Adv Biochem Eng Biotechnol (2017) 159: 129–152

DOI: 10.1007/10_2016_29
© Springer Japan KK 2016
Published online: 22 November 2016

L-CysteineMetabolism and Fermentation

in Microorganisms

Hiroshi Takagi and Iwao Ohtsu

Abstract L-Cysteine is an important amino acid both biologically and commer-

cially. Although most amino acids are industrially produced by microbial fermen-
tation, L-cysteine has been mainly produced by protein hydrolysis. Due to
environmental and safety problems, synthetic or biotechnological products have
been preferred in the market. Here, we reviewed L-cysteine metabolism, including
biosynthesis, degradation, and transport, and biotechnological production (includ-
ing both enzymatic and fermentation processes) of L-cysteine. The metabolic
regulation of L-cysteine including novel sulfur metabolic pathways found in micro-
organisms is also discussed. Recent advancement in biochemical studies, genome
sequencing, structural biology, and metabolome analysis has enabled us to use
various approaches to achieve direct fermentation of L-cysteine from glucose. For
example, worldwide companies began to supply L-cysteine and its derivatives
produced by bacterial fermentation. These companies successfully optimized the
original metabolism of their private strains. Basically, a combination of three
factors should be required for improving L-cysteine fermentation: that is,
(1) enhancing biosynthesis: overexpression of the altered cysE gene encoding
feedback inhibition-insensitive L-serine O-acetyltransferase (SAT), (2) weakening
degradation: knockout of the genes encoding L-cysteine desulfhydrases, and
(3) exploiting export system: overexpression of the gene involved in L-cysteine
transport. Moreover, we found that “thiosulfate” is much more effective sulfur
source than commonly used “sulfate” for L-cysteine production in Escherichia coli,
because thiosulfate is advantageous for saving consumption of NADPH and relat-
ing energy molecules.

H. Takagi and I. Ohtsu

Graduate School of Biological Sciences, Nara Institute of Science and Technology,
8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
e-mail: hiro@bs.naist.jp
130 H. Takagi and I. Ohtsu

Keywords Escherichia coli, Feedback inhibition, Fermentation, Glutaredoxin,

L-Cysteine, L-Cysteine desulfhydrase, L-Cysteine transporter, L-Cysteine/
L-Cystine shuttle system, L-Serine O-acetyltransferase, O-acetyl-L-serine
sulfhydrylase, Thioredoxin, Thiosulfate

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
2 Conventional Processes for L-Cysteine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
2.1 Extraction from Human Hairs and Animal Feathers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
2.2 Asymmetrical Hydrolysis of DL-2-Amino-Δ2-Thiazoline-4-Carboxylic Acid . . . . . . 132
3 L-Cysteine Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.1 L-Cysteine Biosynthesis in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.2 L-Cysteine Biosynthesis in Other Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4 L-Cysteine Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5 L-Cysteine Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
6 Direct Fermentation of L-Cysteine from Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.1 Enhancement of L-Cysteine Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.2 Weakening of L-Cysteine Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
6.3 Enhancement of L-Cysteine Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7 Novel Pathways of L-Cysteine Metabolism in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.1 Thioredoxin/Glutaredoxin-Mediated L-Cysteine Biosynthesis from S-Sulfocysteine 142
7.2 Involvement of the yciW Gene in L-Cysteine
and L-Methionine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
8 L-Cysteine/L-Cystine Shuttle System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
9 Conclusions and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

1 Introduction

L-Cysteine is the most important sulfur-containing compound because it is a

sole metabolic entrance for reduced sulfur into cell metabolism in most organisms,
and it is required for the biosynthesis of sulfur-containing compounds such as
L-methionine, thiamine, biotin, and coenzymes A. It is also needed for Fe/S clusters
of the catalytic domain of some enzymes, such as aconitase, cytochromes, and
degradative L-serine dehydratase of Escherichia coli [1]. In addition, L-cysteine
plays crucial roles in protein folding, assembly, and stability through disulfide-bond
formation. Furthermore, glutaredoxin (Grx) and thioredoxin (Trx) that use an
L-cysteine-containing peptide, L-glutathione, as a cofactor are involved in
protecting cells under oxidative stress conditions. Recently, we have proposed
that the periplasmic L-cysteine protects E. coli cells from hydrogen peroxide,
which is produced by phagocytes, in the environment [2].
In addition to the essential function in cellular metabolism, L-cysteine is also an
important amino acid in terms of its applications in the pharmaceutical (expectorant
agent and freckles preventive medicine), food (dough conditioner, flavor enhancer,
L-Cysteine Metabolism and Fermentation in Microorganisms 131

and dietary supplement), and cosmetic (perm assistant and whitening agent) indus-
tries. L-Cysteine represents a global market of approximately 5,000 tons per year
and is now supplied by a combination of different production technologies, which
include the hydrolysis of keratin, enzymatic synthesis, and fermentation [3–5].
However, extraction methods result in low yields and cause unpleasant odors and
problems of waste treatment [6]. Although L-cysteine is considered a substance that
is generally recognized as safe (GRAS) by the Food and Drug Administration
(FDA), synthetic or biotechnological products are presently preferable for con-
sumers because of the bovine spongiform encephalopathy (BSE) problem in
animal-origin products [7].
The metabolic pathway of L-cysteine has been well studied in E. coli; however,
studies of other microorganisms remain limited. This chapter focuses on the recent
findings on L-cysteine metabolism and advances of biotechnological production
(including both enzymatic and fermentation processes) of L-cysteine. We also
discussed the metabolic regulation of L-cysteine and sulfur compounds including
novel sulfur metabolic pathways found in microorganisms.

2 Conventional Processes for L-Cysteine Production

The protein primary amino acids are all manufactured on an industrial scale. The
production methods fall into three classes: extraction from protein hydrolysates,
fermentation processes, and chemical synthesis. However, most of the amino acids
can now be prepared by fermentation or synthesis, although extraction remains
important for L-histidine, L-leucine, L-cysteine, and L-tyrosine [3, 8].

2.1 Extraction from Human Hairs and Animal Feathers

L-Cysteine has been obtained from acid hydrolysates of keratin, which is the key
structural protein with abundant L-cysteine of human hairs and animal feathers.
Prior to electrolysis, L-cysteine is extracted through a chemical process that
includes treatments with activated charcoal and concentrated hydrochloric acid.
This process is the simplest and easiest method for L-cysteine production, but it has
drawbacks such as a low yield and unpleasant odor and also includes serious
environmental and safety problems, the disposal of hydrochloric acid-containing
waste liquid and the use of animal-derived raw materials, respectively.
132 H. Takagi and I. Ohtsu

COOH COOH
atcB COOH atcC COOH
N N HS HS
S S
NHCONH2 NH2
NH2 NH2
N-Carbamyl-L-cysteine
D-ATC L-ATC L-Cysteine
(L-NCC)

Fig. 1 A metabolic pathway of DL-2-amino-Δ2-thiazoline-4-carboxylic acid (DL-ATC) to L-

cysteine via N-carbamoyl-L-cysteine (L-NCC) in Pseudomonas species. The atcB and atcC
genes encode L-ATC acid hydrolase and L-NCC amidohydrolase, respectively

2.2 Asymmetrical Hydrolysis of DL-2-Amino-Δ2-Thiazoline-

4-Carboxylic Acid

Instead of the hydrolysis of hairs, a bioconversion process for L-cysteine production

has been developed [9]. Some bacteria belonging to the genus Pseudomonas exhibit
activities involved in the asymmetric hydrolysis of DL-2-amino-Δ2-thiazoline-4-
carboxylic (DL-ATC) acid to L-cysteine [10, 11]. The conversion of DL-ATC to
L-cysteine consists of the following three successive steps (Fig. 1): (1) enzymatic
racemization of D-ATC to L-ATC; (2) a ring-opening reaction of L-ATC to
N-carbamoyl-L-cysteine (L-NCC) as intermediate; and (3) hydrolysis of L-NCC to
L-cysteine [12]. The improvement of this enzymatic conversion process, mainly in
terms of the yield, has been reported [9]. This process has been industrialized with
Ajinomoto Co., Inc.
The genes encoding L-ATC hydrolase (atcB) and L-NCC amidohydrolase (atcC)
are cloned and sequenced from Pseudomonas sp. strain BS. The deduced amino
acid sequence of the atcC gene product is highly homologous with other bacterial
L-N-carbamoylases, although that of atcB is novel [13]. The atcB gene is first
identified as a gene encoding an enzyme that catalyzes thiazoline ring-opening
reaction and does not share high homology with other enzymes whose functions
have been reported in detail. Similar results have been also reported using
Pseudomonas sp. strain ON-4a [14].

3 L-Cysteine Biosynthesis

3.1 L-Cysteine Biosynthesis in Escherichia coli

The precursor of L-cysteine is L-serine in most bacteria and plants. L-Serine is

synthesized from 3-phosphoglycerate, the glycolytic intermediate, via a three-step
pathway in enteric bacteria [15]. 3-Phosphoglycerate is first oxidized to
phosphohydroxypyruvate by the reaction of 3-phosphoglycerate dehydrogenase
(PGDH; EC 1.1.1.95, the serA gene product). Subsequent transamination leads to
the formation of phosphoserine, which is dephosphorylated to give L-serine
L-Cysteine Metabolism and Fermentation in Microorganisms 133

L-Serine + Acetyl CoA

cysE L-Homoserine + SuccinylCoA

O-Acetyl-L-serine metA
cysK O-Succinyl-L-homoserine
L-Cysteine metB
L-Cystathionine
transsulfuration
metC
metB
L-Homocysteine

metE or metH
Direct
L-Methionine
sulfhydryration

Fig. 2 Biosynthesis of L-cysteine and L-methionine in Escherichia coli. For L-methionine bio-
synthesis, Corynebacterium glutamicum has the direct sulfhydration pathway other than the
transsulfuration pathway. The cysE, cysK, metA, metB, metC, metE, or metH gene encodes L-serine
O-acetyltransferase (SAT), O-acetyl-L-serine sulfhydrylase-A (OASS-A), L-homoserine
O-succinyltransferase, L-cystathionine γ-synthase/O-succinyl-L-homoserine sulfhydrylase, L-
cystathionine β-lyase (CBL), and L-methionine synthase (vitamin B12-dependent or -indepen-
dent), respectively

Outer membrane
Periplasmic space
Enhancement of biosynthesis inner membrane

Ser + Acetyl-CoA
Sbp

SAT SO42- (internal) CysTWA SO42-

(external)
Activation by
OAS OAS and CysB
Removal of feedback
inhibition by Cys
S2-

YdeD
Cys Yfik
TolC Export

CDs Major transporters

(TNA, CBL,
OASS-A, Enhancement of transport
OASS-B,
MalY)

Pyruvate + NH3 + H2S

Weakening of degradation

Fig. 3 Rational approach for L-cysteine fermentation by enzyme, protein, and metabolic engi-
neering. Ser L-serine, Cys L-cysteine, SAT L-serine O-acetyltransferase, CD L-cysteine
desulfhydrase, TNA tryptophanase, CBL L-cystathionine β-lyase, OASS-A OAS sulfhydrylase-A,
OASS-B OAS sulfhydrylase-B
134 H. Takagi and I. Ohtsu

(catalyzed by phosphoserine aminotransferase and phosphoserine phosphatase,

respectively). The activity of PGDH is inhibited by L-serine [16].
The biosynthesis of L-cysteine from L-serine in enteric bacteria such as E. coli
(Fig. 2) and Salmonella typhimurium proceeds via a two-step pathway. L-Serine
undergoes a substitution of its β-hydroxyl with a thiol in two steps. L-Serine O-
acetyltransferase (SAT; EC 2.3.1.30, the cysE gene product) catalyzes the acetyla-
tion by acetyl-CoA of the β-hydroxyl of L-serine to give O-acetyl-L-serine (OAS).
The second step, the α,β-elimination of acetate from OAS and the addition of H2S
to give L-cysteine, is then catalyzed by OAS sulfhydrylase-A (OASS-A; EC
4.2.99.8, the cysK gene product). The first reaction catalyzed by SAT is the rate-
limiting step of L-cysteine biosynthesis in E. coli (Fig. 3). The enzyme inhibition
constant (Ki) for L-cysteine of E. coli SAT was reported to be on the micromole
order [17]. Some protein engineering approaches for obtaining feedback inhibition-
insensitive SAT have been reported and are described in the section below. The
SAT/OASS-A pathway was believed to be a sole L-cysteine biosynthetic pathway,
and E. coli does not have a reverse transsulfuration pathway (Fig. 2), which
converts L-cystathionine, which is the product of L-methionine metabolism, into
L-cysteine. The cysE mutants in both E. coli and S. typhimurium showed L-cysteine
auxotrophy [18].
The biosynthesis of L-cysteine in E. coli is regulated not only at the level of
enzymatic activity (feedback inhibition) but also at that of gene expression. The
cysteine regulon (known as “cys-regulon”) comprises the gene for biosynthesis of
L-cysteine and those for the uptake and reduction in oxidized sulfur sources, such as
sulfate or thiosulfate. The expression of these genes (except for cysE and cysG) is
under the control of the transcriptional activator CysB. CysB requires the inducer
N-acetyl L-serine (which is formed from OAS by chemical reaction) for activity and
sulfur limitation. Sulfide and thiosulfate act as anti-inducer for CysB. A detailed
discussion of mechanism of cys-regulon can be found elsewhere [17].

3.2 L-Cysteine Biosynthesis in Other Microorganisms

In every organism, L-cysteine and L-methionine are inevitable as sulfur-containing

compounds. Animals intake the amino acids as organic sulfur sources, and catab-
olize them to provide various cellular sulfur compounds. By contrast, most micro-
organisms as well as plants can assimilate environmental inorganic sulfur sources
such as sulfate (SO42
) and thiosulfate (S2O32
) into L-cysteine and the down-
stream metabolite L-methionine. E. coli has two well-known pathways for inorganic
sulfur assimilation (Fig. 4) [19, 20]. One is a sulfate-utilizing “sulfate pathway,”
which consumes two molecules of ATP and four molecules of NADPH to synthe-
size one molecule of L-cysteine. Another is a thiosulfate-utilizing “thiosulfate
pathway,” which requires only one molecule of NADPH [19]. For incorporation
of sulfur atom, both pathways use a common carbon skeleton OAS that is synthe-
sized from L-serine and acetyl-CoA by SAT. OASS-A catalyzes the conversion of
L-Cysteine Metabolism and Fermentation in Microorganisms 135

Thiosulfate Sulfate
pathway pathway
S2O32- out SO42- out

Outer membrane

S2O32- out SO42- out

Periplasm

CysP Sbp

Inner membrane CysU CysW CysU CysW

CysA CysA

Cytoplasm S2O32- in SO42- in

ATP cysDN
APS
L-Serine ATP cysC
Acetyl-CoA cysE PAPS
NADPH cysH
O-Acetylserine SO32-
3NADPH cysIJ (cysG)
S2-
cysM cysK

NrdH, Trxs, or Grxs Degradation

S-Sulfocysteine L-Cysteine Pyruvate,
NADPH tnaA, etc. H2S, and NH3
SO32-

L-Glutathione biosynthesis

Fig. 4 Overall sulfur metabolism in E. coli [19]. Sulfate and thiosulfate are imported from the
outside to the cytoplasm through common transporter CysTWA. GSH glutathione, Trx
thioredoxin, Grx glutaredoxin, APS adenosine 50 -phosphosulfate, PAPS 30 -phosphoadenosine
50 -phosphosulfate

OAS and sulfide (S2
) into L-cysteine. In the sulfate pathway, while in the thiosul-
fate pathway, OAS sulfhydrylase-B (OASS-B; EC 4.2.99.8, the cysM gene product)
produces S-sulfocysteine (SSC) from OAS and thiosulfate. SSC is reductively
divided into L-cysteine and sulfite (SO32
) with NrdH and Grx1 [19]. Sulfate
transport into cell is dominantly mediated by Sbp-CysTWA and thiosulfate trans-
port is dominantly mediated by CysP-CysTWA complex on the inner membrane
[20–22]. It is noteworthy that the inner-membrane channel part consisting of
CysTWA is common for both uptakes (Fig. 4).
Corynebacterium glutamicum and related bacteria are Gram-positive and non-
pathogenic and are known as coryneform glutamic acid-producing bacteria. Their
derivatives have been used in industry for the production of various amino acids
by means of fermentation. The precursor of L-cysteine is also L-serine as in the case
of E. coli. L-Serine is synthesized from 3-phosphoglycerate, the glycolytic inter-
mediate, via a three-step pathway same as E. coli [23]. In C. glutamicum, L-cysteine
is synthesized through basically the same pathway as E. coli. However,
C. glutamicum has the direct sulfhydration pathway (Fig. 2) other than the
transsulfuration pathway for L-methionine biosynthesis [24]. Both SAT and
OASS-A of C. glutamicum have been partially purified and characterized
[25, 26]. The Ki for L-cysteine of C. glutamicum SAT is also estimated to be of
136 H. Takagi and I. Ohtsu

the micromole order [25]. However, detailed characterization of purified

C. glutamicum SAT has not yet been reported. Protein engineering on
C. glutamicum SAT, such as construction of the feedback inhibition-insensitive
mutant enzyme, also remains to be investigated.
Some microorganisms, such as Saccharomyces cerevisiae, Lactococcus lactis
[27], and Pseudomonas putida [28], have been reported to synthesize L-cysteine
from L-methionine through the reverse transsulfuration pathway (Fig. 2). With
regard to S. cerevisiae, previous studies concluded that detectable SAT and
OASS-A do not constitute an L-cysteine biosynthetic pathway and that L-cysteine
is exclusively synthesized via cystathionine by L-cystathionine β-synthase (CBS;
EC 4.2.1.22) and L-cystathionine γ-lyase (CGL; EC 4.4.1.1) [29, 30]. Wheeler
et al. [31] reported that pathogenic bacteria, namely, the Mycobacterium tubercu-
losis complex, have a reverse transsulfuration pathway and can synthesize cysteine
from methionine. However, C. glutamicum, which is taxonomically closely related
to M. tuberculosis, cannot synthesize L-cysteine from L-methionine [25]. L-Cysteine
production by C. glutamicum has been reported, although the productivity was
relatively low (290 mg/L) compared with the productivity of that produced by
E. coli [32].
Some archaea have been reported to synthesize L-cysteine from L-methionine
through reverse transsulfuration from L-homocysteine to L-cysteine [33]. On the
other hand, several archaea are considered to have genes encoding SAT and OASS
[34]. In an extremely thermophilic bacterium, Thermus thermophilus, L-cysteine is
synthesized with OAS and sulfide by the catalysis of OASS-A [35]. This organism
catalyzes the reactions of transsulfuration from L-cysteine to L-homocysteine when
ammonium sulfate is used as the sulfur source, but is unable to catalyze reverse
transsulfuration because of the absence of CBS [35]. It was suggested that OASS-A
purified from T. thermophilus HB8 is responsible for the synthesis of L-cysteine in
this organism cultured with either sulfate or L-methionine given as the sole sulfur
source [36]. The sat1 gene encoding SAT of T. thermophilus HB8 was cloned and
overexpressed in E. coli cells, based on the genome sequence [37]. The predicted
amino acid sequence was homologous to those of the O-acetyltransferase members.
The recombinant enzyme was active over a wide range of temperatures, with
maximum activity at around 75 C. Interestingly, the enzyme was highly activated
by Co2+ or Ni2+, and contained Zn2+ and Fe2+, indicating that the T. thermophilus
SAT is a novel type of enzyme different from this protein family [37].

4 L-Cysteine Degradation

In E. coli, L-cysteine degradation is catalyzed mainly by L-cysteine desulfhydrase

(CD) [38]. Enzymes having CD activity in E. coli have been investigated in
considerable detail (Fig. 3). Awano et al. [39] reported that tryptophanase (TNA;
EC 4.1.99.1, the tnaA gene product) and L-cystathionine β-lyase (CBL; EC 4.4.1.8,
the metC gene product) catalyzed CD reaction and acted on L-cysteine degradation
L-Cysteine Metabolism and Fermentation in Microorganisms 137

Pore diffusion Simple diffusion Efflux pump

Cys Cys

Outer membrane TolC TolC

AcrA
Periplasm Cys Cys Cys Cys AcrE
AcrA EmrA
Cys Cys
H+ MacA
Cys Cys
AcrB
YdeD CydDC AcrF
Inner membrane AcrB EmrB
YfiK Bcr MacB

Cytoplasm
Cys Cys

Major Cys Minor Cys

transporters transporters

Fig. 5 L-Cysteine export systems in E. coli. It is known that the outer membrane channel TolC
forms various types of tripartite efflux pump in Gram-negative bacteria (dashed box), and serves as
an essential element of some mechanisms responsible for tolerance to various compounds,
including hydrophobic antibiotics and organic solvents [43]. TolC interacts with an inner-
membrane transporter, AcrB, which provides export energy, and a protein of the adaptor family,
AcrA, which brings TolC and the inner-membrane transporter together. In addition, the TolC
channel also exports L-cysteine from the periplasm to outside the cell [44]. In contrast, both major
(YdeD and YfiK) and minor (CydDC and Bcr) L-cysteine transporters once export L-cysteine from
the cytoplasm to the periplasm, and then the periplasmic L-cysteine diffuses to outside the cell
through TolC [44]

in E. coli by analyses with CD activity staining and gene disruption. Subsequently,

using the plasmid gene library of E. coli, which contains 4,388 kinds of open
reading frame, three additional proteins having CD activity were identified as
OASS-A, OASS-B, and MalY protein in E. coli [40]. These five CD proteins
(TNA, CBL, OASS-A, OASS-B, and MalY) involved in L-cysteine degradation
are all pyridoxal 50 -phosphate (PLP)-dependent enzymes. Therefore, the CD activ-
ity of these enzymes may be only a side reaction that is often observed in
PLP-dependent enzymes due to the chemistry of PLP. Growth phenotype and
transcriptional analyses suggest that the CD activity of TNA contributes primarily
to L-cysteine degradation in vivo. In fact, TNA, which primarily degrades L-
tryptophan to indole, pyruvate, and ammonia, is induced by the addition of L-
cysteine to the culture medium, suggesting that a novel transcriptional regulation
system is involved in the gene expression. However, it appears that unidentified
proteins with CD activity, which may be induced by L-cysteine, are still present in
E. coli. It is possible that one gene disruption would affect the expression of other
CD proteins and the whole metabolic profile in E. coli [40].
138 H. Takagi and I. Ohtsu

In C. glutamicum, only one CD enzyme has been purified and characterized

[32]. The enzyme was identified as the aecD gene product, a C-S lyase with a
β-elimination activity [41]. This enzyme is also annotated as CBL [42]. Thus, the
CD activity of this enzyme may be a side reaction of CBL as in the case of E. coli.
However, other enzymes having CD activity in C. glutamicum remain to be
clarified. Disruption of the aecD gene was effective for L-cysteine production by
C. glutamicum, as in the case of E. coli [32].

5 L-Cysteine Transport

Export systems for L-cysteine in E. coli were also well studied. Previously, two
transporter proteins, YdeD and YfiK, have been identified as L-cysteine exporters in
E. coli (Fig. 5) [45, 46]. Both YdeD and YfiK are membrane-integral proteins and
belong to different protein families. Overexpression of either YdeD or YfiK led to
parallel secretion of OAS and L-cysteine by E. coli cells [47]. L-Cysteine is also
exported from the E. coli cytoplasm to the periplasm by CydDC, which is an
ATP-binding cassette (ABC)-type transporter required for cytochrome assembly
[48]. CydDC overexpression conferred resistance to high extracellular L-cysteine
concentrations. However, CydDC was shown to have higher transport activity with
L-glutathione than with L-cysteine as the substrate [49].
In addition to these transporters, there are 37 open reading frames assumed to be
drug transporter genes on the basis of sequence similarities in E. coli, although the
transport abilities of most of them have not yet been established [50]. Five families
of drug extrusion translocases have been previously identified based on sequence
similarity, including the major facilitator (MF) family, the small multidrug resis-
tance (SMR) family, the resistance nodulation cell division (RND) family, the ABC
family, and the multidrug and toxic compound extrusion (MATE) family. Among
the 33 putative drug transporter genes tested, Yamada et al. [51] reported that the
multidrug transporter Bcr in the MF family is involved in the export in E. coli cells
(Fig. 5). Amino acid transport assays showed that Bcr overexpression conferring
bicyclomycin and tetracycline resistance specifically promotes L-cysteine export
driven by energy derived from the proton gradient.
Recently, the tolC gene encoding the outer membrane channel TolC was iden-
tified as a novel gene involved in L-cysteine export using a systematic and com-
prehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection)
(Fig. 5) [44]. Gene expression analysis revealed that the tolC gene is essential for
L-cysteine tolerance, which is not mediated by TolC-dependent drug efflux systems
such as AcrA and AcrB, in E. coli cells. It also appears that other outer membrane
porins including OmpA and OmpF do not participate in TolC-dependent L-cysteine
tolerance.
L-Cysteine Metabolism and Fermentation in Microorganisms 139

6 Direct Fermentation of L-Cysteine from Glucose

6.1 Enhancement of L-Cysteine Biosynthesis

As described above, the reaction catalyzed by SAT is the rate-limiting step of

L-cysteine biosynthesis in many microorganisms. Due to feedback inhibition by L-
cysteine of SAT, a high level of production of L-cysteine from glucose has not yet
been successfully achieved in microorganisms. Thus, for the fermentative produc-
tion of L-cysteine, it is necessary to use feedback inhibition-insensitive SATs.
There have been two approaches to obtaining SAT that is less sensitive to
feedback inhibition: (1) the engineering of SAT from E. coli through site-directed
or random mutagenesis [52, 53] and (2) the use of the natural SAT, which is
desensitized to feedback inhibition, from higher plants [54, 55].
Denk and B€ ock [56] first reported that a small amount of L-cysteine (30 mg/L)
was secreted by a revertant from an L-cysteine auxotroph of E. coli. In this
revertant, SAT was less sensitive to feedback inhibition, and the Met residue at
position 256 (Met256) in SAT was replaced with Ile. Because this residue was
supposedly part of the allosteric site for L-cysteine binding, the mutant cysE genes
encoding SAT variants, which were genetically desensitized to feedback inhibition,
were constructed by replacing Met256 with 19 other amino acid residues using site-
directed mutagenesis [52]. It was found that in the L-cysteine non-utilizing E. coli
cells expressing the mutant cysE gene, there was a marked production of L-cysteine
and that stable expression of feedback inhibition-insensitive SAT was necessary for
the overproduction of L-cysteine (at a maximum of 790 mg/L for the Met256Ala
variant). PCR random mutagenesis was introduced into the cysE gene to isolate the
variant SATs that cause overproduction of L-cysteine, due to a much lower level of
feedback inhibition [53]. The production test of L-cysteine and the enzymatic
analysis of SAT variants suggested that the C-terminal region of SAT plays an
important role in the desensitization to feedback inhibition and in the high level of
production of L-cysteine (at a maximum of 990 mg/L for the Met201Arg variant).
The alternative way to obtain feedback inhibition-insensitive SAT is to use the
natural SAT resources from higher plants. In Arabidopsis thaliana, three cDNA
clones encoding organelle-localized SAT have been isolated: SAT-c (a cytosolic
isoform), SAT-m (a mitochondrial isoform), and SAT-p (a plastidic isoform). Noji
et al. [57] found that SAT-m and SAT-p were feedback inhibition-insensitive
isozymes (not inhibited in the presence of up to 100 μM L-cysteine), whereas the
concentrations for 50% inhibition (IC50) of SAT-c and E. coli SAT under the
standard assay condition were 1.8 and 6.0 μM, respectively. As expected, the
expression of two cDNAs encoding SAT-m and SAT-p in the L-cysteine
non-utilizing E. coli cells significantly increased the productivity of L-cysteine
(1,590 and 1,660 mg/L, respectively) [54]. The application of cDNAs encoding
L-cysteine-insensitive SAT from Nicotiana tabacum has been also reported [55].
However, these SATs showed significant decreases in enzymatic activity rela-
tive to the E. coli wild-type enzyme. Further improvements in L-cysteine production
140 H. Takagi and I. Ohtsu

are therefore expected to use an engineered SAT, which shows a higher level of
feedback desensitization and a higher catalytic activity. Previously, the three-
dimensional structure of SAT from E. coli with its inhibitor L-cysteine was deter-
mined by X-ray crystallography at a 2.2-Å resolution [58]. The crystal structure and
the reaction mechanism of SAT from E. coli have shown that SAT is a trimeric
structure, which is likely to interact with another SAT trimer at N-terminal ends,
and that the substrate L-serine and the inhibitor L-cysteine bind to the identical
region in SAT. To decrease the affinity for L-cysteine alone, Kai et al. [59] built the
first structure model of L-serine-binding SAT on the basis of the crystal structure
with bound L-cysteine and compared these two structures (L-serine- and L-cysteine-
binding SAT). The comparison showed that the Cα of Asp92 underwent a substan-
tial positional change upon the replacement of L-cysteine by L-serine. Then, various
amino acid substitutions at positions 89–96 around Asp92 were introduced by
randomized, fragment-directed mutagenesis to change the position of the Asp92.
As a result, SAT variants, which have both extreme insensitivity to inhibition by
L-cysteine (IC50 ¼ 1,100 μM) and extremely high enzymatic activities, were suc-
cessfully obtained [59]. Such structural information is a powerful tool for breeding
an L-cysteine overproducer.
In addition, further improvement in L-cysteine productivity might be possible
using SAT with high stability. Thus, a thermostable SAT of T. thermophilus HB8
[37] should be engineered to desensitize it to feedback inhibition based on our
previous reports [52–54].

6.2 Weakening of L-Cysteine Degradation

A decrease in the degradation activity for the desired amino acid is also a promising
approach to further improving amino acid production by means of fermentation.
The gene disruption for each protein (TNA, CBL, OSAA-A, OASS-B, and MalY)
was significantly effective for overproduction of L-cysteine. The single or quintet
gene mutants transformed with the plasmid containing the cysE gene for feedback-
insensitive SAT exhibited higher L-cysteine productivity than the wild-type strain
[39, 40]. The amounts of L-cysteine produced after 72 h of cultivation increased by
a factor of 1.8–2.3. However, L-cysteine productivity decreased significantly after
96 h of cultivation in all the strains, probably because of the remaining CD enzyme
(s). It is also noteworthy that the quintet gene-disrupted strain (ΔtnaA, ΔmetC,
ΔcysK, ΔcysM, and ΔmalY) in the presence of L-cysteine showed higher CD
activity than that observed in the absence of L-cysteine. It appears that other CDs,
in addition to the five proteins identified, could be induced by L-cysteine in
E. coli [40].
L-Cysteine Metabolism and Fermentation in Microorganisms 141

6.3 Enhancement of L-Cysteine Transport

For effective amino acid production, not only the biosynthetic pathway of target
amino acid but also its efflux from the cell is important. For example, L-lysine
exporter, LysE, is reported to be essential for L-lysine production by C. glutamicum.
In the absence of LysE, L-lysine can reach an intracellular concentration of more
than 1,100 mM, which prevents cell growth [60]. High concentrations of L-cysteine
have been also reported to be inhibitory or even toxic to E. coli cells [61, 62]. To
produce such a toxic compound as L-cysteine by fermentation, the use of an
L-cysteine efflux system could be promising, as in the case of other amino acids
[63]. Yamada et al. [51] reported that the multidrug transporter Bcr is involved in
overproduction of L-cysteine in E. coli. When the bcr gene was overexpressed in
tnaA-disrupted cells expressing the cysE gene for feedback inhibition-insensitive
SAT, the L-cysteine level was approximately fivefold higher than that of the cells
harboring the vector only. It was also concluded that the outer membrane TolC
plays an important role in L-cysteine tolerance probably due to its export ability and
that TolC overexpression is effective for L-cysteine production in E. coli [44]. To
improve L-cysteine production, plasmid pDES carrying the wild-type ydeD gene
and mutant cysE and serA gene, which encodes feedback inhibition-insensitive
SAT and PGDH, respectively (personal communication), was introduced into tnaA-
disrupted cells. The transformant cells produced approximately 20% higher amount
of L-cysteine than cells carrying the vector only.
Although there is insufficient information for achieving L-cysteine fermentation,
two worldwide companies (Wacker Chemie AG, Germany and Ajinomoto Co.,
Inc., Japan) independently began to supply L-cysteine and its derivatives produced
by a microbial fermentation process developed in 2001 and 2014, respectively
(http://kyowa-usa.com/news/2001/06-25, http://www.ajinomoto-usa.com/Pr/Pdf/
Cysteine_Derivatives.pdf). These companies could successfully optimize the orig-
inal metabolism of their private bacterial strains. In particular, Wacker Chemie AG
announced that a large quantity of L-cysteine was secreted into the culture medium
from bacterial cells, as 90% of pure L-cysteine ends up in the final product, which
fulfills the quality standards for foods and pharmaceutical industries (Wacker [64]).
Of all the L-cysteine manufactured worldwide in 2004, only 12% were
fermentation-origin products. Annual growth rate for the market share for microbial
L-cysteine was estimated at 10% [5].
142 H. Takagi and I. Ohtsu

7 Novel Pathways of L-Cysteine Metabolism in Escherichia

coli

7.1 Thioredoxin/Glutaredoxin-Mediated L-Cysteine

Biosynthesis from S-Sulfocysteine

E. coli has two enzymes which assimilate inorganic sulfur sources, OASS-A
(CysK) and OASS-B (CysM) (Fig. 5). While the former enzyme utilizes sulfide
(S2
) as a sulfur donor, the latter enzyme uses thiosulfate (S2O32
). CysK
synthesizes L-cysteine from OAS and sulfide, but the CysM protein differs in
that it can also utilize thiosulfate instead of sulfide. The product formed by the
CysM activity, SSC, is converted into L-cysteine and sulfite by an
uncharacterized reaction [65]. The E. coli sulfite reductase consists of the
alpha subunit protein (the cysJ gene product) and the beta subunit protein (the
cysI gene product). The “sulfate pathway” spends two molecules of ATP and
four molecules of NADPH as a reducing power to make one molecule of
L-cysteine from a sulfate and OAS molecule. On the other hand, the thiosulfate
pathway has the advantage that two molecules of L-cysteine can synthesize from
a thiosulfate molecule by consuming only four molecules of NADPH. As
thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and
NrdH) are known as reductases of peptidyl disulfides [66], overexpression of
these reductases might contribute to improving L-cysteine production by accel-
erating the reduction of SSC.
To test whether these enzymes catalyze the reduction of SSC to L-cysteine, all
His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into
L-cysteine in vitro [19]. Overexpression of Grx1 and NrdH enhanced a 15%–40%
increase in L-cysteine production in E. coli. On the other hand, disruption of cysM
cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting
that its improvement was due to the efficient reduction of SSC. Moreover,
L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI
and ΔcysJ) and the OASS-A gene (ΔcysK) each decreased to about 50% of that in
the wild-type strain. Interestingly, there was no significant difference in L-cysteine
production between wild-type strain and gene deletion mutant of the upstream
pathway of sulfite (ΔcysC or ΔcysH), indicating that sulfite generated from the
SSC reduction is available as the sulfur source to produce additional L-cysteine. It
was finally found that the E. coli L-cysteine producer that co-overexpresses Grx
(NrdH), sulfite reductase (CysI), and OASS-A (CysK) exhibited the highest amount
of L-cysteine produced per cell, proposing that the enhancement of Trx/Grx-
mediated L-cysteine synthesis from SSC is a novel method for improvement of
L-cysteine production [19].
L-Cysteine Metabolism and Fermentation in Microorganisms 143

[L-Cysteine biosynthesis]
Sulfate Sulfite
ATP cysDN

APS
ATP cysC [L-Glutathione biosynthesis]
PAPS L-Glutathione
L-Serine cysH Na2SO4 Cysteine
NADPH
S source
Acetyl-CoA
cysE Sulfite Sulfide
cysIJ
O-Acetyl-L-serine (cysG)
3 NADPH
Na2SO4 Cysteine
Sulfide S source
gshB
cysK Na2SO4 Cysteine L-Glutathione
S source
gshA
L-Cysteine L-γ-Glutamylcysteine
L-Cysteine

Na2SO4 Cysteine
S source
[L-Methionine biosynthesis]
L-Homocysteine

O-Succinyl-L-homoserine YciW? L-Cystathionine

L-Homoserine metB
metA YciW? L-Homocysteine
metC Na2SO4 Cysteine
metE S source
L-Aspartate
L-Methionine
metH

Fig. 6 The possible function of YciW in E. coli by sulfur metabolome [71]. YciW seems to
metabolize L-cysteine and O-succinyl-L-homoserine to L-homocysteine via L-cystathionine. The
graphs of the measured sulfur compound contents are shown in the metabolic pathway map. The
metabolites represented by inverted style are the targets of LC-MS/MS analysis. The content of
each metabolite in wild-type cells carrying the empty vector and overexpressing the yciW gene is
shown by open and closed bar, respectively

7.2 Involvement of the yciW Gene in L-Cysteine

and L-Methionine Metabolism

Transcription of the sulfate pathway genes is known to be upregulated during

cellular L-cysteine shortage by a transcriptional activator CysB (LysR family)
[67]. Using in silico analysis, which are MEME and FIMO program (http://
meme.nbcr.net/meme/), the yciW gene of E. coli was identified as a novel L-cysteine
regulon that may be regulated by the transcriptional activator CysB for sulfur
metabolic genes [68]. To discover a novel CysB-binding motif on the E. coli
genome, the CysB-binding motif was first predicted from the upstream sequences
(300 bp from start codon) of already-known genes for CysB binding (cysD, cysJ,
cysK, and cysP) [69, 70] by the MEME program (Fig. 6). The isolated motif was
consistent with the already-known CysB-binding motif. Subsequently, the
predicted motif (42 bp) was searched for the E. coli W3110 genome (accession
No.: NC_007779.1) using the FIMO program. It was revealed the most plausible
144 H. Takagi and I. Ohtsu

sequence for the CysB-binding motif in the upstream of the yciW gene, which is
suggested to be a new member of the CysB regulons.
Interestingly, yciW conferred tolerance to L-cysteine on E. coli cells, suggesting
that the yciW gene product (YciW) converts L-cysteine to L-methionine or L-gluta-
thione. Actually, the intracellular contents of all sulfur metabolites relatively
increased regardless of yciW overexpression, suggesting that excess L-cysteine
was degraded by enzyme such as L-cysteine desulfhydrases to protect E. coli cells
from L-cysteine toxicity. After 72 h of cultivation, ΔyciW strain carrying pDES
produced about 2.4-fold higher amount of L-cysteine per cell than wild-type strain,
when sulfate was used as sulfur source. Also, when thiosulfate was used, disruption
of yciW significantly increased L-cysteine production of E. coli. In contrast,
overexpression of yciW significantly decreased total amount of L-cysteine in wild-
type cells in the presence of sulfate and thiosulfate. From these results, disruption of
yciW, one of the CysB regulons, enhances L-cysteine production. In silico gene
screening is an effective method for improvement of L-cysteine production [68].
Kawano et al. [71] also analyzed a sulfur index of E. coli cells using LC-MS/MS
combined with thiol-specific derivatization by monobromobimane. The obtained
sulfur index was then applied to evaluate the L-cysteine producer. E. coli cells
overexpressing yciW accumulated L-homocysteine, suggesting that YciW is
involved in the biosynthesis of L-methionine rather than L-glutathione. From the
metabolome analysis of sulfur compounds in E. coli, YciW seems to metabolize
L-cysteine and O-succinyl-L-homoserine to L-homocysteine via L-cystathionine
(Fig. 6).

8 L-Cysteine/L-Cystine Shuttle System

Excessive reactive oxygen species (ROS) are cytotoxic molecules, which result in
the oxidation of DNA, proteins, and cellular membrane lipids [72]. Inside the cell, it
generates from their respiration chain and various redox reactions, which results in
the exchange of one or two electrons to molecular oxygen [73]. Most organisms
acquire various ROS-scavenging strategies for their survival and conservation of
the species. For example, E. coli possesses ROS-scavenging enzymes such as
superoxide dismutase (2O2
+ 2H+ ! O2 + H2O2), catalase (2H2O2 ! O2 + 2H2O),
and peroxidase (ROOR’ + 2e
+ 2H+ ! ROH + R’OH). Superoxide dismutases
exist both in the cytoplasm (SodA and SodB) and the periplasm (SodC), whereas
catalase is present only in the cytoplasm (KatE) but not in the periplasm. Unlike the
cytoplasm, the mechanism underlying the process for scavenging periplasmic H2O2
generated from the SodC reaction has remained unclear [44].
Recently, the E. coli mutants lacking the L-cysteine exporter YdeD or the
L-cystine-binding protein FliY increased sensitivity to H2O2 [2]. It was also
shown that FliY is involved in the uptake of L-cystine from the periplasm to the
cytoplasm. Moreover, the expressions of the ydeD and fliY genes were dramatically
L-Cysteine Metabolism and Fermentation in Microorganisms 145

L-cysteine out

Outer membrane TolC peroxidation

H2O2 H2O

Periplasm L-cysteine out L-cystine out

FliY
Inner membrane YfiK YdeD YecS YdjN
YecC
L-Cysteine/L-Cystine
shuttle system
Cytoplasm
GSH or Trx, Grx ?
L-cysteine in L-cystine in

NADPH

Fig. 7 Proposed L-cysteine/L-cystine shuttle system [2, 74]. The L-cysteine/L-cystine shuttle
system participates in preventing membrane lipids or controlling the level of membrane peroxi-
dation products

increased upon treatment of the cells with H2O2 [2]. From these findings, it was
proposed that E. coli removes the periplasmic H2O2 using L-cysteine supplied to the
periplasm from the cytoplasm by “L-cysteine/L-cystine shuttle system” (Fig. 7)
[2]. In this system, however, it remains questionable: (1) whether ΔydeD mutant
cells actually accumulate H2O2 in the periplasm, (2) what is the FliY-dependent
L-cystine importer, and (3) whether the L-cystine importer cooperatively operates
with YdeD. In addition to them, the question arises as to whether endogenous
L-cysteine de novo synthesized from inorganic sulfur source serves the function of
L-cysteine/L-cystine shuttle system.
Thus, not only L-cysteine exporter such as YdeD but also an L-cystine importer
such as the FliY-dependent protein is critical for protecting cells from oxidative
stress into the periplasm due to quality control of the membrane. Ohtsu et al. [74]
recently identified two different L-cystine importers, YecS and YdjN, which indeed
function against H2O2 stress in E. coli (Fig. 7). These L-cystine importers cooper-
atively function with the L-cysteine exporter YdeD to scavenge the periplasmic
H2O2. Furthermore, endogenous L-cysteine de novo synthesized from inorganic
sulfur source was shown to eliminate the periplasmic H2O2. Thus, it was proposed
that H2O2-inducible L-cysteine/L-cystine shuttle system has an important role for
quality control of the plasma membrane in E. coli. In addition, the yfiK gene
encoding another major L-cysteine transporter, YfiK, is induced by H2O2, although
both the genes encoding Bcr and CydDC are not upregulated.
146 H. Takagi and I. Ohtsu

Table 1 L-Cysteine fermentative production in E. coli

L-Cysteine
Improvement of L-cysteine E. coli host strain; productivity
production genotype (mg/L) Reference
Enhancing biosynthesis JM240; cysEM256I ca. 30 [56]
Enhancing excretion W3110; pYdeD ca. 70 [45]
Enhancing excretion W3110; pYfiK ca. 150 [46]
Enhancing biosynthesis JM39; cysE51 130–280 [52]
+ pcysEM256X
Weakening degradation JM39-8; cysE51 260–490 [53]
Enhancing biosynthesis/Weakening JM39; cysE51 480–670 [53]
degradation + pcysErandom
Enhancing biosynthesis and excre- MG1655; ΔtnaA 890–1,030 [44]
tion/Weakening degradation + pDES
Enhancing biosynthesis and excre- MG1655; ΔtnaA 1,050–1,200
tion/Weakening degradation + pDES + pTolC
Enhancing biosynthesis and excretion BW25113; pDES 1,200–1,230 [19]
Enhancing biosynthesis and excre- BW25113; ΔyciW 1,480–1,720 [68]
tion/Weakening degradation + pDES
Strain JM39 is a cysteine auxotroph. Strain JM39-8 has only 10% of the cysteine desulfhydrase
activity of JM39 [52]
Plasmid pEDS carries the altered serA (Thr410 to Stop codon) and cysE (Thr167Ala) genes and the
wild-type ydeD gene under the control of the ompA gene promoter
Plasmid pcysEM256X were constructed by site-directed mutagenesis to replace Met256 of serine O-
acetyltransferase with 19 other anino acids (X) [52]
Plsmid pcysErandom were constructed by PCR random mutagenesis to isolate serine O-
acetyltransferase variants [53]

9 Conclusions and Future Perspective

Recent advancement in biochemical studies, genome sequencing, structural biol-

ogy, and metabolome analysis has enabled us to use various approaches to achieve
direct fermentation of L-cysteine from glucose. Table 1 summarizes the improved
productivity of L-cysteine fermentation by E. coli cells. In addition to a feedback
inhibition-insensitive SAT, low activity of L-cysteine degradation and high activity
of L-cysteine-export in the host cells are very important for efficient L-cysteine
fermentation. In conclusion, we believe that a combination of the three
abovementioned factors should be required for achieving L-cysteine fermentation:
that is, (1) overexpression of the feedback inhibition-insensitive SAT gene,
(2) knockout of the genes encoding CDs, and (3) overexpression of the gene
involved in L-cysteine export (Fig. 3).
Recently, we found that S. cerevisiae utilizes thiosulfate as a sole sulfur source.
The energetically favored thiosulfate rather than sulfate as sulfur sources is also
more effective for improving growth and ethanol-production rate in yeast cells due
to high levels of intracellular NADPH during thiosulfate utilization [75].
L-Cysteine Metabolism and Fermentation in Microorganisms 147

Thiosulfate is an advantageous sulfur source than sulfate for saving consumption

of NADPH and relating energy molecules. This is probably attributed to less
NADPH consumption for L-cysteine/L-methionine biosynthesis in thiosulfate com-
pared to sulfate. Because reducing balance of one of two sulfur atoms of thiosulfate
is equal to that of sulfide, thiosulfate is much more effective sulfur source than
sulfate for L-cysteine fermentative production in E. coli [19, 68, 71].
We thus propose that thiosulfate compared to commonly used sulfate is effective
sulfur source for biotechnological production of useful compounds other than
ethanol production in S. cerevisiae. According to this idea, we are going to try
production of other useful products using thiosulfate as sulfur source, and also to
elucidate molecular basis of the assimilation pathway and responsible
enzymes [75].

Acknowledgements We are greatly indebted to our coresearchers Drs. Shigeru Nakamori,

Masaru Wada, Yoshimitsu Hamano, and Yusuke Kawano. This review includes the work
supported by a grant from Ajinomoto, Co., Inc., to H.T., by KAKENHI [Grant-in-Aid for
Scientific Research (C) (26450091)] from the Japan Society for the Promotion of Science to I.
O., and by Science and Technology Research Promotion Program for Agriculture, Forestry,
Fisheries and Food Industry (26027AB) to I.O.

References

1. Cicchillo RM, Baker MA, Schnitzer EJ, Newman EB, Krebs C, Booker SJ (2004) Escherichia
coli L -serine deaminase requires a [4Fe-4S] cluster in catalysis. J Biol Chem
279:32418–32425
2. Ohtsu I, Wiriyathanawudhiwong N, Morigasaki S, Nakatani T, Kadokura H, Takagi H (2010)
The L-cysteine/L-cystine shuttle system provides reducing equivalents to the periplasm in
Escherichia coli. J Biol Chem 285:17479–17487
3. Ikeda M (2003) Amino acid production process. In: Scheper T, Faurie R, Thommel J (eds)
Advances in biochemical engineering/biotechnology, vol 79. Springer, Berlin Heidelberg
New York, pp 1–35
4. Leuchtenberger W (1996) Amino acids-technical production and use. In: Roehr M
(ed) Biotechnology, vol 6. VCH, Weinheim, pp 465–502
5. Tamba Berehoiu RM, Popa CN, Popescu S (2013) Assessment of the E 920 additive (L-
cysteine) in relation to some problems of modern food industry. In: Scientific papers series
management, economic engineering in agriculture and rural development, vol 13, pp 413–418
6. Hunt S (1985) Degradation of amino acids accompanying in vitro protein hydrolysis. In:
Barrett GC (ed) Chemistry and biology of the amino acids. Chapman & Hall, London, pp
3763–3798
7. Wada M, Takagi H (2006) Metabolic pathways and biotechnological production of L-cysteine.
Appl Microbiol Biotechnol 73:48–54
8. Hardy PM (1985) The protein amino acids. In: Barrett GC (ed) Chemistry and biology of the
amino acids. Chapman & Hall, London, pp 6–24
9. Ryu OH, Ju JY, Shin CS (1997) Continuous L-cysteine production using immobilized cell
reactors and product extractors. Process Biochem 32:201–209
10. Sano K, Mitsugi K (1978) Enzymatic production of L-cysteine from DL-2-amino-Δ2-thiazoline-
4-carboxylic acid by Pseudomonas thiazolinophium: optimal conditions for the enzyme
formation and enzymatic reaction. Agric Biol Chem 42:2315–2321
148 H. Takagi and I. Ohtsu

11. Sano K, Eguchi C, Yasuda N, Mitsugi K (1979) Metabolic pathway for L-cysteine formation
from DL-2-amino-Δ2-thiazoline-4-carboxylic acid by Pseudomonas. Agric Biol Chem
43:2373–2374
12. Tamura T, Nishino M, Ohmachi T, Asada Y (1998) N-Carbamoyl- L-cysteine as an interme-
diate in the bioconversion from DL-2-amino-Δ2-thiazoline-4-carboxylic acid to L-cysteine by
Pseudomonas sp. ON-4a. Biosci Biotechnol Biochem 62:2226–2229
13. Shiba T, Takeda K, Yajima M, Tadano M (2002) Genes from Pseudomonas sp. strain BS
involved in the conversion of L-2-amino-Δ2-thiazolin-4-carbonic acid to L-cysteine. Appl
Environ Microbiol 68:2179–2187
14. Ohmachi T, Nishino M, Kawata M, Edo N, Funaki H, Narita M, Mori K, Tamura Y, Asada Y
(2002) Identification, cloning, and sequencing of the genes involved in the conversion of d, l-2-
amino-Δ2-thiazoline-4-carboxylic acid to l-cysteine in Pseudomonas sp. strain ON-4a. Biosci
Biotechnol Biochem 66:1097–1104
15. Pizer LI (1963) The pathway and control of serine biosynthesis in Escherichia coli. J Biol
Chem 238:3934–3944
16. Sugimoto E, Pizer LI (1968) The mechanism of end product inhibition of serine biosynthesis. I
Purification and kinetics of phosphoglycerate dehydrogenase. J Biol Chem 243:2081–2089
17. Kredich NM (1996) Biosynthesis of cysteine. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin
ECC, Low KB, Magasanik B, Reznicoff WS, Riley M, Schaechter M, Umbarger JE (eds)
Escherichia coli and Salmonella typhimurium: cellular and molecular biology, 2nd edn. ASM,
Washington, pp 514–527
18. Kredich NM, Tomkins GM (1966) The enzymatic synthesis of L-cysteine in Escherichia coli
and Salmonella typhimurium. J Biol Chem 241:4955–4965
19. Nakatani T, Ohtsu I, Nonaka G, Wiriyathanawudhiwong N, Morigasaki S, Takagi H (2012)
Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine
increases L-cysteine production in Escherichia coli. Microb Cell Fact 11:62. doi:10.1186/
1475-2859-11-62
20. Aguilar-Barajas E, Dı́az-Pérez C, Ramı́rez-Dı́az MI, Riveros-Rosas H, Cervantes C (2011)
Bacterial transport of sulfate, molybdate, and related oxyanions. Biometals 24:687–707
21. Hryniewicz M, Sirko A, Pałucha A, B€ock A, Hulanicka D (1990) Sulfate and thiosulfate
transport in Escherichia coli K-12: identification of a gene encoding a novel protein involved
in thiosulfate binding. J Bacteriol 172:3358–3366
22. Sirko A, Zatyka M, Sadowy E, Hulanicka D (1995) Sulfate and thiosulfate transport in
Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-
binding proteins. J Bacteriol 177:4134–4136
23. Peter-Wendisch P, Stolz M, Etterich H, Kennerknecht N, Sahm H, Eggeling L (2005)
Metabolic engineering of Corynebacterium glutamicum for L-serine production. Appl Environ
Microbiol 71:7139–7144
24. Lee HS, Hwang BJ (2003) Methionine biosynthesis and its regulation in Corynebacterium
glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation. Appl Microbiol
Biotechnol 62:459–467
25. Haitani Y, Awano N, Yamazaki M, Wada M, Nakamori S, Takagi H (2006) Functional
analysis of L-serine O-acetyltransferase from Corynebacterium glutamicum. FEMS Microbiol
Lett 255:156–163
26. Wada M, Awano N, Yamazawa H, Takagi H, Nakamori S (2004) Purification and character-
ization of O-acetylserine sulfhydrylase of Corynebacterium glutamicum. Biosci Biotechnol
Biochem 68:1581–1583
27. Sperandio B, Polard P, Ehrlich DS, Renault P, Guédon E (2005) Sulfur amino acid metabolism
and its control in Lactococcus lactis IL1403. J Bacteriol 187:3762–3778
28. Vermeji P, Kertesz MA (1999) Pathways of assimilative sulfur metabolism in Pseudomonas
putida. J Bacteriol 181:5833–5837
L-Cysteine Metabolism and Fermentation in Microorganisms 149

29. Ono B, Hazu T, Yoshida S, Kawato T, Shinoda S, Brzvwczy J, Paszewski A (1999) Cysteine
biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation. Yeast
15:1365–1375
30. Takagi H, Yoshioka K, Awano N, Nakamori S, Ono B (2003) Role of Saccharomyces
cerevisiae serine O-acetyltransferase in cysteine biosynthesis. FEMS Microbiol Lett
218:291–297
31. Wheeler PR, Coldham NG, Keating L, Gordon SV, Wooff EE, Parish T, Hewinson RG (2005)
Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis com-
plex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria. J Biol
Chem 280:8069–8078
32. Wada M, Awano N, Haisa K, Takagi H, Nakamori S (2002) Purification, characterization and
identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship
to cysteine production. FEMS Microbiol Lett 217:103–107
33. Zhou D, White RH (1991) Transsulfuration in archaebacteria. J Bacteriol 173:3250–3251
34. Kitabatake M, So MW, Tumbula DL, Soll D (2000) Cysteine biosynthesis pathway in the
archaeon Methanosarcina barkeri encoded by acquired bacterial genes? J Bacteriol
182:143–145
35. Yamagata S, Ichioka K, Goto K, Mizuno Y, Iwama T (2001) Occurrence of transsulfuration in
synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus
HB8. J Bacteriol 183:2086–2092
36. Mizuno Y, Miyashita Y, Yamagata S, Iwama T, Akamatsu T (2002) Cysteine synthase of an
extremely thermophilic bacterium, Thermus thermophilus HB8. Biosci Biotechnol Biochem
66:549–557
37. Kobayashi S, Masui R, Yokoyama S, Kuramitsu S, Takagi H (2004) A novel metal-activated L-
serine O-acetyltransferase from Thermus thermophilus HB8. J Biochem 136:629–634
38. Soda K (1987) Microbial sulfur amino acids: an overview. In: Jakoby WB, Griffith OW (eds)
Methods in enzymology, vol 143. Academic, Orlando, pp 453–459
39. Awano N, Wada M, Kohdoh A, Oikawa T, Takagi H, Nakamori S (2003) Effect of cysteine
desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli. Appl
Microbiol Biotechnol 62:239–243
40. Awano N, Wada M, Mori H, Nakamori S, Takagi H (2005) Identification and functional
analysis of Escherichia coli cysteine desulfhydrases. Appl Environ Microbiol 71:4149–4152
41. Rossol I, P€uhler A (1992) The Corynebacterium glutamicum aecD gene encodes a C-S lyase
with α, β-elimination activity that degrades aminoethylcysteine. J Bacteriol 174:2968–2977
42. Kim JW, Kim HJ, Kim Y, Lee MS, Lee HS (2001) Properties of the Corynebacterium
glutamicum metC gene product encoding cystathionine β-lyase. Mol Cells 11:220–225
43. Koronakis V, Eswaran J, Hughes C (2004) Structure and function of TolC: the bacterial exit
duct for proteins and drugs. Annu Rev Biochem 73:467–489
44. Wiriyathanawudhiwong N, Ohtsu I, Li ZD, Mori H, Takagi H (2009) The outer membrane
TolC is involved in cysteine tolerance and overproduction in Escherichia coli. Appl Microbiol
Biotechnol 81:903–913
45. Daßler T, Maier T, Winterhalter C, B€ock A (2000) Identification of a major facilitator protein
from Escherichia coli involved in efflux of metabolites of the cysteine pathway. Mol Microbiol
36:1101–1112
46. Franke I, Resch A, Daßler T, Maier T, B€ock A (2003) YfiK from Escherichia coli promotes
export of O-acetylserine and cysteine. J Bacteriol 185:1161–1166
47. Leinfelder W, Heinrich P (1997) Process for preparing O-acetylserine, L-cysteine and L-
cysteine-related products. German patent number WO 97/15673
48. Pittman MS, Corker H, Wu G, Binet MB, Moir AJG, Poole RK (2002) Cysteine is exported
from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter
required for cytochrome assembly. J Biol Chem 277:49841–49849
49. Pittman MS, Robinson HC, Poole RK (2005) A bacterial glutathione transporter (Escherichia
coli CydDC) exports reductant to the periplasm. J Biol Chem 280:32254–32261
150 H. Takagi and I. Ohtsu

50. Nishino K, Yamaguchi A (2001) Analysis of a complete library of putative drug transporter
genes in Escherichia coli. J Bacteriol 183:5803–5812
51. Yamada S, Awano N, Inubushi K, Maeda E, Nakamori S, Nishino K, Yamaguchi A, Takagi H
(2006) Effect of drug transporter genes on cysteine export and overproduction in Escherichia
coli. Appl Environ Microbiol 72:4735–4742
52. Nakamori S, Kobayashi S, Kobayashi C, Takagi H (1998) Overproduction of L-cysteine and L-
cystine by Escherichia coli strains with a genetically altered serine acetyltransferase. Appl
Environ Microbiol 64:1607–1611
53. Takagi H, Kobayashi C, Kobayashi S, Nakamori S (1999) PCR random mutagenesis into
Escherichia coli serine acetyltransferase: isolation of the mutant enzymes that cause
overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition.
FEBS Lett 452:323–327
54. Takagi H, Awano N, Kobayashi S, Noji M, Saito K, Nakamori S (1999) Overproduction of L-
cysteine and L-cystine by expression of genes for feedback inhibition-insensitive serine
acetyltransferase from Arabidopsis thaliana in Escherichia coli. FEMS Microbiol Lett
179:453–459
55. Wirtz M, Hell R (2003) Production of cysteine for bacterial and plant biotechnology: appli-
cation of cysteine feedback-insensitive isoforms of serine acetyltransferase. Amino Acids
24:195–203
56. Denk D, B€ ock A (1987) L-Cysteine biosynthesis in Escherichia coli: nucleotide sequence and
expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-
excreting mutant. J Gen Microbiol 133:515–525
57. Noji M, Inoue K, Kimura N, Gouda A, Saito K (1998) Isoform-dependent differences in
feedback regulation and subcellular localization of serine acetyltransferase involved in cyste-
ine biosynthesis from Arabidopsis thaliana. J Biol Chem 273:32739–32745
58. Pye VE, Tingey AP, Robson RL, Moody PCE (2004) The structure and mechanism of serine
acetyltransferase from Escherichia coli. J Biol Chem 279:40729–40736
59. Kai Y, Kashiwagi T, Ishikawa K, Ziyatdinov MK, Redkina EI, Kiriukhin MY, Gusyatiner
MM, Kobayashi S, Takagi H. Suzuki E (2006) Engineering of Escherichia coli L-serine O-
acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-
cysteine. Protein Eng Des Sel 19:163–167
60. Viljic M, Sahm H, Eggeling L (1996) A new type of transporter with a new type of cellular
function: L-lysine export from Corynebacterium glutamicum. Mol Microbiol 22:815–826
61. Harris CL (1981) Cystine and growth inhibition of Escherichia coli: threonine deaminase as
the target enzyme. J Bacteriol 145:1031–1035
62. Sørensen MA, Pederson S (1991) Cysteine even in low concentrations, induces transient amino
acid starvation in Escherichia coli. J Bacteriol 173:5244–5246
63. Krämer R (1994) Systems and mechanism of amino acid uptake and excretion in prokaryotes.
Arch Microbiol 162:1–13
64. Wacker Fermentation-Grade (2010) Discovery a new dimension of pureness: vegetarian
cysteine. Wacker Chemie AG, Germany
65. Sekowska A, Kung HF, Danchin A (2000) Sulfur metabolism in Escherichia coli and related
bacteria: facts and fiction. J Mol Microbiol Biotechnol 2:145–177
66. Fernandes AP, Fladvad M, Berndt C, Andresen C, Lillig CH, Neubauer P, Sunnerhagen M,
Holmgren A, Vlamis-Gardikas A (2005) A novel monothiol glutaredoxin (Grx4) from
Escherichia coli can serve as a substrate for thioredoxin reductase. J Biol Chem 280:24544–
24552
67. Kredich NM (1992) The molecular basis for positive regulation of cys promoters in Salmonella
typhimurium and Escherichia coli. Mol Microbiol 6:2747–2753
68. Kawano Y, Ohtsu I, Takumi K, Tamakoshi A, Nonaka G, Funahashi E, Ihara M, Takagi H
(2015) Enhancement of L-cysteine production by disruption of yciW in Escherichia coli. J
Biosci Bioeng 119:176–179
L-Cysteine Metabolism and Fermentation in Microorganisms 151

69. Lochowska A, Iwanicka-Nowicka R, Plochocka D, Hryniewicz MM (2001) Functional dis-

section of the LysR-type CysB transcriptional regulator. Regions important for DNA binding,
inducer response, oligomerization, and positive control. J Biol Chem 276:2098–2107
70. Monroe RS, Ostrowski J, Hryniewicz MM, Kredich NM (1990) In vitro interactions of CysB
protein with the cysK and cysJIH promoter regions of Salmonella typhimurium. J Bacteriol
172:6919–6929
71. Kawano Y, Ohtsu I, Tamakoshi A, Shiroyama M, Tsuruoka A, Saiki K, Takumi K, Nonaka G,
Nakanishi T, Hishiki T, Suematsu M, Takagi H (2015) Involvement of the yciW gene in L-
cysteine and L-methionine metabolism in Escherichia coli. J Biosci Bioeng 119:310–313
72. Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol 57:395–418
73. Riemer J, Bulleid N, Herrmann JM (2009) Disulfide formation in the ER and mitochondria:
two solutions to a common process. Science 324:1284–1287
74. Ohtsu I, Kawano Y, Suzuki M, Morigasaki S, Saiki K, Yamazaki S, Nonaka G, Takagi H
(2015) Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in
the L-cysteine export-dependent manner in Escherichia coli. PLoS One 10(4), e0120619
75. Funahashi E, Saiki K, Honda K, Sugiura Y, Kawano Y, Ohtsu I, Watanabe D, Wakabayashi Y,
Abe T, Nakanishi T, Suematsu M, Takagi H (2015) A finding of thiosulfate pathway for
synthesis of organic sulfur compounds in Saccharomyces cerevisiae and an improvement of
ethanol production. J Biosci Bioeng 120:666–669