Production of β-carotene and Acetate in Recombinant...

Biotechnology and Bioprocess Engineering 17: 1196-1204 (2012)

DOI 10.1007/s12257-012-0272-2

Production of β-carotene and Acetate in Recombinant Escherichia

coli with or without Mevalonate Pathway at Different Culture

Temperature or pH

Anh Do Quynh Nguyen, Seon-Won Kim, Sung Bae Kim, Yang-Gon Seo, In-Young Chung, Dae Hwan Kim, and

Chang-Joon Kim

Received: 24 April 2012 / Revised: 21 June 2012 / Accepted: 30 June 2012

© The Korean Society for Biotechnology and Bioengineering and Springer 2012

Abstract Natural β-carotene has received much attention

as consumers have become more health conscious. Its

production by various microorganisms including metabolically

engineered Escherichia coli or Saccharomyces cerevisiae

has been attempted. We successfully created a recombinant

E. coli with an engineered whole mevalonate pathway in

addition to β-carotene biosynthetic genes and evaluated the

engineered cells from the aspects of metabolic balance

between central metabolism and β-carotene production

by comparison with conventional β-carotene producing

recombinant E. coli (control) utilizing a native methyl-

erythritol phosphate (MEP) pathway using bioreactor

cultures generated at different temperatures or pHs. Better

production of β-carotene was obtained in E. coli cultured at

37oC than at 25oC. A two-fold higher titer and 2.9-fold

higher volumetric productivity were obtained in engineered

cells compared with control cells. Notably, a marginal amount

of acetate was produced in actively growing engineered

cells, whereas more than 8 g/L of acetate was produced in

control cells with reduced cell growth at 37oC. The data

indicated that the artificial operon of the whole mevalonate

pathway operated efficiently in redirecting acetyl-CoA into

isopentenyl pyrophosphate (IPP), thereby improving

production of β-carotene, whereas the native MEP pathway

did not convert a sufficient amount of pyruvate into IPP

due to endogenous feedback regulation. Engineered cells

also produced lycopene with a reduced amount of β-

carotene in weak alkaline cultures, consistent with the

inhibition of lycopene cyclase.

Keywords: recombinant Escherichia coli, engineered

whole mevalonate pathway, bioreactor culture, β-carotene,

acetate

1. Introduction

β-carotene functions as provitamin A and plays an important

role as a pharmaceutical, nutraceutical, animal feed additive,

compound in cosmetic formulations and food colorants [1].

Recently, natural β-carotene has received much attention

because a chemically synthesized version cannot meet the

increased consumer demand for health and well-being.

Traditionally, natural β-carotene is obtained by extraction

from plants or vegetables. However, the natural β-carotene

yield is extremely low because it is present only in trace

amounts and because its extraction recovery is low due to

the rigid cell-wall structure of plants and vegetables [2].

Furthermore, β-carotene production varies seasonally and

geographically. To overcome these yield problems, microbial

production of β-carotene has been tried using green alga

(Dunaliella salina), fungi (Blakeslea trispora and Rhodotorula

Anh Do Quynh Nguyen, Sung Bae Kim, Yang-Gon Seo, Chang-Joon Kim*

Department of Chemical & Biological Engineering and ERI, GyeongsangNational University, Jinju 660-701, KoreaTel: +82-55-772-1787; Fax: +82-55-772-1789E-mail: [email protected]

Seon-Won KimDivision of Applied Life Science (BK21), PMBBRC, GyeongsangNational University, Jinju 660-701, Korea

In-Young ChungDepartment of Electronic and Communications Engineering, KwangwoonUniversity, Seoul 139-701, Korea

Dae Hwan KimSchool of Electrical Engineering, Kookmin University, Seoul 136-702,Korea

RESEARCH PAPER

Production of β-carotene and Acetate in Recombinant Escherichia coli with or without Mevalonate Pathway at… 1197

glutinis), and metabolically engineered yeast (Saccharomyces

cerevisiae) and bacteria (Escherichia coli) [3-8]. Recently,

we successfully created a recombinant E. coli with an

engineered whole mevalonate (MVA) pathway in addition

to β-carotene biosynthetic genes. This recombinant

bacterium produced a higher amount of β-carotene than the

conventional β-carotene producing recombinant E. coli

utilizing only the native methyl-erythritol phosphate

(MEP) pathway [9]. Production was further enhanced by

optimizing the feeding medium in fed-batch cultures of the

engineered E. coli [10]. As will be subsequently described

in detail, a big difference between these novel and

conventional cells concerns the biosynthesis of isopentenyl

pyrophosphate (IPP), a key intermediate metabolite for β-

carotene biosynthesis. The former cells were engineered to

maximize the biosynthesis of IPP by the expressed foreign

mevalonate (MVA) pathway as well as the native MEP

pathway, whereas conventional E. coli utilizes only the

native MEP pathway. The engineered cells utilize acetyl-

CoA in addition to pyruvate and glyceraldehyde-3-phosphate

(G3P), whereas conventional cells utilize pyruvate and G3P

for IPP biosynthesis [11]. The aforementioned metabolites

are glycolytic intermediates that are also used for cell

growth. In particular, pyruvate is a key component that

serves as the major switching point between respiratory

and fermentative metabolism. Intracellular concentration of

pyruvate is elevated in actively growing E. coli in an

aerobic environment, in which carbon flux in the glycolytic

pathway exceeds the required level for the synthesis of

cellular constituents as the threshold growth rate is

approached. In turn, excess pyruvate promotes the formation

of acetate directly or via acetyl-CoA [11-14]. It is desirable

if excess pyruvate is utilized for IPP biosynthesis because

β-carotene production may be improved and acetate

formation may be minimized. However, it is questionable

whether the MEP pathway operates efficiently for the

utilization of pyruvate because it is under the feedback

regulated control in the native host [16]. On the other hand,

it is expected that expression of heterologous MVA pathway

genes results in the redirection of a large amount of excess

pyruvate to IPP via acetyl-CoA. However, excessive

draining of pyruvate may restrict the carbon flux or deplete

this resource necessary for cell growth. These metabolic

imbalances may lead to reduced cell growth and production

[17].

In this study, we aimed to evaluate the aforementioned

engineered E. coli concerning the metabolic balance

between central metabolism and β-carotene production

by comparison with conventional β-carotene producing

recombinant E. coli cells. Bioreactor cultures were generated

at growth-promoting or growth-retarding temperatures.

The engineered cells were also cultured at different culture

pH. Responses were examined by measuring cell growth

and acetate production as an indicator of change in the

central metabolism and β-carotene production. Cell growth,

acetate metabolism, and β-carotene production of these

cells showed different responses to culture temperature, in

which the engineered cells excreted a modest amount of

acetate without changed cell growth, even at the growth-

promoting temperature of 37oC. Notably, accumulation of

lycopene was observed in weak alkaline cultures, whereas

only β-carotene was produced at neutral or weak acidic

cultures. These physiological characteristics of the novel

cells have not been demonstrated previously.

2. Materials and Methods

2.1. Bacterial strains and culture conditions

Two kinds of β-carotene producing recombinant E. coli

cells were used. Control cells were E. coli DH5α containing

the plasmid pT-DHB, which harbors the genes for β-

carotene biosynthesis. These cells represent conventional

recombinant E. coli producing β-carotene. Plasmid pT-

DHB was constructed by cloning crtE, crtB, and crtI of

Pantoea agglomerans, ipiHP1 of Haematococcus pluvialis,

crtY of Pantoea ananatis, and dxs of E. coli into pTrc99A

(Amersham Bioscience, Piscataway, NJ, USA). Engineered

cells (strain 1) were novel to this study, which are E. coli

DH5α containing an additional plasmid pS-NA bearing

genes encoding the enzymes of the whole MVA pathway

beside plasmid pT-DHB. pS-NA was constructed by

cloning mvaE and mvaS from Enterococcus faecalis

ATCC14508; mvaK1, mvaK2, and mvaD from Streptococcus

pneumoniae; and idi from E. coli) into pSTV28 (TaKaRa

Bio, Shiga, Japan). The detailed procedures for construction

of the β-carotene producing cells are described elsewhere

[9]. The blank pSTV28 vector was also introduced into

control cells for comparative study.

2.2. Fermentation conditions

All flask cultures were performed in 500 mL baffled flasks

using a shaking incubator (Jeio Tech, Seoul, Republic of

Korea) at 200 rpm. Glycerol stocks of recombinant E. coli

were inoculated into 50 mL of Luria-Bertani (LB) broth

[18] supplemented with antibiotics (100 mg/L ampicillin,

50 mg/L chloramphenicol) and was grown at 37oC until

the optical density at 600 nm (OD600) reached 0.6. The

seed culture (2.5 mL) was then transferred to 50 mL of

2 × YT medium (16 g/L tryptone, 10 g/L yeast extract, and

5 g/L NaCl) supplemented with antibiotics (100 mg/L

ampicillin, 50 mg/L chloramphenicol), and 30 g/L glycerol

for the main culture.

For bioreactor cultures, 75 mL of seed culture was added

1198 Biotechnology and Bioprocess Engineering 17: 1196-1204 (2012)

to a 3.4 L fermentor (KoBiotech, Incheon, Korea) containing

1.5 L of 2 × YT medium supplemented with 30 g/L

glycerol as well as antibiotics. Cell growth and β-carotene

production were compared by performing bioreactor

fermentations at 37 and 25oC at neutral pH. To investigate

the sole pH effect, the pH was controlled to maintain at 6.8

± 0.1 (neutral pH), 5.9 ± 0.1 (weakly acidic pH), or 7.9 ±

0.1 (weakly alkaline pH) using 2 N H2SO4 and 25%

NH4OH. The dissolved oxygen level was maintained at

greater than 30% air saturation by the addition of pure

oxygen into the air stream and/or manually controlling the

agitation speed.

2.3. Analyses

The optical density at 600 nm (OD600) was measured by an

ultraviolet (UV)-Visible spectrophotometer (Hewlett-Packard,

Palo Alto, CA, USA) to monitor cell growth. The

corresponding dry-cell weight was determined by a

calibration curve relating OD600 to dry-cell weight (g/L).

The β-carotene content in the cells was also determined by

UV-Visible spectrophotometry; 1 mL of culture broth was

taken and appropriately diluted. An orange-colored cell pellet

was obtained by centrifugation at 9,800 × g for 10 min.

After washing once with distilled water, the cell pellet was

resuspended in 1 mL of acetone and incubated at 55oC for

15 min in the dark. During this process, all β-carotene

accumulated in cells was extracted into the acetone and the

resulting cell pellet obtained by centrifugation at 9,800 × g

for 10 min was nearly colorless. β-carotene in the acetone

extract was quantified by measuring the absorbance at

454 nm.

Analytical UV-Visible spectrophotometry is not capable

of determining β-carotene quantitatively in the presence of

lycopene due to interference [19,20]. Because both β-

carotene and lycopene were produced in strain1 cultured at

a weak alkaline pH, quantitative analysis of β-carotene or

lycopene in cell extracts was carried out using a high

performance liquid chromatography (HPLC) system consisting

of a model 515 HPLC pump and a model 486 UV detector

(Waters, Milford, MA, USA). Cell pellets were collected

and then disrupted prior to extraction. Isobutyl acetate was

used instead of acetone as extraction solvent for improved

extraction. The procedure was carried out as follows. Cell

pellets were collected by centrifugation at 9,800 × g for

10 min from 1 mL culture and washed once with 1 mL of

85% methanol. Cell pellets were resuspended in 0.5 mL of

a lysozyme solution (10 mg/mL) in an STE buffer (pH 8.0)

and then incubated at 37oC for 1 h. After centrifugation at

9,800 × g for 10 min, the supernatant was discarded and

the pellet was resuspended in 1.0 or 0.5 mL of isobutyl

acetate depending on the color of the pellet. β-carotene was

completely extracted into the isobutyl acetate. The isocratic

mobile phase of ethanol/methanol/tetrahydrofuran (75:20:5,

v/v/v) for β-carotene analysis or methanol/tert-butyl-metyl-

ether/water (42:54:4, v/v/v) for lycopene analysis was

used. A 20 µL filtered sample obtained using a DISMIC-

13HP 0.2 µm disposable syringe filter (ADVENTEC,

Tokyo, Japan) was injected into a YMC 30 column (250 mm

× 4.6 mm; YMC, Kyoto, Japan) and then eluted with the

mobile phase at 1.0 mL/min at room temperature. β-

carotene and lycopene were detected at a wavelength of

454 and 472 nm, respectively. Authentic β-carotene and

lycopene (Sigma-Aldrich, St. Louis, MO, USA) were used

as standards. Acetic acid and residual glycerol concentration

were determined using a commercial kit (Megazyme,

Wicklow, Ireland).

3. Results and Discussion

3.1. Biosynthesis of IPP and β-carotene in recombinant

E. coli with or without foreign MVA pathways

Essential genes for β-carotene biosynthesis (idi, ispA, crtE,

crtB, crtI, and crtY) as well as two native genes in the MEP

pathway of E. coli (dxs and dxr) were expressed in control

cells and strain 1. Additionally, the IPP biosynthetic pathway

was reconstructed by introducing the MVA pathway in

strain 1. Therefore, the difference between the two cell

types lay in the mechanics of IPP biosynthesis. The foreign

MVA pathway in E. coli can evade endogenous feedback

regulation, whereas the endogenous MEP pathway is under

feedback regulation control. Therefore, IPP biosynthesis

may be maximized in strain 1, whereas there is limited

biosynthesis of IPP in control cells. One of the rate-limiting

steps in the biosynthesis of carotenoids is the supply of IPP

[11,16]. Accordingly, it was anticipated that β-carotene

production would be higher in strain 1 than in control cells,

possibly due to the increased supply of IPP as the building

block for β-carotene production. The biosynthesis of IPP

and β-carotene is shown in Fig. 1 [11].

Acetate metabolism could also be potentially different

between the control and strain 1. The condensation reaction

between pyruvate and G3P leads to IPP and dimethyl allyl

pyrophosphate (DMAPP) in the MEP pathway. The MVA

pathway begins with the conversion of three molecules of

acetyl-CoA to MVA, ultimately leading to IPP. The starting

materials of MVA or MEP pathway are acetyl-CoA,

pyruvate, and G3P, which are intermediates of glycolysis.

Generally, a high growth rate increases the glycolytic flux

to pyruvate in wild-type E. coli cultures at the optimal

growth temperature of 37oC, which results in the imbalance

between glycolytic and tricarboxylic acid (TCA) flux,

resulting in the accumulation of copious amounts of acetate

[12]. Activation of the MEP pathway drains G3P and


pyruvate, and may reduce flux to acetic acid. Furthermore,

the carbon flux from acetyl-CoA to acetate can be

redirected to MVA by expressing the MVA pathway.

Therefore, it is reasonable to anticipate that the amount of

acetate generated by these cells would be lower than that

by wild type E. coli and the production of acetic acid in

strain 1 would be much lower than that in control cells.

Based on these theoretical expectations, the influence of

the metabolic characteristics on the production of β-carotene

and acetic acids at growth-promoting or growth-retarding

conditions was investigated by controlling temperature or

culture pH.

3.2. Preliminary flask culture experiments

Initially, flask cultures of strain 1 were generated to

investigate the effect of culture temperature on β-carotene

production and cell growth. Temperature of 37 and 25oC

was selected as the growth-promoting and growth-retarding

conditions, respectively. The culture pH rapidly dropped

from neutral to 4.5 after 27 h at 37oC. The same pattern of

pH decrease occurred at 25oC but at a slower speed (Data

not shown). Cell growth and β-carotene production stopped

concomitantly at pH <4.5 in both cultures. β-carotene

production appeared to be enhanced during culture at 25oC.

However, this result may have been influenced by the pH

of the culture. We hypothesized that the beneficial effect of

culture at 25oC was due to the slower rate of pH decrease

than at 37oC. Thus, cells had more time to grow and

accumulate β-carotene before succumbing to the acidic pH.

If so, eliminating the influence of pH was needed to

accurately examine the temperature effect on β-carotene

production and cell growth. However, it was impossible to

maintain the pH of these cultures at neutral, despite the

incorporation of several buffer components in flask

experiments. Generally, culture acidification is caused by

secretion of acetic acid in E. coli cultures [12-14]. Therefore,

acetic acid was measured in the next experiment.

3.3. Effect of culture temperature on the biosynthesis of

acetic acid and β-carotene

A bioreactor system can control the cultural temperature or

pH to maintain a suitable point. Thus, the individual effect

of these factors was investigated in bioreactor cultures.

Bioreactor cultures were performed to evaluate the performance

of strain 1 at two different culture temperatures by

maintaining the culture pH at neutral. Fig. 2 shows the

Fig. 1. Biosynthesis of β-carotene in engineered cells (strain 1).IPP, the precursor for the biosynthesis of β-carotene, is synthesizedvia the MEP and MVA pathways from G3P, pyruvate, and acetyl-CoA. Abbreviations are: G3P, glyceraldehyde-3-phosphate; DXP,1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IPP,isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate;GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP,geranyl geranyl diphosphate.

Fig. 2. Time profiles of cell growth, and production of β-caroteneand acetic acid during bioreactor cultures of strain 1 at 37oC (A)and 25oC (B) in 2 × YT medium supplemented with 30 g/Lglycerol. The culture pH was maintained at neutral.


profiles of cell growth and β-carotene production in

cultures of strain 1 at 37 and 25oC. β-carotene production

began when cell growth commenced and ceased after cell

growth stopped at 37oC, whereas the rate of β-carotene

production increased after the cell growth rate slowed after

24 h. Furthermore, production continued even after cell

growth had stopped at 25oC. As shown in Table 1, cells

grown at 37oC produced 119 mg/L of β-carotene after only

20 h, whereas 81 mg/L of β-carotene was produced for

42 h cultivation at 25oC. The result obtained at 37oC was

3.2-fold greater increase in volumetric productivity (Qp)

and a 1.4-fold higher increase in the specific growth rate

(µ) than that at 25oC. These results clearly indicated that β-

carotene production in strain 1 was favored in the actively

growing condition at a temperature of 37oC. This result

was contradictory to previous reports demonstrating a

temperature effect on the production of β-carotene by

recombinant E. coli, in which enhanced production of

carotenoids or β-carotene was obtained during slow growth

at 28oC, rather than active growth at 37oC in flask culture

[21] or bioreactor culture [4]. The main differences between

our system and those in the two previous studies were the

cell type and culture method. We evaluated cultures of

recombinant E. coli with additional MVA pathway (i.e.,

strain 1) in controlled bioreactor culture, whereas the

previous study utilized flask cultures of conventional β-

carotene producing recombinant E. coli utilizing native

MEP pathways only (our control cells). As described

above, we could not evaluate strain 1 in flask cultures at

the two different temperatures because acidification of

culturing medium occurred concurrently. Thus, we suspect

that the dichotomous results of the present and prior studies

are attributable to cell differences.

Bioreactor cultures of control cells were also performed

for comparison. As shown in Fig. 3, biphasic growth was

noted at 37oC, while the rates of glycerol consumption and

β-carotene production remained constant. Cultures generated

at 37oC were almost 2-fold greater in volumetric productivity

(Qp) than that at 25oC, while no significant differences in

the maximum cell growth and β-carotene titer were observed

at the two temperatures (Table 1). This result clearly

showed that 37oC was beneficial for cell growth and

production in the culture of control cells. The findings

supported the conclusion that β-carotene production in both

types of recombinant E. coli was favored during active

growth. Change in culture temperature also influenced the

acetate metabolism of control cells. An elevated amount of

acetate was produced at 37oC – 2 g/L at 6 h and increased

up to 8 g/L thereafter. Once glycerol had been consumed at

18 h, cells reutilized the excreted acetate as a complementary

carbon source. However, acetic acid production was not

notable at 25oC, perhaps due to the slower nutrient uptake

and growth rate. E. coli cells growing at 37oC produce a

high amount of acetic acid as an extracellular product, even

under fully aerobic conditions, because the cells surpass a

threshold-specific rate of nutrient consumption [12]. The

formation of acetate at this growth-promoting temperature

has been suggested to be caused by an imbalance between

glucose metabolism and respiration, a condition in which

the influx of carbon into cells exceeds the biosynthetic

Table 1. Cell growth and β-carotene production in engineered and control cells at different culture temperatures and pH

CellsTemp. (oC)

pHCulture time

(h)µ max

(/h)Xmax

(g/L)Pmax

(mg/L)Qp

(mg/L/h)Yp/x, max

(mg/g-cells)Y p/s

(mg/g-carbon sources)

Strain1 37 7 20 0.37 10 119 6.1 14.5 3.9

Strain1 25 7 42 0.26 9 81 1.9 8.7 2.7

Control 37 7 33 0.34 8 57 2.1 7.1 1.9

Control 25 7 47 0.29 9 47 1.1 5.3 1.6

Strain1 37 8 21 0.47 8 93 4.4 12.8 3.1

Strain1 37 6 24 0.34 12 112 6.2 9.3 3.8

Fig. 3. Time profiles of cell growth, and productions of β-carotene and acetic acid during the bioreactor cultures of controlcells at 37oC (A) and 25oC (B) in 2 × YT medium supplementedwith 30 g/L glycerol. The culture pH was maintained at neutral.


demands and the capacity for energy generation within the

cell; saturation of the TCA cycle and/or the electron

transport chain may be the main cause. At 25oC, the

nutrient uptake rate and oxygen demands are reduced,

thereby reducing the formation of acetic acid [12,22,23].

Acetate is undesirable because it is toxic and inhibits cell

growth, even at concentrations as low as 8 mM (0.5 g/L)

[24]. The toxic effects of acetic acid derive from several

possible processes due to proton or anion accumulation.

The non-dissociated form of acetate can move freely

through the cell membrane and accumulate in the medium.

Some of this extracellular, non-dissociated form re-enters

the cells and dissociates due to the higher pH within the

cytoplasm. Acetate thus acts as a proton conductor and the

process causes a reduction in proton motive force [25].

Furthermore, accumulation of excess free protons may

damage ribosomal RNA as well as DNA, and also

denatures essential enzymes [26]. Therefore, the presently-

observed decrease in the cell growth rate of the control

cells from 0.34 to 0.05/h at 37oC was very likely caused by

the toxicity of the accumulated acetate. The result also

indicated that the MEP pathway does not operate efficiently

and, thus, most pyruvate is directed to the glycolytic

pathways, thereby producing acetate in control cells.

Notably, a small and comparable amount of acetic acid

was produced by strain 1 cultured at 37 or 25oC (Fig. 2). It

was demonstrated previously that strain 1 can biosynthesize

a considerable amount of MVA [8]. The reduced production

of acetate in E. coli over-producing MVA by expression of

heterologous MVA pathway genes was previously reported

[27]. Together with these reports, we suggest that all MVA

pathway enzymes are well expressed and, thus, that the

carbon flux from acetyl-CoA to acetate may have been

redirected to MVA by the expressed enzymes in strain 1

(Fig. 1). If so, why did strain 1 produce a high amount of

acetate in flask cultures? Significant differences in cell

growth and acetate production were observed in flasks and

bioreactor cultures of strain 1. During bioreactor growth, all

of the glycerol, 30 g/L, was exhausted, while approximately

16 g/L glycerol remained in the flasks when growth ceased

at either temperature. Severe oxygen limitation occurs in

shake flasks at the biomass level well below the level

achieved in bioreactors due to the lack of aeration [28,29].

Therefore, prolific production of acetate may lead to the

denaturation of the enzymes for cell growth as well as

MVA pathways, thereby resulting in low production of β-

carotene in flask cultures.

3.4. Lycopen production by strain 1 under weak

alkaline condition

The performance of strain 1 was evaluated in bioreactor

cultures at 37oC using different pH. Maximum concentration

of β-carotene, acetic acid, and cells in culture under slightly

acidic condition (pH 6) was 112 mg/L, 0.3 g/L, and 12 g/

L, respectively, which were not appreciably different from

the corresponding values obtained during culture under the

neutral pH condition (pH 7) (Table 1). However, of note,

the culture broth appeared darker with a stronger orange

color at the weakly alkaline pH of 8 than at a neutral pH.

To clarify the reason for this change, colored compounds

were extracted from cells cultured at both pH conditions

and then UV-VIS spectrophotometric analyses were

performed. The extract from cells cultured at the neutral

pH demonstrated three broad absorptions at 430, 454, and

480 nm, which represented the typical absorption spectrum

of β-carotene [30]. In contrast, absorptions at 454, 472, and

504 nm were evident in extracts from cells cultured under

the weak alkaline condition (Fig. 4). The absorption peaks

at 472 and 504 nm are typical of lycopene [30]. The

metabolites present in these extracts were identified using

HPLC; Fig. 5 shows representative chromatograms of

standards and extracts. The authentic β-carotene and

lycopene eluted at 10 and 95 min, respectively. Extracts

from cells cultured under the weak alkaline condition

revealed two peaks at the retention time of β-carotene and

lycopene, whereas only one peak was observed at the

retention time of β-carotene peak in extracts from cells

cultured under a neutral condition. These results clearly

indicated that lycopene was produced in addition to β-

carotene during culture at weak alkaline pH, whereas

growth at neutral pH resulted in the production of only β-

carotene.

Fig. 6 shows the time profiles of cell growth and productions

of β-carotene and lycopene in cultures generated at the

weak alkaline pH. Cells grew with a concomitant increase

in the production of β-carotene. No further increases in cell

growth and β-carotene production were observed at 12 h,

even though 8.9 g/L of glycerol remained at this time.

Subsequently, lycopene production began and product

Fig. 4. UV/Visible spectra of extracts from strain 1 cultured atweak alkaline pH (A) and neutral pH (B) at 37oC in 2 × YTmedium supplemented with 30 g/L glycerol.


continued to increase until the glycerol was used up.

Notably, cells produced acetic acid from the early log

phase, reaching 4.5 g/L at 15 h, but cells were not able to

utilize acetate for biomass production in spite of the

exhaustion of glycerol at 18 h. This contrasted with the

result that this strain produced only a marginal amount of

acetic acid (<0.1 g/L) and the knowledge that E. coli

generally consumes acetic acid after exhaustion of the

available carbon source in neutral or weakly acidic cultures.

Alkaline pH induces deaminases such as tryptophan

deaminase (Tna A) and serine deaminase producing

pyruvic acids, which can be further degraded to acetic acid

[31,32]. Furthermore, growth of E. coli at external pHs

exceeding 7.8 generates an inverted pH gradient, which

drains the proton potential and effectively excludes acids

from cells [32,33]. This can fully explain the reason why

strain 1 could produce a high amount of acetic acid with no

consumption under the weak alkaline condition. In this

environment, the maximum cell concentration was 7.6 g/L,

which was lower than that obtained in cultures generated at

neutral or weak acidic pH. This provided further evidence

that cells can direct their overall metabolism into pathways

that channel metabolites towards acetic acid instead of

building biomass under weak alkaline condition.

At weak alkaline culture, the obtained total concentration

of carotenoids including β-carotene and lycopene was

150 µM, which corresponded to 68% of the β-carotene

titer (222 µM) obtained in neutral culture. This indicated

that carotenoid production decreased when cells were

cultured at the weak alkaline condition. It is obvious that

alkaline pH stimulated lycopene accumulation, as has been

reported previously [34]. This was probably due to the

decreased activity of lycopene cyclase, which catalyzes the

reaction transferring lycopene into β-carotene. The decreased

enzyme activity would retard the cyclization reaction,

leading to the accumulation of lycopene.

Fig. 5. HPLC chromatograms of extracts from strain 1 cultured at neutral or alkaline pH. (A) Standard lycopene (20 mg/mL); (B)standard β-carotene (60 µg/mL); (C) cell extract from neutral culture; (D) cell extract from weak alkaline culture; (E) mixture containingstandard lycopene and cell extract from weak alkaline culture.


4. Conclusion

We evaluated a recombinant E. coli with an engineered

whole MVA pathway in addition to β-carotene biosynthetic

genes with regard to a metabolic balance between central

metabolism and β-carotene production in bioreactor cultures.

The performance of these novel cells was compared with

that of conventional β-carotene producing recombinant

E. coli. A small amount of acetic acid was produced with

no change in cell growth, even during active growth. In

contrast to this unique characteristic of the engineered cells,

control cells showed the same characteristic of central

metabolism as wild-type E. coli. The highest amount of

β-carotene, 119 mg/L, was obtained in engineered cells

cultured at 37oC, which was 2-folds higher than the

production by control cells, which also corresponded to a

2.9-fold greater increase in volumetric productivity.

The present findings are consistent with the conclusion

that the MVA pathway operates efficiently, redirecting

acetyl-CoA into IPP and thereby producing β-carotene,

whereas the native MEP pathway does not efficiently

convert pyruvate to IPP due to endogenous feedback

regulation. Furthermore, the data supports the view that the

carbon flux between cell growth and β-carotene production

are well-balanced in the engineered cells. The culture

pH was also important for maximizing the production of

β-carotene because engineered cells produced only β-

carotene at neutral pH, whereas some of the intermediate,

lycopene, could not be converted into β-carotene at weak

alkaline pH.

Acknowledgements

This work was supported by Pioneering Research Center

for Nano-morphic Biological Energy Conversion and Storage,

a Korea Science and Engineering Foundation (KOSEF)

grant funded by the Korea government (MEST) (R01-

2008-000-20835-0), and partially supported by the second

stage of the Brain Korea 21 project in 2011.

References

1. Burri, B. J. (1997) Beta-carotene and human health: A review ofcurrent research. Neutri. Res. 17: 547-580.

2. Jaramillo-Flores, M. E., J. J. Lugo-Martinez, E. Ramirez-San-juan, H. Montellano-Rosales, L. Dorantes-Alvarez, and H. Her-nandez-Sanchez (2005) Effect of sodium chloride, acetic acid,and enzymes on carotene extraction in carrots (Daccus carotaL.). J. Food Sci. 70: 136-142.

3. Garcia-Gonzalez, M., J. Moreno, J. C. Manzano, F. J. Florencio,and M. G. Guerrero (2005) Production of Dunaliella salina bio-mass rich in 9-cis-β-carotene and lutein in a closed tubular pho-tobioreactor. J. Biotechnol. 115: 81-90.

4. Kim, S. -W., J. -B. Kim, W. -H. Jung, J. -H. Kim, and J. -K. Jung(2006) Over-production of β-carotene from metabolically engi-neered Escherichia coli. Biotechnol. Lett. 28: 897-904.

5. Mantzouridou, F., T. Roukas, and P. Kotzekidou (2005) Produc-tion of beta-carotene from synthetic medium by Blakesleatrispora in fed-batch culture. Food Biotechnol. 18: 343-361.

6. Malisorn, C. and W. Suntornsuk (2008) Optimization of β-caro-tene production by Rhodotorula glutinis DM28 in fermented rad-ish brine. Biores. Technol. 99: 2281-2287.

7. Saenge, C., B. Cheirsilp, T. T. Suksaroge, and T. Bourtoom(2011) Efficient concomitant production of lipids and carotenoidsby oleaginous red yeast Rhodotorula glutinis cultured in palm oilmill effluent and application of lipids for biodiesel production.Biotechnol. Bioproc. Eng. 16: 23-33.

8. Verwaal, R., J. Wang, J. -P. Meijnen, H. Visser, G. Sandmann, J.A. V. D. Berg, and A. J. J. V. Ooyen (2007) High-level produc-tion of beta-carotene in Saccharomyces cerevisiae by successivetransformation with carotenogenic genes from Xanthophyllomy-ces dendrorhous. Appl. Environ. Microbiol. 73: 4342-4350.

9. Yoon, S. -H, S. -H. Lee, A. Das, H. -K. Ryu, H. -J. Jang, J. -Y.Kim, D. -K. Oh, J. D. Keasling, and S. -W. Kim (2009) Combi-natorial expression of bacterial whole mevalonate pathway forthe production of β-carotene in E. coli. J. Biotechnol. 140: 218-226.

10. Kim, J. H., S. -W. Kim, D. Q. A. Nguyen, H. Li, S. B. Kim, Y. -G.Seo, J. -K. Yang, I. -Y. Chung, D. H. Kim, and C. -J. Kim (2009)Production of β-carotene by recombinant Escherichia coli withengineered whole mevalonate pathway in batch and fed-batchcultures. Biotechnol. Bioproc. Eng. 14: 559-564.

11. Das, A., S. -H. Yoon, S. -H. Lee, J. -Y. Kim, D. -K. Oh, and S. -W.Kim (2007) An update on microbial carotenoid production:Application of recent metabolic engineering tools. Appl. Micro-

Fig. 6. Time profiles of cell growth (A), and productions of β-carotene and lycopene (B) during the bioreactor cultures of strain 1at weak alkaline pH in 2 × YT medium supplemented with 30 g/Lglycerol. The culture temperature was maintained at 37oC.


biol. Biotechnol. 77: 505-512.12. Eiteman, M. A. and E. Altman (2006) Overcoming acetate in

Escherichia coli recombinant protein fermentations. Trends Bio-technol. 24: 530-536.

13. Selvarasu, S., D. S. -W. Ow, S. Y. Lee, M. M. Lee, S. K. -W. Oh,I. A. Karimi, and D. -Y. Lee (2009) Characterizing Escherichiacoli DH5α growth and metabolism in a complex medium usinggenome-scale flux analysis. Biotechnol. Bioeng. 102: 923-934.

14. Vemuri, G. N., T. A. Mining, E. Altman, and M. A. Eiteman(2005) Physiological response of central metabolism in Escher-ichia coli to deletion of pyruvate oxidase and introduction of het-erologous pyruvate carboxylase. Biotechnol. Bioeng. 90: 64-76.

15. Won, W., C. Park, C. Park, S. Y. Lee, K. S. Lee, and J. Lee (2011)Parameter estimation and dynamic control analysis of central car-bon metabolism in Escherichia coli. Biotechnol. Bioproc. Eng.16: 216-228.

16. Martin, V. J. J., D. J. Pitera, S. T. Withers, J. D. Newman, and J.D. Keasling (2003) Engineering a mevalonate pathway inEscherichia coli for production of terpenoids. Nat. Biotechnol.21: 796-802.

17. Pitera, D. J., C. J. Paddon, J. D. Newman, and J. D. Keasling(2007) Balancing a heterologous mevalonate pathway forimproved isoprenoid production in Escherichia coli. Metab. Eng.9: 193-207.

18. Sambrook, J. and D. W. Russel (2010) Molecular Cloning: ALaboratory Manual. 3rd ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, USA.

19. Schierle, J., B. Pietsch, A. Ceresa, and C. Fizet (2004) Methodfor the determination of β-carotene in supplements and raw mate-rials by reversed-phase liquid chromatography: Single laboratoryvalidation. J. AOAC Int. 87: 1070-1082.

20. Torrecilla, J. S., M. Camara, V. Fernandez-Ruiz, G. Piera, and J.O. Caceres (2008) Solving the spectroscopy interference effectsof β-carotene and lycopene by neural networks. J. Agric. Food.Chem. 56: 6261-6266.

21. Sandmann, G., M. Albrecht, G. Schnurr, O. Knorzer, and P. Boger(1999) The biotechnological potential and design of novel caro-tenoids by gene combination in Escherichia coli. Trends Biotech-nol. 17: 233-237.

22. Vemuri, G. N., E. Altman, D. P. Sangurdekar, A. B. Khodursky,and M. A. Eiteman (2006) Overflow metabolism in Escherichiacoli during steady-state growth: Transcriptional regulation and

effect of redox ratio. Appl. Env. Microbiol. 72: 3653-3661.23. Mey, M. D., S. D. Maeseneire, W. Soetaert, and E. Vandamme

(2007) Minimizing acetate formation in E. coli fermentations. J.Ind. Microbiol. Biotechnol. 34: 689-700.

24. Roe, A. J., C. O’Byrns, D. Mclaggan, and I. R. Booth (2002)Inhibition of Escherichia coli growth by acetic acid: A problemwith methionine biosynthesis and homocystein toxicity. Micro-biol. 148: 2215-2222.

25. Warnecke, T. and R. T. Gill (2005) Organic acid toxicity, toler-ance, and production in Escherichia coli biorefining applications.Microb. Cell Fact. 4: 25.

26. Raja, N., M. Goodson, D. G. Smith, and R. J. Rowbury (1991)Decrease DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli. J. Appl. Bac-teriol. 70: 507-511.

27. Tabata, K. and S. -I. Hashimoto (2004) Production of mevalonateby a metabolically-engineered Escherichia coli. Biotechnol. Lett.26: 1487-1491.

28. Dahlgren, M. E., A. L. Powell, R. L. Greasham, and H. A.George (1993) Development of scale-down techniques for inves-tigation of recombinant Escherichia coli fermentations: Acidmetabolites in shake flasks and stirred bioreactors. Biotechnol.Prog. 9: 580-586.

29. Soini, J., K. Ukkonen, and P. Neubauer (2008) High cell densitymedia for Escherichia coli are generally designed for aerobic cul-tivations-consequences for large-scale bioprocesses and shakeflask cultures. Microb. Cell Fact. 7: 26.

30. Anguelova, T. and J. Warthesen (2000) Lycopene stability intomato powders. J. Food. Sci. 65: 67-70.

31. Stancik, L. M., D. M. Stancik, B. Schmidt, D. M. Barnhart, Y. N.Yoncheva, and J. L. Slonczewski (2002) pH-dependent expres-sion of periplasmic proteins and amino acid catabolism inEscherichia coli. J. Bacteriol. 184: 4246-4258.

32. Krulwich, T. A., G. Sachs, and E. Padan (2011) Molecularaspects of bacterial pH sensing and homeostasis. Nat. Rev. Micro.9: 330-343.

33. Zilberstein, D., V. Agmon, S. Schuldiner, and E. Padan (1984)Escherichia coli intracellular pH, membrane potential, and cellgrowth. J. Bacteriol. 158: 246-252.

34. Alper, H., K. Miyaoku, and G. Stephanopoulos (2006) Charac-terization of lycopene-overproducing E. coli strains in high celldensity fermentations. Appl. Microbiol. Biotechnol.72: 968-974.

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