Example: The Production of cx-Galadosidase by Monascus (A Structured Model)

The production of the enzyme a-galactosidase is subject to catabolite repression; when

the mold Monascus is grown on glucose as a carbon and energy source, no a-galactosidase
enzyme is made. However, if both glucose and galactose are present, glucose is consumed
first. When the glucose concentration is reduced to a low leve!, cx-galactosidase is induced
and it is produced about 80 minutes later in batch culture. Similarly, when glucose is added
to a system producing a-galactosidase, enzyme production is repressed over a 40 minute
period. A structured model for the production of the enzyme a-galactosidase by Monascus
has been formulated by Imanaka et al. 36 and is described here as it illustrates the coupling
of substrate transport and regulation of gene expression.
We shall denote extracellular glucose and galactose concentrations as SA and Se; the
interna! galactose concentration is Se; By assuming that galactose inhibits the consumption
of glucose in a competitive manner, the specific growth rate of the mold is assumed to
depend on both substrates in the fol!owing manner:
mASA
= +--- (3.96)
K +S +K_.., S Kss+Ss
SA A K, 8

Transport of galactose into the cell is assumed to be governed by an active transpon

mechanism. The maximum concentration of galactose-transporter binding sites is Ge.
Externa! galactose binds to these sites by an adsorption isothenn that follows a Langmuir
dependence on Se. The rate of galactose transport is described by the following expression.
GsSs ) (3.97)
rate of galactose transport =U ( Km+ 5 8;
58

Here U is a transport rate coefficient in hr" 1, Ge is in g galactose per mg cell mass and the
intracellularconcentration of galactose is in g/mg cells. Intracellular galactose is consumed
ata first order rate given by k 1Se; Thus a mass balance on intracellular galactose gives
d(S8 ;X)
dt
( G8 S8 ;
=U Km+Ss Ss;r-k,Ss;X
lv (3.98)

The effect of glucose repression is modelled by considering that when the externa! glucose
concentration exceeds a critica! value SAc the rate of galactose transport into the cell
immediately ceases, i.e., when SA;:: SAc then U= O. As a consequence, the specific growth
rate m 8 decreases to zero. Experimental data, shown in Figure 3.26 indicates tha_t in the
presence of 5 gmlliter glucose, galactose uptake is negligible. Galactose transport can also
be seen to be constitutive, as there is no time delay in uptake.
 o _.,l:l- - 0 - -

1 0_._~
0 0............... 2 ~~ -;o"-'-'-=.50
T1m~ ( min )

Figure 3.26. Galactose uptake by Monascus. Uptake ofgalactose at 35"C, with a galactose
concentration of 5 gmlliter containing 1 ci 14 C-galactose. Open circles represent cells
grown on giucose, washed and incubated on 5 gmlliter galactose medium. Closed circles
indicare incubation in the presence of 5 gmlliter glucose. No galactose uptake is evident
under glucose repression. (From lmanaka et. al.)

A simple model for enzyme induction by galactose, based on the Monod-Jacob operon
model, can be developed. In the absence of galactose, a repressor (R) acts to inhibit the
synthesis of mRNA for a-galactosidase. When galactose is present, the repressor and
galactose combine to form [RS 8 ] which can no longer bind to the DNA and enzyme synthesis
can occur. The amount of the (unidentified) intracellular repressor is found by considering
it to be produced at a constant rate k2, degraded by a first arder process (k3R) and reacting
reversibly with intracellular galactose to form a complex [RS 8 ;) by mass action kinetics.
The mass balance on repressor R yields
dRX
dr =(k2 -"fsR -k4R S8 ; + k5 [RS8 ])X (3.99)

The action of the repressor on the transcription of DNA and production of mRNA is as
follows. The synthesis of mRNA depends on the concentration of free repressor; when
galactose is present and repressor is bound to it, the concentration of free re pres sor is reduced
and it is no longer as effective in blocking mRNA synthesis. The rate of mRNA synthesis
is assumed to be proportional to the reduction in free repressor from sorne maximum value
R.,. The mRNA is assumed to decay with a first arder rate constant k7 The synthesis of
mRNA (M) is thus described by
dMX
--={k6(Rc-R)-kM}X .(3 .100)

This scheme for regulation of enzyme production is illustrated in Figure 3.27. The rate of
a-galactosidase formed by the mold depends on the concentration of mRNA:
dEX =k MX (3.101 )
8
dt
 DNA

,,--- ------ DNA transcribed in

prevents binding of
/' ---,, absence ot repressor
repressor to operan
I
/ ''
I ',
...
Repressor (R) mRNA (M)

Galactose (8 )
8
Galactosidase

Figure 3.27. Schematic of the gene leve[ regulation of a-galactosidase by galactose. The
repressor R is unknown, but is probably related to glucose or its metabolites.

A complete set of mass balances can now be written, including a balance for repressor-
galactose complex [RS 8 ]. We introduce the individual yield coefficients YA and Y8 , these
being the cell mass yields on glucose and galactose substrates, respectively. In writing the
mass balances, the terms describing the intracellular concentrations have been expanded
viz.
dS8 ;X dS8 ; dX dS8 ;
dt =X dt + Ss; dt =X dt + Ss; (3.102)

Thus the intrace!lular concentrations are effectively diluted by the expanding volume of the
cell.

(3.103)

(3.104)

(3.105)

U=O (3.106)

(3.107)
 dM
dr ={k6(R,. -R)-k.,M}-M where (3.108)

dE
-;=k8M-E (3. 109)

(3.110)

The kinetic and microbial parameters in the above set of equations were determined by
Imanaka et al. from batch and steady state continuous culture data. The intracellular
parameters were estimated, whereas the yields and specific growth rates were obtained from
the experimental data. The values of the constants are listed in Table 3.14.

Table 3.14. Values of constants in the model of Imanaka et al.

Estimated Experimental values
k1 =40 hfI mA ,g = 0.215 hfl
k1 =l g/mg cells-hr mB .g = 0.208 hfl
k3 = l hr"' mA .p = 0.190 h(I
k4 = 0.1 mg cells/g-hr mB .p = 0.162 hr 1
ks = l X 10.J hr" ' K,A.g = l.54 X JO.J gm/ml
~ = 1 hr" ' K,s., = 2.58 X 10.J grnlml
k, =8 hfl KsA.p = l.54 X 10-J gm/ml
ks =4.0 units/g M-hr K.s.p = 3.07 X 10.J gm/ml
kN.p = 6.67 units/g M-hr K = 1.39 X WJ gm/ml
4
SAc = 2.25 X 10' gm/ml
u = 100 h(I YA., = 0.530 grnlgm
Gs = 3.5 g /mg cells Ya., = 0.516 gm/gm
8
Km = 1 X 10 g/mg cells YA.p = 0.377 grnlgm
R: = 0.803 g/mg cells Ys.p = 0.361 gm/gm
The subscripts g and p denote values during the growth phase and enzyme production phase repectively.

The predictions of this model compared to the experimental batch culture data are
illustrated in Figure 3.28. The model can succesfully predict the catabolite repression of
galactose uptake by glucose and the slow rise in the concentration of a-galactosidase once
glucose is consumed and galactose is transported into the cell. The key feature of this model
of enzyme production is the incorporation of a simple model of enzyme repression aceording
to the scheme proposed by Jacob and Monod. It is able to effectively describe the observed
production kinetics. We shall examine sorne more complex models of gene expression in
Section 3.6.
The ability of this model to predict the dynamics of a -galactosidase repression by
glucose is considered in Problem 16. The halflife of mRNA is a key constantin determining
the strength of glucose repression.
 o.~

8 10
o

o 2 4 6 8 10 12 t4 16 o
Cullivolion lime (hr)

Figure 3.28. Time course of cell growth and cx-galactosidase production by Monsacus. The
inoculum contained glucose grown cells. The medium composition was 10 gmll glucose, 3
gmll galactose, 5 gmll NH4 N03 , KH2 P0 4 5 gmll, MgS04 7Hp 1 gmll, yeast extract 0.1 gmll.
Open squares, glucose; closed squares, galactose; open circles, cx-galactosidase; closed
circles, cell mass. The initial conditions for the model were: X= 0.5 gmll; SA = JO gmll; S8
= 3 gmll; S8 =O glgm cells; R = 0.910 glgm cells; M =O glmg cells; E= Ounits/mg
cells; [RS 8;] =O glgm cells.