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Biotechnology Advances xxx (2010) xxx–xxx
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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b i o t e c h a d v
Research review paper
Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the
production of L-threonine
Xunyan Dong a, Peter J. Quinn b, Xiaoyuan Wang a,⁎
a
b
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
Department of Biochemistry, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom
a r t i c l e
i n f o
Article history:
Received 14 May 2010
Received in revised form 17 July 2010
Accepted 26 July 2010
Available online xxxx
Keywords:
Escherichia coli
Corynebacterium glutamicum
L-threonine production
Metabolic engineering
L-threonine biosynthesis
a b s t r a c t
L-threonine is an essential amino acid for mammals and as such has a wide and expanding application in
industry with a fast growing market demand. The major method of production of L-threonine is microbial
fermentation. To optimize L-threonine production the fundamental solution is to develop robust microbial
strains with high productivity and stability. Metabolic engineering provides an effective alternative to the
random mutation for strain development. In this review, the updated information on genetics and molecular
mechanisms for regulation of L-threonine pathways in Escherichia coli and Corynebacterium glutamicum are
summarized, including L-threonine biosynthesis, intracellular consumption and trans-membrane export.
Upon such knowledge, genetically defined L-threonine producing strains have been successfully constructed,
some of which have already achieved the productivity of industrial producing strains. Furthermore,
strategies for strain construction are proposed and potential problems are identified and discussed. Finally,
the outlook for future strategies to construct industrially advantageous strains with respect to recent
advances in biology has been considered.
© 2010 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The metabolic pathway of L-threonine. . . . . . . . . . . . . . . . . . . .
2.1.
Production of L-threonine . . . . . . . . . . . . . . . . . . . . . .
2.2.
Intracellular consumption of L-threonine . . . . . . . . . . . . . . .
2.3.
Export of L-threonine . . . . . . . . . . . . . . . . . . . . . . . .
Regulation mechanism of L-threonine biosynthesis pathway . . . . . . . . .
3.1.
Mechanisms of repression . . . . . . . . . . . . . . . . . . . . . .
3.2.
Mechanisms of inhibition . . . . . . . . . . . . . . . . . . . . . .
Progress in strain construction . . . . . . . . . . . . . . . . . . . . . . .
4.1.
L-threonine producing strains of E. coli . . . . . . . . . . . . . . . .
4.1.1.
Overexpressing the thr operon. . . . . . . . . . . . . . . .
4.1.2.
Enhancing L-threonine export . . . . . . . . . . . . . . . .
4.1.3.
Systems metabolic engineering . . . . . . . . . . . . . . .
4.2.
L-threonine producing strains of C. glutamicum . . . . . . . . . . . .
4.2.1.
Overexpressing genes in the L-threonine biosynthesis pathway
4.2.2.
Reducing the L-threonine consumption . . . . . . . . . . .
4.2.3.
Enhancing the L-threonine export . . . . . . . . . . . . . .
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Engineering the pathway . . . . . . . . . . . . . . . . . . . . . .
5.2.
Enzymes engineering . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Improving production stability . . . . . . . . . . . . . . . . . . . .
5.4.
Extending the substrate spectrum . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Tel./fax: +86 510 85329239.
E-mail address: [email protected] (X. Wang).
0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.07.009
Please cite this article as: Dong X, et al, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of Lthreonine, Biotechnol Adv (2010), doi:10.1016/j.biotechadv.2010.07.009
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X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
6.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
L-threonine has been widely used as a supplement in the food,
pharmaceutical and cosmetics industries. Its demand has been
growing steadily. The most remarkable use of L-threonine is as a feed
additive, as recent studies have acknowledged L-threonine as the
second limiting amino acid in swine feeds after L-lysine and the third
limiting amino acid in poultry feeds after L-lysine and L-methionine
(Ajinomoto, 2009). Application of low protein level formula feeds
supplemented with L-threonine is not only economically advantageous, but also contributes to the relief of crude protein deficiency and
lower nitrogen emissions. Thus, the production of L-threonine is being
rapidly developed (Leuchtenberger et al., 2005). The global production
capacity of L-threonine was about 75,000 tons in 2009 (Ajinomoto,
2009), while the annual demand in the Chinese feed additive industry
alone will exceed 60,000 tons in 2010.
Currently L-threonine is produced mainly by microbial fermentation. Ever since Kinoshita et al. (1958) first reported glutamate
production by Corynebacterium glutamicum, great interest has been
inspired in research into microbial production of L-threonine,
resulting in the industrial prosperity of today. Through breeding
approaches, researchers have developed L-threonine producing
strains of Serratia marcescens (Komatsubara et al., 1978), Escherichia
coli (Furukawa et al., 1988) and C. glutamicum (Morinage et al., 1987).
To date, E. coli mutant strains remain the dominant industrial
producers of L-threonine, followed by C. glutamicum.
The current and future demand for L-threonine provides a strong
impetus to further improve productivity of the bioconversion and
reduce its costs. The most efficient solution is to develop more
productive bacterial strains with reduced growth retardation. Strains
surviving multiple rounds of mutation are genetically undefined and
vulnerable to further changes, occurring especially from random
mutations, which at best will result in marginal increases in yield and
resistance to more stringent process requirements. Obviously, directed
genetic manipulations are more feasible options for subsequent
isolation of mutant strains of genetically defined hyper-producers.
Selection criteria would include reduced by-product formation and
expanded substrate spectra. By all accounts, it is by genetic engineering
that breakthroughs in efficient and clean L-threonine production will be
achieved.
This review covers development of L-threonine producers of E. coli
and C. glutamicum through genetic manipulation over the past
two decades. We focus on the pathways of anabolism and catabolism
of L-threonine as well as its export from the cell. The mechanisms of
regulation of L-threonine production at the molecular level are
considered. Also, progress in strain construction based on metabolic
engineering and systems biology is discussed. The currently employed
strategies are summarized and problems are identified. Future
prospects are illustrated with respect to contemporary knowledge
of biology and by examples in development of hyper-producers of
other amino acids.
2. The metabolic pathway of L-threonine
L-threonine, which belongs to the aspartic family of amino acids, is
synthesized from L-aspartate (Fig. 1). Five enzymes, aspartate kinase,
aspartyl semialdehyde dehydrogenase, homoserine dehydrogenase,
homoserine kinase and threonine synthase are involved in the
biosynthesis of L-threonine (Table 1). Both E. coli and C. glutamicum
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share common L-lysine and L-methionine competing branches, but
C. glutamicum also possesses additional biosynthesis pathways for
both L-lysine (Schrumpf et al., 1991) and L-methionine (Rückert et al.,
2003). L-threonine can be consumed to synthesize L-isoleucine and
glycine in the cell, or be exported out of the cell.
2.1. Production of L-threonine
Aspartate kinase, the first key enzyme in the L-threonine
biosynthesis pathway, phosphorylates aspartate to form aspartyl-P,
thus, serving to direct the carbon flux into the aspartate family of
amino acids.
In E. coli, three aspartate kinase isoenzymes are known, designated
aspartate kinase I, II and III, differing in their genetic determination and
regulation mechanisms (Chassagnole et al., 2001; Viola, 2001).
Aspartate kinase I, the most abundant of the three enzymes, is encoded
by the thrA gene. The expression of thrA is repressed by L-threonine and
L-isoleucine in a covalent manner and the activity of aspartate kinase I is
feed-back inhibited by L-threonine. Aspartate kinase II, the least
abundant isoform, is encoded by the metL gene. The expression of
metL is repressed by L-methionine but the activity of aspartate kinase II
is not inhibited by any amino acid from the aspartate family. Aspartate
kinase III is the product of the lysC gene. The expression of lysC is
repressed by L-lysine and the activity of aspartate kinase III is inhibited
also by L-lysine. Aspartate kinase I and II are bifunctional enzymes with
two catalytic domains, one for aspartate kinase activity and the other for
homoserine dehydrogenase activity (Katinka et al., 1980). Therefore,
they are also named as aspartate kinase I–homoserine dehydrogenase I
and aspartate kinase II–homoserine dehydrogenase II.
In C. glutamicum, aspartate kinase is encoded only by the lysC gene.
Its activity is synergistically inhibited by L-threonine and L-lysine
(Shiio and Miyajima, 1969). Although the enzymes involved in the
biosynthesis pathway of L-threonine are believed to have no
isoenzyme components, deletion of lysC was detrimental but not
lethal to a C. glutamicum strain grown on minimal media (Jetten et al.,
1995).
In both E. coli and C. glutamicum, aspartyl semialdehyde dehydrogenase is encoded by the asd gene. It catalyses deoxidization of
aspartyl-P to form aspartyl semialdhyde (Fig. 1). Expression of asd in
E. coli is subjected to multivalent repression by L-lysine, L-threonine
and L-methionine (Boy and Patte, 1972) but there is no evidence of
feed-back inhibition of its protein product. In C. glutamicum, the
expression of asd is not influenced by any of the aspartic amino acids,
nor is the activity of its product (Cremer et al., 1988).
Homoserine dehydrogenase is the second key enzyme of the
pathway, controlling carbon flux toward L-homoserine synthesis
(Fig. 1). It competes for a common substrate, aspartyl semialdehyde,
with dihydrodipicolinate synthase, which catalyses the first step of
the L-lysine biosynthesis branch. In E. coli, as mentioned above, two
isoenzymes of homoserine dehydrogenase are known, both of which
are present as catalytic domains in the bifunctional aspartate kinase
I–homoserine dehydrogenase I and aspartate kinase II–homoserine
dehydrogenase II. Homoserine dehydrogenase I is subjected to
inhibition by L-threonine (Chassagnole et al., 2001). The level of
homoserine dehydrogenase II has been seldom analyzed because
aspartate kinase II–homoserine dehydrogenase II is present only in
small amounts in vivo. In C. glutamicum, homoserine dehydrogenase is
encoded by hom (Follettie et al., 1988). Expression of hom is slightly
feed-back repressed by L-methionine (Miyajima and Shiio, 1971)
Please cite this article as: Dong X, et al, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of Lthreonine, Biotechnol Adv (2010), doi:10.1016/j.biotechadv.2010.07.009
X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
3
Fig. 1. The biosynthesis pathway of L-threonine. The pathway consists of five enzymatic steps. The first, third, and fourth reactions are catalyzed by the three key enzymes aspartate
kinase, homoserine dehydrogenase, and homoserine kinase, respectively. These enzymes are subjected to feed-back inhibition by L-threonine. There are four competing pathways
that affect the biosynthesis and accumulation of L-threonine, leading to formation of L-lysine, L-methionine, L-isoleucine, and glycine.
and activity of homoserine dehydrogenase is feed-back inhibited by
L-threonine (Miyajima and Shiio, 1970).
The conversion of L-homoserine to L-threonine is carried out by
homoserine kinase and threonine synthase. Homoserine kinase
phosphorylates homoserine to form homoserine-P which is then
dephosphorylated by threonine synthase to form L-threonine (Fig. 1).
Homoserine kinase is the third key enzyme of the pathway; it controls
carbon flux toward L-threonine synthesis. It competes for the common
substrate, homoserine, with acetyltransferase, the first enzyme of the
L-methionine branch. In both E. coli and C. glutamicum, homoserine
kinase is encoded by the thrB gene and threonine synthase by the thrC
gene. In E. coli, thrB and thrC are clustered with thrA in the thr
operon, and their expression is subjected to covalent repression by
L-threonine and L-isoleucine (Theze and Saint-Girons, 1974). The activity
of homoserine kinase is inhibited by L-threonine and L-isoleucine,
whereas the activity of threonine synthase is not subjected to feed-back
inhibition (Chassagnole et al., 2001). In C. glutamicum, expression of thrB
is slightly feed-back repressed by L-methionine and the activity of
Please cite this article as: Dong X, et al, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of Lthreonine, Biotechnol Adv (2010), doi:10.1016/j.biotechadv.2010.07.009
4
X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
Table 1
Enzymes and their coding genes related to the production of L-threonine.
Enzymes
Genes
Function
Aspartate kinase
thrA; metL; lysC
Aspartyl semialdehyde dehydrogenase
Homoserine dehydrogenase
asd
thrA; metL; hom
Homoserine kinase
Threonine synthase
Threonine dehydratase
thrB
thrC
tdcB; ilvA
Threonine dehydrogenase
Threonine aldolase
Serine hydroxymethyl transferase
Permease
tdh
ltaE
glyA
rhtA; rhtB; rhtC; thrE
Phosphorylate aspartate to form aspartyl-P. There are three coding genes
(thrA, metL, lysC) in E. coli, but only one (lysC) in C. glutamicum.
Deoxidize aspartyl-P to form aspartyl semialdehyde.
Remove the carboxyl group of aspartyl semialdehyde. There are two coding
genes (thrA; metL) in E. coli, but only one (hom) in C. glutamicum.
Add a phosphate group to homoserine to form homoserine-P.
Remove the phosphate group of homoserine-P to form threonine.
Consume threonine to produce isoleucine. There are two
coding genes (tdcB; ilvA) in E. coli, but only one (ilvA) in C. glutamicum.
Consume threonine to produce glycine in E. coli.
Consume threonine to produce glycine in E. coli.
Consume threonine to produce glycine in C. glutamicum.
Transport threonine from inside to the outside of cell. There are three coding
genes (rhtA; rhtB and rhtC) in E. coli, but only one (thrE) in C. glutamicum.
homoserine kinase is feed-back inhibited by L-threonine, whereas
threonine synthase is not subjected to feed-back regulation by any
amino acid from the aspartic family (Follettie et al., 1988).
2.2. Intracellular consumption of L-threonine
L-threonine is the precursor for the synthesis of L-isoleucine and
glycine. The first enzyme in the biosynthesis pathway of L-isoleucine
from L-threonine is threonine dehydratase. In C. glutamicum threonine
dehydratase is encoded by the ilvA gene (Mockel et al., 1992). In
addition to ilvA (Umbarger and Brown, 1957), E. coli has another gene,
tdcB, encoding threonine dehydratase, which is expressed under
anaerobic conditions (Umbarger, 1973). The glycine biosynthesis
pathways differ between E. coli and C. glutamicum. In C. glutamicum, a
side activity of serine hydroxymethyl transferase encoded by the glyA
gene fulfills the function of cleaving L-threonine directly into glycine
and acetaldehyde (Simic et al., 2002). The main substrate of this enzyme
is L-serine with which the cleavage activity is 24-fold higher than
with L-threonine. In E. coli, two enzymes, threonine dehydrogenase
encoded by the tdh gene (Bell and Turner, 1976) and threonine
aldolase encoded by the ltaE gene (Liu et al., 1998), have been found
to be responsible for conversion of L-threonine to acetaldehyde and
glycine. However, only when the cellular glycine is lacking will the
threonine aldolase function as a compensatory force.
2.3. Export of L-threonine
Several studies have demonstrated that secretion could be the
limiting factor in amino acid production (Morbach et al., 1996;
Reinscheid et al., 1994). In C. glutamicum, export of L-threonine could
be proceeded by both passive diffusion and carrier-mediated
excretion, the latter accounting for more than 90% of the total efflux
(Palmieri et al., 1996). To date, five permeases have been found in
E. coli (Eggeling and Sahm, 2003). They confer resistance to high
concentration of L-threonine to the producing strains. However, only
three of the permeases show activity in exporting L-threonine out of
cell. They are RhtA, RhtB and RhtC encoded by genes rhtA, rhtB and
rhtC, respectively (Livshits et al., 2003; Kruse et al., 2002). The former
is believed to be a member of the drug/metabolite transporter super
family, while the latter two belong to the RhtB translocator super family
(Eggeling and Sahm, 2003). RhtA catalyses efflux of both L-threonine
and L-homoserine, while RhtB and RhtC are specific exporters for
L -threonine (Diesveld et al., 2009). RhtC has a higher activity than
RhtB. So far, ThrE is the only identified protein showing activity for
exporting L-threonine in C. glutamicum (Simic et al., 2001). It is
encoded by the thrE gene, and can export both L-threonine and L-serine.
ThrE has a low affinity with L-threonine, suggesting its main function
might not be as an exporter for L-threonine.
3. Regulation mechanism of L-threonine biosynthesis pathway
Advances in molecular biology have made the study of metabolic
regulation of amino acid biosynthesis possible at the molecular level. So
far, the mechanisms of feed-back repression of the expression of thrA
and lysC in E. coli, and hom and thrB in C. glutamicum have been
provisionally explained at the genetic level (Grundy et al., 2003; Mateos
et al., 1994; Theze and Saint-Girons, 1974); the mechanisms of feedback inhibition of aspartate kinase III in E. coli and aspartate kinase in
C. glutamicum have been elucidated upon their three-dimensional
protein structures (Kotaka et al., 2006; Yoshida et al., 2007).
Such information serves as a theoretical basis of rational enzyme
modification. Thus allosteric modulation of enzymes could be done
using a genetic approach so that they lose sensitivity to inhibitors
while their activity is unaffected or even enhanced by precise position
modification. Nevertheless, some loss of enzymatic activity was
frequently observed as was also the case in modifying substrate
specificity. The determination of the three-dimensional enzyme
structure from which precise binding sites for small molecule effectors
can be identified may also be helpful in providing positive outcomes.
3.1. Mechanisms of repression
The covalent repression of expression of E. coli thrA by L-threonine
and L-isoleucine can be ascribed to the transcriptional attenuation of the
thr operon, which consists of three structural genes thrA, thrB and thrC
(Theze and Saint-Girons, 1974). A leader sequence thrL containing 178
base pairs precedes the first structural gene thrA. An internal region of
the leader sequence potentially encodes a short peptide with eight
threonine codons and four isoleucine codons, eleven of which are
arranged in tandem. The downstream region of the tandem codons
could participate in the formation of a stem and loop structure by base
pairing (Fig. 2). When the intracellular L-threonine and L-isoleucine are
in excess, ribosome can read through the leader sequence and the stem
and loop structure of a terminator forms, so the transcription of
thr operon is pre-terminated (Fig. 2A). By contrast, when L-threonine
and L-isoleucine are lacking, translation of the short peptide stalls, and
an alternative stem and loop structure forms, preventing the formation
of a terminator, so the transcription of thr operon can proceed (Fig. 2B).
Mutation of a G insertion at position −37 was believed to change the
stability of the terminator, and was confirmed to cause derepression
(Gardner and Reznikoff, 1978; Gardner, 1979).
Expression of E. coli lysC is also repressed by L-lysine through
translational attenuation. When L-lysine is in excess, the leader region
preceding the structural gene lysC can form six helixes via internal
base pairing during transcription (Fig. 3A). Helix 6 contains the ShineDalgarno (SD) sequence and prevents the binding of the ribosome to
mRNA, so that the translation of lysC cannot be initiated (Fig. 3A). On
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X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
5
Fig. 2. Transcriptional attenuations of the thr operon in E. coli. Secondary structures of the leader sequence of mRNA of the thr operon are proposed. The leader sequence encodes a
short peptide containing tandem threonine and isoleucine codons. A. When the intracellular L-threonine and L-isoleucine are in excess, ribosome can read through the leader
sequence and the stem and loop structure of a terminator forms, so the transcription of thr operon is pre-terminated. B. When L-threonine and L-isoleucine are lacking, translation of
the short peptide stalls, and an alternative stem and loop structure forms, preventing the formation of a terminator, so the transcription of thr operon can proceed. The tandem
threonine and isoleucine codons and the stop codon in the leader sequence are underlined. The regions which alternatively participate in the formation of the terminator or the
antiterminator are also underlined. These models were built by using the RNAfold web server (Gruber et al., 2008; Hofacker et al., 1994) and the published information (Gardner
1979).
the other hand, when L-lysine is deficient, the helix 1 and the helix 6
are released and a new helix 7 is formed. The SD sequence now is
available, and therefore the translation of lysC can proceed (Fig. 3B)
(Grundy et al., 2003).
In C. glutamicum, hom and thrB are clustered in the same operon in
the direction of 5′-hom-thrB-3′ on the chromosome with an internal
non-coding space of 10 bp. The feed-back repression of the expression
of hom and thrB by L-methionine might be ascribed to a transcriptional
attenuation-like regulation of the hom-thrB operon, because there is a
long reversibly repeated sequence upstream of hom, which was apt to
form a stem structure with a △G of -16.2 kJ/mol (Mateos et al., 1994).
Expression of the genes subjected to such regulation may need
additional regulatory proteins (Henkin and Yanofsky, 2002; Mateos
et al., 1994). Detailed analysis revealed existence of two promoters in
the hom-thrB operon. The one upstream of hom was believed to initiate
co-transcription of hom and thrB in C. glutamicum. The other promoter
inside the coding region of hom might be functional for individual
transcription of thrB. Transcription from both promoters is arrested by
the same terminator downstream of thrB. From this it was inferred that
the transcription of thrB might be more efficient than that of hom
(Mateos et al., 1994). Subsequent results reported by Diesveld et al.
(2009), however, reached the opposite conclusion. Thus, the transcription mode of hom-thrB operon remains unclear, and the evidence
of transcriptional attenuation needs further investigation.
3.2. Mechanisms of inhibition
E. coli aspartate kinase I–homoserine dehydrogenase I is a homotetramer and each peptide chain contains two catalytic domains, with
the kinase site residing on the N-terminal region and the dehydrogenase
Fig. 3. Translational attenuation of the lysc gene in E. coli. Secondary structures of the leader sequence of mRNA of the lysc gene are proposed. A. When L-lysine is in excess, the leader
region preceding the structural gene lysC can form six helixes during transcription. Helix 6 contains the SD sequence and prevents the binding of the ribosome to mRNA, so the
translation of lysC can not be initiated. B. When L-lysine is deficient, helix 1 and helix 6 are released and a new helix 7 is formed. The SD sequence now is available, and therefore the
translation of lysC can proceed. The SD sequence and the sequence which alternatively participates in the formation of helix 1 or helix 7 are underlined. These structural models were
built by using the RNAfold web server (Gruber et al., 2008; Hofacker et al., 1994) and the published information (Grundy et al. 2003).
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X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
site residing on the C-terminal region. Between the two catalytic
domains an interphase region is located, which is responsible for
allosteric regulation by L-threonine (Fazel et al., 1983). Aspartate kinase
I is partially inhibited by L-threonine, and the inhibitory mechanism is
allosteric and competitive with L-aspartate. Homoserine dehydrogenase
I is also partially inhibited by L-threonine, but the inhibitory mechanism
is non-competitive (Chassagnole et al., 2001). Previously it was reported
that the inhibition of homoserine dehydrogenase I was mediated
through the aspartate kinase I domain (Truffa-Bachi et al., 1974), but
subsequently James and Viola (2002a) constructed chimeric enzymes
and found that monofunctional aspartate kinase I was released from
L-threonine inhibition and monofunctional homoserine dehydrogenase I also lost sensitivity to L-threonine inhibition. Homoserine
dehydrogenase I with an interphase region, however, became even
more sensitive to L-threonine inhibition. Furthermore, confusion of
aspartate kinase III and homoserine dehydrogenase I containing an
interphase region conferred on homoserine dehydrogenase I an additional sensitivity to L-lysine with its L-threonine sensitivity retained (James and Viola, 2002a). Thus the mechanism of inhibition
remains ambiguous until a high-resolution structure of aspartate
kinase I–homoserine dehydrogenase I is available.
The inhibition of E. coli aspartate kinase III by L-lysine is complete,
and the inhibitory mechanism is non-competitive and co-operative
(Chassagnole et al., 2001). The functional E. coli aspartate kinase III is a
homodimer (Fig. 4A). The N-terminal region of each subunit functions
as a catalytic domain, containing binding sites for L-aspartate (Thr-45,
Glu-119, Arg-198, Ser-201) and ATP (Lys-8, Gly-11, Ser-39, Thr-221,
Asp-222, Tyr-227, Arg-232, Lys-257). Inadequate contact of L-aspartate
with E. coli aspartate kinase III is responsible for its overlapping
substrate specifity. The C-terminal region serves as a regulatory domain,
containing two ACT domains, ACT1 (residues 308-384) and ACT2
(residues 386-439) which are arranged perpendicularly. The ACT
(Acronym for aspartate kinase, Chorismate mutase and TyrA) domain
is structurally conserved and involved in binding small-molecule
regulatory ligands found in functionally diverse proteins (Chipman
and Shaanan, 2001). The interface of the two ACT1 domains from
different subunits shapes binding sites for two L-lysine molecules
(Met-318, Ser-321, Phe-324, Leu-325, Ser-338, Val-339, Asp-340, Ser345, Glu-346) (Fig. 4). Mutations of T344M, S345L, T352I have been
confirmed to be associated with partial L-lysine resistance. ACT2 which
does not bind any effector molecule stabilizes the dimer and transmits an
L-lysine
binding signal to the catalytic domain. The mechanism of
inhibition involves tetramerization of two dimers resulting from L-lysine
binding via hydrogen bond of N-terminal (Leu-104, Ser-107) and
concomitant allosteric transition of both regulatory and catalytic
domains. These results in blockage of the ATP binding site and eventually
activity lost. Furthermore, the allosteric transition disrupts one hydrogen
bond at the L-aspartate binding site, which does not affect the substrate
binding ability but may reduce conversion rate (Kotaka et al., 2006).
In E. coli, the entire amino acid sequence of aspartate kinase III is
homologous to the N-terminal region of aspartate kinase I–homoserine
dehydrogenase I and aspartate kinase II–homoserine dehydrogenase II,
and the identity between aspartate kinase III and aspartate kinase
I–homoserine dehydrogenase I is higher than that between aspartate
kinase III and aspartate kinase II–homoserine dehydrogenase II (Cassan
et al., 1986). Bioinformatic analysis revealed that aspartate kinase III
might be the prototype of aspartate kinase I and aspartate kinase II
(Fondi et al., 2007). The duplication of lysC and the following fusion
between the redundant copy of lysC and the gene encoding homoserine
dehydrogenase might give rise to a bifunctional aspartate kinase–
homoserine dehydrogenase; then the duplication and divergence of the
prototype gene encoding the aspartate kinase–homoserine dehydrogenase might give rise to thrA and metL. Conserved residues of aspartate
kinase I, II and III have been identified. They are Lys-8 and Asp-202 for
phosphoryl transferation, Thr-45, Glu-119 and Arg-198 for L-aspartate
binding, Asp-222 and Arg-232 for ATP binding (Kotaka et al., 2006).
The inhibition of E. coli homoserine kinase is complicated. It is
inhibited by substrate, L-homoserine, in the concentration of more
than 1 mM and ATP of more than 3 mM in a hypothetic “preferred
order” manner, by L-threonine in a competitive manner, and by L-lysine
in a non-competitive manner (Chassagnole et al., 2001). So far, no
molecular mechanism has been proposed.
In C. glutamicum, aspartate kinase is a heterotetramer, comprised of
two α subunits and two β subunits. The two types of subunits are
encoded by a single gene lysC, containing an in-frame sequence overlap
(Kalinowski et al., 1990). The N-terminal region of the α subunit
functions as the catalytic domain, while the C-terminal region together
with the β subunit serves as a regulatory domain (Kato et al., 2004). Each
β subunit contains two ACT domains, ACT1 and ACT2, which are
arranged perpendicularly to each other (Fig. 5A). Association of an ACT1
from one β subunit and an ACT2 from the other forms an L-threonine
binding site (Ile-23, Asp-25, Pro-27, Gly-28, Ala-30, Ala-31, Gln-49,
Fig. 4. The detailed structure of L-lysine binding site in the E. coli aspartate kinase III. A. The dimer structure of the regulatory domains is shown. ACT1 and ACT2 domains of chain A
are shown in green and yellow, respectively. ACT1 and ACT2 domains of chain B are shown in blue and purple, respectively. Atoms of the regulatory effector L-lysine are shown in red.
B. The amino acid residues involved in binding L-lysine are shown in the manner of sticks. Residues from chain A and chain B are shown in green and cyan, respectively. The
bound L-lysine molecule is shown in orange. These models are built by using the PyMOL software, Protein Data Bank (accession number 2J0X) and the published information
by Kotaka et al. (2006).
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X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
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Fig. 5. The structure of L-threonine binding site in the β subunit of C. glutamicum aspartate kinase. A. The dimer structure of the β subunits is shown. ACT1 and ACT2 domains of chain
A are shown in yellow and green, respectively. ACT1 and ACT2 domains of chain B are shown in blue and purple, respectively. Atoms of the regulatory effector L-threonine are shown
in red. B. The amino acid residues directly involved in binding L-threonine are shown in the manner of sticks. Residues from chain A are shown in green and residues from chain B in
cyan. The bound L-threonine molecule is shown in orange. These structural models are built by using the PyMOL software, Protein Data Bank (accession number 2DTJ) and the
published information by Yoshida et al. (2007).
Thr-59, Ile-61, Val-124, Ile-126), causing dimerization of two β subunits
(Fig. 5B). Thus the dimer of β subunits has two L-threonine binding sites
and the binding of L-threonine stabilizes the dimer structure. The
L-lysine binding site has not been precisely characterised so far, but is
constrained to an area of around the residue 50 or the residues 105 to
115. Since the β subunit is identical to about 160 residues of C-terminus
of the α subunit, it was postulated that dimerization between the two
rather than homodimerization between two β subunits is the practical
situation responsible for inhibitory regulation. As for L-lysine, both α
subunits and β subunits may contain recognition sites for this amino
acid. The mechanism of inhibition may involve firstly the binding of
L-threonine to produce a stable dimerization of regulatory domains
followed by L-lysine binding leading to a conformational change in
the catalytic domains, causing loss of catalytic activity of aspartate
kinase. Yoshida et al. (2007) analyzed the L-lysine analog S-(2aminoethyl)-l-cysteine resistant C. glutamicum strains and found
that mutations were contained in the regulatory domains of aspartate
kinase.
In C. glutamicum, homoserine dehydrogenase is subjected to feedback inhibition by L-threonine. The wild type homoserine dehydrogenase can be completely inhibited by 2 mM L-threonine. Mutation of
homoserine dehydrogenase by replacing residue Gly378 with Glu
conferred L-threonine resistance (Reinscheid et al., 1991). It was not
inhibited by L-threonine even in concentrations of 25 mM, and the
concentration for half maximal inhibition was 100 mM. Furthermore,
the residue 378 was confirmed to be essential since both the size and
charge of the amino acid at that position affected the inhibitory
regulation of homoserine dehydrogenase. In addition, threonine binding site is located in the C-terminal region of the enzyme (Archer et al.,
1991). C. glutamicum homoserine kinase is also subjected to weak
feed-back inhibition by L-threonine with the concentration for a half
maximal inhibition of 25 mM for wild type (Colon et al., 1995).
Studies on the kinetics of enzymatic activity showed that the
inhibition by L-threonine was competitive and could be relieved as
the concentration of L-homoserine substrate increased (Miyajima et
al., 1968). Due to the competitive mechanism of inhibition that
substrate and inhibitor compete for the same binding site, it is really a
challenging task to construct a desensitized homoserine kinase by
changing its conformation through site mutagenesis, and yet not
affect the catalytic characteristics. The only reported strategy was to
increase homoserine accumulation by overexpression of hom (Colon
et al., 1995). Judging from this fact and from their genetic relationship,
homoserine dehydrogenase and homoserine kinase were postulated to
be functionally correlated (Follettie et al., 1988; Peoples et al., 1988).
4. Progress in strain construction
The strategies for construction of L-threonine producers can be
summarized as follows: (1) overexpressing the genes encoding the
key enzymes of the biosynthesis pathway to condense carbon influx,
(2) weakening the competing branches to save more available
precursors and reduce formation of unwanted products, (3) reducing
intracellular consumption, (4) enhancing L-threonine secretion,
(5) integrating (1) to (4) by systematic approaches.
Overexpressing the genes encoding the key enzymes of the
biosynthesis pathway, especially the deregulated ones, is usually the
most productive strategy. Nevertheless, the outcomes of this strategy
do not always meet with expectations. The efficiency of the approach
depends partly on the localization of the product and its role in
cellular metabolism. For example, application of this strategy only
resulted in a slight increase in L-serine accumulation (Wendisch et al.,
2005), but led to significant enhancements in L-lysine synthesis
(Cremer et al., 1991) and L-threonine production (Eikmanns et al.,
1991; Reinscheid et al., 1994). A computational analysis of the global
metabolic net of E. coli indicated that the L-threonine biosynthesis
pathway along with its conversion into glycine was a part of the highflux backbone of metabolism, which meant that L-threonine played a
crucial role in cellular metabolism (Almaas et al., 2004). Hartman
(2007) reported that the synthesis and uptake of L-threonine was of
great significance for Saccharomyces cerevisiae to maintain cell
stability, as L-threonine could be converted into glycine and
subsequently to initiate de-novo purine synthesis. Curien et al.
(2009) found that in Arabidopsis L-threonine played an integrative
regulatory role in L-aspartate metabolic distribution. All such
information theoretically supports the fact that overexpression of
the genes involved in biosynthesis efficiently leads to increased
L -threonine production, but due to its physiological importance,
accumulation of intracellular L-threonine is severely restricted. In practice, the increase of the genes expression and consequent L-threonine
production are mutually inconsistent (Colon et al., 1995; Eikmanns et
al., 1991).
When L-threonine accumulates to a certain level, the prompt
secretion becomes limiting for production. Even for Gram-negative
E. coli, whose cell envelope is not considered as a permeation barrier
for amino acids, it was reported that the intracellular concentration of
L-threonine exceeded the one observed in medium throughout the
fermentation process, with a 10-fold excess observed in early stages
of the process (Kruse et al., 2002). Gram-positive C. glutamicum
possesses a characteristic cell wall structure, containing an outer layer
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X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
of mycolic acids (Eggeling and Sahm, 2001), the amino acid efflux in
C. glutamicum is therefore significantly impaired. High intracellular
concentrations of L-threonine down-regulate the biosynthesis
enzymes in a feed-back manner, increase the precursor availability
for the consuming pathway, and even inhibit cell growth. Overexpressing the specific permease-coding genes not only accelerates
L-threonine export, conferring strains with L-threonine-resistant phenotype, but also contributes to reduced consumption, since L-threonine
permease and L-threonine consuming enzymes compete for the same
substrate (Simic et al., 2002). Reducing intracellular L-threonine
conversion towards L-isoleucine in E. coli with genotype of relA+ results
in activation of the thr operon via a “stringent response” mechanism in
addition to a derepression effect (Debabov, 2003).
A comprehensive strategy with integration of systems biology, also
referred to as systems metabolic engineering, gives rise to breakthroughs in strain construction. So far, a few E. coli L-threonine hyperproducers have been constructed by this means, and the total
conversion rate of glucose to L-threonine and biomass approximates
to the predicted theoretical values.
4.1. L-threonine producing strains of E. coli
4.1.1. Overexpressing the thr operon
As thrA, thrB, and thrC are clustered in the thr operon, and aspartate
kinase I encoded by thrA exists most abundantly among the three
aspartate kinases, engineering of the L-threonine branch has been
focused on this operon (Table 2). In 1983, Miwa et al. introduced a
recombinant plasmid pBR322-thrAr into a desensitized E. coli mutant
strain βIM4, as a result, L-threonine production was increased by
three-fold reaching 13.4 g/L (Miwa et al., 1983). In 2003, Livshits et al.
(2003) introduced a recombinant plasmid pVIC40 containing the
mutant operon thrA442BC into the desensitized E. coli strain MG442, as
a result, L-threonine production was increased from 8 g/L to 18.4 g/L.
In 2009, Zhang et al. introduced a high-copy number recombinant
plasmid pWYE134 containing a mutated operon thrA345BC under its
native promoter into the wild type strain E. coli W3110. Because the
thrL gene encoding the leader peptide was included, the expression of
the operon was subjected to feed-back repression by L-threonine
and L-isoleucine, but the activity of aspartate kinase I was relieved
from L-threonine inhibition. As a result, L-threonine production was
increased from 0.036 g/L to 9.2 g/L in fed-batch fermentation (Zhang
et al., 2009a).
4.1.2. Enhancing L-threonine export
Kruse et al. (2002) constructed E. coli MG422 (pTrc99A-rhtB),
MG422 (pTrc99A-rhtC) and MG422 (pTrc99A-thrECg) in which either a
homologous or a heterologous gene involved in export of L-threonine
was overexpressed. As a result, L-threonine production was increased by
140%, 200% and 290%, respectively, when compared with the control
strain MG422 (pTrc99A). Livshits et al. (2003) constructed E. coli MG422
rhtA23 (pAYC32-thrArBC), in which rhtA expression was enhanced by a
point mutation (G → A) one base upstream the start codon of rhtA in the
chromosome, L-threonine production was increased from 18.4 g/L to
36.3 g/L.
4.1.3. Systems metabolic engineering
In 2007, Lee et al. constructed an L-threonine hyper-producer,
TH28C (pBRThrABCR3), from a lacI- mutant strain of E. coli W3110 in
which promoters such as Ptac and Ptrc could initiate transcription
constitutively. Several strategies have been used in this construction.
First, the feed-back inhibitions of aspartate kinase I and III were
released through site-directed mutagenesis of their coding genes, thrA
and lysC in chromosome; the feed-back repression to the chromosomal thr operon was released by the promoter substitution with Ptac,
and the deregulated thr operon was overexpressed on an episomal
vector. Secondly, the biosynthesis of L-lysine was blocked by deleting
Table 2
Genetically engineered L-threonine producers.
Strains
Yield (g/L)
Reference
Overexpressing genes in L-threonine synthesis pathway
E. coli
13.4
βIM4(pBR322-thrAr)
18.4
MG422(pAYC32-thrArBC)
r
9.2
W3110(pMD19-thrLA BC)
C. glutamicum
r
DM368-3(pEK-hom -thrB)
1.7
7.7
MH20-22B-(homr-thrB)3
8.2
MH20-22B (pWK-homr-thrB)
ATCC21799(pGC42)
11.8
DM1800(pET-T18homr-thrB-thrE) 2.5a
Reducing L-threonine consumption
C. glutamicum
DM368-2glyA’
DM1800ilvAM
(pET-T18homr-thrB-thrE)
Enhancing L-threonine export
E. coli
MG422(pTrc99A-rhtB)
MG422(pTrc99A-rhtC)
MG422(pTrc99A-thrECg)
MG422 rhtA23(pAYC32-thrArBC)
C. glutamicum
MH20-22B-(homr-thrB)3
(pEC-T18mob2-thrE)
DM368-2glyA’(pEC-T18mob2-thrE)
DM368-3(pEKEx2-rhtCEc)
DM1800(pET-T18homr-thrB-thrE)
(pEKEx2-rhtCEc)
Systems metabolic engineering
E. coli
TH28C (pBRThrABCR3)
MDS-205
a
Miwa et al., 1983
Livshits et al., 2003
Zhang et al., 2009a
Eikmanns et al., 1991
Reinscheid et al., 1994
Reinscheid et al., 1994
Colon et al., 1995
Diesveld et al., 2009
1.3a
4a
Simic et al., 2002
Diesveld et al., 2009
(140% Increase)
(200% Increase)
(290% Increase)
36.3 (100% Increase)
Kruse et al., 2002
Kruse et al., 2002
Kruse et al., 2002
Livshits et al., 2003
8.1 (5% Increase)
Simic et al., 2002
a
1.5 (15% Increase)
3.7 (311% Increase)
6.4 (156% Increase)
Simic et al., 2002
Diesveld et al., 2009
Diesveld et al., 2009
82.4
40.1
Lee et al., 2007
Lee et al., 2009
: Grown on minimal media.
lysA, and the biosynthesis pathway of L-methionine was shut down by
deleting metX, and the L-threonine consumption was reduced by
deleting tdh and down-regulating threonine dehydratase activity
through site-directed mutagenesis. Thirdly, the following modifications were performed according to the transcriptome and in silico flux
analysis; (1) the promoter of ppc encoding the PEP carboxylase was
replaced by a stronger promoter Ptrc to enhance its expression; (2) the
iclR gene encoding the repressor of isocitrate lyase and malate synthase was knocked out to enhance the glyoxylate shunt; (3) the tdcC
gene encoding the uptake-carrier was deleted to cut down L-threonine
uptake; (4) genes rhtA, rhtB and rhtC were overexpressed on the same
episomal vector as for the thr operon to accelerate L-threonine secretion.
Fourthly, the native promoter of acs encoding acetyl-CoA synthetase
was substituted with the stronger promoter Ptrc to enhance its
expression. The constructed strain TH28C (pBRThrABCR3) could
produce 82.4 g/L L-threonine in 50 hours’ fed-batch fermentation. The
L-threonine /glucose conversion rate was 39.3%. No lactate was formed
and the accumulation of acetate was 2.35 g/L (Lee et al., 2007).
In 2009, Lee et al. constructed a plasmid-free L-threonine hyperproducer, MDS-205, from a reduced-genome strain E. coli MDS42. In
MDS-205, the thr operon together with its native promoter was
substituted with the L-threonine resistant thrArBC operon under the
stronger promoter Ptac on the chromosome, and the Ptac repressorcoding gene, lacI, was deleted. The tdh gene was deleted to reduce the
L-threonine consumption. The L-threonine uptake facilitator-coding
genes, tdcC and sstT, were substituted with a mutant exporter gene
rhtA23, which not only blocked the L-threonine uptake but also
enhanced its export. E. coli MDS-205 showed robust growth and
performed even better in high cell-density fermentations. It could
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produce 40.1 g/L L-threonine in 30 hours’ batch-fermentation. The
L-threonine /glucose conversion rate reached 39.3% (Lee et al., 2009).
4.2. L-threonine producing strains of C. glutamicum
4.2.1. Overexpressing genes in the L-threonine biosynthesis pathway
In C. glutamicum, overexpression of lysC or its L-threonine resistant
allele lysCr, can only achieve high L-lysine production, but not L-threonine
(Cremer et al., 1991). C. glutamicum strains carrying a lysC r on the
chromosome were proven to be appropriate base strains for metabolic
engineering, because adequate carbon-influx into the biosynthesis
pathways of aspartic family of amino acids is the very prerequisite
(Colon et al., 1995; Eikmanns et al., 1991; Reinscheid et al., 1994). Genes
lysC and asd are clustered on the chromosome. Mutagenesis of lysC
(S301Y) which conferred S-(2-aminoethyl)-l-cysteine-resistance on
aspartate kinase was found to enhance the downstream asd expression,
probably due to the accidental formation of a stronger internal promoter
(Kalinowski et al., 1991). But individual overexpression of asd on an
episomal vector had no effect on L-lysine production (Cremer et al.,
1991). In most cases, overexpression of hom and thrB in these base
strains resulted in increased L-threonine production and dramatically
reduced L-lysine accumulation.
Overexpression of hom or/and thrB can be effective in L-threonine
production only on the basis of sufficient carbon influx to the
synthesis of aspartic family of amino acids. In C. glutamicum (lysC r)
strains, overexpression of the L-threonine resistant homr allele
alone led to L-threonine accumulation; however, the intermediate
metabolite, L-homoserine, also accumulated. Although high concentrations of L-homoserine can relieve the competitive inhibition of
homoserine kinase (encoded by the downstream thrB) by L-threonine,
it has been shown that proportional overexpression of homr and thrB
could effectively contribute to L-threonine accumulation (Colon et al.,
1995; Reinscheid et al., 1994). Nevertheless, in respect of practical
operations, when the wild-type hom was overexpressed on a highcopy number plasmid, positive transformants were easy to obtain, but
as for the mutant homr, the transformation was particularly difficult.
The successful approach was to overexpress the homr allele
moderately by using a low-copy number plasmid or integrating
additional copies of the gene to the chromosome. The reason is not
clear yet, but it might be that certain intermediate metabolite
accumulation was toxic for cell growth (Reinscheid et al., 1994).
Eikmanns et al. (1991) introduced a recombinant plasmid
containing the homr-thrB operon into C. glutamicum DM368-3 (AECr,
AHVr). L-threonine production doubled from 0.8 g to 1.7 g. In 1994,
Reinscheid et al. integrated 3 additional copies of the homr-thrB
operon into the chromosome in the L-lysine producing strain
C. glutamicum MH20-22B. The constructed strain could produce
7.7 g/L L -threonine, and L -lysine production was dramatically
reduced from 30.4 g/L to 8.5 g/L. However, three by-products (2.5 g/
L L-homoserine, 4.1 g/L L-isoleucine, and 2.3 g/L glycine) were also
accumulated in the medium. The similar results were obtained when
the homr-thrB operon was overexpressed on a low copy number plasmid
in C. glutamicum MH20-22B (8.2 g/L L-threonine, 8.3 g/L L-lysine,
2.7 g/L L -homoserine, 4.5 g/L L -isoleucine, and 2.0 g/L glycine)
(Reinscheid et al., 1994).
In 1995, Colon et al. introduced a recombinant plasmid, on which the
homr allele was expressed constitutively under its native promoter and
thrB was expressed inductively under the Ptac promoter, into the L-lysine
producing strain C. lactofermentum ATCC21799 (AECr). The constructed
strain could produce 11.8 g/L L-threonine, and L-lysine production was
reduced dramatically from 22.0 g/L to 0.8 g/L. L-homoserine was not
accumulated in the medium (b 0.1 g/L), but glycine and L-isoleucine
accumulated up to 4.6 g/L and 1.9 g/L, respectively (Colon et al., 1995).
In 2009, Diesveld et al. constructed C. glutamicum DM1800-T by
introducing a recombinant plasmid, pET-T18homr-thrB-thrE, into the
L-lysine producing strain DM1800 (pyc P458S, lysC T311I). 2.5 g/L L-
9
threonine was produced in the medium originally containing 4%
glucose (Diesveld et al., 2009).
Overexpression of thrC only led to marginal increase in L-threonine
accumulation (Eikmanns et al., 1991).
4.2.2. Reducing the L-threonine consumption
In 2002, Simic et al. constructed C.glutamicum DM368-2glyA',
in which the promoter of glyA was mutated to weaken the conversion
of L-threonine to glycine. L-threonine production was increased to 1.3 g/L,
and glycine accumulation was reduced from 0.5 g/L to 0.3 g/L in minimal
media, compared with the parental strain C.glutamicum DM368-2 (lysC r,
homr ) (Simic et al., 2002).
To reduce the L-threonine consumption, the promoter of ilvA in
DM1800-T was mutated, resulting in a new strain, DM1800-96-T,
characterised by an increased L-threonine production from 2.5 g/L to
4 g/L (Diesveld et al., 2009).
4.2.3. Enhancing the L-threonine export
Introduction of a recombinant plasmid containing thrE into a
DM368-2glyA’ strain resulted in increase of L-threonine production
from 1.3 g/L to 1.5 g/L, and decrease of the glycine accumulation.
When the recombinant plasmid containing thrE was introduced into a
MH20-22B-(homr-thrB)3 strain, L-threonine production increased
from 5.8 g/L to 8.1 g/L with reduction in accumulation of L-lysine,
glycine and L-isoleucine (Simic et al., 2002).
In 2009, Diesveld et al. overexpressed the E. coli genes rhtA, rhtB,
rhtC and yeaS in different C. glutamicum strains. Except for rhtB, all
other genes resulted in the increased L-threonine production. The
best result was obtained when rhtC gene was expressed which
increased L-threonine production in C. glutamicum DM368-3 (AECr,
AHVr) from 0.9 g/L to 3.7 g/L and in DM1800 (pET-T18homr-thrB-thrE)
from 4 g/L to 6.4 g/L without L-homoserine accumulation (Diesveld et
al., 2009).
5. Outlook
5.1. Engineering the pathway
Systems biology opens up a new chapter for strain construction, that
is “post genome study” (Lee et al., 2005). The approach, including
transcriptomics, proteomics, metabolomics and computational modeling, etc, is more efficient and more comprehensive than the traditional
“step-by-step” molecular biology. Numerous L-threonine producers
have been developed using conventional breeding approaches. Though,
these strains are not sufficiently productive or are deficient in other
respects. “Post genome study” of these strains directly unveiled the key
factors influencing L-threonine synthesis, some of which were inconceivable by conventional methods/knowledge. Combining practical
experience with theoretical analysis provides a more rational basis for
the selection of essential gene targets to be engineered. For example,
through transcriptomic analysis, the glyoxylate shunt has been found to
play a role in L-threonine synthesis (Lee et al., 2003; Lee et al., 2007),
which was further confirmed by experiments (Lee et al., 2007). In E. coli
this anaplerotic pathway is not turned on under aerobic conditions on
glucose. Through proteomic analysis, Kim and co-workers found that
the accumulation of precursor contributed significantly to the increased
production of L-threonine (Kim et al., 2004), which was consistent with
practice. Through in vitro modeling, Chassagnole and co-workers
determined kinetic parameters and interactions between intermediates
and catalyzing enzymes throughout the L-threonine biosynthesis
pathway under realistic cellular conditions (Chassagnole et al., 2001),
which suggested basis for predicting metabolic flux changes that may be
caused by genetic manipulations. Through the analysis of an in silico
kinetic model of E. coli, Rodríguez-Prados and co-workers suggested that
the deletion of pyruvate kinase was sufficient to increase L-threonine
production, with up-modulation of phosphotransferase system,
Please cite this article as: Dong X, et al, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of Lthreonine, Biotechnol Adv (2010), doi:10.1016/j.biotechadv.2010.07.009
10
X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
homoserine dehydrogenase, aspartyl semialdehyde dehydrogenase and
aldolase (Rodríguez-Prados et al., 2009).
Nevertheless, most of such systematic information has been
acquired from E. coli L-threonine producers. Thus, systems metabolic
engineering has only been applied to constructing L-threonine producers of E. coli strains. On the other hand, systems biology approaches
have only recently been applied to C. glutamicum because of limited
availability of tools. L-lysine producing strains of C. glutamicum have
been intensively analyzed and improved by utilizing post genome
technologies (Krömer et al., 2004; Sindelar and Wendisch, 2007).
Furthermore, L-valine hyper-producers of C. glutamicum have already
been successfully developed by successive genetic manipulations
(Blombach et al., 2008). Lack of strong promoters in C. glutamicum
apparently hinders the development of L-threonine producers. The most
widely used promoters, Ptac and Ptrc, employed in gene overexpression
were shown to function efficiently in C. glutamicum (Nesvera and Pátek,
2008) but to a much weaker extent than in E. coli. Some relevant
biological information has just been discovered in recent years such as
identification of glyA (Simic et al., 2002), novel pathways for L-lysine
(Schrumpf et al., 1991) and L-methionine (Rückert et al., 2003)
synthesis branches, and regulation of TCA and glyoxylate cycles in
C. glutamicum (Bott, 2007; Han et al., 2008). The glyA gene, for example,
whose product mainly fulfills methyltransferation from L-serine to
tetrahydrofolic acid, represents the generation of active C-1 block
molecules for cellular activities and is essential for cell growth because
its deficiency causes growth retardation (Wendisch et al., 2005). The
reason why C. glutamicum employs both split pathways for the L-lysine
and L-methionine branches remains obscure and so far no modifications
aiming at limitation of the two branches, so as to increase L-threonine
production, have yet been reported. Thus, to construct a genetically
determined L-threonine hyper-producer of C. glutamicum, a more
demanding strategy is required.
5.2. Enzymes engineering
To date, the widely adopted enzyme modification schemes can be
divided into two categories. One is to sequence the coding gene for a
proven inhibition resistant homolog, identifying positive mutations by
sequence comparison with the wild-type allele, and then only apply
positive mutations to the wild-type allele to obtain a desensitized
mutant. The other is to utilize bioinformatic methods to analyze
sequences of the alleles from different species to roughly identify
the location of the regulatory and catalytic domains according to the
principle of sequence conservation, and then mutate or delete the
deduced coding sequence for the regulatory domain with the
expectation that the result will be a desensitized mutant. Significant
achievements have been made in applying both schemes.
In the future, enzymes engineering based on synthetic biology will
be a more rational strategy for novel strain constructions than simply
overexpressing or knocking out the coding genes (Heinemann and
Panke, 2006; Picataggio, 2009). Some key enzymes in L-threonine
biosynthesis pathway, such as aspartate kinase III and aspartyl
semialdehyde dehydrogenase of E. coli, and aspartate kinase of
C. glutamicum, have been purified and their structures analyzed
(Hadfield et al., 1999; Kotaka et al., 2006; Yoshida et al., 2007). Based
on the structure information, biochemical characteristics such as Km,
Ki and Kcat of the key enzymes can be modified by genetic
manipulation to optimize the L-threonine production. In addition, it
was proposed that “substrate channeling”, a process in which the
intermediate produced by one enzyme is directly transferred to the
next enzyme or active site without being released into the solution
(Spivey and Ovádi, 1999), might be the trigger of evolution of E. coli
aspartate kinase I–homoserine dehydrogenase I and aspartate kinase
II–homoserine dehydrogenase II (Fondi et al., 2007); in vitro
“substrate channeling” has been developed for oxaloacetate between
aspartate aminotransferase and malate dehydrogenase (Anderson,
1999), and for aspartyl-P between aspartate kinase and aspartyl
semialdehyde dehydrogenase (James and Viola, 2002b), suggesting
that it might be possible to construct artificial, bifunctional enzymes
in vivo for high catalytic efficiency.
5.3. Improving production stability
The greatest disadvantage of genetically engineered producing
strains is that their productivity is unstable, usually because of the loss
of recombinant plasmids (Nudel et al., 1989; Wang et al., 1999). This is
especially the case with some specific genes, the overexpression of
which lead to accumulation of metabolites unfavorable for cell growth
(Reinscheid et al., 1994). Thus in the fermentation course of a
plasmid-containing producing strain, the presence of antibiotics may
be required to provide selective pressure to maintain the plasmid
stability. This is obviously undesirable for industrial production and
problematic for food/drug-grade L-threonine production. Without
selective pressure stringent requirements for cultivation, such as a
strictly defined temperature (Wang et al., 1999), may be demanded.
The conventional way to solve this problem is to delete a certain nonessential gene on the chromosome, then express the same gene on the
plasmid, e.g. thrC, or knock out a wild-type gene and introduce a
truncated-promoter allele of the gene to increase copy-number of an
insert, or reduce the flux of the downstream pathway, i.e. knockingout ilvA (Debabov, 2003). These methods have been proven to be
effective for maintaining plasmid stability, due to the intrinsic need of
the cells for enough L-threonine and L-isoleucine for normal
propagation. These strategies are well suited to the concept of
engineering physiological functionality, the importance of which has
recently been reviewed (Zhang et al., 2009b).
As the technologies of gene deletion and replacement have been
highly developed, and promoters of different strengths have been
widely studied, both in E. coli and C. glutamicum (Pátek et al., 1996), gene
overexpression can now also be realized by chromosomal engineering
without introducing any heterologous gene mark. Thus derepression
can be achieved by promoter substitution, and disinhibition can be
carried out by gene replacement with a mutant allele. Since an increase
in enzyme activity is not necessarily proportional to copy number, and
nor is production to enzymatic activity, plasmid-free strategies may
be considered. Some successful cases have been reported. A plasmidfree L-lysine producer of C. glutamicum has been constructed by
chromosomal mutation of lysC plus up-modulation of the dapA
promoter (de Graaf et al., 2001). Furthermore, a plasmid-free L-valine
producer of C. glutamicum has been constructed by chromosomally upand down-modulation of promoters of related genes together with gene
knockout (Holátko et al., 2009), in addition to the L-threonine producer
of E. coli MDS-205 (Lee et al., 2009).
5.4. Extending the substrate spectrum
Extending the substrate spectrum of producing strains is industrially desirable to reduce the cost and increase production flexibility.
Oligosaccharide and polysaccharide substrates are more abundant
and cheaper than the directly usable monosaccharides. However,
strains of different species possess different sugar uptake and
metabolic systems, and are not able to utilize all kinds of sugars. As
for E. coli, glucose, lactose, and succinate, etc, can be utilized as carbon
and energy source, but sucrose is occasionally excluded (Bockmann
et al., 1992; Jahreis et al., 2002). In the case of C. glutamicum, glucose,
myo-inositol and ethanol, etc, can be utilized, but lactose is excluded
(Arndt and Eikmanns, 2008; Arndt et al., 2008; Krings et al., 2006). In
addition, both these species can not directly utilize polysaccharides.
Genetically engineering the sugar uptake and metabolic system of
the producing strains by introducing homologous or heterologous
genes can confer an extended substrate spectrum on particular strains
(Adham et al., 2001; Jojima et al., 2010; Jolkver et al., 2009; Rittmann
Please cite this article as: Dong X, et al, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of Lthreonine, Biotechnol Adv (2010), doi:10.1016/j.biotechadv.2010.07.009
X. Dong et al. / Biotechnology Advances xxx (2010) xxx–xxx
et al., 2008). A few successful trials in this direction have been
reported. An L-threonine producer of an E. coli strain which was
initially unable to assimilate sucrose acquired such ability after being
equipped with a sucA-sucB operon (Debabov, 2003). An L-lysine
producer of C. glutamicum strain was able to utilize lactose as the
carbon and energy source by introducing the heterologous lactose
permease and β-galactosidase coding genes of L. delbrueckii subsp.
Bulgaricus (Barrett et al., 2004). Another L-lysine producer of
C. glutamicum was even able to directly utilize homopolysaccharide
after transfection with the α-amylase gene, amy, of Streptomyces
griseous (Seibold et al., 2006).
6. Conclusion
In this review, which focused on the main L-threonine producing
species, E. coli and C. glutamicum, we summarized the genetic and
enzymatic information related to the L-threonine production, as well
as the strategies employed in strain construction by presenting
representative studies. All the above-mentioned strains are defined
genetically. Although most of the genetically engineered L-threonine
producers are less competitive in productivity than conventionally
mutated and selected clones in which L-threonine productivity could
be as high as 100 g/L in E. coli and 57.7 g/L in C. glutamicum carrying a
recombinant plasmid (Ikeda, 2003), knowledge generated from these
studies is of considerable significance to better understand cellular
metabolism and for future strain development, especially for
construction of L-threonine producers with minimum genetic alterations from the wild-type strains.
At present, E. coli strains dominate as L-threonine producers, with
their advantage of robust growth. As comprehensive understanding
(Feist and Palsson, 2008) and discovery of new methods (Tyo et al.,
2009) proceed, there is still scope for improvement of highly
productive E. coli strains. Nevertheless, E. coli strains are not suitable
for pharmaceutical-grade amino acid production because they
synthesize endotoxins (Wang and Quinn, 2010). With the rapid
development of tools applicable for C. glutamicum (Kirchner and
Tauch, 2003; Xu et al., 2010), e.g. Kanamycin/SacB double selective
system (Schäfer et al., 1994), Cre/loxP knockout and integration
system (Suzuki et al., 2005; Suzuki et al., 2007), and even a data
warehouse, CoryneRegNet, facilitating post genome study (Baumbach
et al., 2006), the food-safety-grade C. glutamicum strains show
considerable promise as industrial L-threonine producers.
Acknowledgments
This work was supported by Chinese 863 National High-Tech
Research and Development Plan Project (No. 2007AA02Z229 and
No. 2007AA02Z230), the 111 Project (No. 111-2-06) and the Basic
Research Programs of Jiangsu Province (BK2009003).
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Please cite this article as: Dong X, et al, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of Lthreonine, Biotechnol Adv (2010), doi:10.1016/j.biotechadv.2010.07.009