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High-efficiency secretion of #-mannanase in Bacillus subtilis through protein synthesis and secretion optimization Yafeng Song, Gang Fu, Huina Dong, Jianjun Li, Yuguang Du, and Dawei Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05528 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017
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Journal of Agricultural and Food Chemistry
High-efficiency secretion of β-mannanase in Bacillus subtilis through protein synthesis and secretion optimization Yafeng Song1, 2, Gang Fu1, 2, Huina Dong1, 2, Jianjun Li3, 4,*, Yuguang Du3, 4 Dawei Zhang1, 2,*
1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China 2 Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China 3 National Key Laboratory of Biochemical Engineering, National Engineering Research Center for Biotechnology (Beijing), 100190, China 4 Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, 100190, China
*Corresponding author.
[email protected] (J.Li) +86-10-82545039;
[email protected] (D. Zhang) +86-022-24828749
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Abstract
2
The manno endo-1, 4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random
3
hydrolysis of internal (1→4)-β-mannosidic linkages in the mannan polymers. A codon
4
optimized β-mannanase gene from Bacillus licheniformis DSM13 was expressed in
5
Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide
6
sequences cloned from B. subtilis were placed upstream of the target gene, the highest
7
activity of β-mannanase was observed using SPlipA as a signal peptide. Then, a
8
1.25-fold activity of β-mannanase was obtained when another copy of groESL operon
9
was inserted into the genome of host strain. Finally, five different promoters were
10
separately used to enhance the synthesis of the target protein. The results showed that
11
promoter Pmglv, a modified maltose-inducible promoter, significantly elevated the
12
production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of
13
β-mannanase in the supernatant when using locust bean gum as substrate reached
14
2207 U/mL. This work provided a promising β-mannanase production strain in
15
industrial application.
16
Keywords: β-mannanase, Bacillus subtilis, signal peptides, promoters, chaperones,
17
signal peptidase
18
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Introduction
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β-mannanase (endo-1,4-β-D-mannan mannosidase, EC. 3.2.1.78), an extracellular
21
enzyme, is responsible for randomly cleaving the backbone of mannan-based
22
polysaccharides, releasing short β-1,4-mannooligomers as well as new chain ends1. It
23
plays an important role in the hydrolysis of hemicellulose, which is estimated to be
24
the second most abundant polysaccharide in nature because mannans constitute the
25
predominant component of hemicellulose fractions in various plant tissues2.
26
Furthermore, it displayed synergistic interactions with β-mannosidase and
27
α-galactosidase 3, due to the elaborate chemically distinctness and interlocking of
28
polysaccharides within the plant cell walls. Being one of the most significant
29
mannan-degrading enzymes, β-mannanases are widely distributed among plants,
30
marine mollusks, fungi and some species of bacteria, and many have been purified
31
and characterized4-8. The broad substrate specificities and resistance to harsh
32
conditions of some β-mannanases cloned from different species permit their industrial
33
applications, ranging from food, feed, paper and pulp biobleaching, bioactive
34
oligosaccharides production in addition to second generation biofuels9. Owing to the
35
importance and demand for mannooligosaccharides, β-mannanases have been
36
extensively researched. These efforts comprise the enzyme cloning, purification and
37
identification of the X-ray crystal structures to facilitate the elucidation of catalytic
38
mechanisms and engineering of improved enzymes with regard to product specificity
39
and catalytic efficiency.
40
β-mannanase (ManB) from Bacillus licheniformis DSM13 (ATCC14580) which
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belongs to glycoside hydrolase family 26
42
alkali-stability under a wide range of conditions, especially when substrates were
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high-molecular-weight mannans consisting of more than six mannose monomers11.
44
The half-life time of activity (τ1/2) of ManB remained as high as approximately 80
45
hours, when incubating temperature increased up to 50oC. In addition, it also kept
46
stable within pH ranging from 5-12 after incubation for 30 min at 50oC. In many cases,
47
genes encoding β-mannanases originated from different bacteria and fungi species
48
were
49
respectively12-14. Similarly, β-mannanase (ManB) has once been expressed in E. coli
50
by applying OmpA signal peptide directing its secretion11. However, only half of the
51
enzymes secreted into the medium and half stayed within the periplasmic space. This
52
elevated costs for downstream purification process and hampered its commercial
53
implementation.
heterologously
expressed
in
, performed well in thermo-stability and
Escherichia
coli
and
Pichia
pastoris,
54
B. subtilis, a Gram-positive bacterium, is widely used in expressing a variety of
55
enzymes, therapeutic proteins and metabolites15-20 due to the Generally Recognized
56
As Safe (GRAS) status, which gives it a priority in food, beverage, feed additive,
57
pharmaceuticals applications. The most significant advantage of this bacterium is to
58
secrete proteins directly into the culture medium at high concentrations19,
59
Furthermore, genetic engineering strategies and high-efficiency transformation
60
methods22-25 make the genetic manipulation more accessible, which prompt the
61
optimizations of this host strain. Although B. subtilis has been used to express
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mannanase, neither the regulation of transcription and translation nor the secretion
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pathway has been optimized18,
26-29
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protease-deficient strain, and Brevibacillus brevis have been employed to express
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mannanase Man23, but with only two vectors being investigated to improve its
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production30. In another example, lacking systematic optimizations, a shuttle vector
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carrying an endo-1,4-beta-mannosidase from Bacillus pumilus was expressed in B.
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subtilis 1A751, leading to a low level of enzyme production (8.65U/mL)31. Therefore,
69
a combination of above-mentioned approaches was conducted to verify whether it
70
could reduce specific bottlenecks encountered by β-mannanase during the secretion
71
process, to further increase the final production.
. For instance, B. subtilis WB600, a
72
In the present study, we attempted to increase the synthesis and secretion of
73
β-mannanase in B. subtilis. The β-mannanase gene from B. licheniformis DSM13 was
74
codon optimized after the removal of the native signal peptide, followed by the
75
expression in pMA5. In the following optimization processes, we firstly selected the
76
most suitable signal peptide among 6 candidates, which could direct the secretion of
77
most β-mannanases into the medium. The second step was the identification of the
78
bottlenecks that hindered the secretion and release of high concentration proteins.
79
This was achieved by expressing our target protein in host strains that overexpressed
80
genes encoding bottleneck candidates, such as secretion components, chaperones or
81
signal peptidase. The final step was intended to increase the flux of the protein
82
amounts by efficient synthesis. In this phase, several constitutive promoters and
83
inducible promoters were evaluated and compared. As a result, the combination of
84
these strategies (Figure 1) considerably increased the final production of β-mannanase.
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Furthermore, this improvement not only prompted the potential industrial application
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of β-mannanase, but also implied the feasibility of this strategy being applied to
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increase the production of many other heterologous proteins in B. subtilis.
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Materials and Methods
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Strains, plasmids, and cultivation conditions
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Plasmids and bacteria strains used in this study were listed in Table 1 and Table 2.
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E. coli strains were cultured at 37°C with 220 rpm orbital shaking in lysogeny broth
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(LB) medium [1% tryptone, 0.5% yeast extract, 0.5% NaCl]. B. subtilis cells
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harboring appropriate plasmids were cultivated at 37°C in 2×SR medium [3%
94
tryptone, 5% yeast extract and 0.6% K2HPO4, pH 7.2] in 250-mL triangular flasks.
95
Appropriate antibiotics were supplemented to the medium when required: 100 µg/mL
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ampicillin for E. coli, 40 µg/mL kanamycin and 10 µg/mL chloramphenicol for B.
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subtilis. Only B. subtilis 1A751 derivative strains with genes encoding Sec-secretion
98
components, signal peptidases, and molecular chaperones integrated into the genome
99
(Table 2) were cultured with media adding 10 µg/mL chloramphenicol as well as 2%
100
(w/v) of xylose. Strains containing inducible promoters were separately cultivated in
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medium with the presence of inducers under different concentrations: 0, 0.001, 0.01,
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and 0.1 mM of IPTG, and 0, 0.5, 1.0, 2.0% (w/v) of maltose. Cells of B. subtilis were
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pre-cultured in LB medium overnight and inoculated to OD600 of 0.01, followed by 4
104
hours of incubation in 2×SR medium. Different final concentrations of IPTG were
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then added to the medium with strains containing Pgrac, respectively. For the strains
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carrying Pmglv, maltose was added to the medium immediately after the inoculation.
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Cell growth was monitored by measurement of the optical density at 600 nm (OD600).
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Standard deviations are based on a minimum of three statistically independent
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experiments.
110
General manipulation
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The native signal peptide of β-mannanase (NCBI accession number AAU39670)
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from B. licheniformis DSM13 was removed and the rest sequences were codon
113
optimized for B. subtilis. The sequence of the native signal peptide being removed
114
was: MKKNIVCSIFALLLAFAVSQPSYA. Plasmids were constructed by applying
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prolonged overlap extension (POE)-PCR32 method. β-mannanase and linearized
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vector fragment were prepared by PCR amplification using PrimerSTAR Max DNA
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Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and
118
primers (ms/ma and vs/va, respectively), according to the instructions of the
119
manufacturer. POE-PCR amplification mixture comprises Q5 DNA polymerase,
120
dNTP mix, amplified β-mannanase and linearized vector fragments. The PCR
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protocol was as follows: denaturation at 98oC for 30 s, followed by 30 cycles of
122
denaturation at 98oC for 5 s and then annealing at 55oC for 10 s and extension at 72°C
123
for 4 min. The POE-PCR products were directly transformed into competent E. coli
124
DH5α, and positive colony samples were selected to be further validated by
125
sequencing the fragments (Genwiz, China).
126
Other plasmids were constructed following the same method. Primers used to
127
amplify the signal peptide fragments and promoter fragments, as well as other
128
oligonucleotides used in this study were listed in Table S1. The genome of B. subtilis
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168 served as the template to amplify signal peptide
fragments including SPamyL,
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SPlipA, SPnprB, SPnprE, SPphoD, SPywbN, and promoter fragments including PaprE, and P43.
131
The fragment lengths of PaprE and P43 were 200 bp and 275 bp, respectively. Pgrac
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containing the promoter, lacO sequences and the regulator protein lacI was 1548 bp,
133
and originated from the plasmid pHT43 plasmid33. Pmglv was a modified maltose
134
inducible promoter34, 35, which was constructed by researchers in our lab (unpublished
135
work). Constructed plasmids were transformed into B. subtilis under the standard
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competent transformation method.
137
SDS-PAGE analysis
138
Supernatant samples were harvested and separated by centrifugation (12000g, 10
139
minutes, 4oC) after fermentation in flasks for different hours to perform sodium
140
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (NuPAGE 10 %
141
Bis-Tris Gel, Commassie Brilliant Blue, Invitrogen Life Technologies, USA). Cell
142
precipitate was resuspended with 50 µL lysis buffer (50 mM Tris/HCl, 150 mM NaCl,
143
pH 8.0) per OD600. Prestained Protein Ladder, purchased from Invitrogen Life
144
Technologies, was applied to determine the molecular weights of the proteins with
145
prominent bands.
146
Enzyme assays
147
Dinitrosalicylic acid (DNS) method was performed to determine the
148
β-mannanase activity by measuring the amount of reducing sugar released from locust
149
bean gum (LBG). The substrate, 0.5% LBG (Sigma G0753) was dissolved in acetic
150
acid-sodium acetate buffer, pH 5.5. 40 µL of appropriate dilutions of culture
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supernatants (i.e. the secreted β-mannanase) and 40 µL of the substrate were mixed
152
and incubated at 37oC for 30 minutes. 100 µL of DNS was added to the enzyme
153
reaction mixture and boiled for 10 minutes to stop the reaction. The mixture was then
154
cooled on ice for 5 minutes and diluted with 320 µL of double distilled water before
155
measuring the absorbance at 540 nm. One unit (U) of β-mannanase activity is defined
156
as the amount of enzyme that liberates 1 µmol of reducing sugar (using D-mannose as
157
a standard) per minute under the given experimental conditions.
158
Results and Discussion
159
Host and vector selection
160
Compared to E. coli and yeast, two extensively used protein expression hosts, B.
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subtilis possesses its own advantages in producing proteins such as its strong capacity
162
of
163
well-characterized secretion pathways25, 36. In some cases, when E. coli serves as the
164
host, large fraction of proteins might be remained within cytoplasmic or periplasmic
165
space of the cells, making the downstream separation and purification much more
166
complex. When comparable secretion capacity occurs between the two generally
167
recognized as safe species, i. e. yeast and B. subtilis, the much higher growth rate of
168
the latter gives it a priority in industrial application. Therefore, we used B. subtilis
169
1A751, a protease-deficient strain, as the host to express β-mannanase.
secreting
high
concentration
proteins
into
the
culture
medium
and
170
The multicopy plasmid pMA5 was employed as the parent plasmid to express
171
β-mannanase37. The fragment of the target protein was successfully placed
172
downstream of promoter PhpaII, and upstream of T4 terminator, resulting in
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pMA5-Man. Thereafter, a series of pMA5-Man vectors with different signal peptide
174
fragments were constructed, to facilitate the target protein secretion.
175 176
Increased secretion of β-mannanase by selecting the most efficient signal peptide
177
Typically, to secrete β-mannanase directly into the culture medium, screening
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one efficient signal peptide that well functioned in B. subtilis was necessary. There are
179
several well-characterized secretion pathways elucidated in B. subtilis, especially the
180
general secretory (Sec) pathway, and the twin-arginine (Tat) translocation pathway.
181
We selected four signal peptides (SPamyL, SPlipA, SPnprB, and SPnprE) from Sec
182
pathway38-40 and two (SPphoD and SPywbN) from Tat pathway41, 42, which were reported
183
to highly direct the secretion of different enzymes.
184
Six different signal peptide fragments were inserted between PhpaII and
185
β-mannanase gene of pMA5-Man, respectively. Then they were transformed into the
186
B. subtilis 1A751. After 72 h of fermentation, most of the enzymes could secrete into
187
the medium. We could easily recognize that all of the signal peptides could drive the
188
protein secrete into the medium since the prominent bands corresponding to the size
189
of 38kDa were observed on the SDS-PAGE gel (Figure 2A), which were in
190
accordance with the theoretical molecular weight of the mature protein. Among the
191
four Sec pathway signal peptides, SPlipA (1A7512) was the most efficient one (enzyme
192
activities listed in Table S2), with almost a twofold activity of SPnprB (1A7514), which
193
turned out to be the least efficient. SDS-PAGE result of the supernatant samples
194
(Figure 2A) also showed that both the two Tat-dependent signal peptides (SPphoD,
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SPywbN) could direct the secretion of β-mannanase, but at relatively low levels.
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Substantial fractions of protein precursors remained within the cytoplasmic of the
197
cells harboring Tat-dependent signal peptides. This was observed from further
198
analysis of the cell samples by SDS-PAGE (Figure 2B). In addition, those remained
199
proteins had a larger size than the mature proteins, implying that most of the Tat
200
signal peptides could not be successfully removed by the signal peptidases. However,
201
when Sec-dependent signal peptides performed, only small fractions of proteins
202
remained in the cytoplasm (Figure 2B), with signal peptides being removed. Thus, we
203
concluded that β-mannanase tended to be highly secreted under the direction of
204
Sec-pathway signal peptide SPlipA. Therefore, pMA5-2Man (SPlipA) was used for the
205
following experiments.
206 207
Increase the secretion of β-mannanase by systematic gene overexpression
208
Since both the enzyme activity detection of the cytoplasmic extracts and the
209
SDS-PAGE result of the cell fraction samples (Figure 2B) indicated that some
210
β-mannanases retained in the cytoplasm, we concluded that only the most suitable
211
signal peptide itself could not guarantee their maximum production in B. subtilis, but
212
some other bottlenecks within the secretion pathway needed to be addressed.
213
Insufficiency of each component of the secretion machinery might decrease the final
214
secretion amounts of β-mannanases. It was reported that the secretion level of
215
α-amylase was remarkably improved by systematic overexpression of Sec-pathway
216
related genes to screen the main bottleneck candidates43. Thus it was suspected this
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process to be a necessary step to distinguish the components that limited β-mannanase
218
secretion, considering the different sequences between α-amylase and β-mannanase.
219
Therefore, we transformed pMA5-2Man (SPlipA) into B. subtilis 1A751 derivative
220
strains containing the overexpression genes encoding elements of Sec secretion
221
machinery, respectively. Each single copy of these overexpressed genes integrated
222
into the B. subtilis chromosome at amyE locus via double crossing-over43, and under
223
the strict control of promoter Pxyl. Candidate recombinant strains were listed in Table
224
2. B. subtilis 1A751S1 (secY), 1A751S2 (secE), 1A751S3 (secG), 1A751S4 (secYEG),
225
1A751S5 (secDF), 1A751S6 (secA), 1A751S7 (ffh), 1A751S8 (scr), 1A751S9 (hbs),
226
1A751S10 (SRP), and 1A751S11 (ftsY) were strains which overexpressed genes of the
227
Sec-translocon components and B. subtilis 1A751P1(sipS), 1A751P 2(sipT), 1A751P3
228
(sipU), 1A751P4 (sipV), 1A751P5 (sipW), 1A751P6 (partial dnaK operon), 1A751P7
229
(groESL operon) and 1A751P8 (prsA) were strains that overexpressed different signal
230
peptidases and molecular chaperones.
231
These candidates were selected because of their importance in secretion process.
232
Among the Sec translocase complex17, SecYEG constituted the secretion channel, and
233
SecDF served as the accessory protein. SecA provided energy for crossing membrane
234
by catalyzing the hydrolysis of ATP. The signal recognition particle (SRP) consisted
235
of a GTPase named Ffh, two histone-like proteins (Hbs) and scRNA (scr), and FtsY
236
was the SRP receptor. To some extent, well cooperation or not between these elements
237
and β-mannanase considerably influenced the secretion process, as well as the
238
ATP-dependent molecular chaperones, such as DnaK and GroESL. Among the seven
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type I signal peptidases, five (sipS, sipT, sipU, sipV, and sipW) were identified to
240
locate on the chromosome. Either the presence of SipS or SipT was sufficient for
241
processing of precursor proteins and cell viability44.
242
After bacteria being inoculated into the 2×SR fermentation medium and cultured
243
at 37oC for 4 hours in 250-mL triangular flasks, xylose was added to a final
244
concentration of 2% (m/v) to induce the overexpression of the relevant genes in Sec
245
secretion pathway. Fermentation cultures were harvested to obtain supernatant
246
samples to detect the β-mannanase activities and perform the SDS-PAGE after 72 h of
247
incubation. Compared to B. subtilis 1A751 (pMA5-2Man), no strains that
248
overexpressed genes had increased the enzyme activities for several times. No
249
dramatic effects on the target protein secretion led to the enzyme yields almost
250
keeping equivalent to levels of the control strain B. subtilis 1A7512 (pMA5-2Man)
251
(Figure 3A and Table S2). These strains were B. subtilis 1A751S1 (secY), 1A751S2
252
(secE), 1A751S6 (secA), 1A751S8 (scr), 1A751P3 (sipU), 1A751P4 (sipV), 1A751P5
253
(sipW), 1A751P6 (partial dnaK operon), 1A751S7 (ffh), 1A751S10 (SRP), and
254
1A751P8 (prsA).
255
When separately overexpressed genes (secY, secE, and secG) encoding the
256
channel of Sec translocase complex45, there were no apparent changes in the
257
β-mannanase secretion levels. However, combinatorial overexpression of the channel
258
genes (secYEG) enhanced the secretion to 116% compared to B. subtilis 1A7512
259
(pMA5-2Man). This implied that balanced amounts of the translocase components
260
were critical for the increased number of the functional SecYEG channels, thus
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enhancing the target protein secretion. In addition, overexpression of the translocase
262
accessory protein SecDF (1A751S5), the SRP receptor FtsY (1A751S11), had positive
263
effects on the secretion, but the improvements were not very obvious (Figure 3B).
264
Despite the fact that Ffh could assist the forming of stable SRP complex, no elevated
265
level of production was observed.
266
Not only the translocase complex plays an important role in its secretion, but also
267
many other factors affect this process. For example, the signal peptidases (SPPases)
268
were responsible for the cleavage of the N-terminal signal peptides, so that mature
269
proteins could secrete into the medium. The SipS (1A751P1) and SipT (1A751P2)
270
overexpression indeed enhanced the secretion of β-mannanase, as could be seen from
271
Figure 3A and 3B. This was consistent with the previous conclusion that SipS or SipT
272
served as the dominant SPPases 46. We supposed that overexpression of these SPPases
273
could intensify the cleavage of the signal peptides, accelerate the transportation, thus
274
reducing the accumulation of these cytoplasmic proteins. Even though increased
275
amounts of secretory β-mannanases were obtained, a small portion of mature proteins
276
still existed within the cells (Figure 4). Therefore, other secretion bottlenecks still
277
have to be discovered.
278
The overexpression of groESL operon, intracellular chaperones which were in charge
279
of mediating protein folding, decreasing aggregation, and maintaining pre-proteins in
280
translocation-competent conformations, highly increased (125% activity of the control)
281
the secretion amount of β-mannanase (Figure 3). This was similar with the result that
282
GroESL actively contributed to the Sec-dependent export process, which has been
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demonstrated in E. coli47. Partial dnaK operon also encoded intracellular chaperones,
284
but their overexpression almost had no positive effects on the secretion. This might
285
attribute to its preference for binding to certain polypeptide sequences48, and the
286
major function of DnaK was in response to stress affecting the Sec translocon. An
287
extracytoplasmic chaperone, PrsA, which could increase the α-amylase secretion by
288
several times43, 49, failed to assist β-mannanase with the secretion. This could be
289
probably due to the tremendously different primary sequences and folding states
290
between the two target proteins. Until now, even though we know the fact that these
291
chaperones participate in protein translocation, more knowledge is needed to explain
292
their abilities to interact with substrates and assist the folding of certain target proteins.
293
Based on that, it would be much easier to further facilitate the target protein secretion.
294
Increase the secretion of β-mannanase by using strong promoter
295
Overexpression of genes including translocon components (secYEG, secDF, ftsY),
296
SPPases (sipS, sipT), and chaperones (groESL operon), could increase the secretory
297
production of β-mannanases to different levels, with the highest (groESL operon)
298
being 125% of the control strain (Figure 3A). However, a small fraction of mature
299
enzymes still remained within the cells after strengthening the secretion pathway
300
(Figure 4). It seemed that these remained cytoplasmic proteins could not be avoided.
301
Therefore, we shifted to enhance the synthesis of enzymes by optimizing the level of
302
transcription and translation so that more proteins could be secreted. Using strong
303
promoters was one of the most efficient methods to increase the production, when no
304
obvious secretion bottlenecks hindering the transportation.
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We selected three constitutive promoters and two inducible promoters to
306
determine the most suitable one. Promoter PhpaII was the original promoter in pMA5,
307
detected to be a relatively strong promoter50. Promoter PaprE was reported to highly
308
drive the expression of the downstream gene of α-Amylase when bacteria were
309
under stationary phase20. Promoter P43 was a well-characterized constitutive strong
310
promoter, and widely used in many protein expressions in B. subtilis30, 51, 52. The
311
inducible promoters included IPTG inducible promoter Pgrac (Figure 5A), and a
312
modified maltose inducible promoter Pmglv (Figure 5B). With the concentration of the
313
inducers increasing, the production of secreted β-mannanase increased. According to
314
the results of the SDS-PAGE, when the concentration of IPTG and maltose were 0.1
315
mM and 2% (m/v), the promoters performed better (Figure 5A and 5B) than either
316
higher or lower concentration.
317
To determine the most efficient promoter, these strains containing different
318
promoters (1A751P7, 1A751SPP1, 1A751SPP2, 1A751SPP4, 1A751SPP5) were
319
cultured in the 2×SR medium at 37oC for 72 h, with the optimized concentrations of
320
inducers. The enzyme activity produced by 1A751SPP5 (Pmglv) was the highest 2207
321
U/mL (Figure 6A and Table 3 and Table S2), with almost a 3.1-fold activity of the
322
control strain 1A751P7 (groESL operon, PhpaII). This was probably because maltose
323
served as both the inducer and carbon sources, which promoted bacteria growth, as
324
could be inferred from the higher OD600 (Figure 6B) of this strain than the control one
325
(1A751P7). Compared to other strains, the period of stationary phase of 1A751SPP5
326
(Pmglv) was much longer, which kept the cells continuously producing, accumulating
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and subsequently secreting β-mannanase into the medium. The function of maltose
328
was evaluated by incubating the control strain 1A751P7 in the medium with 2% of
329
maltose (1A751P7-a) and 0.1 mM IPTG (1A751P7-b). The enzyme activity of
330
β-mannanase secreted by 1A751P7-a increased to 124% of the control strain (Table 3),
331
whereas adding IPTG did not increase the secretion of β-mannanase. 1A751SPP2
332
(P43) secreted the second highest amount of β-mannanase (Table S2), which was
333
about 1.8-fold activity of the control one. The constitutive promoter P43 was much
334
stronger than PhpaII and PaprE, even though PaprE could keep at high strength during the
335
stationary phase. 1A751SPP4 (Pgrac) could only reach 90% enzyme activity of the
336
control strain, which was still lower than the strains with promoter P43.
337
All of the strains carrying constitutive promoters could drive the transcription
338
and translation of the β-mannanase, but to extremely different levels. Promoter Pmglv
339
could prodigiously improve the secretion yields of β-mannanase. In this research,
340
selection of the suitable signal peptide, overexpression of secretion components or
341
chaperones to reduce the secretion bottlenecks, selection of the strong promoters to
342
enhance the transcription and translation, were combined to optimize and improve the
343
final production of β-mannanase to 2207 U/mL. Considering the potential application
344
of this enzyme in the feed industry, enzyme assay was conducted at 37oC, which was
345
not the optimal temperature. If the temperature was set between 50oC and 60oC, we
346
supposed that higher enzyme activity would be obtained. Nevertheless, the enzyme
347
activity produced by flask fermentation was already much higher than other reported
348
B. subtilis strains30,
31
. It is quite promising that a much higher production of
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349
β-mannanase would be obtained once this strain cultivated in fed-batch and further
350
optimization of fermentation process was conducted. In conclusion, not only the
351
valuable promoter Pmglv could be used for the production of β-mannanase, but also
352
these strategies could be adapted to improve other protein expression systems in B.
353
subtilis.
354 355
Abbreviations Used
356
SP:
357
Dinitrosalicylic acid;
358
Acknowledgement
Signal
peptide;
IPTG:
Isopropyl
β-D-1-thiogalactopyranoside;
DNS:
359
This work was supported by the National Nature Science Foundation of China
360
(31370089,21506244), State Key Development 973 Program for Basic Research of
361
China (2013CB733600), National High Technology Research and Development
362
Program of China (863 Program) (No. 2014AA093511), Nature Science Foundation
363
of Tianjin City (CN) (16JCYBJC23500, 15JCQNJC09500) and the Key Projects in
364
the Tianjin Science & Technology Pillar Program 11ZCZDSY08600.
365
Supporting Information
366
Table S1. Oligonucleotides used in this study.
367
Table S2. Enzyme activities of β-mannanases secreted by different strains of Bacillus
368
subtilis.
369
Notes
370
This work has been included in a patent application by Tianjin Institute of Industrial
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Biotechnology, Chinese Academy of Sciences.
372 373
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different expression hosts. J. Microbiol. Biotechnol. 2014, 24, 1405-12.
509 510
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Figure captions
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Figure 1. Scheme of combined strategies to improve the secretion of β-mannanase.
513
Approaches include improvements of protein synthesis and secretion capacity. The
514
former was performed by application of strong promoters and codon optimization of
515
the target gene. The latter was intended to strengthen the transportation of mature
516
proteins, with signal peptides selection performed on plasmids and overexpression of
517
secretion machinery components on the chromosome.
518
Figure 2. Comparison of efficiency of different signal peptides. The molecular weight
519
of mature β-mannanase is about 38 kDa. Column Ct: plasmids without the gene of
520
β-mannanase. A: SDS-PAGE analysis of β-mannanase in the supernatant secreted by
521
B. subtilis. Four Sec-dependent signal peptides (SPamyL, SPlipA, SPnprB, and SPnprE) and
522
two Tat-dependent (SPphoD, SPywbN) signal peptides were used, and SPlipA was the most
523
efficient. B: SDS-PAGE analysis of β-mannanase of the cell samples. The two
524
Tat-dependent signal peptides could not be removed successfully, leading to the size
525
of proproteins larger than the mature ones.
526
Figure 3. Effects of overexpression genes on β-mannanase secretion. A: Relative
527
activities of β-mannanase that secreted by different B. subtilis strains (1A751S1,
528
1A751S2, 1A751S3, 1A751S4, 1A751S5, 1A751S6, 1A751S7, 1A751S8, 1A751S9,
529
1A751S10, 1A751S11, 1A751P1, 1A751P2, 1A751P3, 1A751P4, 1A751P5,
530
1A751P6, 1A751P7, 1A751P8 overexpressed secY, secE, secG, secYEG, secDF, secA,
531
ffh, scr, hbs, SRP, ftsY, sipS, sipT, sipU, sipV, sipW, partial dnaK operon, groESL
532
operon, prsA, respectively.) Column control was set as 100% activity, and the strains
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533
were B. subtilis 1A7512, carrying SPlipA. Error bars represent standard deviations of
534
biological triplicates. B: SDS-PAGE analysis of β-mannanase in the supernatant
535
secreted by B. subtilis strains that overexpressed different genes.
536
Figure 4. SDS-PAGE analysis of β-mannanase cell samples of B. subtilis strains that
537
overexpressed different genes. Strains B. subtilis 1A751S1, 1A751S2, 1A751S3,
538
1A751S4,
539
1A751S11, 1A751P1, 1A751P2, 1A751P3, 1A751P4, 1A751P5, 1A751P6, 1A751P7,
540
1A751P8, overexpressed gene of secY, secE, secG, secYEG, secDF, secA, ffh, scr, hbs,
541
SRP, ftsY, sipS, sipT, sipU, sipV, sipW, partial dnaK operon, groESL operon, prsA,
542
respectively.
543
Figure 5. Optimization of the concentration of inducers to improve the β-mannanase
544
production. A: SDS-PAGE analysis of β-mannanase in the supernatant secreted by B.
545
subtilis 1A751SPP4 (Pgrac), with the concentration of IPTG increased from 0 to
546
0.1mM. B: SDS-PAGE analysis of β-mannanase in the supernatant secreted by B.
547
subtilis 1A751SPP5 (Pmglv), with the concentration of maltose increased from 0 to 2%
548
(m/v).
549
Figure 6. Comparison of β-mannanase amounts produced by strains with different
550
promoters. A: SDS-PAGE analysis of β-mannanase in the supernatant secreted by
551
strains with constitutive promoters (PhpaII, PaprE, P43) and inducible promoters (Pgrac,
552
Pmglv). B: Growth curve of B. subtilis strains with promoter PhpaII (Control) and Pmglv.
1A751S5, 1A751S6,
1A751S7, 1A751S8,
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Table 1 Plasmids used in this study Plasmids
Genotype and/or relevant characteristics
pMA5
E. coli/B. subtilis shuttle vector, PhpaII, Apr, Kmr
BGSC
pMA5-Man
pMA5 derivative, β-mannanase, T4 terminator
This work
pMA5-1Man
pMA5-Man derivative, SPamyL,
This work
pMA5-2Man
pMA5-Man derivative, SPlipA,
This work
pMA5-3Man
pMA5-Man derivative, SPnprB,
This work
pMA5-4Man
pMA5-Man derivative, SPnprE,
This work
pMA5-5Man
pMA5-Man derivative, SPphoD,
This work
pMA5-6Man
pMA5-Man derivative, SPywbN,
This work
pMA5-2Man1
pMA5 derivative, PaprE, β-mannanase
This work
pMA5-2Man2
pMA5 derivative, P43, β-mannanase
This work
pMA5-2Man4
pMA5 derivative, Pgrac, β-mannanase
This work
pMA5-2Man5
pMA5 derivative, Pmglv, β-mannanase
This work
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Table 2 Strains used in this study Strains
Genotype and/or relevant characteristics
Sources
E. coli DH5α
F−∆lacU169(-80d lacZ∆M15) supE44 hsdR17 recA1 gyrA96 endA1
Invitrogen
B. subtilis 1A751
eglS∆102 bglT/bglS∆EV aprE nprE his
BGSC
B. subtilis 1A7511
1A751 derivative, pMA5-1Man, Kmr
This work
B. subtilis 1A7512
1A751 derivative, pMA5-2Man, Kmr
This work
B. subtilis 1A7513
1A751 derivative, pMA5-3Man, Kmr
This work
B. subtilis 1A7514
1A751 derivative, pMA5-4Man, Kmr
This work
B. subtilis 1A7515
1A751 derivative, pMA5-5Man, Kmr
This work
B. subtilis 1A7516
1A751 derivative, pMA5-6Man, Kmr
This work
B. subtilis 1A751S1
1A751 derivative, amyE::Pxyl-secY, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S2
1A751 derivative, amyE::Pxyl-secE, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S3
1A751 derivative, amyE::Pxyl-secG, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S4
1A751 derivative, amyE::Pxyl-secYEG, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S5
1A751 derivative, amyE::Pxyl-secDF, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S6
1A751 derivative, amyE::Pxyl-secA, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S7
1A751 derivative, amyE::Pxyl-ffh, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S8
1A751 derivative, amyE::Pxyl-scr, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P1
1A751 derivative, amyE::Pxyl-sipS, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P2
1A751 derivative, amyE::Pxyl-sipT, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P3
1A751 derivative, amyE::Pxyl-sipU, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P4
1A751 derivative, amyE::Pxyl-sipV, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P5
1A751 derivative, amyE::Pxyl-sipW, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P6
1A751 derivative, amyE::Pxyl-dnaK, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P7
1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S9
1A751 derivative, amyE::Pxyl-hbs, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751S10
1A751 derivative, amyE::Pxyl-SRP, Cmr, pMA5-2Man, Kmr
This work
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Strains
Genotype and/or relevant characteristics
Sources
B. subtilis 1A751S11
1A751 derivative, amyE::Pxyl-ftsY, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751P8
1A751 derivative, amyE::Pxyl-prsA, Cmr, pMA5-2Man, Kmr
This work
B. subtilis 1A751SPP1
1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man1(PaprE) , Kmr
This work
B. subtilis 1A751SPP2
1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man2 (P43) , Kmr
This work
B. subtilis 1A751SPP4
1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man4 (Pgrac) , Kmr
This work
B. subtilis 1A751SPP5
1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man5 (Pmglv) , Kmr
This work
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Table 3 Relative activity of β-mannanase secreted by different recombinant strains Strains
Promoters
Relative
1A751P7
PhpaII
100
1A751P7-a
PhpaII
124
1A751P7-b
PhpaII
102
1A751SPP1
PaprE
32
1A751SPP2
P43
179
1A751SPP4
Pgrac
87
1A751SPP5
Pmglv
310
activity
of
B. subtilis 1A751P7-a and B. subtilis 1A751P7-b represent that the same strain B. subtilis 1A751P7 cultured in medium adding 2% (m/v) of maltose and 0.1mM IPTG, respectively.
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Figure graphics
Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Figure 6
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Journal of Agricultural and Food Chemistry
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2 3 4
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