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A cell population structuring model to estimate recombinant strain growth in a closed system for subsequent search of the mode to increase protein accumulation during protealysin producer cultivation

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Biofabrication 4 (2012) 019601 (1pp)

Erratum: A cell population structuring model to estimate recombinant strain growth in a closed system for subsequent search of the mode to increase protein accumulation during protealysin producer cultivation

S P Klykov et al 2011 Biofabrication 3 045006

S P Klykov 1, 3 , V V Kurakov 1 , V B Vilkov 1 , I V Demidyuk 2 ,

T Yu Gromova 2 and D A Skladnev 1

1 PHARM-REGION, Ltd, c.1, b.10, Baryshikha street, Moscow, 125222, Russia

2 Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq. 2, Moscow 123182, Russia

Received 25 November 2011 Published 23 February 2012 Online at stacks.iop.org/BF/4/019601

There was an error in the published version of figure 4 . The correct figure is shown below.

of figure 4 . The correct figure is shown below. 3 Author to whom any correspondence

3 Author to whom any correspondence should be addressed.

Figure 4. Dependence of enzymeactivity on growth time. Growth time, hours, is the obscissa. Activity P, Units ml 1 , is the ordinate axis. Experimental activity in Control 2005. Model calculation of the activity in Control 2005.

Model calculation

of the activity in Control 2010. Predicted activity in Experiment 1 according to Control 2010 results. It is assumed that specific rate of product destruction is equal to the specific destruction rate in Control 2010. Experimental activity in Experiment 1. Predicted activity in Experiment 1 according to Control 2010 results. It is assumed that product destruction does not occur.

It is assumed that product destruction does not occur. Experimental activity in Control 2010. 1758-5082/12/019601
It is assumed that product destruction does not occur. Experimental activity in Control 2010. 1758-5082/12/019601

Experimental activity in Control 2010.

does not occur. Experimental activity in Control 2010. 1758-5082/12/019601 + 01$33.00 1 © 2012 IOP

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Biofabrication 3 (2011) 045006 (12pp)

A cell population structuring model to estimate recombinant strain growth in a closed system for subsequent search of the mode to increase protein accumulation during protealysin producer cultivation

S P Klykov 1, 3 , V V Kurakov 1 , V B Vilkov 1 , I V Demidyuk 2 ,

T Yu Gromova 2 and D A Skladnev 1

1 PHARM-REGION, Ltd, c.1, b.10, Baryshikha street, Moscow, 125222, Russia

2 Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq. 2, Moscow 123182, Russia

Received 14 December 2010 Accepted for publication 12 September 2011 Published 25 October 2011 Online at stacks.iop.org/BF/3/045006

Abstract In this paper we have proposed a new structured population growth model, further developing a model previously proposed by the authors. Based on this model, optimal growth characteristics of the recombinant strain Escherichia coli BL-21 (DE3) [pProPlnHis 6 ] were determined, which allowed us to increase the output of metalloproteinase by 300%. We have experimentally demonstrated the applicability of the new model to cell cultures with implanted plasmids and the potential practical use for an output increase of a wide variety of biosynthesis processes.

(Some figures in this article are in colour only in the electronic version)

Notation

LGP

GIP

S

X

OD

τ

P

dP/ dτ

Q = −dS/ dτ Q O 2

logarithmic growth phase; growth inhibition phase;

substrate concentration, g l 1 ;

biomass concentration, OD units or g l 1 ; unit of an optical density;

time, hour;

products-metalloproteinase, units P ml 1 ; absolute rate of product synthesis, units of P(ml h) 1 ; absolute rate of substrate consumption; oxygen mass exchange rate, mmole O 2 /(volume units per time units);

3 Author to whom any correspondence should be addressed.

J

stochiometric factor of energy substrate ( S) oxidation, Joule of S/mmoleO 2 ;

μ

specific growth rate of biomass X, (hour) 1 ;

q = Q/X or

q = (1/X) dP/ dτ specific rate of substrate utilization or product synthesis, units of S(units of X h) 1 or units of P(ml units of

X h) 1 ;

trophic coefficient, amount of energy substrate consumed for the synthesis of a biomass unit, Joule of S/ Joule of X or units of S/ units of X;

amount of energy substrate accumulated

in biomass X during cultivation on a synthetic medium, Joule of S/ Joule of

X or units of S/units of X;

a

f

Biofabrication 3 (2011) 045006

S P Klykov et al

energy maintenance coefficient, the rate of substrate consumption for maintaining viability of one biomass unit per a unit of time, Joule of S(Joule of X h) 1 or units of S(units of X h) 1 ; (1) Parameter describes a delay of the biomass growth rate; (2) specific rate of accumulation of stable cells, h 1 ; maximum biomass concentration, when all the energy generated during cultivation is consumed for cell viability maintenance; biomass concentration in the end of

exponential growth phase and beginning of growth inhibition phase; X st concentration of the biomass of zero age cells (stable), the content of resting cells, OD units or g l 1 ;

m

A = m/a

X p

X Lim

X div = X X st

concentration of proliferation biomass, OD units or g l 1 ; time of exponential growth phase termination, hour; concentration of stable cells at the end of exponential growth phase, OD units or g l 1 ;

ratio of X st to biomass X, relative content of stable cells in the biomass, synchronization degree, part of 1; initial biomass concentration in LGP corresponding the beginning of popu- lation structuring, OD units or g l 1 ; final biomass concentration, at which R = 1 (when energy consumption is limited);

k r div , k r st , k s div , k s st constants of metabolite and substrate biochemical reaction rates, [units of P (or S) / (ml units of biomass per one hour)];

R

X Lim st

τ Lim

X l

X nal

P Lim

P

0

or tivity at the end of LGP and beginning of GIP, units of P ml 1 ; metabolite concentration or activity in LGP, when biomass structuring occurs at X = X l , units of P ml 1 .

ac-

metabolite concentration

1. Introduction

Studies on microorganism growth and biosynthesis of metabolites have given rise to a great number of mathematical models describing the dynamics of biomass growth and nutrient substrate consumption. Many models of microorganism growth are based on J Monod’s growth model or its variants [ 1, 2 ]. The basic idea of these models consists in the restriction or limitation of the specific growth rate of a cell population either by concentrations of a limiting substrate or of a biosynthetic end-product. However, limited application areas and numerous exceptions are the usual drawbacks of such models that necessitate the construction of a new model.

2

The basic disadvantage of the J Monod model described in [2] is an ambiguity about the physical sense of its parameters (or even absence of this sense). The works cited above also describe the so-called logistic curves characterizing all phases of microorganism growth. However, the model data have no wide practical application in designing biotechnological production technologies yet. The Volterra model considers a variant of the logistic curve describing the phase when all nutrients are completely utilized and the cell population growth reaches the stationary phase and/ or the phase of cell destruction [2]. The fact that the models mentioned do not consider the factors influencing cell growth, for instance oxygen mass- exchange, is a common disadvantage [ 2]. A number of mathematical models are also available to describe product biosynthesis. Among these, the Leudeking– Piret model is one of the most well known [ 3]. One of the characteristic features of the Leudeking–Piret model is that factors either related or not related to cell growth contribute to the kinetics of metabolite production. For the cases when the studied substance is a final product of metabolism related to energy consumption, the first member of the Leudeking–Piret equation describes biomass cell growth, while the second one characterizes the amount of energy consumed for cell viability maintenance. However, if the target products are not related to biomass energy metabolism, the Leudeking–Piret equation is difficult to interpret. Time dependence of metabolite concentration in the batch process may be rather complicated. In the growth medium several substances, which then are exposed to further transformations, can be accumulated. In some cases, a complicated kinetics of metabolite production may indicate modifications in the mechanism of cell metabolism under the influence of growth condition changes. The kinetics of acetate, butyrate, acetone and butanol production by Clostridium acetobutilicum [ 2 ] serves as a good example of these processes. The production of metabolites can also be accompanied by their chemical transformations in the growth medium. Such complicated processes, thus, should require the inclusion of these reactions, if they are well studied, into a mathematical model. Undoubtedly, this would make the mathematical description more sophisticated. Currently, the models of biomass growth, substrate consumption and metabolite synthesis are, as a rule, subdivided into two groups: structured and unstructured models [ 2]. The structured model implies the availability of more than one component to describe the structure of a cell population and its viability. The unstructured model suggests that the cell population is homogeneous and only one component, for example, biomass X, is used for its characterization. Since unstructured models are rather simple, they are often used for research. In all of the above-mentioned models of cell growth, energy consumption and metabolite synthesis are calculated by different equations describing the amounts of consumed substrates and synthesized products. From our point of view, this approach is not correct, since both substrates and products

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S P Klykov et al

are substances, which undergo various transformations during fermentation, and thus should be described by one and the same equation. Free access of oxygen to cells during cultivation plays the key role in aerobic processes. Since oxygen deficiency slows cell growth down resulting in the reduction of aerobic microorganism biomass output and synthesis of the majority of metabolites, it is important to provide oxygen inflow so that the concentration of the dissolved oxygen in the substrate would not be lower than 10% of a complete oxygen saturation level. In [ 4], a biotechnological method providing optimum conditions for cell growth has been described. The method suggests that during cultivation microorganism cultures receive additional feeding from a balanced nutrient medium concentrate. The inflow rate and the amount of feeding concentrate exclude both the lack of nutritious substrates, and growth inhibition owing to high concentration of nutrient com- ponents. In this case, oxygen mass exchange rate in the culture remains constant and oxygen concentration tends to zero. The Pirt–Marr equation,

Q = −dS/dτ = adX/dτ + mX, (1)

proposed for the calculation of the energy substrate consumption rate, has been modified for the analysis of energy consumed by cells for growth and viability maintenance. This was done by expressing Q through the oxygen mass exchange rate as follows:

(2)

This yields the initial equation (3) for the unstructured mathematical model of cell growth limited by oxygen consumption. All the equation constituents are expressed in energy units:

JQ O 2 = adX/dt + mX. (3)

The solution of the basic equation ( 3 ) allows for the formulation of fundamental laws of culture growth under conditions of a limited oxygen supply rate. Unlike earlier existing conceptions, it is shown that a linear decrease in absolute growth rate of the biomass and hyperbolic reduction in the specific growth rate is a function of biomass concentration, and that the energy substrate consumption rate specified by oxygen mass exchange rate is constant. Methods for defining the parameters of the unstructured model proposed included growth efficiency and energy substrate consumption ( m, a and A = m/ a), which were not previously used in any practical way to estimate periodic culture growth. Parameter A describes a delay of the total biomass growth rate. Studies on the effect of Salmonella culture growth rate on cell survival under adverse external influences [5 ] showed that during GIP, if there is a lack of dissolved oxygen, the accumulation of stable cells occurs at a constant specific rate equal to that of the growth delay (A = m/a). The share of stable cells within the population is obviously equal to that of nonproliferating cells, which consume energy only for viability maintenance. Methods for the definition of parameters of the structured model describing substrate consumption and metabolite biosynthesis on the basis of

Q = JQ O 2 .

3

preliminary calculated parameters of the unstructured model were designed. Thus, the proposed structured model assumes that within a growing population there are two groups of cells essentially differing in their physiology. Group I represents newly generated (young) cells and group II contains cells being in a state of active proliferation. Although the cells of group I are often called ‘resting’ cells [ 1], in our opinion these are the cells of ‘zero age’ [ 2], i.e. the cells being in phase G 1 or in phase V as designated for eukaryotes and prokaryotes, respectively. Group I cells exhibit minimal physiological functions, and for each cell these functions are constant. A characteristic feature of the cells is that they consume energy substrates only for their viability maintenance . In [ 5] these cells are called ‘stable’. All these discussions give good theoretical basis for the dynamics of the structure formation of microbial populations limited by the lack of oxygen in terms of energy consumption and cell viability maintenance. An analysis of the experimental and literature data made it possible to propose a new structured model of cell population growth [ 68 ]. On the basis of the model, consumption of substrates used for cell construction and synthesis of metabolites in the cultures consisting of two groups of cells differing in energy consumption was described. The performed experiments and analysis of the literature data have proved that the proposed model is adequate for the description of a wide range of microorganisms (from obligate aerobes up to obligate anaerobes), synthesis of various products (antibiotics, organic acids, alcohol, poly- β -butyric acid, polysaccharides, etc) and consumption of substrates (glucose, nitrogen and phosphorus) [ 6, 7]. The present paper shows the feasibility of the structured and unstructured models for estimation of the growth of recombinant microorganism strains and expression of foreign proteins in them. An analytical solution of constitutive equations (1)–( 3) for the unstructured model of biomass growth is shown in [ 4]. Equations ( 1)–(6) presented in table 1 describe changes in the consumption of energy substrate S, growth of biomass X, absolute growth rate of a total biomass dX/ dτ and specific growth rate μ. In [ 8], it was shown that quantity of nonproliferating zero age cells consuming energy only for viability maintenance changes in direct proportion to the change of energy consumption for viability maintenance, i.e. adX st = dS st (3a). On the other hand, apparently dS st / dτ = mX st (3b) since cells of zero age (stable cells) consume an energy substrate only for viability maintenance. Superposition of the last two equations gives equation ( 28), table 1. It is also obvious that if m 0, then, according to equation ( 1), the rate of energy substrate consumption Q is proportional to the absolute biomass growth rate dX/ dτ , and exponential cell population growth is observed. For GIP, m = 0. Successful research in the field of recombinant products performed during the last 30 years has led to an increase of their manufacturing. Taking into account all of the above- mentioned, we have found it interesting to use the proposed

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S P Klykov et al

Table 1. Basic equations for GIP.

Function

Equation

##

12

 

3

Q

=

dS = f( X)

dτ

Q = a dX + mX Q = JQ O 2 JQ O 2 = adX /dτ + mX = mX p dS st = adX st

dτ

 

(1)

Q

= f (Q O 2 )

 

(2)

Q

= f ( X )

(3)

dS st = f (dX st )

(3a)

dS st /dτ = f ( X st )

dτ

   

(3b)

dX

f (X)

(4)

=

 

dS st /dτ = mX st dX = A(X p X)

dτ

μ

= dX × X = f (X)

dτ

1

μ = A X p 1

X

(5)

X

= f (τ)

X

= X p (X p X Lim ) exp[ A(τ τ Lim ) ]

 

(6)

× X st = f (τ)

dτ

X

st

dX

st

1

dX

st

dτ

× X

1

st = m

a

=

A

st

X st = X Lim exp[ A(τ τ Lim ) ]

 

(7)

(8)

R

R =

K

p ×

2

K

μ

A + A

μ

+ 2

(9)

(10)

R

=

= f (X)

f (μ)

R =

A 2 X

A 2 (X p

XX 2 )

 

K

 

K = dX

dτ

× dX st dτ

=

A 2 X Lim × ( X p X Lim )

st

 

(11)

τ final

τ final

τ final =

τ final =

1

A ln

X p X

X p X

A 1 ln X final

X

st

Lim

Lim

nal + τ Lim

+ τ Lim

(12)

(13)

X

final

X final = X p

2

+ 2 X

1

2

p 4X Lim (X p X Lim )

st

(14)

X

final

 

X final = X p +

A 1 A 2 X p 4K

2

 

(15)

2

 

μ final

μ final = A

nal 1

X

X

p

 

(16)

dS

dτ

= f (X st ,X div ) construction substrate consumption rate

dS = k

dτ

 

st

s

X st + k

div

s

X div

(17)

dP

dτ

= f (X st ,X div ) product synthesis rate

dτ

dP = k X st + k

st

p

div

p

X div

(18)

P

= f (X)

 
 

div

st

product concentration

P = P Lim + k

p

A

X p ln X p X Lim

X p X

(X X Lim ) 1 +

X

Lim

(19)

X

p

X

P

= f (X)

 
   

div

) +

 

p

   

X Lim

 

product concentration

+

1

P = P Lim + k

A

X p ln X p X Lim

X p X

(X

 

(20)

A (k st k div )X

st

Lim XX Lim

X p X

 
   

S = S Lim k

div

s

A

X p ln X p X Lim

X p X

(X X Lim )

 

S

= f (X) construction substrate concentration

 

(21)

1

A (k

st

s

k

div

s

)X

st

Lim XX Lim

X p X

 

q

= f(R)

 

q = k div + k st k div

R

 

(22)

X

st

Lim

X

st

Lim = 2 X

2

Lim X

p ( X p X Lim )

2

 

(23)

X

st

Lim

 

X

st Lim = ( X p X nal ) X nal / (X p X Lim )

(24)

models for the description and intensification of the growth of genetically modified strains and biosynthesis of recombinant metabolites. For testing the proposed model, E. coli strain BL-21 (DE3) [pProPlnHis 6 ] [ 9], a producer of protealysin (metalloproteinase belonging to the family of thermolysins) [ 10 12 ] was used. Enzymes of this group can serve as potential drugs; in addition, they have a considerable innovative potential for biotechnological application [13]. Currently, recombinant producers [14 19 ] are used for thermolysin-like proteases. In the proposed paper, an attempt to increase the efficiency of metalloproteinase expression by recombinant E. coli strain BL-21 (DE3) [pProPlnHis 6 ] owing to the increase of oxygen mass exchange rate in a reactor followed by a consequent correction of nutrient medium composition has been made. The correction of medium composition is required due to a significant increase of the rates of all metabolic

processes in the reactor. In this case, additional nutrients are required. This is also stipulated by the increase of energy used for maintaining the producer viability during biosynthesis of substances genetically foreign for a cell host. Thus, the objective of the proposed paper is to demonstrate novel structured and unstructured models of recombinant strain growth and metabolite biosynthesis with the purpose to intensify cultivation processes and to increase the target product output.

2. Materials and methods

2.1. Escherichia coli strain BL-21 (DE3) [pProPlnHis 6 ] a producer of protealysin

This was previously constructed on the base of E. coli strain BL-21 (DE3) widely used for recombinant protein production [ 9]. This strain exhibits lysogenic properties with respect to

4

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S P Klykov et al

prophage λ DE3 bearing prophage T7 RNA pol gene under the control of promotor lacUV5, IPTG, galactose and glucose being the inducers and repressors, respectively.

2.1.1. Total proteolytic activity. TPA was determined from the ability of the strain to hydrolyse of azocasein [ 10 ]. For this purpose 50 μl of the enzyme solution were added to 100 μl of 1% azocasein solution in 20 mM Tris-HCl (pH 8,0). The mixture was incubated at 37 S for 15 min. The reaction was ceased by adding 200 μl of 10% trichloracetic acid solution. The sediment was separated by centrifuging at 9500 g for

5 min. 50 μl of 5 M NaOH were added to 250 μl of the

supernatant. Light absorption was measured at 450 nm on a plate photometer. The amount of the enzyme increasing the light absorption by 1 OD units per 1 min was considered as 1 unit of the activity. Enzyme activity was determined after the destruction of the biomass and metalloprotease formation. For this purpose, the cells were centrifuged and then resuspended in 20 mM Tris-HCl, pH 8,0. The cells suspension at a concentration

of approximately 30 g l 1 was exposed to ultrasonication for

1.5–2 min 5–7 times and incubated at 4 S for 24–48 h.

2.2. Cultivation of Escherichia coli BL-21 (DE3) [pProPlnHis 6 ]

A standard scheme of culture preparation was used: producer-

collection culture on Petri dishes-inoculation flasks-reactor. The cultivation was performed in a reactor with a working volume of 5–5.5 l, in a liquid medium, containing (g l 1 ):

tripton—15; yeast extract—7.5; sodium chloride—1.0; potassium di-phosphate—5.0; potassium monophosphate— 2.5; glucose-3.0; ampicillin—0.1; antifoam—1.0 ml, at pH 7,0 ± 0,2. At this stage 2 control cultivations (Control 2005 and Control 2010) were performed. Experimental cultivation (experiment 1) was conducted at high oxygen inflow rate under stirring and broken glucose feeding according to pH and pO 2 detector signals. Flasks were inoculated with a small amount of inoculate taken with a microbiological loop from the surface of the bacterium layer in Petri dishes. Each of the five flasks containing 0.2 l of the inoculation material was used to inoculate the reactor containing 4–4.5 l of the nutrient medium with all the necessary additives. Stirrer rotations and the amount of oxygen consumed were adjusted automatically according to the signal of pO 2 detector.

Prior to inoculation, a small amount (2 ml l 1 ) of 50%

glucose solution was poured into the reactor. Before the initial amount of glucose had been consumed, pH was maintained

at 7,0 with 15% NaOH solution according to the call of the

pH detector. After the consumption of the initial amount of glucose, it was fed in low doses through a peristaltic pump according to the pH detector signal. The rate of pumping was adjusted to provide glucose feeding (dry weight) within the

range from 1 t o 3 g (l h) 1 . Alkaline feeding was stopped to avoid glucose overdose. IPTG (0.01 g l 1 ) was introduced into Control 2005 and Control 2010 reactors after microbial suspension had reached optical density equal to 2.5 and

2 OD units, respectively. The inductor was placed into

experiment 1 reactor at OD units equal to 7 OD units.

5

2.3. Description of a model for the estimation of Escherichia coli strain BL-21 (DE3) [pProPlnHis 6 ] growth and metalloproteinase biosynthesis

The basic equations of the proposed model are presented in table 1 .

2.3.1. Unstructured model equations. Equations ( 1)–(6),

except for (3a) and (3b), deduced in [ 5] represent basic equations of the unstructured model of microorganism growth.

The presented equations are used for

analysis of oxygen mass exchange in the cultural medium, k d ; analysis of physiological producer constants, m, a, A,

μ max , τ Lim , X Lim , X p .

Oxygen mass exchange can be measured by other well- known methods and selected for cultivation. If this parameter is unknown, it can be calculated using equations (2 ) and ( 3 ) of the unstructured model. Parameters of the unstructured model τ Lim , X Lim , X p depend on the selected oxygen mass exchange. All the parameters depend on nutrient medium composition and cultivation conditions. Then all the parameters of the unstructured model are used for calculating the parameters of the structured model and biosynthesis constants. Experimental biomass concentration parameters are used for the estimation of biomass growth according to the unstructured model. Sampling is performed at equal time intervals τ = τ i +1 τ i = const, at which biomass concentration changes, X τ = X τ + τ X τ . According to equation ( 3) for GIP the following is deduced:

(25)

X τ + τ = X τ + X

X τ + X = X p (X p X τ ) exp (A τ),

or

X τ + τ = X p (X p X τ ) exp (A τ)

X = X p X τ (X p X τ ) exp (A τ)

(26)

(27)

(28)

(29)

at τ = const (30) equation ( 29 ) represents a linear regression function of X τ

X = ( 1 exp (A τ))X p ( 1 exp (A τ))X τ .

(31)

In the point of intersection with the ordinate axis, at X = 0, equation ( 31) is converted as follows:

X = ( 1 exp (A τ))X p = 0 X, (32)

where 0 X is the point where the regression line crosses the ordinate axis (31). From this it follows that

1 X/X p = exp (A τ). (33)

X = (X p X τ )( 1 exp (A τ))

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S P Klykov et al

Expression (33) can be presented in the following form:

(34)

Similarly to formula (34) for A of logarithmic growth phase (LGP), equations for μ max can be deduced:

A = −[ln( 1 0 X/X p ) ] / τ.

X τ + τ = X τ + X = X τ exp max τ),

(35)

from which

X = [exp max τ) 1] X τ ,

(36)

and

μ max = [ Ln( 1 + M) ] / τ,

(37)

where M = [exp( μ max τ ) 1] is the inclination angle tangent of the straight line X = f ( X) for LGP. In the work given, μ max was calculated by a standard technique [ 8]. In this case, dependence of LGP natural

logarithms, X, on time τ was built and the tangent of the inclination angle of the obtained straight line was determined. These values were compared to the results obtained with equation ( 37 ). For the analysis of biomass growth, the following parameters are determined first: biomass concentration, X Lim , and time, τ Lim , corresponding to LGP termination and beginning of GIP; hypothetical maximum biomass concentration, X p , when all energy transformed by the system

is consumed for biomass viability maintenance; A = m/a is

the specific growth delay rate of the biomass during GIP.

2.3.2. Equations for the structured model. Equation (7)

was theoretically proved in [ 68 ]. This equation indicates that in any population limited in energy consumption, X st ,

a specific rate of the accumulation of nonproliferating cells

consumed energy only for viability maintenance is constant

and equal to A. This is due to the fact that the energy consumed for cell viability maintenance causes a total reduction in the growth rate of proliferating cells, X div , and, as a consequence, of the population growth rate as a whole. In this case, nonproliferating cell accumulation is directly proportional to concentration X st at any instant time. Equation (8) represents an integrated form of equation (7). This equation shows exponential character of X st accumulation

in time.

Equations (9)–(16) were deduced in [58 ]. Equations (9) and (10) represent a share of nonproliferating cells in the population. In [8], it was shown that R = X st /X also describes the degree of synchronization of

zero age cultures. All physiological functions of zero age cells are minimal, and cell resistance to adverse external influences

is maximal owing to temporarily inhibited metabolism. It is

shown [ 5, 7 , 8] that if growth of the biomass is limited only by

energy inflow, all proliferating cells are converted into stable ones after which further proliferation ceases. Thus, the whole amount of energy is consumed for cell viability maintenance.

It

is obvious that equality R = 1 is true for this case. Physical meaning of parameter K in equation (11) consists

in

the fact that with growth limitation strengthening (increase

the duration of growth phase), the effect from one and the same

work of biochemical mechanisms underlying cell processes

6

continuously decreases, i.e. the number of proliferating cells reduces over time. At the same time, there is an accelerated increase in the quantity of stable cells consumed energy only for viability maintenance (see equations (7) and (8)). The latter statement should probably be understood to reflect the increase of stable cell numbers due not only to the termination of early cell proliferation cycles, but also to the progressive failure of stable cells to proliferate; otherwise, the cells could start proliferation again in the absence of limits. Equations (12)–(16) describe the time of culture growth termination and specific growth rate at the moment, when the discussed unstructured model of cell growth and biosynthesis during GIP, as it is presented in table 1, does not work. In this case, other equations are required (see below). Equations (17), (18) and (19)–(21) were considered in [ 68 ]. The physiological processes occurring in proliferating and stable cells differ greatly and are diametrically opposed. Therefore, subdivision of population cells into proliferating ( X div ) and stable ( X st ) ones makes it possible to use equations (17), (18), and table 1 to describe the consumption rate of substrates utilized for cell construction and metabolite synthesis:

dP( or S)/dτ = k div

P,S X div + k P,S X st ·

st

We assume that metabolites are synthesized only by proliferating cells. Nonproliferating cells, as a rule, destroy these products. Therefore, signs of the constants for metabolite synthesis and degradation are opposite. The same should be stated for substrates utilized for cell construction. If stable cells do not influence synthesis of metabolites (and substrate utilization), i.e. k P,S = 0, then the synthesis is carried out by proliferating cells and can be described by the integrated equation (19). If both proliferating cells and zero age cells participate in the synthesis, then the accumulation of metabolites is described by equation (20). Similarly equation (21) is proposed for the consumption of substrates used for cell construction. An analysis of equations (17)–(21) shows that metabolite synthesis proceeds with rate constants, whose signs are opposite to each other if the influence of the stable cells on the product output is not equal to 0. This means that groups of proliferating and stable cells behave differently during product biosynthesis: the former group synthesizes metabolites, the latter destroys them. It was found out that if cell growth process is limited not only by energy consumption but also by any substrate participating in cell construction, then the increase of biomass yield may suddenly stop. Studies on the dynamics of biomass accumulation limited by substrates utilized for cell construction [ 8] showed that this parameter can be presented as

R nal = k div /(k div k st ). (38)

The required final value of R nal is expressed by an identical

equation, if it is necessary to terminate the biosynthesis process in order to avoid the destruction of metabolite products by

active cells when k

= 0. The situation, when maximum

st

st

P

of biomass accumulation does not coincide with that of metabolite accumulation, i.e. the latter occurs earlier than the

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S P Klykov et al

former, is actually rather common. For this case, equation (15) would have the following appearance:

X

p +

A 1 A 2 X

2

( 1/R nal ) 4K

p

 

.

 

2

(39)

Equations (12)–(14) and (16) in this case are transformed according to (39). In the studies of growing cultures, especially, of Pseudomonas and Yersinia pestis , by cytorefractometric methods [ 20 ], we have repeatedly noticed that a certain number of nonproliferating cells are always present in the exponential growth phase. As the cell growth reaches GIP, the number of such cells increases significantly. Equations (12)–(14) and (16) then can be transformed according to (39). In the given work, biosynthesis parameters were determined for cells in GIP. The calculated values were used to estimate the conformity of the data obtained for the cells in GIP. As is well known, biosynthesis of metabolites often occurs during GIP. In [8], the factors affecting cell population structuring during LGP were investigated. Equations describing metabolite accumulation in this growth phase are as follows:

(40)

(41)

and

X final =

X st = 2X l ( 1 X l /X),

R = 2X l ( 1/X X l /X 2 )

P = P 0 + (k div max )(X X l )

(42)

where

(43)

is the initial biomass concentration in LGP, corresponding to the beginning of cell population structuring.

+ 2[ (k st k div )/μ max ] X l [ln(X/X l ) + X l /X 1]

2

X l = X Lim /X p

2.4. Technique for the estimation of bacterium growth and metabolite biosynthesis

Parameters X theor for LGP were calculated according to standard exponential equation (1, 2): X theor = X 0 exp[ μ max τ ] ,where μ max and X 0 were determined from LnX experiment = f ( τ ) calculated from the data presented in figure 1. μ max was also calculated from equation (37 ) and compared with the parameter calculated according to the above equations. For this purpose, X for the cells in LGP was estimated according to experimental data X experiment from which corresponding values X were calculated for each of the specified sampling interval. For each previous value of the experimental biomass concentration change, X, i.e. X τ = X (τ + τ) X τ ., dependence X τ = f ( X τ ) was then built according to equation (36) and μ max was determined from ( 37 ) (see figure 2 ). Parameters X theor for GIP were calculated on the base of experimental data X experiment (figure 1), according to which values X for sampling within specified time τ , were obtained as a difference between previous and subsequent X, i.e. X τ = X (τ + τ) X τ . Then, dependence X τ = f ( X τ ) was drawn, and corresponding constants 0 X, X p , A were

7

Table 2. Estimated model parameters.

Control Control Experiment 2005 2010 2010

Parameter

 

X 0 ,[OD units]

0,20

0,65

1,1

μ max, [ 1,2], [h 1 ]

0,740 0,550

0,649

μ

max

,(37),[h 1 ]

0,821 0,438

0,622

 

0,330

0,381

0,395

0,75

4

7

4,5

4

3

4,33

13,33

24,5

1

1

1

1,224

4,210

7,90

0,21

1,68

2,86

A,[h 1 ] X Lim ,[OD units] τ Lim , [h] X p ,[OD units] τ ,[h] 0 X ,[OD units] X Lim St ,[OD units] K ,[(OD units) 2 h 2 ] P Lim ,[units ml 1 ]

k div ,[units (ml OD units h) 1 ] 0,0535

k st ,[units (ml OD units h) 1 ] X l ,[OD units] P 0 ,[units ml 1 ]

R nal = R forPmax (38)

0,0840 2,2792 7,7942

0,04

0,7

0,200

1,225

0,200

0

0,164 0,045

0,13

1,20

2,0

0

0,22

0,20

1

0,549

0,826

determined. 0 X is an intersection of the regression line (31) and ordinate axis; X p is an intersection of the regression line (31) and abscissa axis; A is calculated from formula (34) (see figure 2 ). The boundary point of two growth phases ( X Lim , τ Lim ) is determined from the intersection of straight lines (31) and (36) (see figure 2). Biosynthesis parameters are calculated from equations (10) and (22). For this purpose X Lim is pre- liminarily estimated by equation (23). Then the values of the biosynthesis specific rate are calculated. (q P ) τ is estimated from [ P/ τ ] τ = [ P (τ i+ τ) P τ ] / τ and ( q p ) τ

= (1/ X τ )[ P/ τ ] τ . On the basis of X p , X Lim , A, τ Lim

previously estimated from equation (8) dependence X theor = f ( τ ) is deduced (figure 1 ). X Lim calculated above makes it possible to obtain dependence R = f 1 ( τ ) = f 2 ( X). Then the

R τ (figure 3), at R =

0, cuts off a line segment equal to k P div on the ordinate axis.

Inclination angle tangent of this line is equal to k

P ,

straight line ( q P ) τ = k P div + k

st

st

st k div

P

P

st k div

P

from which k can be calculated. The integrated accumulation of metabolite P theor is estimated according to equation (19) or (20): if k = 0, then

equation (19) is used, if k

= 0, equations (20) and (21) are

suitable. Experimental results on E. coli strain BL-21 (DE3) [pProPlnHis 6 ], X, metalloproteinase P synthesis and the corresponding calculated parameters X theor and P theor for LGP were compared with the model values. The latter were obtained from the calculated parameters for GIP,

, and from μ max determined

preliminary for LGP. Equation ( 40 ) for calculating the number of stable cells was used. For the description of metabolite accumulation during LGP, equation ( 42), where P 0 is the product concentration at the moment, when biomass structuring supposed to occur during LGP, is used. Parameters P 0 and P Lim were calculated from the results of cell cultivation in fermentations Control 2005 and Control 2010 and are presented in table 2. As was noted above, often there is a situation when biomass growth stops growing while metabolite biosynthesis

X p , X Lim , X

st

P

st

P

P

st

st

Lim , A, k div

P

,

k

st

P

Biofabrication 3 (2011) 045006

S P Klykov et al

Biofabrication 3 (2011) 045006 S P Klykov et al Figure 1. Control 2005 cultivation. Experimental biomass,

Figure 1. Control 2005 cultivation. Experimental biomass, X , and biomass calculated according to unstructured model X theor , stable (nonproliferating) cells, X st , proliferating cells, X div . Growth time, hours, is the abscissa axis. X , OD units, is the ordinate axis.

is the abscissa axis. X , OD units, is the ordinate axis. X theor , X

X theor ,

X st ,

X div .

1,2

1,1

1,0

0,9

0,8

0,7

0,6

0,5

0,4

0,3

0,2

0,1

0,0

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0

Figure 2. Control 2005 cultivation. Determination of unstructured model parameters, A, μ max, τ Lim , X Lim , X p , 0 X . Parameter values are

presented in table 2. X , OD units, is the abscissa axis. X , OD units /hour, is the ordinate axis.

axis. X , OD units / hour, is the ordinate axis. Experimental data for LGP, Linearization

Experimental data for LGP,

/ hour, is the ordinate axis. Experimental data for LGP, Linearization for GIP, Linearization for LGP,

Linearization for GIP, Linearization for LGP,

Calculation data for GIP. X LGP = 1,273X ; X GIP = −0,2814X + 1,224.

continues for some time. It was found that the absolute rate of product synthesis can be described also by equation (18). However, if no changes of R occur, then X, X st and X div remain constant. The integrated accumulation of biosynthesis metabolites in this case can be expressed by the following equation:

P = P finalGIP + k div X

div finalGIP (τ τ finalGIP )

(44)

where P nalGIP is the final concentration of the product, which

is determined from equation (20), table 1 , X

are the corresponding final concentrations that can either be calculated using equations (6), (8), (38) and ( 39 ) or determined experimentally.

+ k st X

st nalGIP τ nalGIP ),

div

finalGIP and X finalGIP

st

8

3. Results and discussion

The possibility to increase the output of the target product synthesized by E. coli strain BL-21 (DE3) [pProPlnHis 6 ] is determined by the presence of the following genes:

(1) ompT responsible for the inhibition of protease activity for product output increase. (2) lon responsible for the inhibition of intracellular ATP- dependent protease for product output increase. (3) lacUV5 being a promoter repressed by glucose.

Gene 1 and 2 determine the selection of nitrogen component of the nutrient medium, acid casein hydrolysate. Amino acids

Biofabrication 3 (2011) 045006

S P Klykov et al

0,15

0,10

0,05

0,00

-0,05

-0,10

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0

Figure 3. Control 2005 cultivation. Determination of parameters k div and k st according to the structured model. Parameter values are

presented in table 2. R , portion 1, is the abscissa axis. q, units P /(ml OD hour), is the ordinate axis. Linearization for LGP. q = –0,0476R + 0.0535.

axis. Linearization for LGP. q = –0,0476 R + 0.0535. Experimental data for GIP, are the

Experimental data for GIP,

are the source of nitrogen in the caseine hydrolysate. lacUV5 determines the mode of glucose feeding of the producer with induced product expression. It is efficient to introduce glucose into the nutrient medium at small doses (1–3 g (1 h) 1 ) in a form of 50% solution according to the pH detector signal. Such mode of glucose feeding allows for the producer to remain in

a stated of glucose limitation. In this state, glucose does not induce lac-operon repression. Experimental results obtained during the cultivation of producer X, metalloproteinase P biosynthesis and the corresponding parameters X theor and P theor are presented in figures 14 . Parameters of producer biomass and metalloproteinase changes calculated according to the model are also depicted in the figures. The parameters of growth and biosynthesis shown in table 2 were calculated from experimental data X (figure 1 ) and P (figure 4 ), according to the technique described above. A mode of calculating the parameters presented in table 2

is shown in figures 2 and 3 , the Control 2005 experiment being

taken as an example. Parameters of the other processes were calculated by the similar mode. The producer growth estimation was performed from a limited number of points (from 4 to 6). This provides the limited accuracy of the related analyses possible for these cases. For LGP, calculation of μ max by two well-known modes showed practically similar values (table 2 ). However, even at the accuracy given, the description of growth and biosynthesis both during GIP and LGP has appeared comprehensible to sum up the results allowing for forecasting metalloproteinase biosynthesis processes. Nutrient medium composition and cultivation conditions have been respectively corrected. Enzyme activity in the intermediate samples of all three experiments is depicted in figure 4. The area of gray color in

the center of figure 4 represents an expectation value of the enzyme activity for experiment 1 performed at high oxygen mass exchange rate and increased content of nutrients. The upper curve limiting this area is built using a product synthesis constant corresponding to the Control 2010 process on the assumption that product degradation does not take place. The rest of the parameters correspond to the characteristics of the experiment 1 process. The lower curve differs from the upper one because degradation constant k st equal to the value determined for the Control 2010 process is taken into consideration. The enzyme activity values in the control experiments, Control 2005 and Control 2010, were obtained when cultivation was performed at a low oxygen exchange rate, and the decreased content of nutrients is depicted in figure 4. These values were used as the basis for modeling experiment 1 processes. As is seen from figure 4, the data for experiment 1 satisfactorily fall into the area marked in gray. This correspondence has been forecasted. Results of mathematical description of three points of experiment 1 were obtained by a trial and error method using the constants of product synthesis and destruction (see table 2 ) at the minimized root- mean-square deviation. Definition by the method described in section 2.4 is unsuitable for the given case, since quantity of points for the analysis (three points) is not enough. In order to receive a comprehensible result by the method proposed, a minimal quantity of the points should be no less than 4. For the Control 2005 process, product destruction constant is equal to 0. For the Control 2010 process, product destruction constant, k st , makes about half of the product synthesis constant, k div . This means that more intensive growth

9

Biofabrication 3 (2011) 045006

S P Klykov et al

Biofabrication 3 (2011) 045006 S P Klykov et al Figure 4. Dependence of enzyme activity on

Figure 4. Dependence of enzyme activity on the growth time. Growth time, hours, is the abscissa. Activity P , units ml 1 , is the ordinate

axis. Experimental activity in Control 2005.

2010.

It is assumed that specific rate of product destruction is equal to the specific destruction rate in Control 2010. Experimental activity in

destruction rate in Control 2010. Experimental activity in Model calculation of the acvitity in Control 2005.

Model calculation of the acvitity in Control 2005. Experimental activity in Control

acvitity in Control 2005. Experimental activity in Control Predicted activity in Experiment 1 according to Control

Predicted activity in Experiment 1 according to Control 2010 results.

activity in Experiment 1 according to Control 2010 results. Model calculation of the activity in Control

Model calculation of the activity in Control 2010.

Experiment 1.

Predicted activity in Experiment 1 according to Control 2010 results. It is assumed that product destruction does not

occur.

( a ) (b ) ( c )
( a )
(b )
( c )

Figure 5. ( a) The change of decimal logarithm of the concentration of mammalian cell HUVEC-population corresponding to the experimental and model data (according to the proposed model); ( b) experimental data on the quantity of cells of different age cycle; ( c) model data (according to the proposed model) on the quantity of cells of different age cycle. Reproduced by permission [ 25], coordinated by Dr J Tyson.

of the producer in Control 2010 has not been provided with the required additional amount of nutrients that has caused degradation of target protein being a potential source of carbon and nitrogen for cells. During experiment 1, a successful attempt to prevent the undesirable degradation of the protein by increasing the content of nutrients in the initial medium and duly addition of glucose as an energy source has been undertaken. Thus, the product destruction constant, k st , was lowered approximately four-fold. Knowing the amount of energy inflow to the system, which can be calculated from equations (1)–(3), using the dynamics of glucose solution inflow expressed in terms of heat of combustion and Xp expressed in the units of biomass

heat combustion, it is possible to calculate m(3) and a values easily. If the oxygen mass exchange rate has not been preliminarily determined by any other way, it is possible to calculate this parameter using equations ( 2) and ( 3). Equations ( 1)–(6) for the estimation of total biomass are used to be applied within the unstructured model of cell population growth and substrate consumption. In our paper, we pioneered the use of equations (7) and (8) for the structured model with the purpose of obtaining a combined solution of equations (17), (18) and (22) (also shown for the first time), in a form of equations (19)–(21). The pioneer solution of equation (22) had been confirmed with international patent PCT [21 ].

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Biofabrication 3 (2011) 045006

S P Klykov et al

One of the reviewers’ questions is as follows: Why do we use the biosynthesis of a recombinant strain to demonstrate our model? The point is that biosynthesis processes, which are described in the above-stated international Patent and the Patent of the Russian Federation, are ordinary biosynthetic

processes carried out by usual ‘not recombinant’ strains of bacteria, yeasts and fungi. Within the last 30 years, the role of recombinant strains has sharply increased in science and industry. Indeed, the recombinant strains are mainly used now. This circumstance has determined the choice of the research object. The experimental and model data were analyzed using f -tests included in the Excel Program [22 24 ]. For this purpose, experimental data for Control 2005 and Control 2010 were divided by the corresponding data, calculated according to the model for Control 2005 and Control 2010. If the model adequately describes the experiment, the obtained average value of the ratio of the experimental and model data (their quotients) will be randomly grouped near 1. Indeed, according to the f -test for two experiments carried out by

a ‘conventional’ method (Control 2005, Control 2010), the

ratio of the average value is equal to 0.96 ± 0.42, confidence probability being 95%. Thus, the Fisher criterions is equal to 1.644, 3.695 and 2.248 for various combinations of the number sequences for the analysis that is less than the critical criterion equal to 5.05,

the discretion range number being equal to 5. Hence, the model is adequate for the experimental data. For the model and experimental data of experiment 1,

a similar analysis was made using two-selective f -tests at

various dispersions as well. For this purpose, experimental

data for experiment 1 were divided by the corresponding data calculated according to the model for experiment 1 and were compared with the number sequence of the ratio of the average values of the experiment / model for Control 2005 and Control 2010. The Fisher criterion is equal to 7.37, that is less than the critical criterion equal to 19.3, the discretion range number being equal to 2. The ratio of the average value is equal to 0.99 ± 0.15, the confidence probability being 95%. Thus, there is no reason to consider the observed difference between the number sequences statistically significant. This provides evidence to the fact that both the values belong to one and the same general data set and that the model

is adequate.

Experimental confirmation of the conclusions of our theory was published in February 2011 by the American researchers Dr John J Tyson et al [ 25]; it is shown in the last drawing in this work and figure 5 of this paper. Figure 5 fully

confirms the predictions obtained by our model for changes in the proportion of non-dividing (resting) cells (see equation for R). It is clear from the graph, which is designated by letter ‘B’

in the figure, with our proposed model describing the authors’

data better than the model offered by the authors themselves.

The fundamental equation for the unstructured model is the Marr–Perth equation (1) for the phase of slower growth in the integral form (6). It can be easily obtained if the relevant work is done with equations (1) and (4) under our methodology.

The basic equations for the non-structured model are the following: (7) integral equation for X, expressed by equation (6), and equation (17) and/ or (18). All other equations are auxiliary, derived from the basic equations, and are applied depending on the objectives set by a researcher or an engineer. Moreover, from equations ( 1), (4), (6), (7) a general equation was obtained:

d n X div

n = K

A

2 (1)

(n1) n!

d(X st )

(X st ) (n+1)

C,

(45)

where n are the whole numbers, order of the derivative of a function. Moreover, C = 1 if n = 1 and C = 0 if n 2. The factor ‘C’ has the following physical sense: in GIP each stable cell can become dividing again 1 time only, i.e. C ( n=1) = 1; each such subsequent transition for each

separate line of cell is impossible, i.e. C ( n=2,

) = 0. This

expression is a generalizing equation for any component of the biomass and for total biomass and, thus, unifies structured and unstructured models. To describe the biomass growth for any proposed model, the major differential equation is (45). To describe the cycle of the main substances a major differential equation is (17) and/ or (18).

These equations describe all the known diversity of the processes with S-like growth curves and changes in the concentrations of substances in closed systems, which is an entirely new and previously unknown fact. It is known that a physical law means a generalization of a numerical relationship between the objects of the real physical world that is running under specified conditions for the class of the objects and does not follow from any of the previously discovered laws. There is no reason not to admit the two described equations for the GIP as laws for GIP. The data obtained were used for the selection of techniques to increase the protein expression by genetically modified microorganisms.

4. Conclusions

(1) Growth characteristics of the recombinant strain and metalloproteinase biosynthesis were calculated on the basis of structured and unstructured population models. The results obtained allowed for the selection of cultivation conditions providing the increase of target product output in order to perform one more series of the experiment to design new more intensive cultivation regimens; (2) It was shown that the producer cultivated in the presence of different nitrogen sources and at different modes of glucose feeding exhibits different growth and biosynthetic properties. Also, it was demonstrated that metalloproteinase shows different biosynthetic properties during cultivation on nutrient media of different compositions. (3) Preferable substrates and cultivation regimens were selected to optimize growth properties of the producer and to increase the target product (metalloproteinase) output.

11

Biofabrication 3 (2011) 045006

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Acknowledgments

This work was supported in part by the Russian Foundation for Basic Research (project no 09-04-00734). The authors cordially thank Dr V V Derbyshev for valuable advice given during the designing of the unstructured model and Dr V A Samoilenko and Ms T V Yakshina for assistance in experiment performance.

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