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Applied and Environmental Microbiology, June 1999, p. 2439-2445, Vol. 65, No. 6
Institute of Food and Agricultural Sciences,
Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida 32611
Received 23 November 1998/Accepted 14 March 1999
Escherichia coli B has been engineered as a biocatalyst
for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce
high levels of Erwinia chrysanthemi endoglucanase (encoded
by celZ) as a periplasmic product and that this enzyme can
function with commercial fungal cellulase to increase ethanol
production. In this study, we have demonstrated two methods that
improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli
glycolytic gene promoters (gap and eno) were
tested and found to be less effective than the original
celZ promoter. By screening 18,000 random fragments of
Zymomonas mobilis DNA, a surrogate promoter was identified
which increased celZ expression up to sixfold. With this
promoter, large polar inclusion bodies were clearly evident in the
periplasmic space. Sequencing revealed that the most active surrogate
promoter is derived from five Sau3A1 fragments, one of
which was previously sequenced in Z. mobilis. Visual
inspection indicated that this DNA fragment contains at least five
putative promoter regions, two of which were confirmed by primer
extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of
active enzyme and facilitated secretion or release of over half of the
activity into the extracellular environment. With the most active
construct, of a total of 13,000 IU of active enzyme per liter of
culture, 7,800 IU was in the supernatant. The total active
endoglucanase was estimated to represent 4 to 6% of cellular protein.
The cost of cellulase enzymes is one
of the principal economic bottlenecks for the commercialization of
lignocellulosic biomass-to-ethanol technology (13, 16, 18, 29,
32). Three different approaches have been proposed to reduce the
cost of cellulase for the bioethanol industry: (i) improved on-site
production of cellulase (16, 32), (ii) optimized
reconstitution of cellulase components from different sources into a
more effective artificial cellulase system (12, 43, 44), and
(iii) development of improved ethanologenic biocatalysts which supply a
portion of the cellulase needed for the direct microbial conversion of
cellulose into ethanol (6, 17). Our lab has focused on the
latter approach.
Recombinant strains of Escherichia coli B have been
engineered which produce ethanol efficiently from all sugar
constituents of lignocellulose by using the Zymomonas
mobilis genes encoding the pyruvate-to-ethanol pathway
(pdc and adhB) (20, 30). Genes for
cellulose hydrolysis are now being added (27, 47). The solubilization of cellulose involves three classes of enzymes: endoglucanase, exoglucanase, and Further improvement of cellulose utilization will require extracellular
secretion or release of endoglucanase and exoglucanase activities.
E. coli is very limited in its ability to secrete proteins
into the extracellular environment (34). Recombinant proteins such as endoglucanases, which are secreted by their source organisms, are typically accumulated in the periplasmic space in
E. coli (11, 26, 47). In related gram-negative
bacteria, however, at least three different types of protein secretion
systems have been described (type I, type II, and type III) (25,
34). Considerable progress has been made in our understanding of
the type II secretion system (5, 14, 15, 23-25, 28, 34, 37), the most widely used system for protein secretion. Type II
secretion involves a two-step process in which a pro-protein containing
an N-terminal signal peptide is exported to the periplasm and processed
into a mature enzyme by using a highly conserved Sec pathway. The
mature enzyme is then secreted into the extracellular environment
through the peptidoglycan and outer membrane. Seven type II secretion
systems have been cloned from gram-negative bacteria (2, 15, 24,
28, 34, 35, 38, 41), and all contain 12 to 15 gene products which
share homology. Although heterologous proteins are often exported to
the periplasm by the Sec system, the genes which complete extracellular
secretion are highly species specific. This is true even for closely
related enteric bacteria such as Erwinia chrysanthemi and
E. carotovora (15, 24, 35). Two type II secretion
systems, encoded by pul genes from K. oxytoca and
the out genes from E. chrysanthemi, have been
reconstituted in K-12 strains of E. coli (15,
34). The E. chrysanthemi EC16 out gene
products have been shown to secrete EC16 pectate lyase in recombinant
E. coli (15). In EC16, this system also appears
to facilitate the secretion of the endoglucanase EGZ (3).
Our laboratory has previously cloned celZ from E. chrysanthemi P86021 (4) and demonstrated that the
encoded enzyme (EGZ) can function with commercial fungal cellulase to
improve ethanol production (47). In these studies, EGZ was
accumulated in the E. coli B periplasm and required cell
disruption (solvents, detergent, or heat) for release. Total EGZ
production was approximately 3 IU per ml of broth.
In this study, we have identified a strong promoter to enhance the
expression of celZ endoglucanase in E. coli B and
demonstrated that this enzyme can be effectively secreted (or released
into the environment) by using the out genes from E. chrysanthemi EC16, thus eliminating the need for cell disruption.
Organisms and culture conditions.
Bacterial strains and
plasmids used in this study are listed in Table
1. E. coli DH5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Enhancement of Expression and Apparent Secretion of
Erwinia chrysanthemi Endoglucanase (Encoded by
celZ) in Escherichia coli B
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-glucosidase (32). The
requirement for the addition of -glucosidase has been eliminated by
the functional insertion of genes for cellobiose utilization from a
related organism, Klebsiella oxytoca M5A1. These K. oxytoca genes encode a phosphoenolpyruvate-dependent phosphotransferase system and phosphocellobiase (22).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
was used as
the host for plasmid construction. The celZ gene was
previously cloned in our laboratory from E. chrysanthemi
P86021 (4, 47). The out genes were cloned by He
et al. (15) from E. chrysanthemi EC16.
TABLE 1.
Strains and plasmids used in this study
Genetic methods.
E. chrysanthemi out genes (pCPP2006)
were conjugated into E. coli by using pRK2013 for
mobilization (10, 28). Standard methods were used for all
plasmid constructions (1, 40). Small-scale plasmid isolation
was performed by the TELT procedure (1). Large-scale plasmid
isolation was performed with a Promega Wizard Kit. DNA fragments were
isolated from gels with a Qiaquick Gel Extraction Kit from Qiagen.
E. coli and Z. mobilis chromosomal DNAs were
isolated as described by Cutting and Horn (8) and Yomano et
al. (49), respectively. Purified chromosomal DNA from E. coli DH5 served as a template for the amplification of
two glycolytic gene promoters by using the following primer pairs: gap promoter, 5'-CGAATTCCTGCCGAAGTTTATTAGCCA-3'
and 5'-AAGGATCCTTCCACCAGCTATTTGTTAGTGA-3'; and
eno promoter, 5'-AGAATTCTGCCAGTTGGTTGACGATAG-3'
and 5'-CAGGATCCCCTCAAGTCACTAGTTAAACTG-3'.
Primer extension analysis of transcriptional initiation. Promoter regions were identified by mapping the transcriptional start sites with a primer within the celZ gene. RNA was isolated from cells in late exponential phase by using a Qiagen RNeasy kit. Cells were treated with 400 µg of lysozyme per ml in TE containing 0.2 M sucrose (25°C, 5 min) prior to lysis. After ethanol precipitation, RNA was dissolved in 20 µl of Promega avian myeloblastosis virus (AMV) reverse transcriptase buffer (50 mM Tris-HCl, pH 8.3; 50 mM KCl; 10 mM MgCl2; 0.5 mM spermidine; 10 mM dithiothreitol). IRD41-labeled primer (5'-GACTGGATGGTTATCCGAATAAGAGAGAGG-3'; LI-COR, Inc.) was added. The sample was denatured at 80°C for 5 min, annealed at 55°C for 1 h, and purified by alcohol precipitation. Annealed samples were dissolved in 19 µl of AMV reverse transcriptase buffer containing 500 µM deoxynucleoside triphosphates and 10 U of AMV reverse transcriptase and then incubated for extension (1 h at 42°C). Products were treated with 0.5 µg of DNase-free RNase A per ml, precipitated, dissolved in loading buffer, and compared to parallel dideoxy sequences by using the LI-COR model 4000-L DNA sequencer.
Endoglucanase activity. Expression of celZ was initially evaluated by using the Congo red procedure of Wood and Bhat (48). Recombinant clones were transferred to gridded CMC plates and incubated for 18 h at 30°C prior to staining. Endoglucanase-positive clones formed yellow zones on a red background. The diameters of these zones were recorded as a relative measure of celZ expression.
EGZ activity was also determined in vitro by using CMC as a substrate. Appropriate dilutions of cell-free culture broth (extracellular activity) or broth containing cells treated with ultrasound (total activity) were assayed at 35°C in 50 mM citrate buffer (pH 5.2) containing CMC (20 g/liter). Conditions for optimal enzyme release for 3- to 4-ml samples were determined to be four pulses at full power for 1 s each (model W-220F cell disruptor; Heat System-Ultrasonics, Inc., Plainview, N.Y.). Enzyme reactions were terminated by heating in a boiling water bath (10 min). Reducing sugars were measured by using the dinitrosalicylic acid reagent (48) with glucose as a standard. Enzyme activity (IU) is expressed as micromoles of reducing sugar released per minute or as a percentage of total activity. Results are averages of two or more determinations.Ultrastructural analysis. For ultrastructural analysis, fresh colonies from Luria agar plates were fixed in 2% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7) and then in 1% osmium tetroxide, followed by treatment with 1% uranyl acetate in distilled water. Samples were dehydrated in ethanol and embedded in Spurr's plastic (42). Ultrathin sections were examined with a Zeiss EM-10CA electron microscope.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of a low-copy-number promoter probe vector with celZ as the reporter. To facilitate the isolation of strong promoters, a low-copy-number vector was constructed with a pSC101 replicon and a BamHI site immediately preceding a promoterless celZ gene (pLOI2171). Plasmid pLOI1620 was used as a source of celZ and is a pUC18 derivative with expression from consecutive lac and celZ promoters. The BamHI site in this plasmid was eliminated by digestion and Klenow treatment (pLOI2164). The celZ gene was isolated as a promoterless NdeI fragment after Klenow treatment. The resulting blunt fragment was digested with HindIII to remove downstream DNA and then ligated into pUC19 (HindIII to HincII) to produce pLOI2170. In this plasmid, celZ is oriented opposite to the direction of lacZ transcription and was weakly expressed. The BamHI (amino terminus)-SphI (carboxyl terminus) fragment from pLOI2170 containing celZ was then cloned into the corresponding sites of pST76-K, a low-copy-number vector with a temperature-sensitive replicon, to produce pLOI2171 (Fig. 1). Expression of celZ in this vector was extremely low, facilitating its use as a probe for strong promoters.
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Expression of celZ from E. coli gap and
eno promoters.
Chromosomal DNA from DH5 was used as
the template to amplify the gap and eno promoter
regions by PCR. The resulting fragments of approximately 400 bp each
were digested with EcoRI and BamHI and cloned
into the corresponding sites in front of a promoterless celZ
in pLOI2171 to produce pLOI2174 (gap promoter) and pLOI2175 (eno promoter). As a control, the
EcoRI-SphI fragment from pLOI2164 containing the
complete celZ gene and native E. chrysanthemi
promoter was cloned into the corresponding sites of pST76-K to produce pLOI2173.
to
compare EGZ production. For both strains of E. coli,
endoglucanase activities were lower on CMC plates with E. coli glycolytic promoters than with pLOI2173 containing the native
E. chrysanthemi celZ promoter (Table
2). Assuming activity is related to the
square of the radius of each zone (Fick's law of diffusion), EGZ
production with glycolytic promoters (pLOI2174 and pLOI2175) was
estimated to be 33 to 65% lower than in the original construct.
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Cloning Z. mobilis DNA fragments as promoters for
celZ expression.
Previously, we have used random
fragments of Z. mobilis DNA as an effective source of
surrogate promoters for the high-level expression of heterologous genes
in E. coli (7, 19). A similar approach was used
to find promoters for Erwinia celZ expression. Z. mobilis chromosomal DNA was extensively digested with
Sau3A1. The resulting fragments were ligated into pLOI2171
at the BamHI site and transformed into E. coli
DH5 to generate a library of potential promoters. Resulting colonies
were transferred to gridded CMC plates and stained for endoglucanase
activity after incubation (Table 2). Approximately 20% of the 18,000 clones tested were CMC positive. The 75 clones which produced larger
zones than the control, pLOI2173, were examined further by using
E. coli B. In general, recombinants of DH5 produced
larger zones than recombinants of strain B. However, the relative
promoter strength in each host was similar for most clones. Based on
zone size with CMC plates, four clones appeared to express
celZ at approximately 6-fold-higher levels than the
construct with the original E. chrysanthemi celZ gene
(pLOI2173) and at 10-fold-higher levels than the E. coli glycolytic promoters.
Production and apparent secretion of endoglucanase.
Eight
plasmid derivatives of pST76-K (pLOI2177 to pLOI2184) were selected
from group I and group II (Table 2) and assayed for total
endoglucanase activity in strain B (Table
3). The four plasmids with the largest
zones on CMC plates also had the highest endoglucanase activities
(pLOI2177, pLOI2180, pLOI2182, and pLOI2183). The activities were
approximately sixfold higher than that of the unmodified
celZ (pLOI2173), which is in excellent agreement with our
estimate by using the square of the radius of the cleared zone on CMC
plates. Figure 2 shows a comparison of
activity estimates from plates and in vitro enzyme assays for strain B
containing a variety of different promoters, with and without the
out genes. Although there is some scatter, a direct
relationship is clearly evident, which validates the plate method for
estimating relative activity. The original construct in pUC18, a
high-copy-number plasmid, was also included for comparison (pLOI2164).
This construct with consecutive lac and celZ
promoters produced less EGZ activity than three of the low-copy-number
plasmids with surrogate promoters (pLOI2177, pLOI2182, and pLOI2183).
The DNA fragment containing celZ and the most effective
surrogate promoter was isolated from pLOI2183
(EcoRI-SphI) and inserted into pUC19 with
transcription oriented opposite to that of the lac promoter
(pLOI2307). With this high-copy-number plasmid, endoglucanase activity
was further increased twofold.
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Sequence of Z. mobilis fragment with strong promoter
activity.
The sequences of the four strongest surrogate promoters
(pLOI2177, pLOI2180, pLOI2182, and pLOI2183) were determined. To
facilitate this process, each was fused with pUC19 at the
PstI site. The resulting plasmids, pLOI2196, pLOI2197,
pLOI2198, and pLOI2199, were produced at high copy numbers (ColE1
replicon) and could be sequenced in both directions by using M13 and T7
sequencing primers. All four plasmids contained identical pieces of
Z. mobilis DNA and were siblings. Each was 1,417 bp in
length and contained four internal Sau3A1 sites. DNA and
translated protein sequences (six reading frames) of each piece were
compared to the current database. Only one fragment exhibited a strong
match in BLAST searches (281-bp internal fragment); it was 99%
identical in DNA sequence to part of the Z. mobilis hpnB
gene which is proposed to function in cell envelope biosynthesis
(39). Primer extension analysis revealed both a single major
start site, 67 bp upstream from the Sau3A1/BamHI
junction site with celZ, and a second minor start site
farther upstream (Fig. 3). Sequences in
the 
10 and 35 regions were compared to the conserved
sequences for E. coli sigma factors (45, 46).
The dominant promoter region (ca. 85% of the total start site)
appears similar to a 
70 promoter, whereas the secondary
promoter site resembles a 38 promoter.
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Periplasmic inclusion bodies. Little difference in cell morphology was observed between recombinants and the parental organism by light microscopy. Under the electron microscope, however, small polar inclusion bodies were clearly evident in the periplasm of strain B(pLOI2164) and are presumed to contain EGZ (Fig. 4). With strain B(pLOI2307), which produced twofold-higher activity, these polar bodies were very large and occupied up to 20% of the total cell volume. The large size of these polar bodies suggests that activity measurements may underestimate total EGZ production. Typically, polar inclusion bodies were smaller in constructs which also contained the out genes (not shown). No periplasmic inclusion bodies were evident in strain B(pUC19), which served as a control.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Despite extensive investigation of technology for the
bioconversion of lignocellulose to fuel ethanol, the cost of cellulase is still one of the major barriers for commercialization (13, 16,
18, 29, 32). The development of inexpensive enzymatic methods for
cellulose hydrolysis has great potential for improving the efficiency
of substrate utilization and the economics of the process. Prior to
fermentation, insoluble substrates such as cellulose must be converted
into a soluble form which can be transported and metabolized. Fungal
cellulase is commercially produced on a large scale and is used in food
processing, detergents, and pulping (32). Although
industrial experience with these enzymes makes it likely that they will
also be used in cellulose-to-ethanol processes, it should be possible
to develop ethanologenic biocatalysts that provide at least part of the
required cellulase activities as a recombinant coproduct with ethanol.
Ethanologenic derivatives of E. coli B have been previously
engineered to metabolize cellobiose and cellotriose by inserting genes
encoding the phosphoenolpyruvate-dependent phosphotransferase system
and phospho--glucosidase from K. oxytoca M5A1
(22), eliminating a need for the least-stable component of
fungal cellulase, -glucosidase (27). However, enzymes
which solubilize polymeric cellulose must be present in the
extracellular environment to be effective.
Previous studies have identified E. chrysanthemi EGZ as an effective endoglucanase which can be used in combination with commercial fungal cellulase to improve ethanol production from cellulose (47). The gene encoding this enzyme was expressed well (ca. 3,000 IU per liter), but higher levels of expression would be desirable. The native E. chrysanthemi promoter was more effective in E. coli than either of two E. coli glycolytic promoters (gap and eno). However, Z. mobilis DNA served as an excellent source of surrogate promoters. Sau3A1 fragments with strong promoter activity were identified which provided a sixfold increase in EGZ production in a low-copy-number vector and a twofold increase in a high-copy-number vector. In both cases, most of the activity was cell associated (periplasmic, intracellular, or bound). Relative activity correlated with the size of periplasmic inclusion bodies. Based on activity, EGZ is calculated to represent 4 to 6% of the total cellular protein in the most active construct. However, this may be an underestimate of total EGZ protein since inclusion bodies represented approximately 20% of the total cell volume. It is possible that some of the proteins retained in the inclusion bodies are misfolded or partially degraded.
Native EGZ was secreted or released from the periplasm to the extracellular environment by the out gene products in E. chrysanthemi (21), and the out gene products have been previously demonstrated to secrete recombinant E. chrysanthemi pectate lyases in E. coli (15). Addition of the out genes increased the EGZ activity produced in all E. coli recombinants up to twofold and facilitated the apparent secretion of approximately one-half of the activity into the extracellular environment. The increase in EGZ activity could result from improved folding or reduced degradation. At this time, the basis for incomplete secretion or release remains unknown but may be growth associated. Preliminary experiments revealed that over 90% of the EGZ activity was extracellular in early exponential phase. The fraction of extracellular activity slowly declined and continued to decline during stationary phase, although total activity continued to increase. It is possible that the stability or synthesis of the Out proteins or Sec proteins limited EGZ export during the late-exponential and stationary phases, while the unregulated promoters driving EGZ production continued to function.
The ability of these E. coli strains to secrete or release high levels of EGZ demonstrates an approach which can be used to produce EGZ as a coproduct with ethanol in ethanologenic strains of E. coli B and eliminates the need for additional process steps for the release of endoglucanase activity (47). Although this approach may not be readily used with genes from other hosts, it does demonstrate the potential for E. coli to be further developed as a host for recombinant protein secretion. Considerable progress has been made in defining the basis for protein recognition and secretion in the analogous systems from seven different organisms (2, 5, 14, 15, 23-25, 28, 34, 35, 37, 38, 41). Structural information for extracellular secretion by type II systems does not appear to be localized within a particular region and may involve the entire structure of the secreted protein (14). Recognition of target proteins for secretion appears to reside in the outC and outD gene products (24). With continued progress in defining this relationship it should be possible to secrete large amounts of recombinant products for biotechnology applications by using E. coli as a host.
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ACKNOWLEDGMENTS |
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We thank A. Collmer for sharing plasmid pCPP2006 containing the out genes from E. chrysanthemi EC16 and G. Posfai for sharing the low-copy-number vector, pST76-K.
This research was supported in part by grants from the U.S. Department of Agriculture, National Research Initiative (98-35504-6177), and the U.S. Department of Energy, Office of Basic Energy Science (DE-FG02-96ER20222).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Cell Science, IFAS, P.O. Box 110700, University of Florida, Gainesville, FL 32611. Phone: (352) 392-8176. Fax: (352) 846-0969. E-mail: lingram{at}micro.ifas.ufl.edu.
Florida Agricultural Experiment Station journal series no.
R-06614.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, June 1999, p. 2439-2445, Vol. 65, No. 6
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