J. Biol. Macromol. 8(2), 28-37 (2008)

Review
Ribosome-independent Peptide Synthesis in Nature and Their Application to
Dipeptide Production
Shin-ichi Hashimoto
Technical research laboratories, Kyowa Hakko Kogyo Co., Ltd.,
Kyowa-cho, Hofu-shi 747-8522, Japan
Received May 26, 2008; Accepted June 30, 2008
While the ribosome system is the major equipment for peptide bond formation in nature,various ribosome-independent peptide-bond forming activities also exist. These activities are not
only responsible for the syntheses of specific peptides but also involved in the biosynthesis of cofactors and nucleic acid and in the processes of protein breakdown. In this review, the ribosome-independent activities are overviewed and recent attempts to apply the activities in dipeptide
production are introduced.
Key words: peptide bond, nonribosomal peptide synthetase, L-amino acid α-ligase, dipeptide

Introduction
Proteins (and peptides) are major
components of organisms and play central roles
in living creatures. Since they are configured
by peptide bonds, peptide bond is one of themost abundant chemical bond in organisms.
Proteins are synthesized by the ribosome system,
which is ubiquitously equipped by cellular organisms. The system is very sophisticated andexcellent as it can synthesize any sequence ofamino acids with marvelous accuracy. While
the ribosome system is the major player of peptide bond formation in nature, it is not the only
player. Activities other than the ribosome system, which combine amino acids to form peptide
bonds, certainly exist. In this article, these ribosome-independent peptide-bond forming activities are reviewed and their application in the
production of dipeptides, the simplest peptides.
Existence of peptide bonds in nature
There is no question that the majority
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of peptide bonds in nature belong to protein.
However, various compounds other than proteinthat contain peptide bond(s) also exist.
Purine biosynthesis takes the peptide-bondcontaining
intermediate,2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamideo]-succinate (1, 2). Coenzyme A,an essential co-factor, contains two peptide
bonds (3-5). Folate in organisms is modified
by polyglutamate (6). Glutathione is a tripeptide (γ-Glu-Cys-Gly), distributed in a wide
range of organisms, and plays a central role in
keeping redox balance in the cell (7). Carnosine(β-Ala-His)
andanserine(β-Ala-methylhistidine) are dipeptides, found inmuscle or brain of mammal, bird, or fish (8, 9).
Eubacteria contain short peptides containing
D-Ala in their cell surface peptidoglycan (10,11).
Some kinds of microorganisms havebeen known to produce polymers build up withamino acid(s); poly-γ-glutamate by Bacillus andseveral other species, ε-poly-lysine by Streptomyces albulus, and cyanophicin (co-polymer of
Asp and Arg) by Cyanobacteria (12).
There are numerous bioactive oligo-

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Nonribosomal peptide synthesis

peptides and derivatives of oligopeptide produced by microorganisms such as penicillin,
vancomycin, and gramicidin S. Dipeptides
containing unusual amino acids have also beenreported to be produced by microorganisms suchas bacilycin (Ala-anticapsin) by Bacillus subtilis,
bestatin (3-amino-2-hydroxyl-4-phenylbutanoic
acidyl - Leu) by Streptomyces olivoreticuli (13).
Two molecules of amino acids can form two
peptide bonds, resulting in a cyclic structure(diketopiperazine).
Diketopiperazines are
known to be found in microbial metabolite suchas albonoursin (a derivative of cyclo (Phe-Leu))by Streptomyces noursei and thaxtomin (a derivative of cyclo (Trp-Phe)) by Streptomyces acidiscabies (13).
It should be mentioned that peptide
bond formation occurs not only in biosynthesisof some compounds but also in degradation ofprotein (14). In eukaryotes, proteins with

N-terminal primary destabilizing residues are
subjected to ligatation with ubiquitins followed
by degradation mediated by the 26S proteasome
complexes. In eubacteria, specific amino acids
such as Phe are added to the N-terminal residueof proteins that are degraded via the pathway
governed by N-end rule.
These examples show the versatility
and widespread importance of peptide bond inorganisms. Interestingly, formations of thesenonproteinous peptide bonds do not depend on
the ribosome system. Specific enzymes are
responsible for the formation of peptide bonds,
some of which will be introduced in the following section.
Peptide-bond forming activities
To add an amino acid to other aminoacid or peptide, the amino acid should be activated. The activation reaction requires ATP

Shin-ichi Hashimotoand two kinds of active intermediates have been
known (Table). One is aminoacyl-AMP, which
is formed by the following reaction; amino acid+ ATP → aminoacyl-AMP + pyrophosphate(PPi). The other is aminoacylphosphate, which
is formed by the following reaction; amino acid+ ATP → aminoacylphosphate + ADP. Peptide-bond forming activities taking aminoacyl-AMP are represented by the ribosomal
system and nonribosomal peptide synthetase.
In the ribosome system, an aminoacyl residue istransferred from aminoacyl-AMP to the corresponding t-RNA to form a stable intermediateaminoacyl-tRNA. In a narrow sense, peptide
bond formation in the ribosome system occurs at
the peptidyltransfer reaction on rRNA (15).
But in this review, peptide-bond forming activity
is defined the activity including activation ofamino acids. It should be worthy for noticingthat some nonribosomal peptide-bond forming
activities also use aminoacyl-tRNA. On the30

other hand, glutathione synthase and L-aminoacid α-ligase are the examples of the activities
via aminoacylphosphate. Several typical enzymes other than ribosomal system are briefly
reviewed below.
Aminoacyl-tRNA dependent nonribosomal peptide-bond forming activities
The peptidoglycan in Streptococcus
pneumoniae contains “stem peptide” composed
of
up
to
five
amino
acids,
Ala-γ-D-Glu-Lys-D-Ala-D-Ala, of which thelysyl residue ε-amino group is substituted by adipeptide branch of Ala or Ser followed by Ala.
MurM is responsible for the addition of Ala or
Ser to the stem peptide Lys. Recently it was
revealed that MurM transfers Ala or Ser from
Ala-tRNAAla or Ser-tRNASer, respectively, to thestem peptide (11). BppA1 in Enterococcus
faecalis and FemXAB in Staphylococcus aureus
are supposed to catalyze the similar reactions

Fig. 1 Schematic view of peptide-forming reaction by NRPS.
Diamonds and triangles indicate amino acids.

31

Nonribosomal peptide synthesis(16, 17).
In Escherichia coli, proteins, which are
to be degraded and bearing an N-terminal Arg(or Lys), recruit Phe or Leu to their N-terminal
and then enter into N-end rule pathway of proteolysis (18). The recruit is carried out by
leucyl/phenylalanyl-tRNA protein transferase.
The enzyme transfers Leu and Phe residue from
Leu-tRNAleu and Phe-tRNAphe, respectively, to
the N-terminal of the target protein (19).
NRPS (Nonribosomal peptide synthetase)
NRPSs are responsible for the syntheses of a wide array of therapeutically important peptides produced by microorganisms suchas vancomycin, gramicidin S, and cyclosporine.
ACV synthetase, which catalyzes the formationof tripeptide backbone of penicillins and cephalosporins, belongs to NRPS. NRPSs are huge,
multifunctional proteins. They are made up of aseries of modules, each of which takes charge ofadding one amino acid to a growing peptide.
Each module contains at least three enzymaticunits called domains (Fig. 1). An adenylation
domain (A-domain) recognizes the substrateamino acid and activates it as an aminoacyl-AMP. The selectivity-conferring “nonribosomal” code of the A-domain has been determined. The activated amino acid is transferred to 4’-phophopantetheine moiety of thethiolation domain (T-domain) with the release of
AMP. Then the adjacent condensation domain(C-domain) catalyzes the formation of the peptide bond. Finally, the thioesterase domain(Te-domain) catalyzes the release of the productpeptide from the enzyme protein. NRPS havealso been reported to be involved in the synthe-

sis of cyclo-dipeptide. For more details on
NRPS, please refer to the comprehensive reviews (20, 21).
γ-Glutamyl-cysteine synthetase and glutathione
synthetase
As glutathione is the most abundant
non-protein thiol compound widely distributed
in organisms, the biosynthesis of the tripeptide
have been opened as early as in 1950s. Biosynthesis of glutathione occurs in two steps; Glu+ Cys + ATP → γ-Glu-Cys + ADP + Pi and
γ-Glu-Cys + Gly + ATP → glutathione + ADP +Pi.
The former reaction is catalyzed by
γ-glutamyl-cysteine synthetase and the latter one
is catalyzed by glutathione synthetase. Substrate specificities of both enzymes are limited totheir natural substrates and structural analogs(22).
L-amino acid α-ligase (Lal)

Tabata and coworkers discovered agene coding for a novel enzyme named L-aminoacid α-ligase (Lal) from Bacillus subtilis’s genome (23). Lal catalyzes α-dipeptide formation from L-amino acids in the presence of ATP(Fig. 2). It exhibits so wide substrate specificity as 44 kinds of dipeptide can be synthesized,
but never reacts with D-amino acids. It neverforms tri- or longer peptides. From the aminoacid sequence and biochemical data, Lal is considered to belong to ATP-dependent carboxylate-amine/thiol ligase superfamily (24) as wellas γ-glutamyl-cyteine synthetase and glutathione
stnthetase.
Cyanophysin synthetase and γ-polyglutamate

Fig. 2 Schematic view of peptide-forming reaction by Lal.
Diamonds and triangles indicate amino acids. Partially opened circles indicate Lal.

Shin-ichi Hashimoto

synthetase
Cyanophycin [multi-Arg-poly(Asp)] isa branched peptide, uniquely found in cyanobacteria. Cyanophycin synthetase, encoded on
cphA, is responsible for the biosynthesis of thepolymer. It was demonstrated that the purified
enzyme catalyzed polymer synthesis from Arg,
Asp, and a primer polymer concomitant withhydrolysis of ATP to ADP (25). Based on thebiochemical data and its amino acid sequence,the enzyme is regarded to take aminoacylphosphate intermediates (25, 26).
Poly-γ-glutamate is produced by several Bacillus strains and a few other organisms.
The genes governing the synthesis of the polymer have been identified in B. anthracis (capB
and C) and B. subtilis (pgsB and C). Whilebiochemical characteristics of the enzymes havealso been revealed, argumentation on the active
intermediate (aminoacylphosphate or aminoacyl-AMP) still exists (27, 28). The aminoacid sequences of CapB and PgsB share significant homology with Mur ligases of other bacteria, which take aminoacylphosphate.
AlbC
Streptomyces noursei and S. alburus
have been known to produce an antibiotic compound, albonoursin, a dehydrated derivative of
cyclo(Phe-Leu) (29, 30). A recombinant S.
lividans expressing a gene from S. noursei,
named albC, was found to synthesize cyclo(Phe-Leu) (31). The amino acid sequence of
AlbC has no homology with any known NRPSs
and dose not contain an ATP grasp motif, thus,
the reaction mechanism of the enzyme remains
unclear.
Application of peptide-bond forming activites
in dipeptide production(1) Dipeptides’ situation
L-α-Dipeptides are the smallest peptides but versatile compounds. They havesome characteristics which are absent in thecorresponding amino acids. For instance, Gln

32

is unstable in solution, but its dipeptide, Ala-Gln,is very stable and acts as Gln source once it wasincorporated into a human body (32, 33). Tyr
is hardly soluble but Ala-Tyr is 23-folds soluble.
Certain kinds of dipeptides are also known tohave specific physiological functions; Tyr-Arg
has an analgesic effect (34), Val-Tyr has an antihypertensive effect (35), etc. Despite of theseusefulness, commercial use of dipeptides is limited due to their unavailability at low cost.
There have been a variety of methods
to synthesize dipeptides, which are categorizedin two types; chemical synthesis and enzymaticsynthesis. Chemical synthetic way, originatedfrom Emil Fisher’s work in 1901, generally requires protection and deprotection steps. Any
kinds of dipeptide can be synthesized by chemical methods (36). However, because of themulti steps and the necessity of protection, theirproduction costs are so high that they can not beapplied in wider commercial use. Enzymatic
method has also a lot of variations but commonly employs the reverse reaction of a protease or
peptidase to connect amino acids (37, 38). To
suppress the forward reaction, hydrolysis of thepeptide bond, and to direct the order of the twoamino acids, protection of amino- or carboxygroup of the amino acids is required as well as
chemical synthetic method. Thus, applicationof enzymatic methods has also been limited.
The ideal way to synthesize dipeptides is connecting unmodified amino acids in an irreversible reaction.
(2) Emerging new technologies for dipeptide
production
Progress in the research on peptide-bond forming activities have been opened
the possibility to synthesize a designed dipeptide
from unprotected amino acids. Two lines ofstudies have been reported; NRPS utilizing method and Lal utilizing method.
NRPS method

33

Nonribosomal peptide synthesis

The modular architecture of NRPS
prompted to engineer the enzyme to synthesize adesigned dipeptide. Doeckel and Marahiel (39)designed synthetic templates for dipeptide formation by combining parts of bac (coding forbacitracin-biosynthetic NRPS in Bacillus licheniformis) and tyc (coding for tyrocidine-biosynthetic NRPS in B. brevis) genes.
The artificial enzyme was expressed in Escherichia coli, purified, and incubated with Ile, Leu,
and ATP. Ile-Leu was formed as expected.
Using similar strategy, dimodular NRPSs which
synthesized Asp-Phe were constructed (40).
A-PCP or A-PCP-C domains of Asp-activating
module from surfactin synthetase of B. subtilis
and A-PCP, C-A, A-PCP, or A domains of Phe
activating modules from tyrocidine synthetasewere fused. These artificial synthetases had
Asp-Phe synthesizing activity, but their activities
were significantly different, indicating the importance of the strategy to construct a hybrid
NRPS. Knowledge on NRPS and know-how
for engineering it have been accumulating rapidly. Theoretically, any dipeptide can be synthesized by a designed NRPS (41). However,since C and Te domains have been revealed tohave some preference on their substrates, careful
design and some trial and error shall be needed

to get a highly active enzyme. These difficulties may cause the very low productivities in
productions with whole cells.
Lal method
Considering the broad substrate specificity and simplicity of Lal, this enzyme was
expected to realize a novel process, which would
overcome the drawbacks of the current dipeptide
manufacturing processes (chemical synthesis orprotease/peptidase dependent methods). Twotypes of the process have been invented; theresting cell reaction process and the direct fermentation process (Fig. 3).
The resting cell reaction process is a
coupling
reaction
of
Lal
and
an
ATP-regeneration reaction. Detergent treated E.
coli cells expressing Lal from B. subtilis andpolyphosphate kinase from Rhodobacter sphaeroides was reported to produce several kinds of
dipeptides (Ala-Met, Ala-Val, Ala-Ile, Ala-Leu,
Gly-Met, and Gly-Phe) by incubating the corresponding amino acids and polyphosphate (42).
Ala-Met gave the highest titer, 127.9 mM (28g/L), from 200 mM each of Ala and Met. Any
dipeptides within the product spectrum of Lal
can be produced by changing the substrate amino acids.

Fig. 3 Lal-based dipeptide production systems.
Diamonds and triangles indicate amino acids.

Shin-ichi Hashimoto

Because Lal takes unprotected usual
amino acids as the substrates, Lal-expressingorganism would produce some dipeptides without adding the amino acids. This conceptualidea of the direct fermentation was tested, but
simply expressing Lal in E. coli resulted in noaccumulation of dipeptides (43). Two obstacles were assumed; the relatively low affinityof Lal for amino acids and the dipeptide degrading activity of the host cells. To overcome
these problems, enhancing the metabolic flux tothe substrate amino acids and reducing the degradation activity, are necessary. Ala-Gln fermentation is a successful example (43). For theformer purpose, Gln biosynthesis was deregulated and alanine dehydrogenase (Ald) from B.
subtilis was co-expressed with Lal. Reductionof dipeptide degradation was achieved by the
combinatorial disruption of genes for several
dipeptidases (PepA, PepB, pepD, and PepN) andthe dipeptide-import system (Dpp). Lal and
Ald were expressed in the host strain under astationary phase specific promoter to escape theharmful effect of Lal expression on cell growth.
Fed-batch cultivation of the recombinant strain
on a glucose-ammonium medium resulted in theaccumulateion of Ala-Gln (100 mM) in the cultivation supernatant.
No tripeptides orD-amino acid containing dipeptides were detected.
Some other producer strains for
Ala-Met, and Thr-Phe, respectively, were also
reported (44). Obviously, the direct fermentation method is the most cost-effective for dipeptide manufacturing since it dose not need eventhe substrate amino acids. However, there has
been little insight into balancing two metabolic
fluxes or the intracellular fate of dipeptides,
those of which are important for the constructionof the producer strain. The unexpected factthat Lal expression harmed cell growth should
also be managed.
Perspectives
It is astonishing that nature prepares somany ways to form peptide bonds as describedabove. The activities are different in the flex-

34

ibility, accuracy, reaction rate, and energy cost.
The ribosome system is most flexible and highly
accurate, but needs a huge and complex enzyme
system. NRPS is also a huge enzyme and lessflexible but can be designed to synthesize any
peptide. Lal is a simple enzyme but lesser
flexible. On the other hand, the high-energy
phosphate bonds consumed to form one peptide
bond are 4 (2 x ATP → AMP) in NRPS and 1(ATP → ADP) in Lal. It would be interesting
to know how organisms have appointed eachactivity for each job. While there are various
kinds of peptide-bond forming activities, they
can be divided into two types based on the reaction intermediate. Evolutional relation between
the two types may be another interesting point.
From the view point of application, nonribosomal peptide-bond forming activities should be
useful for peptide production. Fermentative
production of dipeptide using Lal is the pioneering example. Pursuing these possibilities
will open the new filed of peptide application.
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Communicated by Araki Kazumi