Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: Carbohydrate phosphotransferase system

Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Av, Universidad 2001, Col, Chamilpa, Cuernavaca, Morelos, 62210, México.
Microbial Cell Factories (Impact Factor: 4.22). 04/2010; 9(1). DOI: 10.1186/1475-2859-9-21
Source: DOAJ
ABSTRACT
Background
Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis.

Results
Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (μ) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the μ decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in μ, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).

Conclusions
The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.

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RESEARCH
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Research
Metabolic engineering for the production of
shikimic acid in an evolved
Escherichia coli
strain
lacking the phosphoenolpyruvate: carbohydrate
phosphotransferase system
Adelfo Escalante*
1
, Rocío Calderón
1
, Araceli Valdivia
1
, Ramón de Anda
1
, Georgina Hernández
1
, Octavio T Ramírez
2
,
Guillermo Gosset
1
and Francisco Bolívar
1
Abstract
Background: Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this
work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an
evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS
-
) but with
capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK,
aroL, pykF or pykA and the expression of plasmid-coded genes aroG
fbr
, tktA, aroB and aroE, on SA synthesis.
Results: Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose
consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial
high specific growth rate (μ) and specific glucose consumption rate (qs), but low level production of aromatic
intermediates. During the second growth phase the μ decreased, whereas aromatic intermediate production reached
its maximum. The double aroK
-
aroL
-
mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to
7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol.
Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in μ, qs, SA
production, and yield; whereas TACY increased by 33% (0.5 mol/mol).
Conclusions: The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic
intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and
transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of
0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in
strain PB12.SA22 to increase the production of aromatic compounds in the PTS
-
background. Results indicate
differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the
first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic
precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.
Background
The shikimic acid (SA) pathway is the common route
leading to the biosynthesis of aromatic compounds in
bacteria and in several eukaryotic organisms such as
ascomycetes fungi, apicomplexans, and plants [1,2]. In
Escherichia coli, the first step in this pathway is the con-
densation of the central carbon metabolism (CCM) inter-
mediates phosphoenol pyruvate (PEP) and erythrose 4-
phosphate (E4P) into 3-deoxy-D-arabinoheptulosonate
7-phosphate (DAHP) by the DAHP synthase (DAHPS)
* Correspondence: adelfo@ibt.unam.mx
1
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología,
Universidad Nacional Autónoma de México (UNAM). Av. Universidad 2001, Col.
Chamilpa, Cuernavaca, Morelos, 62210, México
Full list of author information is available at the end of the article
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isoenzymes AroF, AroG, and AroH, coded respectively by
the aroF, aroG and aroH genes (Figure 1).
DAHP is converted to 3-dehydroquinate (DHQ) by
dehydroquinate synthase, coded by aroB. DHQ dehy-
dratase, coded by aroD, catalyzes the transformation of
DHQ into 3-dehydroshikimic acid (DHS). This com-
pound is reduced to SA by the shikimate dehydrogenase,
coded by aroE. In turn, SA is transformed to shikimate-3-
P (SHK-3P) by the shikimate kinase isoenzymes I and II,
coded by the aroK and aroL genes, respectively; SHK-3P
is then transformed to chorismic acid (CHA) (Figure 1).
SA is used as the precursor for the synthesis of a large
number of chemicals [3-5] and nowadays has gained
importance as the starting compound for the chemical
synthesis of the neuraminidase inhibitor oseltamivir
phosphate ((3R,4R,5S)-4-acetylamino-5-amino-3 (1-eth-
ylpropoxy)-1-cyclohexene-1-carboxylic acid, ethyl ester
phosphate (1:1)) known as Tamiflu
®
and produced by
Roche Pharmaceuticals. This compound is currently
employed as an antiviral drug for the treatment of both
common seasonal influenza A and B virus infections [6,7]
and for the treatment of both the avian virus type H5N1
and A/H1N1 influenza infections. The latter has been
considered a new pandemic [8,9]. It has been estimated
that in the case of a global pandemic of influenza, the
present capacity of Tamiflu
®
production could be insuffi-
cient to protect large populations, particularly in devel-
oping countries [7,8]. Thus, alternative biotechnological
strategies with engineered strains to produce SA have
gained relevance.
Several metabolic engineering approaches have been
developed to obtain SA from E. coli by biotechnological
processes as an alternative to its limited and costly extrac-
tion procedures from plants such as Illicium anisatum or
I. verum [3,5,9-11]. Previously developed approaches
involve E. coli derivatives with several genetic modifica-
tions in the CCM and SA pathways. CCM modifications
comprise inactivation of the PTS operon (ptsHIcrr),
expression of non-PTS glucose transporters like glucose
facilitators and transformation with plasmids carrying
Figure 1 Central carbon metabolism and shikimic acid pathways in E. coli PB12 strain lacking the PTS. Glucose transport and phosphorylation
are performed by GalP and Glk, respectively [27]. Abbreviations: Glc, glucose; GalP, galactose permease; Glc-6-P, glucose-6-P; Glk, glucokinase; PEP,
phosphoenol pyruvate; PYR, pyruvate; Ac-CoA, acetyl coenzyme-A; TCA, tricarboxylic acid cycle; OAA, oxaloacetate; PPP, pentose phosphate pathway;
E4P, erythrose-4-P; DAHP, 3-deoxy-D-arabinoheptulosonate-7-P; DHQ, 3-dehydroquinic acid; DHS, 3-dehydroshikimic acid; SA, shikimic acid; S3P, shi-
kimate-3-P; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismic acid; QA, quinic acid; PCA, protocatehuic acid; GA, gallic acid. Genes and cod-
ed enzymes: tktA, transketolase I;pykF, pyruvate kinase I;pykA, pyruvate kinase II;ppsA, phosphoenolpyruvate synthase; aroF, aroG, aroH, DAHP synthase
isoenzymes F, G and H, respectively; aroB, DHQ synthase; aroD, DHQ dehydratase; aroE, shikimate dehydrogenase; aroK, shikimate kinase I; aroL, shiki-
mate kinase II; aroA, EPSP synthase; aroC, chorismate synthase; aroZ, dehydroshikimate dehydratase; pobA, p-hydroxy-benzoate hydroxylase [32]. Con-
tinuous arrows represent unique reactions catalyzed by one or more enzymes; dotted lines or arrows represent two or more enzymatic reactions or
incomplete characterized reactions.
DAHP DHQ
DHS
Shikimic acid pathway
aroD
CHASHK-3P EPSP
aroA aroC
Aromatic
aminoacids
aroK,
aroL
aroE
SA
aroB
QA PCA GA
aroE aroZ
Central carbon metabolism
in the PTS
-
Glc
+
strain PB12
aroF
aroG
aroH
PTS
pobA
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the tktA and ppsA genes, coding for transketolase I and
PEP synthase, respectively, to increase the availability of
intermediates E4P and PEP, respectively [3,4,12-18]. The
main modifications in the SA pathway include the partial
or total blockage of the SA flux into CHA. This has been
achieved by decreasing or completely eliminating the
synthesis of SHK-3P -by inactivating aroK and aroL
genes- with the subsequent SA accumulation (Figure 1).
These modifications are commonly complemented with
the transformation of plasmid-coded feedback resistant
(fbr) AroF or AroG proteins (AroF
fbr
and AroG
fbr
, respec-
tively), required to avoid possible feedback inhibition in
the first step of the aromatic pathway catalyzed by
DAHPS isoenzymes. The rate-limiting enzyme DHQ
synthase, and shikimate dehydrogenase, which is feed-
back inhibited by SA [3,15], catalyze two reactions that
can be improved with the goal of increasing the synthesis
of SA. It has been proposed that high extracellular SA
concentration drives the transport of this compound into
the cells by the SA transporter ShiA (shiA). Higher intrac-
ellular SA accumulation reverts the reaction catalyzed by
aroE to synthesize DHS, resulting in "hydroaromatic
equilibration" and by-productby formation, such as
quinic (QA) and gallic acids (GA) (Figure 1). Inactivation
of the ShiA transporter has been used as a strategy to
reduce the intracellular accumulation of DHS, QA, and
GA [3,4,15,19,20]. Engineered E. coli strains with several
of the genetic modifications described above have been
successfully applied to produce 71 g/L of SA with a yield
of 0.27 mol SA/mol glc and total aromatic compound
yield (TACY) (including SA, DHS and QA) of 0.34 mol
aromatic compounds/mol glc in 1-L fed-batch cultures
using mineral broth with 15 g/L yeast extract and glucose
addition to maintain a 55-170 mM concentration [4]. The
effects of carbon and phosphate limitations in chemostat
cultures on SA production have been studied elsewhere
[15,19].
Our group has been involved in the characterization of
E. coli strains lacking the phosphoenolpyruvate: carbohy-
drate phosphotransferase system (PTS
-
), such as strain
PB12 (PTS
-
glc
+
), which has been selected as an evolved
strain for growth rate recovery in a chemostat with glu-
cose fed at progressively faster rates [21,22]. This strain
utilizes galactose permease (GalP) and glucokinase (Glk)
to transport and phosphorylate glucose into glucose-6-P,
respectively (Figure 1). In addition, most of the glycolytic
and other CCM genes are upregulated in this derivative
as compared to its parental strains [21-25]. Further char-
acterization of this evolved strain has shown increased
PEP availability that can be redirected into the aromatic
pathway, as compared to isogenic PTS
+
strains. PB12
strain has been modified for the high yield production of
aromatic compounds such as L-phenylalanine [26,27] and
L-tyrosine [28].
In this work, we report the construction of a SA over-
producing derivatives from the E. coli PB12 strain by
inactivation of the aroL and aroK genes and expressing in
plasmids different combinations of aroG
fbr
, tktA, aroE,
and aroB genes. The effects of single inactivation of either
pyruvate kinase (Pyk) I or II, coded respectively by pykF
and pykA, were also evaluated. This strategy was used to
achieve additional availability of PEP for the synthesis of
aromatic compounds and SA in the E. coli PB12 PTS
-
glc
+
background.
Results and discussion
Inactivation of the genes coding for shikimate kinases I and
II, and expression of the aroG
fbr
, tktA, aroB and aroE genes
in plasmids in the PB12 strain background
The capacity of the E. coli PB12 (PTS
-
glc
+
) strain to pro-
duce SA was evaluated in 500 mL batch cultures in 1 L
fermentors grown in mineral broth supplemented with 25
g/L of glucose and 15 g/L of yeast extract. Specific growth
rate (μ), glucose consumption (qs), SA production and
Table 1: Growth kinetic parameters for strain PB12 and SA-producing derivatives.
Strain/derivative μa
(h-1)
qsb
(millimol glc g DW-1 h-1)c
PB12 0.48 ± 0.02 5.17 × 10
-6
± 4.07 × 10
-7
PB12.SA11 0.41 ± 0.00 2.03 × 10
-6
± 4.79 × 10
-7
PB12.SA21 0.42 ± 0.02* 2.5 × 10
-6
± 8.55 × 10 × 10
-7
*
PB12.SA22 0.42 ± 0.01* 1.93 × 10
-6
± 5.9 × 10 × 10
-7
*
PB12.SA31 0.32 ± 0.02 7.76 × 10
-6
± 9.67 × 10
-8
PB12.SA41 0.45 ± 0.03 2.58 × 10
-6
± 5.39 × 10
-7
Values are the average of two independent experiments.
a
μ, specific growth rate;
b
qs, specific glucose consumption rate;
c
DW, dry cell weight.
Mean values within each column with the same superscript (*) (P < 0.05) do not differ significantly with respect to the immediate parental
strain (see Methods).
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yield, as well as DAHP, DHS and GA concentrations were
evaluated during 50-h cultures (Table 1, Figure 2). Strain
PB12 reached an OD
600 nm
of 32, after 8 h of fermentation
with the consumption of 98.7% of added glucose. From
this time (8 h) to the end of the fermentation (50 h), a
decrease in biomass concentration was observed (Figure
2). Analysis of culture supernatants showed that as
expected, strain PB12 did not accumulate DAHP (Table 2,
Figure 3).
Similarly to strain PB12, cultures of strain PB12.SA11
(aroL- strain expressing aroGfbr, tktA and aroB from two
different plasmids) (see Methods, Figure 1 and Table 3)
also showed an exponential growth phase during the first
8-h cultivation interval as detected for strain PB12 (Fig-
ure 2). However, a significant decrease (P < 0.05), deter-
mined by the Tukey's Honestly Significant Difference
(HSD) test, was observed (see Methods) in both μ and qs
values (86% and 39%, respectively), when compared to
those recorded for strain PB12 (Table 1). From this
moment (8 h) the strain remained stationary. DAHP,
DHS, SA, and GA production was detected during the
exponential growth phase (Figures 3 and 4). Interestingly,
relatively constant concentration levels of all aromatic
intermediates were observed after glucose was com-
Table 2: Aromatic metabolites production and yields determined for strain PB12 and SA-producing derivatives.
Strain SA
(g/L)
SA yield
(mol SA/mol
glc)
DAHP
(g/L)
DHS
(g/L)
GA
(g/L)
TACY1
(mol aromatic
compounds/mol
glc)
PB12 ND --- 0.044 ± 0.07 ND ND 0.00
PB12.SA11 2.82 ± 0.01 0.11 ± 0.00 1.71 ± 0.07 2.79 ± 0.21 0.21 ± 0.06 0.28
PB12.SA21 5.07 ± 0.00 0.21 ± 0.00 0.52 ± 0.00 2.49 ± 0.06* 0.14 ± 0.00 0.33*
PB12.SA22 7.05 ± 0.06 0.29 ± 0.00 0.81 ± 0.04 1.46 ± 0.14 0.08 ± 0.01 0.37*
PB12.SA31 4.35 ± 0.57 0.22 ± 0.04 3.03 ± 0.00 2.12 ± 0.02 0.23 ± 0.04 0.50
PB12.SA41 1.00 ± 0.36 0.03 ± 0.02 0.14 ± 0.00 0.79 ± 0.01 ND 0.07
Values are the average of two independent experiments.
1
TACY, Total aromatic compound yield (combined DAHP, DHS, SA and GA molar
yields); ND, Non-detected. Mean values within each column with the same superscript (*) (P < 0.05) do not differ significantly with respect to
the immediate parental strain (see Methods).
Figure 3 DAHP and DHS concentrations in PB12 and SA-produc-
ing derivatives.
A
B
PB12
S PB12.SA11
T PB12.SA21
PB12.SA22
z PB12.SA31
PB12.SA41
Figure 2 Biomass and glucose concentrations in PB12 and SA-
producing derivatives.
A
B
PB12
S PB12.SA11
T PB12.SA21
PB12.SA22
z PB12.SA31
PB12.SA41
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pletely consumed. DAHP, DHS, and SA accumulated dur-
ing the first 26 h of cultivation; thereafter their
concentration remained constant at around 1.71, 2.8, and
2.8 g/L, respectively (Table 2,). SA yield on glucose was
0.11 mol SA/mol. GA concentration was lower than the
other aromatic intermediates; however, as in the case of
DHS, this strain produced higher GA concentrations
(approximately 0.3 g/L, Table 2, Figure 4), than the other
PB12 derivatives. It has been proposed that GA is formed
by the oxidation of DHS into a diketo intermediate proto-
catehuic acid (PCA) followed by its spontaneous aromati-
zation. Alternatively, this compound may result from the
dehydration of DHS followed by hydroxylation of the
intermediate PCA [29] (Figure 1). GA accumulation dur-
ing SA production has not been reported in either batch
or fed-batch cultures [4], but it has been detected in batch
and chemostat cultures under carbon-limited conditions
[15].
Plasmid-coded AroGfbr DAHPS avoided feedback
inhibition of the first reaction of the SA pathway by the
phenylalanine present in the yeast extract included in the
medium or produced by the cell. It has been reported that
DAHPS activity in vivo is limited by PEP and E4P avail-
ability and that maximum specific activity of DAHPS is
reached when the concentration of both intermediates is
increased [13,17]. It has also been reported that the pres-
ence of a plasmid-coded copy of tktA (coding for transke-
tolase I) causes an increase in E4P availability in strain
PB12 [21,22,25,30-32]. In addition, it is expected that the
Table 3: Strains and plasmids used and developed in this work.
Strain/derivative Relevant characteristics Reference
E. coli JM101 supE, thi, Δ(lac-proAB), F' [45]
E. coli JM101 aroK
-
E. coli JM101 aroKΔ::cm This work
E. coli aroB
-
E. coli K12 strain BW25113 ΔaroB::kan
(JW3352)
[48]
E. coli aroE
-
E. coli K12 strain BW25113 ΔaroE::kan
(JW3242)
[48]
E. coli PB28 PB12 ΔpykA::cat ΔpykF::gen [31]
E. coli PB12 JM101 Δ(ptsH-I-crr)::kan glc
+
[18]
PB12.SA1 PB12 ΔaroL This work
PB12.SA11 PB12.SA1 pJLBaroG
fbr
tktA pTOPOaroB This work
PB12.SA2 PB12 ΔaroL ΔaroK::cm This work
PB12.SA21 PB12.SA2 pJLBaroG
fbr
tktA pTOPOaroB This work
PB12.SA22 PB12.SA2 JLBaroG
fbr
tktA pTOPOaroB aroE This work
PB12.SA3 PB12.SA2 ΔpykF::gen This work
PB12.SA31 PB12.SA3 pJLBaroG
fbr
tktA pTOPOaroB aroE This work
PB12.SA4 PB12.SA2 ΔpykA::gen This work
PB12.SA41 PB12.SA4 pJLBaroG
fbr
tktA pTOPOaroB aroE This work
E. coli TOP10 F
-
mcrA Δ(mrr-hsdRMS-mcrBC)
φ80lacZΔM15 ΔlacX74 recA1 araD139
Δ(ara-leu)7697 galU galK rpsL endA1nupG
Invitrogen
Plasmids
pJLBaroG
fbr
tktA pJLBaroG
fbr
(aroG
fbr
expressed from the
lacUV5 promoter, lacIq and tet genes (Tc
r
),
pACYC184 replication origin) derivative,
containing the tktA gene with its native
promoter.
[21,47]
pCR
®
-Blunt II-TOPO
®
P
lac
lacZ-α ORF T7 promoter ccdB kan (Km
r
)
Zeocin pUC origin.
Invitrogen
pTOPO aroB pCR
®
-Blunt II-TOPO
®
containing the aroB
gene
This work
pTOPO aroB aroE pTOPOaroB derivative containing the aroB
and aroE genes
This work
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presence of aroB in a multicopy plasmid (Figure 1) will
reduce the possible accumulation of DAHP [3,4].
Inactivation of aroL gene as part of a SA production
strategy has been previously described in the E. coli
W3110 aroL
-
strain (W3110 shik1) in chemostat cultures
that resulted in maximum SA yields on glucose of 0.02
and 0.05 mol/mol under carbon and phosphate limited
conditions that resulted in a maximum SA yield on glu-
cose of 0.2 mol/mol and 0.05 mol/mol, respectively
[15,19]. In the present study, strain PB12.SA11 yielded
0.11 mol SA/mol glc; however, maximum yields of SA on
glucose of 0.27 and 0.33 mol/mol have been reported for
another E. coli strain (PTS
-
glf, glk, aroF
fbr
, tktA, aroE,
aroK
-
aroL
-
) in 1-L and 10-L fed-batch cultures, respec-
tively [4], suggesting that the ΔaroL phenotype is in itself
insufficient to achieve high SA yields.
Strain PB12.SA21 (aroL
-
aroK
-
strain expressing plas-
mid coded aroG
fbr
tktA and aroB genes) (Figure 1 and
Table 3) showed an exponential growth phase during the
first 8 h fermentation interval and a stationary stage simi-
lar to what was observed for PB12 and PB12.SA11 strains
(Figure 2). No significant differences (P < 0.05) were
observed in μ and qs values between PB12.SA21 and
PB12.SA11 strains as a consequence of the inactivation of
the aroL and aroK genes (Table 1). DAHP, DHS, SA, and
GA production was also detected during the exponential
growth phase, but important differences were observed
(Figures 3 and 4). Compared to the PB12.SA11 derivative,
maximum concentrations of DAHP and GA in the
PB12.SA21 strain were significantly lower (P < 0.05),
whereas no significant difference (P < 0.05) was observed
in the maximum concentration of DHS (Table 2, Figure
3). Furthermore, SA production was observed through-
out all the process (Figure 4). After 50 h of cultivation,
about 5.1 g/L of SA were detected with a yield on glucose
of 0.21 mol/mol. This result represents a significant
increase (P < 0.05) (80%) in both SA concentration and
yield, as a consequence of the double aroK
-
aroL
-
muta-
tions (Table 2, Figure 4). The concentrations of DAHP,
DHS (Figure 3) and SA obtained in strain PB12.SA11, as
compared to the ones recorded for the PB12.SA21 deriva-
tive, indicate an efficient flow of aromatic intermediates
from DAHP to SA. However, based on the still relatively
high DHS concentration observed, it appears that this
strain can further convert part of the remaining DHS to
improve SA concentration and yield.
Cultures of strain PB12.SA22 (aroK
-
aroL
-
strain
expressing aroG
fbr
, tktA, aroB and aroE from two differ-
ent plasmids) (Table 3, Figure 1) showed no significant
differences (P < 0.05) in μ and qs values with respect to
those for strain PB12.SA21 (Table 1). Glucose was totally
consumed in both strains only after 38 h of cultivation
(Figure 2). DHS was detected in a significantly (P < 0.05)
lower concentration than the previous derivative and SA
reached the highest concentration compared to all other
derivatives (Table 2, Figure 4). At the end of the fermenta-
tion, 7.1 g/L of SA were detected with a yield on glucose
of 0.29 mol SA/mol (39% increase in yield with respect to
the previous derivative) and a TACY value of 0.378 mol
aromatic compounds/mol glc (Table 2, Figure 4). Intro-
duction of a copy of the aroE gene in the multicopy plas-
mid pTOPO resulted in a more efficient conversion of
DHS into SA, probably as a consequence of a responsible
for the synthesis of DHS from SA [3,4,15,19,20]. Accord-
ingly, very small amounts of GA were produced during
the cultivation of this strain (Table 2, Figure 4).
Inactivation of the genes coding for pyruvate kinases I and
II in the PB12SA.22 strain
Disruption of the pykF gene in strain PB12.SA22 gener-
ated the PB12.SA31 derivative (aroL
-
, aroK
-
, pykF
-
strain
expressing aroG
fbr
, tktA, aroB and aroE genes from two
different plasmids) (Figure 1 and Table 3). Cultures of this
strain showed the characteristic two-phase growth
behavior observed for the previous derivatives (Figure 2),
although significant (P < 0.05) differences were observed
in μ and qs values as compared to the PB12.SA22 pykF
+
parental strain (Table 1). In addition, maximum biomass
concentration after 8 h of fermentation was only 33%
with respect to the one recorded for PB12.SA22 and, con-
trary to all other analyzed strains, glucose was not com-
Figure 4 SA and GA concentrations in PB12 and SA-producing
derivatives.
A
B
PB12
S PB12.SA11
T PB12.SA21
PB12.SA22
z PB12.SA31
PB12.SA41
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pletely consumed after 50 h (Figure 2). DHS, GA, and
specially DAHP final concentrations were higher than
those obtained with the pykF
+
parental strain, whereas
the final SA production and yield were lower (Table 2).
Importantly, the TACY value in this strain was 0.50 mol
aromatic compounds/mol glc (i.e., a 33% increment with
respect to PB12.SA22), the highest yield obtained when
compared to all previous PB12.SA derivatives (Table 2).
Inactivation of the pykA gene in strain PB12.SA22 gen-
erated the derivative PB12.SA41 (aroL
-
, aroK
-
, pykA
-
strain expressing aroG
fbr
, tktA, aroB and aroE genes from
two different plasmids) (Table 3, Figure 1). Cultures of
this strain showed no-significant differences (P < 0.05) in
μ and qs values with respect to those recorded for the
parental strain PB12.SA22 pykA
+
(Table 1). This strain
reached an OD
600 nm
of 14 after 20 h of fermentation;
however, an important decrease in growth was observed
from this moment to the end of the fermentation (Figure
2). This strain also showed the lowest production of
DAHP, DHS, and SA as compared to all other variants,
and no GA was detected (Table 2, Figures 3 and 4).
Pyruvate kinase isoenzymes Pyk I and Pyk II play a key
role in the glycolytic pathway, especially in overall carbon
metabolism in strains lacking PTS [33,34]. Pyk activity,
together with phospho-fructokinase I and glucokinase,
control the carbon flux through the glycolytic pathway
and catalyze the essentially irreversible trans-phosphory-
lation of PEP and ADP into PYR and ATP [33]. It has
been previously reported that inactivation of pykF in
strain PB12 (PTS
-
glc
+
) results in an apparently slight
increase in the specific activity of Pyk A enzyme (13.5%)
[34]. Likewise, carbon flux analysis in this strain has
shown a flux increase through the Pyk AF enzymes in the
absence of PTS as compared to the wild-type strain
(JM101 PTS
+
) [23]. Furthermore, transcriptome analyses
in strain PB12 and in a phenylalanine overproducing
PB12 derivative have shown a slight upregulation of pykA
with respect to the wild type strain JM101, suggesting
that the overall activity of PyK isoenzymes present in the
PB12 strain is sufficient to convert PEP into PYR, at least
at similar rates as in JM101 [31,35]. These results suggest
that single inactivation of the pykF or pykA gene could be
an attractive strategy to increase the amount of PEP avail-
able for DAHP synthesis, without compromising the syn-
thesis of PYR and its flux to acetyl-CoA.
Interruption of either the pykF or pykA gene in the E.
coli strain PB12.SA22 demonstrated a differential role of
Pyk isoenzymes in overall cellular metabolism in this
strain which produces aromatic compounds. Disruption
of pykF in strain PB12.SA31 negatively affected growth,
glucose consumption, and SA accumulation with respect
to the PB12.SA22 pykF
+
parental strain. Importantly, the
TACY value increased to 0.50 mol aromatic compounds/
mol glc in the pykF
-
strain, which is 33% higher than the
total yield observed for the parental strain PB12.SA22.
These results suggest that pykF inactivation apparently
increases PEP availability, which in turn is channeled into
the aromatic pathway, resulting in a higher TACY value.
Higher DAHP concentrations produced by the
PB12.SA31 derivative also indicate that in this genetic
background, DHQ synthase could be one of the limiting
steps for SA production. This explanation contravenes
the fact that this strain was transformed with a plasmid-
carrying aroB; however, a previous report on the pro-
teomic response to pykF inactivation in E. coli BW25113
strain demonstrated the upregulation of all the genes of
the SA pathway, with the exception of aroB, during the
production of aromatic amino acids [33]. Therefore,
increasing the expression of the aroB gene, by substitu-
tion of its natural promoter for a stronger one, could be a
viable strategy to improve SA concentrations in strain
PB12.SA31.
Pyk activity plays a key role in cellular metabolism by
connecting glycolysis with amino acid and lipid biosyn-
thetic pathways [34,36,37]. Consequently, one remarkable
characteristic of Pyk isoenzymes is their allosteric
response to several effectors involved not only in central
carbon metabolism but also in global cellular metabo-
lism, among them, the glycolytic intermediate PEP
[34,38,39]. It has been proposed that Pyk isoenzymes are
involved in catabolite repression in E. coli glucose fer-
mentations [40]; however, no information is available to
correlate the specific role of individual Pyk isoenzymes in
global bacterial metabolism, particularly in strains
devoted to the production of aromatic compounds. Our
results demonstrate that inactivation of the pykA gene in
strain PB12.SA41 caused a negative effect on the produc-
tion of aromatic compounds, probably due to an
increased growth rate (Table 1). In addition, SA accumu-
lation and TACY were substantially reduced in this strain
as compared to the PB12.SA31 (pykF
-
) and PB12.SA22
(pykA
+
pykF
+
) strains. The lack of pykF clearly reduced μ
and qs values in relation to the parental PB12.SA22 strain.
In addition, glucose was not completely consumed in
strain PB12.SA31 after 50 h, as compared to strain lack-
ing pykA, where it was completely consumed after 25 h
(Figure 2). Furthermore, the accumulation of aromatic
compounds was the highest in the strain lacking pykF,
while in the strain PB12.SA22, 37% of glucose was con-
verted into aromatic compounds; this amount increased
to 50% in strain PB12.SA31. Altogether, the result of dif-
ferentially inactivating the kinases I and II suggest that
the PykF isoenzyme may have a more relevant role in
global cellular processes than PykA in the derivatives
constructed under the growth conditions tested here,
since it seems that the absence of pykF apparently allows
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higher accumulation of PEP than the absence of pykA.
Importantly, pykF is apparently transcribed when grow-
ing on glucose from at least three different promoters in
strains JM101 and PB12, while pykA is apparently only
transcribed from two [41]. These results are in agreement
with previous observations which suggest that PykF plays
a more important role than PykA in strain JM101 (PTS
+
)
and other derivative strains lacking PTS, when growing
on glucose as the only carbon source [34].
Conclusions
E. coli PB12 (PTS
-
glc
+
) strain was used as the host for the
synthesis of SA. The derivative PB12.SA22 was obtained
by inactivation of both aroL and aroK genes, and trans-
formed with plasmids carrying aroG
fbr
tktA, aroB, and
aroE genes. This strain was capable of efficiently channel-
ing carbon from metabolites participating in the CCM
into the aromatic pathway for the synthesis of SA. Fer-
mentor cultures of PB12.SA22 strain in mineral broth
complemented with 25 g/L glucose and 15 g/L yeast
extract resulted in the production of 7 g/L of SA with a
yield of SA on glucose of 0.29 mol/mol and a TACY of
0.38 mol aromatic compounds/mol glc. Importantly, glu-
cose was totally consumed in strain PB12.SA22 after 48 h
of fermentation. It is known that PTS
-
strains are capable
of utilizing higher concentrations of glucose (100 g/L)
[42,43] and different carbon sources simultaneously with
glucose [29,31]. Therefore, experiments with higher glu-
cose concentrations, including fed-batch fermentations
should be performed to increase SA concentrations. In
fact, preliminary results, in which glucose concentration
in the medium was increased to 100 g/L, in a 500 mL
batch fermentor cultures with strain PB12.SA22, allowed
the production of 14 g/L of SA (unpublished results).
Single inactivation of either the pykF or pykA gene was
performed to further increase PEP availability for SA pro-
duction in strain PB12.SA22. Inactivation of these genes
demonstrated differential roles of Pyk isoenzymes in final
growth, glucose consumption, and production of aro-
matic intermediates and SA. The pykF
-
mutation present
in strain PB12.SA31 substantially affected biomass con-
centration, glucose consumption, and SA production,
suggesting a more important role of the PykF isoenzyme
in comparison to PykA, in these growing conditions. The
production of SA was reduced in this strain as compared
to strain PB12.SA22; however, it is notable that TACY
reached a value of 0.5 mol aromatic compounds/mol glc,
which was 33% higher than the one obtained in the
parental pykF
+
strain. As far as we know, there are no
reports in which the utilization of a double PTS
-
, pykF
-
derivative has been used to improve the production of SA
and its aromatic precursors [3,4,16-18,44]. Further
genetic modifications will be undertaken in this pykF
-
derivative, such as the substitution of the aroB natural
promoter for another that allows its upregulation to avoid
the accumulation of the aromatic intermediate DAHP in
order to increase the production of SA. In addition, car-
bon flux could still be further modulated by reducing the
expression of pykA in the strain lacking pykF, to obtain a
higher accumulation of PEP to be channeled into the SA
pathway. The pykA gene in these E. coli derivatives, as
mentioned, is expressed from two different promoters
when glucose is utilized as the only carbon source [41].
Therefore, it could be possible to construct derivatives
lacking one of these two promoters to reduce the tran-
scription of pykA with the goal of increasing PEP concen-
tration.
This study demonstrated for the first time the simulta-
neous inactivation of PTS and pykF as part of a strategy to
improve SA production and its aromatic precursors in E.
coli, with the resulting high yield of 0.5 mol aromatic
compounds/mol glc.
Methods
Bacterial strains and plasmids
Bacterial strains and plasmids used in this work are listed
in Table 3. E. coli PB12, a derivative of strain JM101 [45],
was used as the parental strain to originate the interrup-
tions in aroL and aroK as well as the single interruption of
pykF or pykA. Amplification of target genes was per-
formed with Pfu DNA polymerase (Fermentas, Glen Bur-
nie, USA), according to recommendations by the supplier,
in a GeneAmp PCR System thermocycler (Perkin Elmer
Cetus, Norwalk, USA). Primer sets employed for amplifi-
cation of target genes are listed in Table s1 (see Addi-
tional file 1). The size of the PCR products was
determined by agarose gel electrophoresis. When
required, amplicons were purified by cutting the desired
band from the agarose gels and processed with a gel PCR
purification kit (Marligen Biosciences, Urbana-Pike-
Ijamsville, USA). The obtained derivative strains were
transformed with plasmids carrying the aroG
fbr
, tktA,
aroB, and aroE genes (see below) for the construction of
SA producing strains.
Inactivation of the aroL gene
PB12.SA1 strain (aroL
-
derivative) (Table 3) was obtained
by the one-step inactivation procedure of chromosomal
genes by PCR products [46]. Primer sets used are listed in
Table s1 (see Additional file 1). The aroL gene was
replaced by the ΔaroL::cat cassette. Selection was per-
formed in chloramphenicol (Cm) containing Luria Ber-
tani (LB) plates. Inactivation of the aroL gene in
chloramphenicol resistant (Cm
r
) colonies was confirmed
by PCR and the size of the PCR product was determined
by agarose gel electrophoresis. The Cm cassette was
deleted from the ΔaroL::cat construction, as previously
described [46], to facilitate subsequent gene inactivation;
the aroL
-
genotype was confirmed by PCR.
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Inactivation of the aroK gene
Strain PB12.SA2 (aroL
-
, aroK
-
derivative) (Table 3) was
constructed in a two-step procedure. First, the aroK gene
of E. coli JM101 strain was replaced by the ΔaroK::cat
cassette [46]. Selection was performed in Cm containing
plates and the inactivation of aroK in Cm
r
colonies was
confirmed by PCR. Second, strain PB12.SA1 was the
recipient of P1 phage lysate grown on the JM101 aroK
strain; the aroK
-
genotype was confirmed by PCR.
Inactivation of the pykF gene
PB12.SA22 strain was the recipient of P1 phage lysate of
E. coli PB28 (ΔpykF::gen) strain (Table 3). Transductants
were selected on gentamicin (Gm) plates and the inacti-
vation of pykF in Gm
r
colonies was confirmed by PCR;
the size of the PCR product was determined by agarose
gel electrophoresis. The resultant strain (aroL
-
, aroK
-
,
pykF
-
derivative) was named PB12.SA31.
Inactivation of the pykA gene
PB12.SA4 strain (aroL
-
, aroK
-
, pykA
-
derivative) (Table 3)
was constructed by a modification of the one-step inacti-
vation procedure of chromosomal genes by PCR prod-
ucts [46]. Briefly, template plasmids pKD3, pKD4, or
pKD13, used to amplify FRT-resistance gene-FRT cas-
sette, only allowed the use of Cm or kanamycin (Km) as
selection markers [46]; however, PB12.SA2 carried both
resistance genes as a consequence of previous genetic
modifications [31]. For this reason, a primer set was
designed (Table s1, see Additional file 1), for priming the
Gm
r
cassette flanked by the entire FRT sequence and
homology regions for the pykA gene. The Gm
r
cassette
was amplified using chromosomal DNA from PB12.SA3
as template and the expected product was confirmed by
PCR. Purified PCR products were used to inactivate pykA
in strain JM101. Selection of the resultant ΔpykA::gen
mutant was achieved on Gm containing plates and the
inactivation of pykA in Gm
r
colonies was confirmed by
PCR; the size of the PCR products was determined by
agarose gel electrophoresis. The ΔpykA::gen construction
was then P1 phage transduced to PB12.SA3; Gm
r
colonies
were selected and screened.
Transformation of derivative strains with plasmid pJLBaroG
fbr
tktA
The construction of plasmid pJLB aroG
fbr
tktA (Table 3)
has been previously reported [21,47]; this vector was used
to transform all SA producing derivative strains. Positive
clones were selected by growing colonies on LB plates
supplemented with tetracycline (Tet).
Cloning the aroB gene and transformation with plasmid
pTOPOaroB
The aroB gene (1484 bp) was obtained by PCR using
chromosomal DNA from E. coli JM101 strain as template
and the primers FwaroB and RvaroB (Table s1, see Addi-
tional file 1). PCR reaction was performed with Pfu poly-
merase; the size of the PCR product was determined by
agarose gel electrophoresis and cloned directly into the
pCR
®
-Blunt II-TOPO
®
vector (Invitrogen, Carlsbad, USA)
leading to the construction of the pTOPOaroB plasmid
(Table 3). This vector was used to transform competent
TOP10 cells (Invitrogen) and selection was performed on
25 μg/mL of zeocin-containing LB plates. Functionality of
the cloned aroB gene was tested by restoring growth of an
aroB
-
E. coli mutant [48] in M9 minimal medium plates
supplemented with zeocin, as a consequence of the com-
plementation of the SA pathway in this mutant strain.
Cloning the aroE gene and transformation with plasmid
pTOPOaroB aroE
The aroE gene (835 bp) was obtained by PCR using chro-
mosomal DNA from E. coli JM101 strain as template and
primers FwaroE and RvaroE (Table s1, see Additional file
1). PCR amplification was performed as described for the
aroB gene; the size of the PCR product was determined
by agarose gel electrophoresis. Amplified aroE gene and
plasmid pTOPOaroB were both digested with Bam HI
endonuclease. This vector was treated with calf intestine
phosphatase (Fermentas) and ligated with the digested
aroB product using T4 DNA ligase (Fermentas), trans-
formed into TOP10 competent cells and selection was
performed on zeocin-containing LB plates. Functionality
of the cloned aroE gene was tested by restoring growth of
an aroE
-
E. coli mutant [48] in M9 minimal medium
plates supplemented with zeocin.
Cultivation media and growth conditions
Shake flask cultures inoculated with frozen stocks of each
strain were performed in 125 mL baffled flasks contain-
ing 10 mL of LB supplemented with the respective antibi-
otics as required: 30 μg/mL Km, 15 μg/mL Gm, 20 μg/mL
Cm or 30 μg/mL Tet (Table 3 shows specific antibiotic
resistances). Cultures were incubated overnight in a
shaker (New Brunswick Scientific, Edison, USA) at 37°C,
300 rpm. An aliquot of 150 μL from each culture was
used to inoculate a 250 mL baffled flask with 50 mL of
fermentation medium, whose composition has been pre-
viously reported for the production of SA, and grown as
described above. This medium contained 25 g/L of glu-
cose, 15 g/L of yeast extract [4] and the required antibiot-
ics. Biomass concentrations were determined and
calculations were performed to adjust inoculum size to
an OD
600 nm
of 0.35. Batch cultures were performed in
duplicate in an Applikon autoclavable glass Bio Reactor
(Schiedam, The Netherlands) 1 L fermentor (500 mL of
working volumen of fermentation medium supplemented
with the required antibiotics). This device was connected
to an Applikon ADI 1010 BioController and ADI 1025
controllers to monitor temperature, pH, impeller speed
and dissolved oxygen (DO). Batch fermentations were
run for 50 h at 37°C, pH 7.0 (maintained by addition of
3.0% NH
4
OH). An impeller speed of no less than 500 rpm
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Escalante et al. Microbial Cell Factories 2010, 9:21
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Page 10 of 12
was used to maintain DO levels at 20% air saturation.
Gene expression of cloned genes was induced by adding
0.1 mM IPTG at the onset of fermentation.
Analytical procedures
Biomass concentrations were monitored every hour dur-
ing the first 8 h of culture; after this point they were mon-
itored every 6 h until the end of the fermentation.
Samples (1.5 mL) were withdrawn from each reactor and
cell turbidity was determined spectrophotometrically at
600 nm (Beckman DU
®
-70 Spectrophotometer, Palo Alto,
USA). Samples for the determination of SA, DHS, QA,
and GA were prepared by centrifuging at 12,000 rpm for
1 min (Eppendorff Centrifuge 5410, Brinkman Instru-
ments Inc., Westubury, USA) 1 mL of fermented broth to
remove cells and filtered through 0.45 μM nylon mem-
branes (Millipore, Brazil). SA, DHS, QA, and GA concen-
trations were determined by HPLC using a Waters system
(600E quaternary pump, 717 automatic injector, 2410
refraction index, and 996 photodiode array detectors,
Waters, Milford, USA), equipped with an Aminex HPX-
87H column (300 × 7.8 mm; 9 μm) (Bio-Rad, Hercules,
USA) maintained at 50°C. The mobile phase was 5 mM
H
2
SO
4
, with a flow rate of 0.5 mL/min, at 50°C. All
metabolites were detected with a photodiode array detec-
tor at 210
nm
. DAHP concentrations were determined by
the thiobarbituric acid assay [49]. This method does not
distinguish between DAHP and DAH, so in this work,
DAHP levels corresponded the sum of both compounds
[26]. Glucose concentration was assessed by a biochemi-
cal analyzer (YSI 2700 Select, Yellow Springs, USA).
Calculations
The specific glucose (S) consumption rate (qs) was calcu-
lated during the exponential growth phase as the differ-
ential change in S with time (t) normalized to the biomass
concentration . A predetermined correlation
factor (1 OD
600
corresponded to 0.37 g/L of dry cellular
weight) [50] was used to transform OD
600
values into cell
concentrations for qs calculation. TACY determinations
were based on the combined molar yields of DAHP, DHS,
SA, and GA [4].
In order to determine whether the observed differences
between growth, qs, and aromatic intermediate produc-
tion (DAHP, DHS, SA and GA) in strain PB12 and in
PB12.SA derivatives were significant (P < 0.05), an analy-
sis of variance (ANOVA) and the multiple comparison
test of Tukey's Honestly Significant Difference (HSD)
were performed using the XLSTAT program V2009.5.01
http://www.xlstat.com
.
Additional material
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AE and FB participated in the design of this study. AE and RC participated in
the construction of ΔaroK, ΔAroL, ΔpykF mutants and data analysis. RC was
involved in the construction of pTOPO aroB aroE vector and fermentations. AV
participated in the construction of the ΔpykA mutant and fermentations. RA
was responsible for the fermentations. GH performed HPLC determinations
and data analysis. AE, OR, GG, and FB participated in the analysis of the results,
as well as in writing and critical review of the manuscript. All authors have read
and approved the manuscript.
Acknowledgements
We thank Alfredo Martínez, Shirley Ainsworth and Marcela Sánchez for critical
reading of the manuscript, Mercedes Enzaldo for technical support and Paul
Gaytán, Jorge Yañéz and Eugenio López for primer synthesis and DNA
sequencing. This work was supported by FONSEC/SSA/IMSS/ISSSTE/CONACyT
Grants 44126, 126793 and DGAPA-PAPIIT, UNAM Grants IN213508, IN224709.
Author Details
1
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología,
Universidad Nacional Autónoma de México (UNAM). Av. Universidad 2001, Col.
Chamilpa, Cuernavaca, Morelos, 62210, México and
2
Departamento de
Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad
Nacional Autónoma de México (UNAM). Av. Universidad 2001, Col. Chamilpa,
Cuernavaca, Morelos, 62210, México
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Received: 15 September 2009 Accepted: 12 April 2010
Published: 12 April 2010
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Cite this article as: Escalante et al., Metabolic engineering for the produc-
tion of shikimic acid in an evolved Escherichia coli strain lacking the phos-
phoenolpyruvate: carbohydrate phosphotransferase system Microbial Cell
Factories 2010, 9:21
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    • "Interruption of the SA pathway by inactivation of the aroK and aroL genes imposes an auxotrophic requirement for aromatic AAs and probably other metabolites derived from CHA on the cell; these effects were successfully reversed by the addition of YE to the fermentation media. As the chemical complexity of YE or peptone significantly interferes in the study of carbon flux through the CCM and SA pathway metabolic networks, no studies to date have been reported on the application of metabolic models to identify possible targets for the application of further ME strategies focused on the improvement of SA production in fermentation culture using complex production media (Chandran et al., 2003; Escalante et al., 2010; Chen et al., 2012; Rodriguez et al., 2013; Cui et al., 2014). The application of omics, such as GTA, in SA-producing conditions, including YE, as reported for the strain P12.SA22, provides valuable information on the role of diverse transporter systems and other pathways involved in carbon supply from YE to SA synthesis (Tolalpa et al., 2014 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several “intuitive” metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.
    Full-text · Article · Sep 2015 · Frontiers in Bioengineering and Biotechnology
  • Source
    • "To increase the intracellular concentration of these two metabolites, we modulated central carbon metabolism by engineering the glucose transport system (De Anda et al., 2006), overexpressing tpiA (Choi et al., 2010), edd/eda (Liu et al., 2013), talA/tktB (Song et al., 2006), ppsA (Farmer and Liao, 2001), and pckA (Farmer and Liao, 2001), and knocking out gdhA (Alper et al., 2005) and pykF (Toya et al., 2010). Using galactose permease system to replace phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) to transport glucose has been reported to increase the concentration of phosphoenolpyruvate (PEP) and G3P (Escalante et al., 2010; Zhang et al., 2013) _ENREF_28. Other targets (except the gdhA deletion) are directly related with G3P, PEP, or pyruvate metabolism. "
    [Show abstract] [Hide abstract] ABSTRACT: Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0g/L β-carotene in fed-batch fermentation. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jun 2015 · Metabolic Engineering
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    • "In addition, the reaction catalyzed by the enzyme quinate/shikimate dehydrogenase (coded by ydiB) was also reported as limiting in the development of L-TYR production strains [58]. Either the overexpression of some of these genes by plasmid-cloned copies [28,66], their co-expression in a modular operon under control of diverse promoters [50,58,67], or their expression by chromosomal integration of additional gene copies and promoter engineering by chromosomal evolution [68] , have relieved to a great extent these rate-limiting steps typically encountered during the development of SHK and AAA overproducing strains (Table 1). To date, genetically modified E. coli strains can overproduce SHK from glucose with yields in the range of 0.08 to 0.42 mol SHK / mol glucose under diverse culture conditions [28,50,686970. "
    [Show abstract] [Hide abstract] ABSTRACT: The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives.
    Full-text · Article · Dec 2014 · Microbial Cell Factories
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