Also it is unknown whether other by-products are synthesised either. The phenomena described abovecaused that kMEV had to be tuned depending on the proportion oflactose and nitrogen in the medium (Table 3). It can be regardedas a disadvantage of the model. Nevertheless, these drawbacksare related to its high level of simplicity.

Despite all this, the model describes the process quite well(Fig. 4) and a fairly good fit for almost all independent runs wasobtained (Fig. 5). The best agreement between the experimentaldata and simulated curves was obtained for the runs, in whichthe concentrations of both C- and N-source did not achieve theextreme values. In the runs, in which N-source was highly over-dosed, as in run N5 (12 g l1) or extremely limited as in run N1(2 g l1), the goodness of fit was significantly worse. The samesituation took place when the extreme concentrations of lactosewere used. Both for run L1 (5 g l1) and L4 (40 g l1) the fit wasworse than for run L2 and L3. These worse results were causedby the possible significant changes in metabolism of the fungusexerted by the overdose or deficiency of lactose or yeast extract.

Finally, the utility of the model was proved, when it was usedfor the description of the fed-batch run FB1 (Fig. 6). This runalso confirmed the partial growth association of product for-mation with biomass growth. Although lactose was added and


Fig. 6. Comparison of the experimental data and simulated curves for the fed-batch culture, kMEV was tuned to the value 7.4 106 g MEV l g LAC1 g X1 h1.

Fig. 7. Changes of absolute-relative sensitivity of model parameters for lactose (a), organic nitrogen (b), mevinolinic acid (c) and biomass (d) in time (data from Z1runs simulation). In order to use one Y-axis in the graph some sensitivities were multiplied by a constant shown in the graph.


Fig. 8. Chromatogram with by-products detected in the broth in run L4.

became available for the fungus, practically no biomass growthwas observed due to this additional feeding. At the same timelovastatin titre increased significantly. In this run the highestconcentration of lovastatin was obtained and the model suc-cessfully predicted all experimental data, which proves thatthe form of the equations used to express participation of thenon-growth-associated lovastatin formation is proper. The dis-crepancy between the experimental data and the model curve,especially for lactose and mevinolinic acid, whose curves arelocated above the experimental points after the third feed (Fig. 6),is probably connected with the deterioration of the biomass qual-ity in the late hours of the process. This is often observed infilamentous fungi and can be more accurately modelled withthe use of the structured modelling concept, when biomass isdivided into zones of different physiological activity (Bizukojcand Ledakowicz, 2003).

The performed sensitivity analysis of the model parametersenabled a more detailed analysis of the properties of the model(Fig. 7). The sensitivity of parameters confirmed the assump-tions of the model. Lactose was mainly utilised for biomassgrowth, as YX/LAC and saturation constants reflected their uptake.Nevertheless, KMEVLAC , i.e. the saturation constant with regard tomevinolinic acid synthesis also remained an important parame-ter for lactose uptake. Nitrogen uptake and biomass formationrelated parameters (YX/N, max, and saturation constants) wenttogether and they were both the most sensitive with regard tonitrogen and biomass curves.

The time distribution of sensitivities of max and kMEV withregard to mevinolinic acid is very characteristic. In the earlyhours of the run the sensitivity ofmax achieved its highest value(at t = 60 h), while kMEV increased within all time domain up tothe end of the run (Fig. 7c). It reflected the partial association ofmevinolinic acid biosynthesis with biomass growth.

To sum up all considerations, this model is the first attempt tomodel the kinetics of lovastatin biosynthesis. Although it is very

simplified and does not take into account all phenomena con-nected with the biosynthesis of lovastatin, it is accurate enoughto become a background for the further investigations of theprocess. Also it is the first attempt to make a detailed and system-atic analysis of the influence of C- and N-source concentration,C/N ratio and initial biomass concentration on the kinetics oflovastatin biosynthesis.

Acknowledgements

The authors wish to acknowledge Prof. Isao Fujii for his kinddonation of the sample of (+)-geodin. This work was financedfrom grant no. 3 T09C 013 28 realised from 2005 to 2007(Ministry of Scientific Research and Information Technology,Poland).

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A macrokinetic modelling of the biosynthesis of lovastatin by Aspergillus terreusIntroductionMaterials and methodsStrain and mediaAnalytical methodsModelling tools

ResultsThe run of mevinolinic acid formation at different process conditions

ModellingVerbal formulation of the modelModel equationsDetermination of model parameters

Verification of the modelThe application of the model for the fed-batch runDiscussionAcknowledgementsReferences

Kinetic Statins Copia

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