Assessing post-induction cellular response in a recombinant E. coli lactose inducible system by monitoring β-galactosidase levels; A paper written by me and Jeff Lewis

1. BIOTECHNOLOGY LETTERS
Volume 17 No.lO (0ct.1995) p.1025-103~
Received as revised 18th August
ASSESSING POST-INDUCTION CELLULAR RESPONSE IN A
RECOMBINANT E. COLI LACTOSE-INDUCIBLE SYSTEM BY MONITORING
~-GALACTOSIDASE LEVELS
Clark E. Hartsock I111,Jeffrey K. Lewis2, Ian Leslie/, Joseph A. Pope Jr./ ,
Larry B. Tsai,2 Raj Sachdev1, and Shi-Yuan Meng.2
/Fermentation Process Development
2DepartmentofMicrobiologyandAppl~dMicrobialGenetics
Amgen Inc.
Thousand Oaks, CA 91320-1789
SUMMARY
A synthetic lactose-inducible promoter was chosen to study host cell responses to the
over-expression of heterologous genes. Fermentations were conducted to compare the
effect of induction strategies on the synthesis of 13-galactosidase versus the production of
recombinant protein. The levels of lactose, IPTG and glucose during induction were
manipulated to adjust the utilization of lactose as the inducer and/or the carbon source. In
addition, the involvement of the gal operon in lactose metabolism was also explored in
order to optimize lactose transport and utilization during induction.
INTRODUCTION
There have been numerous successful cases of using E. coli to produce
heterologous proteins; the high yields, at grams per liter of fermentation broth, provide an
economical advantage over other expression systems. As more peptide therapeutics are
produced in E. coli, it is important to understand the process of diverting resources to
heterologous protein synthesis inside the cell. Information collected from these kinds of
studies will be useful in improving expression levels and cell growth. How well the cell
is adapted to an inducing condition is generally considered a prime factor in determining
its productivity. This implies that different induction strategies are expected to elicit
different cellular responses. Commonly used induction strategies in the production of
heterologous proteins involve the control of fermentation parameters and/or medium
components (Brosius 1988; Denhardt and Colasanti 1988). A system that minimizes the
effect of induction conditions is therefore more favorable for studying cellular response to
over-expression of heterologous genes.
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2. In this study we used a system that comprises an expression vector carrying a
recombinant gene under the control of a synthetic lactose-inducible promoter and a host
cell whose chromosome, in addition to the wild type lacl gene, carries a copy of the/ac/q
gene. Important features of this system include: (1) the addition of lactose or isopropyl-13-
D-thiogalactoside (IPTG)'to the fermentation medium induces both the plasmid-carried
promoter (Plac-p)for heterologous gene expression and the host cell chromosomal wild
type lac operon promoter (Plac-c); (2) Plac-p is a much stronger promoter than Plac-c
and it is not subject to catabolite repression; (3) the influence of chromosomal lacY
activity on plasmid promoter transcription can be negated by Using IPTG to by-pass the
requirement of the lactose permease; (4) a Iaclq gene is integrated into the host cell
chromosome for tighter regulation of the Plac-pon a multicopy plasmid; (5) the effects of
over-production of a recombinant protein on the expression of the lac operon can be
monitored by measuring lacZ expression with the ~-galactosidase ~-Gal) assay.
Comparison of I~-Gallevels between strains with or without a product gene on the
plasmid can serve as a model to study cellular response to high level heterologous protein
production.
MATERIALS AND METHODS
Bacterial strain and expression vector. The E. coli K12 strain FM15, derived from
W1485 (F-, prototrophic), carries an insertion of laclqgene in its chromosome to provide
sufficient promoter repression on a multicopy plasmid. The expression vector is a
kanamycin resistant plasmid with multiple cloning sites downstream of a synthetic
lactose-inducible promoter and followed by a transcription terminator sequence. The
product genes #1, #2 or #3 have been individually inserted between the P/ac-p promoter
and the transcription terminator. The plasmid without the insertion of any product gene
serves as a control. Promoter Plac-pcontains features of a strong promoter recognized by
sigma 70 RNA polymerase and a lacO operator sequence for regulation.
Fermentation. Fed-batch fermentations were grown to an OD600 of about 55. Induction
was then initiated by starting a lactose feed either with or without glucose, or by adding
IPTG with a glucose feed. The specific lactose feed rate was kept constant regardless of
induction strategy. Dissolved oxygen was maintained at or above 50% of saturation. The
pH was controlled at 7.0. Temperature was kept constant throughout the fermentations.
Each induction condition was performed in triplicate.
Glucose and lactose assay: Glucose and lactose assays were performed on dedicated
YSI model 2700 Select analyzers (Yellow Springs Instrument Co., Inc., Yellow Springs,
OH).
B-galactosidaseassay: Assay ofB-galactosidase was pefformed according toMiller,
1972.
1026

3. Protein gel analysis. SDS-polyacrylamidegel electrophoresis was performed using 3-
27% gradient gels (Integrated Separation System, Natick, MA). The expression level was
quantified by densitometry using Bioimage Scanner with Visage software (Millipore,
Ann Arbor, MI).
RESULTS AND DISCUSSION
Four induction conditions were tested to evaluate the cellular responses to product
gene #1 expression: (1) use of lactose as inducer and the sole carbon source in the
medium; (2) use of IPTG as inducer with glucose as the carbon source; (3) use of lactose
as inducer (and carbon source) together with glucose as the carbon source; (4) addition of
galactose 30 minutes before induction with lactose. These fermentations were designed
to encompass various induction methods for lac-derived promoter/repressor systems.
Furthermore, to examine if the results would be affected by the nature of the product
protein and the level of product expression, the effects of two other product genes (#2 and
#3) on host cell lacZ expression were compared with product gene #1. Fermentations
under each of these conditions were also performed with an isogenic strain carrying the
expression vector without a product gene.
The effect of product gene #1 expression on the host cell lacZ gene expression. The
expression of lacZ was monitored by measuring the amount of 13-Galactivity. The 13-Gal
activity is significantly lower (3 to 10 fold difference based on induction strategy) in the
strain with product gene #1 than the control strain (Figure 1). This provides evidence that
normal lac operon expression was impeded because of the diversion of cellular resources
A 0-Gal Units B
~..o..=~ 6000 1,,,Z~ ~- 5000
i / k 4000
/2r----.-~ ~- 3000
-~ ..... 0 ..... 0... ~- 2000
........ 7' ooo
P , , , , i 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (hrs) Time (hrs)
r'1 Lactose ........O........ Glucose + Lactose
.... O.... Galactose Shot + Lactose .... ~,.... IPTG
Figure 1. Comparison of post-induction 0-Gal levels of strains with or without product gene #1
expression under various induction conditions. (A) Strain without product gene expression. (B)
Strain with product gene #1 expression.
1027

4. to synthesize product #1. The [3-Gal activities of cells with product gene #1 expression
were all lower than 600 units regardless of the induction strategy used (Figure 1B). IPTG
was found to be the most effective inducer for the lac operon in cells that do not express
product gene (Figure 1A). This may reflect the fact that protein synthesis is an integral
function of the cell; IPTG may well be a stronger inducer than lactose in regard to Plac-c
activation, but for an over-burdened cell, there may not be enough resources to carry out
normal 13-galactosidase synthesis. In other words, the major limiting factor for host cell
gene expression during heterologous gene induction is likely to be a lack of resources.
Investigations into cellular responses to this condition are continuing. Although the lac
operon is subject to catabolite repression, the glucose utilization was very efficient and
the glucose concentration in the medium was kept lower than 0.03 g/L (detector baseline)
throughout the runs. With such low glucose level conditions the effect of catabolite
repression was probably negligible (Figure 1A).
Lactose transport and utilization were affected in the strain with product #1
synthesis. An accumulation of lactose in the fermentation medium was observed when
the host cells were synthesizing product #1. In contrast the control strain showed no such
accumulation (Figure 2A). This implies that during heterologous gene expression lactose
transport and utilization may be less efficient. In agreement with this idea, lacZ
expression is higher in a cell that is not expressing a product gene (Figure 1). The
amount of lactose accumulated in the medium peaked during the first two hours of
induction and gradually subsided to the background level during the following four hours.
4
3
o 2
.-J 1
¢..
~2
A
I I I I
2 4 6 8 10
Time (hrs)
Without Product
Lactose
0
[] ........O,.......
600
500 -
400 -
300 -
200 -
100--
0 2
B
~.,,O ¢
4 6 8 10
Time (hrs)
IPTG
Glucose + Lactose
.... O---- Galactose Shot + Lactose
Figure 2. (A) Comparison of post-induction medium lactose levels of strains with or without
product gene #1 expression under various induction conditions. (B) Comparison of post-induction
PrGal levels in strain with product gene #1 expression. (Expanded view of Figure 1B)
1028

5. Interestingly, the addition of galactose 30 minutes before lactose inductionprevented the
initial accumulation of lactose in the medium. For host cells expressing product gene #1,
lactose accumulated for at least the fast two hours of induction (Figure 2A). This
transient accumulation of lactose may be the result of competition for cellular resources
between the/ac operon and the product gene. After two hours the cells were able to
utilize the lactose more efficiently bringing the lactose levels down to levels comparable
to the control strain (Figure 2A). In addition, the instantaneous expression of the product
gene decreases after two hours (data not shown). These data suggest that with subsidence
of product gene expression more resources become available for lac expression. The
optical density profiles for both strains are similar (data not shown) implying that cell
density is not likely the cause of the different lactose profiles. Another observation
shown in Figure 2A is that the addition of glucose during induction did not repress the lac
operon (Figure 1); instead, it increased lactose utilization. However, this increased
lactose utilization in the presence of glucose could not be explained as the result of more
lacZ expression (Figure 2B).
Lactose accumulation can be adjusted by induction of the gal operon. Extra-cellular
lactose can be transported into the cytoplasm by at least three permease systems (Adhya,
1987), where it is broken down into glucose and galactose by 13-galactosidase. Glucose
goes through glycolysis and the metabolism of galactose is carried out by enzymes
encoded by the gal operon (Weickert and Adhya, 1993 ). Cytoplasmic accumulation of
lactose, glucose or galactose might have a feedback effect on lactose transport and
utilization. One way to increase lactose utilization is to induce the gal operon before
cellular resources are diverted to product synthesis. This was accomplished by the
addition of galactose 30 minutes before induction with lactose. The result of this strategy
was dramatic: no lactose accumulation was observed for galactose potentiated cells after
induction with lactose (Figure 2A), and the galactose potentiated cells had higher [~-Gal
activity (Figure 2B).
Strains expressing different product genes having different expression levels exhibit
similar [3-galactosidase profiles. To ascertain whether the effect on cellular 13-Gal
activity was product dependent we tested two additional product genes in the same
expression system. Based on the hypothesis that over-expression of the product gene
diverts cellular resources away from the expression of endogenous genes, one would
expect that the amount of product gene synthesized could be an indicator of resources left
for host cell gene expression. Therefore, the level of ~-Gal activity could be inversely
1029

6. proportional to the expression level of the product gene. However, when lactose
induction with glucose feed was tested for three different product genes, #1, #2 and #3,
as shown in Figure 3, the [~-Galactivities of all three strains with different product genes
were reduced to about the same level despite their difference (about 10 fold) in
expression levels. This suggests that beside resource diversion to heterologous gene
expression there are other factors contributing to lower host cell gene expression. For
example, the accumulation of proteins of different properties could have very different
effects on host cell physiology.
6000
5000-
•~ 4000-
3000-
2000-
&
1000-
. . . . . . . . . . . .
0 2 4
~me(hm)
Product #1 ---O--- Product #3
.... ~ .... Product#2 Wit~= Product
,I,
. . . . . 111
6 8
Figure 3. Comparison of post-induction 13-Gallevels of strains with different product gene
expressionversus strain without product gene expression under glucose plus lactose induction
condition.
The goal of designing expression systems and production protocols in an
industrial setting is to maximize the diversion of cellular resources to heterologous gene
expression while minimizing the effect on normal cellular metabolism. This study
demonstrates that expression of the lac operon (specifically lacZ) is affected by
heterologous gene expression. The next challenge will be understanding the mechanisms
involved in cellular resource allocation and their role in heterologous gene expression.
REFERENCES
Adhya, S. (1987): The galactose operon. In: Escherichia coli and Salmonella
typhimurium; cellular and molecular biology. F.C. Neidhardt, chief ed. pp. 1503-
1512, Washington, D.C: American Society for Microbiology,
Brosius, J. (1988). Biotechnology 10, 205-25.
Denhardt, D.T. and Colasanti, J. (1988). Biotechnology 10, 179-203.
Miller, J.H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
Weickert, M.J. and Adhya, S. (1993). Mol. Microbiol. 10, 245-51.
1030