Appl Microbiol Biotechnol (2006) 70: 564–572
DOI 10.1007/s00253-005-0111-x
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Junya Narita
.
Kenji Okano
.
Toshihiro Tateno
.
Takanori Tanino
.
Tomomitsu Sewaki
.
Moon-Hee Sung
.
Hideki Fukuda
.
Akihiko Kondo
Display of active enzymes on the cell surface
of
Escherichia coli
using PgsA anchor protein
and their application to bioconversion
Received: 26 June 2005 / Revised: 20 July 2005 / Accepted: 20 July 2005 / Published online: 20 August 2005
# Springer-Verlag 2005
Abstract We have developed a novel Escherichia coli cell
surface display system by employing PgsA as an anchoring
motif. In our display system, C-terminal fusion to PgsA
anchor protein from Bacillus subtilis was used. The en-
zymes selected for display were α-amylase (AmyA) from
Streptococcus bovis 148 and lipase B (CALB) from Can-
dida antarctica. The molecular mass values of AmyA and
CALB are approximately 77 and 34 kDa, respectively.
The enzymes were displayed on the surface as a fusion
protein with a FLAG peptide tag at the C terminus. Both
the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion
proteins were shown to be displayed by immunofluores-
cence labeling using anti-FLAG antibody. The displayed
enzymes were active forms, and AmyA and CALB ac-
tivities reached 990 U/g (dry cell weight) and 4.6 U/g (dry
cell weight), respectively. AmyA-displaying E. coli cells
grew utilizing cornstarch as the sole carbon source, while
CALB-displaying E. coli cells catalyzed enantioselective
transesterification, indicating that they are effective whole-
cell biocatalysts. Since a target enzyme with a size of
77 kDa and an industrially useful lipase have been suc-
cessfully displayed on the cell surface of E. coli for the
first time, PgsA protein is probably a useful anchoring motif
to display various enzymes.
Introduction
In recent years, there has been considerable progress in the
development of expression systems for the display of het-
erologous peptides and proteins on the surface of micro-
organisms (Benhar 2001). Microbial cell surface display
has a wide range of biotechnological and industrial ap-
plications in areas such as whole-cell biocatalysis for bio-
conversion, biosensing, biosorption, immobilization, oral
vaccine development, and combinatorial peptide library
screening (Lee et al. 2003). In gram-negative bacteria, sur-
face display systems based on various anchoring strategies
have been reported, with outer membrane proteins (Chang
and Lo 2000; Lee et al. 2004), pili and flagella (Westerlund-
Wikström et al. 1997), modified lipoproteins (Georgiou
et al. 1996), ice nucleation proteins (Jung et al. 1998),
and autotransporters (Veiga et al. 2003) among the an-
chors used. Escherichia coli is an attractive host because
various genetic tools and mutant strains are available, and
its high transformation efficiency makes it ideal for screen-
ing a peptide or protein libraries after surface display. In the
current E. coli cell surface display systems, however, there
remain problems such as the size limitation of the target
protein, misfolding, mislocalization, and destabilization of
the outer membrane (Georgiou et al. 1997; Wan et al. 2002).
The size limitation and misfolding in particular are sig-
nificant problems for broader applications. In most of the
previous studies, peptides and small proteins have been
displayed. The largest target protein reported to have been
displayed so far is a bacterial lipase with a molecular size of
49.9 kDa using OprF as an anchor motif (Lee et al. 2005).
However, for instance, to use E. coli cells for bioconversion
and biosensing, it is necessary to display various enzymes,
J. Narita
.
T. Tanino
.
H. Fukuda
Division of Molecular Science,
Graduate School of Science and
Technology, Kobe University,
Nada-ku, Kobe, 657-8501, Japan
K. Okano
.
T. Tateno
.
A. Kondo (*)
Department of Chemical Science and Engineering,
Faculty of Engineering, Kobe University,
1-1 Rokkodaicho, Nada-ku,
Kobe, 657-8501, Japan
e-mail: akondo@kobe-u.ac.jp
Tel.: +81-78-8036196
Fax: +81-78-8036206
T. Sewaki
BioLeaders Japan Corporation,
Saito Asagi,
Ibaraki, 567-0085, Japan
M.-H. Sung
Department of Bio and Nanochemistry,
Kookmin University,
Songbuk-gu,
Seoul, 136-702, South Korea