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Improving trehalose synthase activity by adding the C-terminal domain of trehalose synthase from Thermus thermophilus
Accepted Manuscript Improving trehalose synthase activity by adding the C-terminal domain of trehalose synthase from Thermus thermophilus Yan Li, Ziwei Wang, Yue Feng, Qipeng Yuan PII: DOI: Reference:
S0960-8524(17)30857-X http://dx.doi.org/10.1016/j.biortech.2017.05.189 BITE 18215
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
9 March 2017 27 May 2017 29 May 2017
Please cite this article as: Li, Y., Wang, Z., Feng, Y., Yuan, Q., Improving trehalose synthase activity by adding the C-terminal domain of trehalose synthase from Thermus thermophilus, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.05.189
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1
Improving trehalose synthase activity by adding the C-terminal domain of
2
trehalose synthase from Thermus thermophilus
3
Yan Li, Ziwei Wang, Yue Feng, Qipeng Yuan⃰
4
State Key Laboratory of Chemical Resource Engineering, Beijing University of
5
Chemical Technology, No. 15 East Road of North Third Ring, Chao Yang District,
6
Beijing 100029, China
7 8 9
Abstract
10
The conversion of maltose into trehalose by trehalose synthase (TreS) is a simple, fast,
11
high substrate specificity and low-cost approach to produce trehalose. TreS from
12
Thermus thermophilus (TtTreS) is a thermostable enzyme, whose C-terminal domain
13
(TtTreS-C) was previously shown to improve the thermostability of a TreS enzyme
14
through direct linking. The aim of this work was to study In this study, we studied the
15
activities of four other TreS enzymes from different sources linked with or without
16
TtTreS-C. The results showed that a flexible linker peptide between TreS enzymes
17
and TtTreS-C is essential for their activity enhancement. Moreover, the specific
18
activities of the four enzymes were also improved by linking to the TtTreS-C
19
fragment. Together, our study provides novel insights into the functions of the
20
C-terminal domain of TtTreS, and would facilitate its future application in enzyme
21
engineering.
22
1. Introduction
23
Trehalose (R-D-glucopyranosyl-1, 1-R-D-glucopyranoside) is a non-reducing 1
24
disaccharide found in various organisms including bacteria, algae, fungi, yeasts and
25
plants (Elbein et al., 2003; Richards et al., 2002; Zhang et al., 2010). It serves as a
26
source of energy, protects proteins and cellular membranes from a variety of
27
environmental stresses, including desiccation, dehydration, high temperature, freezing,
28
et al (Asker et al., 2009; Elbein et al., 2003; Li et al., 2012; Ryu et al., 2010; Wei et al.,
29
2013; Xiuli et al., 2009). Some bacteria and yeasts such as Kluyveromyces marxianus
30
were sensitive to alcohol, osmotic and oxidative stress, which correlated with the
31
increases in the cell trehalose concentrations (Erdei et al., 2011). Chang-Joon Kim’s
32
group overexpressed trehalose biosynthetic genes (otsBA), which could help the cells
33
tolerate the toxicity of crude glycerol for direct use (Nguyen et al., 2013). Due to
34
these properties, trehalose is widely applied as additives, stabilizers, and sweeteners in
35
food, cosmetics and pharmaceutical industries (Guo et al., 2000).
36
Among different approaches to synthesize trehalose, enzymatic synthesis has
37
been most widely used, which has been recently reviewed (Schiraldi et al., 2002). A
38
putative UDP-glucose 4-epimerase in Pyrococcus horikoshiii was cloned, expressed
39
and purified from Escherichia coli in Lee’s group’s study. It could be coupled with
40
trehalose sytnase (TreT) to regenerate UDP-Gal from UDP (Chung et al., 2012). The
41
conversion of maltose into trehalose, catalyzed by trehalose synthase (TreS), is a
42
simple, fast, high substrate specificity and low-cost approach (Liang et al., 2013; Wu
43
et al., 2011), much better than starch bioprocessing which involves two steps. The
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TreS pathway has been revealed in several organisms, such as Pseudomonas putida,
45
Corynebacterium glutamicum, Streptomyces coelicolor, Thermus thermophilus and 2
46
Thermotoga maritime.
47
Trehalose synthase from Thermus thermophilus (TtTreS) is a thermostable
48
enzyme from the Thermus strain. The enzyme contains a unique C-terminal domain
49
apart from the active domain (Silva et al., 2003). Wang et al have identified that this
50
extra C-terminal domain plays a key role in maintaining the thermophilicity and
51
thermostability of TtTreS (Wang et al., 2007). This previous report showed that the C
52
terminus of TtTreS (designated as TtTreS-C here after) also modulates the side
53
reaction to reduce glucose production under high temperature conditions. When added
54
with the C-terminal domain of TtTreS, the thermostability of a cold-active TreS from
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Deinococcus radiodurans (DrTreS) was greatly improved. However, they did not test
56
the impact on the specific activity of a TreS enzyme by adding the TtTreS-C, although
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they detected less byproduct during the reaction catalyzed by DrTreS with the
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TtTreS-C than DrTreS itself.
59
Previously, we have optimized the activity of trehalose synthases from different
60
species have been optimized and determined the optimal temperature has been
61
determined, pH value, reaction time and substrate concentration for TtTreS (Li et al.,
62
2015). In the beginning of this study, The whole C-terminal domain of TtTreS has
63
been deleted as in the study of Wang et al. Surprisingly, the truncated forms of TtTreS
64
(containing 1-552, 1-566, 1-666, 1-766 or 1-866 amino acids) all showed decreased
65
activities compared with the full-length TtTreS enzyme (data not shown). We also
66
tested The enzyme reactions of all the truncated forms of TtTreS enzymes also have
67
been tested under different temperatures, however, none of the truncated forms of 3
68
enzymes showed normal activities in these situations (data not shown). Thus, it
69
suggested that the TtTreS-C might not only play a role in thermophilicity and
70
thermostability of TtTreS, but also function in the activity of TtTreS possibly through
71
stabilizing the active domain, which was not studied in Wang’s study. In this study,
72
trehalose synthase genes with only the active domains from four different sources
73
have been expressed in Escherichia coli (Fig.1). Our results showed that linking
74
TtTreS-C to the C-termini of the four TreS enzymes could all increase the enzymatic
75
activities of them. In addition, a linker peptide between TreS and TtTreS-C is essential
76
for the activity enhancement, suggesting that spatial organization of the two domains
77
is important for the impact of TtTreS-C on the activity of the active domains of TreSs
78
(Fig.1). Together, our study provides novel insights into the functions of the
79
C-terminal domain of TtTreS and would facilitate its future application in enzyme
80
engineering.
81
2. Materials and methods
82
2.1. Bacterial strains and media
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E. coli DH5α (from Strata gene) was used for plasmid construction. E. coli
84
Rosetta (DE3) (from the E. Coli Genetic Stock Center) was used for protein
85
expression and enzyme assays. Strains used in this study are summarized in Table 1.
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The trehalose synthase genes were obtained from Pseudomonas putida
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NBRC14164, Corynebacterium glutamicum ATCC 13032, Streptomyces coelicolor
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ATCC 23899, Thermus thermophilus HB27 and Thermotoga maritime MSB8.
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LB medium contains 10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl. 4
90
The TB minimal medium contains 12 g/liter tryptone, 24 g/liter yeast extract, 4
91
ml/liter glycerol, 2.31 g/liter KH2PO4, and 16.43 g/liter K2HPO4·3H2O. When needed,
92
ampicillin, was added to the medium at 100 µg/ml, respectively.
93
2.2. Amplification of the trehalose synthase genes by PCR and plasmid construction
94
The genomic DNA of Pseudomonas putida NBRC 14164, Corynebacterium
95
glutamicum ATCC 13032, Streptomyces coelicolor ATCC 23899, Thermus
96
thermophilus HB27 and Thermotoga maritime MSB8 were obtained from the
97
University of Georgia, Athens, GA. The nucleotide sequences of the trehalose
98
synthase (TreS) were originally retrieved from GenBank with the BLAST program
99
(Fig. 2). These trehalose synthases have different N-terminal domains and also low
100
identity amino acid sequences. Trehalose synthases from two thermophilic bacteria
101
and two normal bacteria have been tried to meature the impact of the C-terminal
102
domain of trehalose synthase from Thermus thermophilus. All the genes were
103
amplified with PCR using PrimeSTAR DNA polymerase (TaKaRa) and the genomic
104
DNAs as templates.
105
PCR amplification consisted of an initial denaturation step at 98 °C for 2 min,
106
followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s,
107
and extension at 72 °C for a specific time, which is decided by the target gene length.
108
Plasmids and primers used in this study are listed in Table 1 and Table 2,
109
respectively. The plasmids pET-22b was utilized for gene cloning and protein
110
expression. The PpTreS, PpTreS565, CgTreS, ScTreS and TmTreS were short for
111
TreS from Pseudomonas putida NBRC 14164 genomic DNA, Corynebacterium 5
112
glutamicum ATCC 13032 genomic DNA, Streptomyces coelicolor ATCC 23899
113
genomic DNA and Thermotoga maritime MSB8 genomic DNA, respectively. The
114
TtTreS-C was short for C-terminal of TreS from Thermus thermophilus HB27
115
genomic DNA.
116
The amplified DNA fusion fragments from four sources were subsequently
117
digested and thereafter ligated into expression vector pET-22b respectively, to
118
construct twelve recombinant plasmids (Table 1), which were then transformed into
119
the competent E. coli DH5α. The restriction enzymes and ligase were purchased from
120
Thermo Scientific. The competent E. coli DH5α and E. coli Rosetta (DE3) were
121
purchased from TransGen.
122
2.3. Protein expression and purification
123
E. coli Rosetta (DE3) was transformed with the corresponding expression
124
plasmids. The obtained transformants were inoculated in 4 ml LB medium containing
125
100 µg/ml of ampicillin and grown aerobically at 37 °C overnight. 500 µl overnight
126
cultures were inoculated into 50 ml fresh TB medium, left to grow at 37 °C until the
127
optical density at 600 nm (OD600) reached 0.6, and then the E. coli Rosetta (DE3)
128
transformants were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG)
129
and shaken at 16 °C overnight. The cells were harvested 30 h after induction and
130
broken using ultrasonic cell disruptor. After centrifugation, the supernatants were used
131
as crude enzyme for enzymatic assays. The crude extracts of the recombinant
132
enzymes were further purified by using the following steps: The protein was first
133
purified with affinity chromatography using a Ni-NTA column (Qiagen) 6
134
pre-equilibrated with the lysis buffer and eluted with the lysis buffer supplemented
135
with 300 mM imidazole, which was followed by purification with gel filtration using
136
a Superdex-200 (GE Healthcare) column equilibrated with a buffer containing 10 mM
137
Tris–HCl pH 8.0, 200 mM NaCl, and 5 mM DTT. The purified protein was analyzed
138
by SDS–PAGE. The fractions containing the target protein were pooled and
139
concentrated to 20 mg/ml.
140
2.4. Enzymatic assays
141
The reaction was carried out in a 21 ml system containing 20 ml 20% maltose and
142
1 ml crude or purified enzymes in 1×PBS buffer (pH 7.4). Four different sources of
143
trehalose synthases have been reacted at the optimal temperature which has been
144
obtained in the previous study (Li et al., 2015). When four sources of recombinant
145
TreS-TreS-C activity was meatured, different temperatures were attempted to obtain
146
the optimal temperature. After 10 min, the trehalose production was measured by
147
High Performance Liquid Chromatography analysis (HPLC). Trehalose synthase
148
enzyme activity was defined under the condition of the optimal reaction temperature,
149
with excessive maltose solution as the substrate. The activity consuming 1 µmol
150
maltose or generating 1 µmol trehalose in 1 min was defined as one unit of enzyme
151
activity. The protein amount was determined by software Image J grayscale scanning.
152
2.5. HPLC analysis
153
Trehalose (SIGMA), was used as the standard. Both standard and samples were
154
analyzed and quantified by HPLC (HITACHI) with a reverse-phase Venusil PS NH
155
column (Agela) and a differential refraction detector. Solvent A was water with 87% 7
156
acetonitrile. The column temperature was set to 25 °C. The flow rate was 1 ml/min.
157
Quantification of trehalose was based on the peak areas.
158
2.6. Optimization of the reaction conditions
159
For the recombinant E. coli harboring pET-22b-PpTreS, pET-22b-CgTreS,
160
pET-22b-ScTreS,
pET-22b-TmTreS,
pET-22b-PpTreS565-linker-TtTreS-C,
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pET-22b-CgTreS-linker-TtTreS-C,
162
pET-22b-TmTreS-linker-TtTreS-C, respectively, the enzymatic reaction conditions
163
were optimized as follows. The reaction was carried out under the temperature of
164
20 °C, 30 °C, 40 °C or 50 °C, respectively. To define the optimal temperature, the
165
enzyme was incubated in various temperatures for 10 h with 10% maltose, and
166
trehalose amount was measured under the condition described above.
167
3. Results and discussion
168
3.1. Effect of TtTreS-C on enzymatic activity of TreS enzymes
pET-22b-ScTreS-linker-TtTreS-C
and
169
TreS enzymes from four different sources were adopted in this research. In order
170
to test whether linking the TtTreS-C would increase the activity of the single-domain
171
TreSs, we linked tThe active domains of four TreS enzymes from different sources to
172
TtTreS-C have been linked, with or without a flexible linker peptide. The sequence of
173
the flexible linker peptide was GGGSGS, modified from the review article of
174
J.Sivaraman (Li et al., 2015). These chimera proteins have been expressed in E.coli,
175
and their crude enzymatic activities were compared with the four native enzymes.
176
First, different enzyme reaction temperatures (containing 20, 30, 40, 50 °C) have been
177
tested in this study, and the reactions were subsequently carried out under the optimal 8
178
temperature of 20 °C, 20 °C, 20 °C or 30 °C, respectively for the four enzymes from
179
different sources. After a reaction of 10 min, the trehalose production was measured
180
by High Performance Liquid Chromatography analysis (HPLC) and the results
181
showed that although linking the four enzymes directly to TtTreS-C did not increase
182
the enzymatic activity of TreSs, the chimera proteins with linker peptides between the
183
enzymes and TtTreS-C all showed increased activities compared to the native
184
enzymes (Fig. 3).
185
This suggested that the linker peptide between TreS and TtTreS-C has an
186
important role in the activity enhancement. In order to maintain steric configuration
187
and catalytic ability of trehalose synthase, medium-sized flexible linker peptide was
188
adopted in this study. On the other hand, the results also showed that the TtTreS-C not
189
only plays a role in thermophilicity and thermostability of TtTreS, but also function in
190
the activity of TtTreS. Taken together, these results suggested that TtTreS-C did affect
191
the enzymatic activities of TreSs, and the spatial organization of the active domain
192
and TtTreS-C is important for the impact of TtTreS-C on the activity of the active
193
domains of TreSs. Thus, in the following studies, only chimera proteins with linker
194
peptides were used in the comparison with native enzymes.
195
3.2. TtTreS-C increases the specific activities of TreSs
196
By transforming the eight recombinant plasmids (four native enzyme plasmids
197
and four chimera protein plasmids with linkers) into E.coli, we first determined tThe
198
optimal temperature of the enzyme reaction for each construct has been determined
199
firstly. Under the optimal temperatures, the specific activities of each transformants 9
200
have been obtained (Fig. 4). The results showed that linking the four enzymes to
201
TtTreS-C with peptide linkers could also increase the specific activities of TreS
202
enzymes. The specific activities of trehalose synthases from Pseudomonas putida,
203
Corynebacterium glutamicum, Streptomyces coelicolor and Thermotoga maritime
204
were increased 1.91-fold, 1.38-fold, 1.94-fold and 1.64-fold by fusion with TtTreS-C,
205
respectively. This suggested that the enzyme properties of the four enzymes were
206
modified by the TtTreS-C linked.
207
3.3. Reaction temperature optimization of trehalose biosynthesis
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Furthermore, the enzymatic reaction conditions for trehalose synthase production
209
were optimized with the recombinant E. coli harboring the four native enzyme
210
plasmids and four chimera protein plasmids, respectively. When the crude enzyme
211
was added with 10% maltose solution, we noticed the results show that the conversion
212
efficiency began to decrease when the temperature was raised up. No additional
213
trehalose accumulation was observed up to high temperature in the above described
214
experiments (Fig. 5). Therefore, the optimal temperatures of the four chimera
215
enzymes were determined to be 20 °C, 20 °C, 20 °C and 30 °C, respectively (Fig. 5).
216
In the study of Wang et al, the recombinant DrTS-TtTS∆N enzyme had a higher
217
thermostability than DrTS in their reactions. However, in our this study, the optimal
218
temperatures of the four chimera enzymes were not increased under our reaction
219
conditions. We suggest that it This phenomenon might result from the different
220
reaction conditions used in our and their studies. Moreover, it also indicated that the
10
221
TtTreS-C might exhibit different effects on different proteins, either increasing the
222
thermostability or the specific activities of target enzymes.
223
4. Conclusions
224
In the current study, we first tested the heterologous expression of TreS genes
225
from four different sources with or without TtTreS-C has been tested firstly. The
226
enzymatic reaction temperature for the trehalose bioconversion was optimized
227
respectively. The results showed that both the conversion and the specific activities of
228
TreS enzymes were increased after linking to the TtTreS-C. The present study could
229
shed light on the further investigation of the efficient expression of TreS genes and
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optimization of enzymatic reactions. In addition, linker-based connection between the
231
active domains of trehalose synthases and TtTreS-C has an important impact on the
232
enzymatic activity, suggesting that spatial organization of the two domains is
233
important for the impact of TtTreS-C on the activity of the active domains of TreSs.
234
Future more linker types could be tested to get better improvement of the activity of
235
TreS enzymes.
236 237
Acknowledgments
238
The authors would like to acknowledge the financial support of the National
239
Natural Science Foundation of China (Nos. 21636001 and 31670766) and the
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Fundamental Research Funds for the Central Universities (buctrc201613, ZY1629).
241 242
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14
304 305
Fig.1.
306
CgTreS-linker-TtTreS-C, ScTreS-linker-TtTreS-C and TmTreS-linker-TtTreS-C. And
307
then they were subsequently digested and thereafter ligated into expression vector
308
pET-22b respectively, to construct eight recombinant plasmids and were transformed
309
into E.coli Rosetta (DE3).
Expression
of
enzymes
310
15
about
PpTreS565-linker-TtTreS-C,
PpTreS CgTreS ScTreS TmTreS
...................................MAKRSRPA.AFIDDPL......WYKDAVIYQLHIKSF MTDTSPLNSQPSADHHPDHAARPVLDAHGLIVEHESEEFPVPAPAPGEQPWEKKNREWYKDAVFYEVLVRAF ..................................MTVNEPVPD.TFEDTPAGDRHPDWFKRAVFYEVLVRSF ...........................................................VDVVLRERSIEEY
30 72 37 13
PpTreS CgTreS ScTreS TmTreS
FDANNDGIGDFAGLISKLDYIAELGVNTLWLLPFYPSPRRDDGYDIAEYKAVHPDYGSLADARRFIAEAHKR YDPEGNGVGSLKGLTEKLDYIQWLGVDCIWIPPFYDSPLRDGGYDIRNFREILPEFGTVDDFVELVDHAHRR QDSNGDGIGDLKGLTAKLDYLQWLGVDCLWLPPFFKSPLRDGGYDVSDYTAVLPEFGDLADFVEFVDAAHQR RTIIGNEVDEIKKLAEPLKGKKVLHVNATAYGGGVAEILHN.LVPLMRSVGLDARWRVIEAPDEFFNVTKKF
102 144 109 84
PpTreS CgTreS ScTreS TmTreS
GLRVITELVINHTSDQHPWFQRARHAKRGSKARDFYVWSDDEQKYDGTRIIFLDTEKSNWTWDPVAGQYFWH GLRVITDLVMNHTSDQHAWFQESRRDPTGPYG.DFYVWSDDPTLYNEARIIFVDTEESNWTYDPVRGQYFWH GMRVIIDFVMNHTSDQHPWFQESRKNPDGPYG.DYYVWADDDTRYADARIIFVDTEASNWTHDPVRGQYYWH HNTLQGADIEISEEEWNLYEEVCRKNAELIQDEELFVIHDSQP...AAVRKFVDLNDRKWIWRCHIDLSTPN
174 215 180 153
PpTreS CgTreS ScTreS TmTreS
RFYSHQPDLNFDNPQVLNAVIKVMRFWLDLGVDGLRLDAIPYLIERDGTNNENLPETHTVLKAIRAEIDANY RFFSHQPDLNYDNPAVQEAMLDVLRFWLDLGLDGFRLDAVPYLFEREGTNGENLKETHDFLKLCRSVIEKEY RFFSHQPDLNYENPAVQEEMLAALKFWLDLGVDGYRLDAVPYLYAEEGTNCENLPASHAFLKRVRREIDAQY MKVWQKFSQYLEG...YNRLVFHLEEYFPQGWK.ERSIAFPPSIDPLSEKNRDLDEDTIRKTLERLEIDPER
246 287 252 221
PpTreS CgTreS ScTreS TmTreS
PDRMLLAEANQWPEDTRPYFGE.GEG.DECHMAFHFPLMPRMYMALAMEDRFPITDILRQTPEIPANCQWAI PGRILLAEANQWPQDVVEYFGEKDKG.DECHMAFHFPLMPRIFMGVRQGSRTPISEILANTPEIPKTAQWGI PDTVLLAEANQWPEDVVDYFGDYSTGGDECHMAFHFPVMPRIFMAVRRESRYPVSEILAKTPAIPSGCQWGI PLITVVARFDPWKD....................LFSAIDVYRLVKKEIPEVQLAVVSAMAADDPE..GWFF
316 358 324 271
PpTreS CgTreS ScTreS TmTreS
FLRNHDELTLEMVTDRERDYLWNYYAEDRRARINLGIRRRLAPLLQRDRRRIELLTSLLLSMPGTPTLYYGD FLRNHDELTLEMVSDEERSYMYSQFASEPRMRANVGIRRRLSPLLEGDRNQLELLHGLLLSLPGSPVLYYGD FLRNHDELTLEMVTDEERDYMYAEYAKDPRMRANIGIRRRLATLLDNDRDQIELFTALLLALPGSPILYYGD FEK.....VLRYAGTDEDIKFCTNLKGVGNKEVNAIQRATTVALHTATREGFGLVISEALYKR...VPVVAR
388 430 396 335
PpTreS CgTreS ScTreS TmTreS
ELGMGDNIYLGDRDGVRTPMQWSPDRNGGFSRADPQRLVLPPIMDPLYGYQTVNVEAQSHDPHSLLNWTRRM EIGMGDNIWLHDRDGVRTPMQWSNDRNGGFSKADPERLYLPAIQNDQYGYAQVNVESQLNRENSLLRWLRNQ EIGMGDNIWLGDRDAVRTPMQWTPDRNAGFSTCDPGRLYLPAIMDPVYGYQVTNVEASMASPSSLLHWTRRM PVGG...VKIQVKHGENGYLAWEREDLAGYVVKLIKDEELRRKMGE...KGRQTVVENFIITVHLKNYLKLF
460 502 468 401
PpTreS CgTreS ScTreS TmTreS
LAVRKQQKAFGRGTLRTLTPSNRRILAYIREYTDADGHTEVILCVANVSRAAQAAELELSQYADKVPVEMLG ILIRKQYRAFGAGTYREVSSTNESVLTFLREHKG.....QTILCVNNMSKYPQAVSLDLREFAGHTPREMSG IEIRKQNPAFGLGTYTELPSSNPAVLAFLREYED.....DLVLCVNNFARFAQPTELDLREFAGRHPVELFG LDLLR...................................................................
532 569 535 406
PpTreS CgTreS ScTreS TmTreS
GSAFPPIGQLPFLLTLPPYAFYWFLLA.AHDRMPSWHIQATEGLPELTTLVLRKRMEELLEAPARDTLQTTI GQLFPTIAEREWIVTLAPHGFFWFDLT..ADEKDDME................................... GVRFPAIGELPYLLTLGGHGFYWFRLTRVASRIGRRA................................... ........................................................................
603 604 572 406
311 312
Fig.2. The amino acid sequence of CgTreS, ScTreS, TmTreS and the front partial
313
amino acid sequence of PpTreS. By comparing, their sequence were different. Lane 1:
314
The partial sequence of trehalose synthase gene from Pseudomonas putida NBRC
315
14164; Lane 2: The partial sequence of trehalose synthase gene from
316
Corynebacterium glutamicum ATCC 13032; Lane 3: The partial sequence of 16
317
trehalose synthase gene from Streptomyces coelicolor ATCC 23899; Lane 4: The
318
partial sequence of trehalose synthase gene from Thermotoga maritime MSB8.
319
17
320
321 322
Fig. 3. Crude trehalose synthase activity in recombinant E.coli Rosetta (DE3)
323
harboring pET-22b-PpTreS, pET-22b-PpTreS565-TtTreS-C,
324
pET-22b-PpTreS565-linker-TtTreS-C, pET-22b-CgTreS, pET-22b-CgTreS-TtTreS-C,
325
pET-22b-CgTreS-linker-TtTreS-C, pET-22b-ScTreS, pET-22b-ScTreS-TtTreS-C,
326
pET-22b-ScTreS-linker-TtTreS-C, pET-22b-TmTreS, pET-22b-TmTreS-TtTreS-C and
327
pET-22b-TmTreS-linker-TtTreS-C respectively by measuring at the same reaction
328
conditions. The amount of trehalose were measured by high performance liquid
329
chromatography.
330
18
331 332
Fig. 4. The activities of TreS, which from different sources, with or without
333
C-terminal of trehalose synthase from Thermus thermophilus were showed. The data
334
were generated from three independent experiments.
335
19
336 337
Fig. 5. Trehalose production in recombinant E.coli Rosetta (DE3) harboring
338
pET-22b-PpTreS, pET-22b-PpTreS565-linker-TtTreS-C (a), pET-22b-CgTreS,
339
pET-22b-CgTreS-linker-TtTreS-C (b), pET-22b-ScTreS,
340
pET-22b-ScTreS-linker-TtTreS-C (c), pET-22b-TmTreS, and
341
pET-22b-TmTreS-linker–TtTreS-C (d) respectively by varying reaction temperatures.
342
The amount of trehalose under different temperatures were measured by high
343
performance liquid chromatography.
344
20
345
Table 1
346
The details of strains and plasmids. Plasmid or strain Strains E coli DH5α
E coli Rosetta (DE3)
E coli Rosetta (DE3) -1 E coli Rosetta (DE3) -2 E coli Rosetta (DE3) -3 E coli Rosetta (DE3) -4 E coli Rosetta (DE3) -5 E coli Rosetta (DE3) -6 E coli Rosetta (DE3) -7 E coli Rosetta (DE3) -8 E coli Rosetta (DE3) -9
E coli Rosetta (DE3) -10 E coli Rosetta (DE3) -11 E coli Rosetta (DE3) -12 Plasmids pET-22b pET-22b-PpTreS
Descriptiona
Reference or sources
F-ψ80dlacZ△ (lacZYA-argF) U169 endA1 recA1 hsdR17 (rk-, mk+) supE44λ-thi-1 gyrA96 relA1 phoA F-ompT hsdSB(rB-, mB-) galdcmlacY1 (DE3) pRARE (argU, argW, ilex, glyT, leuW, proL) (Cmr) E coli Rosetta (DE3) harboring pET-22b-PpTreS E coli Rosetta (DE3) harboring pET-22b-CgTreS E coli Rosetta (DE3) harboring pET-22b-ScTreS E coli Rosetta (DE3) harboring pET-22b-TmTreS E coli Rosetta (DE3) harboring pET-22b-PpTreS565-TtTtreS-C E coli Rosetta (DE3) harboring pET-22b-CgTreS-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-ScTreS-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-TmTreS-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-PpTreS565-linker-TtTtre S-C E coli Rosetta (DE3) harboring pET-22b-CgTreS-linker-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-ScTreS-linker-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-TmTreS-linker-TtTreS-C
TransGen
TransGen
This study This study This study This study This study This study This study This study This study
This study This study This study
pT7 expression vector, Novagen PBR322origin, Ampr pET-22b vector containing PpTreS This study 21
from pseudomonas putida pET-22b-CgTreS pET-22b vector containing CgTreS from Corynebacterium glutamicum pET-22b-ScTreS pET-22b vector containing ScTreS from Streptomyces coelicolor pET-22b-TmTreS pET-22b vector containing TmTreS from Thermotoga maritime pET-22b-PpTreS565-TtTre pET-22b vector containing PpTreS S-C from pseudomonas putida and TtTreS-C from Thermus thermophilus pET-22b-CgTreS-TtTreS-C pET-22b vector containing CgTreS from Corynebacterium glutamicum and TtTreS-C from Thermus thermophilus pET-22b-ScTreS-TtTreS-C pET-22b vector containing ScTreS from Streptomyces coelicolor and TtTreS-C from Thermus thermophilus pET-22b-TmTreS-TtTreS-C pET-22b vector containing TmTreS from Thermotoga maritime and TtTreS-C from Thermus thermophilus pET-22b-PpTreS565-linker- pET-22b vector containing PpTreS TtTreS-C from pseudomonas putida and TtTreS-C from Thermus thermophilus with linker pET-22b-CgTreS-linker-TtT pET-22b vector containing CgTreS reS-C from Corynebacterium glutamicum and TtTreS-C from Thermus thermophilus with linker pET-22b-ScTreS-linker-TtT pET-22b vector containing ScTreS reS-C from Streptomyces coelicolor and TtTreS-C from Thermus thermophilus with linker pET-22b-TmTreS-linker-Tt pET-22b vector containing TmTreS TreS-C from Thermotoga maritime and TtTreS-C from Thermus thermophilus with linker 347
a
Amp r, ampicillin resistant.
348
22
This study This study This study This study
This study
This study
This study
This study
This study
This study
This study
349
Table 2
350
The details of primer sequences. Name of Primers
Primer sequences
PpTreS-f PpTreS-r CgTreS-f
5’-GGGAAAGGATCCGGCCAAGCGTTCCCGCC-3’ 5’-GGGAAACTCGAGTGACTCTCCCCAGGTACTGATC-3’ 5’-GGGAAACCATGGGCACTGATACCTCTCCGTTGAATTCTCA3’ 5’-GGGAAAAAGCTTTTCCATATCGTCCTTTTCATCGGC-3’ 5’-GGGAAAGGATCCGCACCGTCAACGAGCCCGTAC-3’ 5’-GGGAAAAAGCTTAGCGCGGCGGCCG-3’ 5’-GGGAAAGAATTCGGATGTTGTGTTGAGAGAGCGAAGC-3’ 5’-GGGAAAGTCGACCCTCAACAGATCTAAGAAGAGTTTCAG ATAGTT-3’ 5’-GGGAAAGAATTCGGCCAAGCGTTCCCGCCCGGC-3’
CgTreS-r ScTreS-f ScTreS-r TmTreS-f TmTreS-r PpTreS565(TtTreSC)-f PpTreS565(TtTreSC)-r TtTreS-C(PpTreS56 5)-f TtTreS-C(PpTreS56 5)-r CgTreS(TtTreS-C)-f CgTreS(TtTreS-C)-r TtTreS-C(CgTreS)-f TtTreS-C(CgTreS)-r ScTreS(TtTreS-C)-f ScTreS(TtTreS-C)-r TtTreS-C(ScTreS)-f TtTreS-C(ScTreS)-r TmTreS(TtTreS-C)-f TmTreS(TtTreS-C)-r TtTreS-C(TmTreS)-f TtTreS-C(TmTreS)-r PpTreS565L(TtTreS -C)-f PpTreS565L(TtTreS -C)-r LTtTreS-C(PpTreS5
5’-CGGGGGCGGGCTCCTCGGCGGGCATGCGGTCATGGGCG-3 ’ 5’-CGCCCATGACCGCATGCCCGCCGAGGAGCCCGCCCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGTCGACGACTGATACCTCTCCGTTGAA-3’ 5’-CGGGGGCGGGCTCCTCGGCTTCCATATCGTCCTTTTCA-3’ 5’-TGAAAAGGACGATATGGAAGCCGAGGAGCCCGCCCCCG-3 ’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGAATTCGACCGTCAACGAGCCCGTACC-3’ 5’-CGGGGGCGGGCTCCTCGGCGGATGCGACTCGGGTGAGC-3 ’ 5’-GCTCACCCGAGTCGCATCCGCCGAGGAGCCCGCCCCCG-3 ’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGAATTCGGATGTTGTGTTGAGAGAGCG-3’ 5’-CGGGGGCGGGCTCCTCGGCCCTCAACAGATCTAAGAAG-3 ’ 5’-CTTCTTAGATCTGTTGAGGGCCGAGGAGCCCGCCCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGGATCCGGCCAAGCGTTCCCGCCCGGCAGCC-3’ 5’-GGCGGCGGCTCCGGCTCCGGGCATGCGGTCATGG-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCG-3’ 23
65)-f LTtTreS-C(PpTreS5 65)-r CgTreSL(TtTreS-C)f CgTreSL(TtTreS-C)r LTtTreS-C(CgTreS)f LTtTreS-C(CgTreS)r ScTreSL(TtTreS-C)f ScTreSL(TtTreS-C)r LTtTreS-C(ScTreS)f LTtTreS-C(ScTreS)r TmTreSL(TtTreS-C) -f TmTreSL(TtTreS-C) -r LTtTreS-C(TmTreS) -f LTtTreS-C(TmTreS) -r
5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’ 5’-GGGAAACCATGGGACTGATACCTCTCCGTTGAATTCTCAG CCG-3’ 5’-GGCGGCGGCTCCGGCTCCTTCCATATCGTCCTTT-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’ 5’-GGGAAAGGATCCGACCGTCAACGAGCCCGTACCTGACAC CTTCGAG-3’ 5’-GGCGGCGGCTCCGGCTCCAGCGCGGCGGCCGATG-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’ 5’-GGGAAAGGATCCGGATGTTGTGTTGAGAGAGCGAAGCAT AGAGG-3’ 5’-GGCGGCGGCTCCGGCTCCCCTCAACAGATCTAAG-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCGCCCCC GAG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’
351 352
24
353
Highlights
354 355 356 357 358 359 360 361
We tested the heterologous expression of TreS genes with or without TtTreS-C in Escherichia coli. Linking TtTreS-C to the C-termini of TreS enzymes could all increase the enzymatic activities. A linker peptide between TreS and TtTreS-C is essential for the activity enhancement. The specific activities of TreS enzymes were also improved by TtTreS-C.
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S0960-8524(17)30857-X http://dx.doi.org/10.1016/j.biortech.2017.05.189 BITE 18215
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
9 March 2017 27 May 2017 29 May 2017
Please cite this article as: Li, Y., Wang, Z., Feng, Y., Yuan, Q., Improving trehalose synthase activity by adding the C-terminal domain of trehalose synthase from Thermus thermophilus, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.05.189
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Improving trehalose synthase activity by adding the C-terminal domain of
2
trehalose synthase from Thermus thermophilus
3
Yan Li, Ziwei Wang, Yue Feng, Qipeng Yuan⃰
4
State Key Laboratory of Chemical Resource Engineering, Beijing University of
5
Chemical Technology, No. 15 East Road of North Third Ring, Chao Yang District,
6
Beijing 100029, China
7 8 9
Abstract
10
The conversion of maltose into trehalose by trehalose synthase (TreS) is a simple, fast,
11
high substrate specificity and low-cost approach to produce trehalose. TreS from
12
Thermus thermophilus (TtTreS) is a thermostable enzyme, whose C-terminal domain
13
(TtTreS-C) was previously shown to improve the thermostability of a TreS enzyme
14
through direct linking. The aim of this work was to study In this study, we studied the
15
activities of four other TreS enzymes from different sources linked with or without
16
TtTreS-C. The results showed that a flexible linker peptide between TreS enzymes
17
and TtTreS-C is essential for their activity enhancement. Moreover, the specific
18
activities of the four enzymes were also improved by linking to the TtTreS-C
19
fragment. Together, our study provides novel insights into the functions of the
20
C-terminal domain of TtTreS, and would facilitate its future application in enzyme
21
engineering.
22
1. Introduction
23
Trehalose (R-D-glucopyranosyl-1, 1-R-D-glucopyranoside) is a non-reducing 1
24
disaccharide found in various organisms including bacteria, algae, fungi, yeasts and
25
plants (Elbein et al., 2003; Richards et al., 2002; Zhang et al., 2010). It serves as a
26
source of energy, protects proteins and cellular membranes from a variety of
27
environmental stresses, including desiccation, dehydration, high temperature, freezing,
28
et al (Asker et al., 2009; Elbein et al., 2003; Li et al., 2012; Ryu et al., 2010; Wei et al.,
29
2013; Xiuli et al., 2009). Some bacteria and yeasts such as Kluyveromyces marxianus
30
were sensitive to alcohol, osmotic and oxidative stress, which correlated with the
31
increases in the cell trehalose concentrations (Erdei et al., 2011). Chang-Joon Kim’s
32
group overexpressed trehalose biosynthetic genes (otsBA), which could help the cells
33
tolerate the toxicity of crude glycerol for direct use (Nguyen et al., 2013). Due to
34
these properties, trehalose is widely applied as additives, stabilizers, and sweeteners in
35
food, cosmetics and pharmaceutical industries (Guo et al., 2000).
36
Among different approaches to synthesize trehalose, enzymatic synthesis has
37
been most widely used, which has been recently reviewed (Schiraldi et al., 2002). A
38
putative UDP-glucose 4-epimerase in Pyrococcus horikoshiii was cloned, expressed
39
and purified from Escherichia coli in Lee’s group’s study. It could be coupled with
40
trehalose sytnase (TreT) to regenerate UDP-Gal from UDP (Chung et al., 2012). The
41
conversion of maltose into trehalose, catalyzed by trehalose synthase (TreS), is a
42
simple, fast, high substrate specificity and low-cost approach (Liang et al., 2013; Wu
43
et al., 2011), much better than starch bioprocessing which involves two steps. The
44
TreS pathway has been revealed in several organisms, such as Pseudomonas putida,
45
Corynebacterium glutamicum, Streptomyces coelicolor, Thermus thermophilus and 2
46
Thermotoga maritime.
47
Trehalose synthase from Thermus thermophilus (TtTreS) is a thermostable
48
enzyme from the Thermus strain. The enzyme contains a unique C-terminal domain
49
apart from the active domain (Silva et al., 2003). Wang et al have identified that this
50
extra C-terminal domain plays a key role in maintaining the thermophilicity and
51
thermostability of TtTreS (Wang et al., 2007). This previous report showed that the C
52
terminus of TtTreS (designated as TtTreS-C here after) also modulates the side
53
reaction to reduce glucose production under high temperature conditions. When added
54
with the C-terminal domain of TtTreS, the thermostability of a cold-active TreS from
55
Deinococcus radiodurans (DrTreS) was greatly improved. However, they did not test
56
the impact on the specific activity of a TreS enzyme by adding the TtTreS-C, although
57
they detected less byproduct during the reaction catalyzed by DrTreS with the
58
TtTreS-C than DrTreS itself.
59
Previously, we have optimized the activity of trehalose synthases from different
60
species have been optimized and determined the optimal temperature has been
61
determined, pH value, reaction time and substrate concentration for TtTreS (Li et al.,
62
2015). In the beginning of this study, The whole C-terminal domain of TtTreS has
63
been deleted as in the study of Wang et al. Surprisingly, the truncated forms of TtTreS
64
(containing 1-552, 1-566, 1-666, 1-766 or 1-866 amino acids) all showed decreased
65
activities compared with the full-length TtTreS enzyme (data not shown). We also
66
tested The enzyme reactions of all the truncated forms of TtTreS enzymes also have
67
been tested under different temperatures, however, none of the truncated forms of 3
68
enzymes showed normal activities in these situations (data not shown). Thus, it
69
suggested that the TtTreS-C might not only play a role in thermophilicity and
70
thermostability of TtTreS, but also function in the activity of TtTreS possibly through
71
stabilizing the active domain, which was not studied in Wang’s study. In this study,
72
trehalose synthase genes with only the active domains from four different sources
73
have been expressed in Escherichia coli (Fig.1). Our results showed that linking
74
TtTreS-C to the C-termini of the four TreS enzymes could all increase the enzymatic
75
activities of them. In addition, a linker peptide between TreS and TtTreS-C is essential
76
for the activity enhancement, suggesting that spatial organization of the two domains
77
is important for the impact of TtTreS-C on the activity of the active domains of TreSs
78
(Fig.1). Together, our study provides novel insights into the functions of the
79
C-terminal domain of TtTreS and would facilitate its future application in enzyme
80
engineering.
81
2. Materials and methods
82
2.1. Bacterial strains and media
83
E. coli DH5α (from Strata gene) was used for plasmid construction. E. coli
84
Rosetta (DE3) (from the E. Coli Genetic Stock Center) was used for protein
85
expression and enzyme assays. Strains used in this study are summarized in Table 1.
86
The trehalose synthase genes were obtained from Pseudomonas putida
87
NBRC14164, Corynebacterium glutamicum ATCC 13032, Streptomyces coelicolor
88
ATCC 23899, Thermus thermophilus HB27 and Thermotoga maritime MSB8.
89
LB medium contains 10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl. 4
90
The TB minimal medium contains 12 g/liter tryptone, 24 g/liter yeast extract, 4
91
ml/liter glycerol, 2.31 g/liter KH2PO4, and 16.43 g/liter K2HPO4·3H2O. When needed,
92
ampicillin, was added to the medium at 100 µg/ml, respectively.
93
2.2. Amplification of the trehalose synthase genes by PCR and plasmid construction
94
The genomic DNA of Pseudomonas putida NBRC 14164, Corynebacterium
95
glutamicum ATCC 13032, Streptomyces coelicolor ATCC 23899, Thermus
96
thermophilus HB27 and Thermotoga maritime MSB8 were obtained from the
97
University of Georgia, Athens, GA. The nucleotide sequences of the trehalose
98
synthase (TreS) were originally retrieved from GenBank with the BLAST program
99
(Fig. 2). These trehalose synthases have different N-terminal domains and also low
100
identity amino acid sequences. Trehalose synthases from two thermophilic bacteria
101
and two normal bacteria have been tried to meature the impact of the C-terminal
102
domain of trehalose synthase from Thermus thermophilus. All the genes were
103
amplified with PCR using PrimeSTAR DNA polymerase (TaKaRa) and the genomic
104
DNAs as templates.
105
PCR amplification consisted of an initial denaturation step at 98 °C for 2 min,
106
followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s,
107
and extension at 72 °C for a specific time, which is decided by the target gene length.
108
Plasmids and primers used in this study are listed in Table 1 and Table 2,
109
respectively. The plasmids pET-22b was utilized for gene cloning and protein
110
expression. The PpTreS, PpTreS565, CgTreS, ScTreS and TmTreS were short for
111
TreS from Pseudomonas putida NBRC 14164 genomic DNA, Corynebacterium 5
112
glutamicum ATCC 13032 genomic DNA, Streptomyces coelicolor ATCC 23899
113
genomic DNA and Thermotoga maritime MSB8 genomic DNA, respectively. The
114
TtTreS-C was short for C-terminal of TreS from Thermus thermophilus HB27
115
genomic DNA.
116
The amplified DNA fusion fragments from four sources were subsequently
117
digested and thereafter ligated into expression vector pET-22b respectively, to
118
construct twelve recombinant plasmids (Table 1), which were then transformed into
119
the competent E. coli DH5α. The restriction enzymes and ligase were purchased from
120
Thermo Scientific. The competent E. coli DH5α and E. coli Rosetta (DE3) were
121
purchased from TransGen.
122
2.3. Protein expression and purification
123
E. coli Rosetta (DE3) was transformed with the corresponding expression
124
plasmids. The obtained transformants were inoculated in 4 ml LB medium containing
125
100 µg/ml of ampicillin and grown aerobically at 37 °C overnight. 500 µl overnight
126
cultures were inoculated into 50 ml fresh TB medium, left to grow at 37 °C until the
127
optical density at 600 nm (OD600) reached 0.6, and then the E. coli Rosetta (DE3)
128
transformants were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG)
129
and shaken at 16 °C overnight. The cells were harvested 30 h after induction and
130
broken using ultrasonic cell disruptor. After centrifugation, the supernatants were used
131
as crude enzyme for enzymatic assays. The crude extracts of the recombinant
132
enzymes were further purified by using the following steps: The protein was first
133
purified with affinity chromatography using a Ni-NTA column (Qiagen) 6
134
pre-equilibrated with the lysis buffer and eluted with the lysis buffer supplemented
135
with 300 mM imidazole, which was followed by purification with gel filtration using
136
a Superdex-200 (GE Healthcare) column equilibrated with a buffer containing 10 mM
137
Tris–HCl pH 8.0, 200 mM NaCl, and 5 mM DTT. The purified protein was analyzed
138
by SDS–PAGE. The fractions containing the target protein were pooled and
139
concentrated to 20 mg/ml.
140
2.4. Enzymatic assays
141
The reaction was carried out in a 21 ml system containing 20 ml 20% maltose and
142
1 ml crude or purified enzymes in 1×PBS buffer (pH 7.4). Four different sources of
143
trehalose synthases have been reacted at the optimal temperature which has been
144
obtained in the previous study (Li et al., 2015). When four sources of recombinant
145
TreS-TreS-C activity was meatured, different temperatures were attempted to obtain
146
the optimal temperature. After 10 min, the trehalose production was measured by
147
High Performance Liquid Chromatography analysis (HPLC). Trehalose synthase
148
enzyme activity was defined under the condition of the optimal reaction temperature,
149
with excessive maltose solution as the substrate. The activity consuming 1 µmol
150
maltose or generating 1 µmol trehalose in 1 min was defined as one unit of enzyme
151
activity. The protein amount was determined by software Image J grayscale scanning.
152
2.5. HPLC analysis
153
Trehalose (SIGMA), was used as the standard. Both standard and samples were
154
analyzed and quantified by HPLC (HITACHI) with a reverse-phase Venusil PS NH
155
column (Agela) and a differential refraction detector. Solvent A was water with 87% 7
156
acetonitrile. The column temperature was set to 25 °C. The flow rate was 1 ml/min.
157
Quantification of trehalose was based on the peak areas.
158
2.6. Optimization of the reaction conditions
159
For the recombinant E. coli harboring pET-22b-PpTreS, pET-22b-CgTreS,
160
pET-22b-ScTreS,
pET-22b-TmTreS,
pET-22b-PpTreS565-linker-TtTreS-C,
161
pET-22b-CgTreS-linker-TtTreS-C,
162
pET-22b-TmTreS-linker-TtTreS-C, respectively, the enzymatic reaction conditions
163
were optimized as follows. The reaction was carried out under the temperature of
164
20 °C, 30 °C, 40 °C or 50 °C, respectively. To define the optimal temperature, the
165
enzyme was incubated in various temperatures for 10 h with 10% maltose, and
166
trehalose amount was measured under the condition described above.
167
3. Results and discussion
168
3.1. Effect of TtTreS-C on enzymatic activity of TreS enzymes
pET-22b-ScTreS-linker-TtTreS-C
and
169
TreS enzymes from four different sources were adopted in this research. In order
170
to test whether linking the TtTreS-C would increase the activity of the single-domain
171
TreSs, we linked tThe active domains of four TreS enzymes from different sources to
172
TtTreS-C have been linked, with or without a flexible linker peptide. The sequence of
173
the flexible linker peptide was GGGSGS, modified from the review article of
174
J.Sivaraman (Li et al., 2015). These chimera proteins have been expressed in E.coli,
175
and their crude enzymatic activities were compared with the four native enzymes.
176
First, different enzyme reaction temperatures (containing 20, 30, 40, 50 °C) have been
177
tested in this study, and the reactions were subsequently carried out under the optimal 8
178
temperature of 20 °C, 20 °C, 20 °C or 30 °C, respectively for the four enzymes from
179
different sources. After a reaction of 10 min, the trehalose production was measured
180
by High Performance Liquid Chromatography analysis (HPLC) and the results
181
showed that although linking the four enzymes directly to TtTreS-C did not increase
182
the enzymatic activity of TreSs, the chimera proteins with linker peptides between the
183
enzymes and TtTreS-C all showed increased activities compared to the native
184
enzymes (Fig. 3).
185
This suggested that the linker peptide between TreS and TtTreS-C has an
186
important role in the activity enhancement. In order to maintain steric configuration
187
and catalytic ability of trehalose synthase, medium-sized flexible linker peptide was
188
adopted in this study. On the other hand, the results also showed that the TtTreS-C not
189
only plays a role in thermophilicity and thermostability of TtTreS, but also function in
190
the activity of TtTreS. Taken together, these results suggested that TtTreS-C did affect
191
the enzymatic activities of TreSs, and the spatial organization of the active domain
192
and TtTreS-C is important for the impact of TtTreS-C on the activity of the active
193
domains of TreSs. Thus, in the following studies, only chimera proteins with linker
194
peptides were used in the comparison with native enzymes.
195
3.2. TtTreS-C increases the specific activities of TreSs
196
By transforming the eight recombinant plasmids (four native enzyme plasmids
197
and four chimera protein plasmids with linkers) into E.coli, we first determined tThe
198
optimal temperature of the enzyme reaction for each construct has been determined
199
firstly. Under the optimal temperatures, the specific activities of each transformants 9
200
have been obtained (Fig. 4). The results showed that linking the four enzymes to
201
TtTreS-C with peptide linkers could also increase the specific activities of TreS
202
enzymes. The specific activities of trehalose synthases from Pseudomonas putida,
203
Corynebacterium glutamicum, Streptomyces coelicolor and Thermotoga maritime
204
were increased 1.91-fold, 1.38-fold, 1.94-fold and 1.64-fold by fusion with TtTreS-C,
205
respectively. This suggested that the enzyme properties of the four enzymes were
206
modified by the TtTreS-C linked.
207
3.3. Reaction temperature optimization of trehalose biosynthesis
208
Furthermore, the enzymatic reaction conditions for trehalose synthase production
209
were optimized with the recombinant E. coli harboring the four native enzyme
210
plasmids and four chimera protein plasmids, respectively. When the crude enzyme
211
was added with 10% maltose solution, we noticed the results show that the conversion
212
efficiency began to decrease when the temperature was raised up. No additional
213
trehalose accumulation was observed up to high temperature in the above described
214
experiments (Fig. 5). Therefore, the optimal temperatures of the four chimera
215
enzymes were determined to be 20 °C, 20 °C, 20 °C and 30 °C, respectively (Fig. 5).
216
In the study of Wang et al, the recombinant DrTS-TtTS∆N enzyme had a higher
217
thermostability than DrTS in their reactions. However, in our this study, the optimal
218
temperatures of the four chimera enzymes were not increased under our reaction
219
conditions. We suggest that it This phenomenon might result from the different
220
reaction conditions used in our and their studies. Moreover, it also indicated that the
10
221
TtTreS-C might exhibit different effects on different proteins, either increasing the
222
thermostability or the specific activities of target enzymes.
223
4. Conclusions
224
In the current study, we first tested the heterologous expression of TreS genes
225
from four different sources with or without TtTreS-C has been tested firstly. The
226
enzymatic reaction temperature for the trehalose bioconversion was optimized
227
respectively. The results showed that both the conversion and the specific activities of
228
TreS enzymes were increased after linking to the TtTreS-C. The present study could
229
shed light on the further investigation of the efficient expression of TreS genes and
230
optimization of enzymatic reactions. In addition, linker-based connection between the
231
active domains of trehalose synthases and TtTreS-C has an important impact on the
232
enzymatic activity, suggesting that spatial organization of the two domains is
233
important for the impact of TtTreS-C on the activity of the active domains of TreSs.
234
Future more linker types could be tested to get better improvement of the activity of
235
TreS enzymes.
236 237
Acknowledgments
238
The authors would like to acknowledge the financial support of the National
239
Natural Science Foundation of China (Nos. 21636001 and 31670766) and the
240
Fundamental Research Funds for the Central Universities (buctrc201613, ZY1629).
241 242
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Li, Y., Sun, X., Feng, Y., Yuan, Q., 2015. Cloning, expression and activity
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Liang, J., Huang, R., Huang, Y., Wang, X., Du, L., Wei, Y., 2013. Cloning, 12
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Nguyen, A.D.Q., Kim, Y.G., Kim, S.B., Kim, C., 2013. Improved tolerance of
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NDP-glucoses. Enzyme Microb. Technol. 47, 249–256.
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Schiraldi, C., Di Lernia, I., De Rosa, M., 2002. Trehalose production: exploiting novel approaches. Trends Biotechnol. 20, 420–425.
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da Costa, M.S., 2003. Osmotic Adaptation of Thermus thermophilus RQ-1:
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Wei, Y., Liang, J., Huang, Y., Lei, P., Du, L., Huang, R., 2013. Simple, fast, and
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Wu, T.-T., Lin, S.-C., Shaw, J.-F., 2011. Integrated process for the purification and
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Zhang, Y., Zhang, T., Chi, Z., Wang, J.-M., Liu, G.-L., Chi, Z.-M., 2010. Conversion
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of cassava starch to trehalose by Saccharomycopsis fibuligera A11 and
301
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302 303
14
304 305
Fig.1.
306
CgTreS-linker-TtTreS-C, ScTreS-linker-TtTreS-C and TmTreS-linker-TtTreS-C. And
307
then they were subsequently digested and thereafter ligated into expression vector
308
pET-22b respectively, to construct eight recombinant plasmids and were transformed
309
into E.coli Rosetta (DE3).
Expression
of
enzymes
310
15
about
PpTreS565-linker-TtTreS-C,
PpTreS CgTreS ScTreS TmTreS
...................................MAKRSRPA.AFIDDPL......WYKDAVIYQLHIKSF MTDTSPLNSQPSADHHPDHAARPVLDAHGLIVEHESEEFPVPAPAPGEQPWEKKNREWYKDAVFYEVLVRAF ..................................MTVNEPVPD.TFEDTPAGDRHPDWFKRAVFYEVLVRSF ...........................................................VDVVLRERSIEEY
30 72 37 13
PpTreS CgTreS ScTreS TmTreS
FDANNDGIGDFAGLISKLDYIAELGVNTLWLLPFYPSPRRDDGYDIAEYKAVHPDYGSLADARRFIAEAHKR YDPEGNGVGSLKGLTEKLDYIQWLGVDCIWIPPFYDSPLRDGGYDIRNFREILPEFGTVDDFVELVDHAHRR QDSNGDGIGDLKGLTAKLDYLQWLGVDCLWLPPFFKSPLRDGGYDVSDYTAVLPEFGDLADFVEFVDAAHQR RTIIGNEVDEIKKLAEPLKGKKVLHVNATAYGGGVAEILHN.LVPLMRSVGLDARWRVIEAPDEFFNVTKKF
102 144 109 84
PpTreS CgTreS ScTreS TmTreS
GLRVITELVINHTSDQHPWFQRARHAKRGSKARDFYVWSDDEQKYDGTRIIFLDTEKSNWTWDPVAGQYFWH GLRVITDLVMNHTSDQHAWFQESRRDPTGPYG.DFYVWSDDPTLYNEARIIFVDTEESNWTYDPVRGQYFWH GMRVIIDFVMNHTSDQHPWFQESRKNPDGPYG.DYYVWADDDTRYADARIIFVDTEASNWTHDPVRGQYYWH HNTLQGADIEISEEEWNLYEEVCRKNAELIQDEELFVIHDSQP...AAVRKFVDLNDRKWIWRCHIDLSTPN
174 215 180 153
PpTreS CgTreS ScTreS TmTreS
RFYSHQPDLNFDNPQVLNAVIKVMRFWLDLGVDGLRLDAIPYLIERDGTNNENLPETHTVLKAIRAEIDANY RFFSHQPDLNYDNPAVQEAMLDVLRFWLDLGLDGFRLDAVPYLFEREGTNGENLKETHDFLKLCRSVIEKEY RFFSHQPDLNYENPAVQEEMLAALKFWLDLGVDGYRLDAVPYLYAEEGTNCENLPASHAFLKRVRREIDAQY MKVWQKFSQYLEG...YNRLVFHLEEYFPQGWK.ERSIAFPPSIDPLSEKNRDLDEDTIRKTLERLEIDPER
246 287 252 221
PpTreS CgTreS ScTreS TmTreS
PDRMLLAEANQWPEDTRPYFGE.GEG.DECHMAFHFPLMPRMYMALAMEDRFPITDILRQTPEIPANCQWAI PGRILLAEANQWPQDVVEYFGEKDKG.DECHMAFHFPLMPRIFMGVRQGSRTPISEILANTPEIPKTAQWGI PDTVLLAEANQWPEDVVDYFGDYSTGGDECHMAFHFPVMPRIFMAVRRESRYPVSEILAKTPAIPSGCQWGI PLITVVARFDPWKD....................LFSAIDVYRLVKKEIPEVQLAVVSAMAADDPE..GWFF
316 358 324 271
PpTreS CgTreS ScTreS TmTreS
FLRNHDELTLEMVTDRERDYLWNYYAEDRRARINLGIRRRLAPLLQRDRRRIELLTSLLLSMPGTPTLYYGD FLRNHDELTLEMVSDEERSYMYSQFASEPRMRANVGIRRRLSPLLEGDRNQLELLHGLLLSLPGSPVLYYGD FLRNHDELTLEMVTDEERDYMYAEYAKDPRMRANIGIRRRLATLLDNDRDQIELFTALLLALPGSPILYYGD FEK.....VLRYAGTDEDIKFCTNLKGVGNKEVNAIQRATTVALHTATREGFGLVISEALYKR...VPVVAR
388 430 396 335
PpTreS CgTreS ScTreS TmTreS
ELGMGDNIYLGDRDGVRTPMQWSPDRNGGFSRADPQRLVLPPIMDPLYGYQTVNVEAQSHDPHSLLNWTRRM EIGMGDNIWLHDRDGVRTPMQWSNDRNGGFSKADPERLYLPAIQNDQYGYAQVNVESQLNRENSLLRWLRNQ EIGMGDNIWLGDRDAVRTPMQWTPDRNAGFSTCDPGRLYLPAIMDPVYGYQVTNVEASMASPSSLLHWTRRM PVGG...VKIQVKHGENGYLAWEREDLAGYVVKLIKDEELRRKMGE...KGRQTVVENFIITVHLKNYLKLF
460 502 468 401
PpTreS CgTreS ScTreS TmTreS
LAVRKQQKAFGRGTLRTLTPSNRRILAYIREYTDADGHTEVILCVANVSRAAQAAELELSQYADKVPVEMLG ILIRKQYRAFGAGTYREVSSTNESVLTFLREHKG.....QTILCVNNMSKYPQAVSLDLREFAGHTPREMSG IEIRKQNPAFGLGTYTELPSSNPAVLAFLREYED.....DLVLCVNNFARFAQPTELDLREFAGRHPVELFG LDLLR...................................................................
532 569 535 406
PpTreS CgTreS ScTreS TmTreS
GSAFPPIGQLPFLLTLPPYAFYWFLLA.AHDRMPSWHIQATEGLPELTTLVLRKRMEELLEAPARDTLQTTI GQLFPTIAEREWIVTLAPHGFFWFDLT..ADEKDDME................................... GVRFPAIGELPYLLTLGGHGFYWFRLTRVASRIGRRA................................... ........................................................................
603 604 572 406
311 312
Fig.2. The amino acid sequence of CgTreS, ScTreS, TmTreS and the front partial
313
amino acid sequence of PpTreS. By comparing, their sequence were different. Lane 1:
314
The partial sequence of trehalose synthase gene from Pseudomonas putida NBRC
315
14164; Lane 2: The partial sequence of trehalose synthase gene from
316
Corynebacterium glutamicum ATCC 13032; Lane 3: The partial sequence of 16
317
trehalose synthase gene from Streptomyces coelicolor ATCC 23899; Lane 4: The
318
partial sequence of trehalose synthase gene from Thermotoga maritime MSB8.
319
17
320
321 322
Fig. 3. Crude trehalose synthase activity in recombinant E.coli Rosetta (DE3)
323
harboring pET-22b-PpTreS, pET-22b-PpTreS565-TtTreS-C,
324
pET-22b-PpTreS565-linker-TtTreS-C, pET-22b-CgTreS, pET-22b-CgTreS-TtTreS-C,
325
pET-22b-CgTreS-linker-TtTreS-C, pET-22b-ScTreS, pET-22b-ScTreS-TtTreS-C,
326
pET-22b-ScTreS-linker-TtTreS-C, pET-22b-TmTreS, pET-22b-TmTreS-TtTreS-C and
327
pET-22b-TmTreS-linker-TtTreS-C respectively by measuring at the same reaction
328
conditions. The amount of trehalose were measured by high performance liquid
329
chromatography.
330
18
331 332
Fig. 4. The activities of TreS, which from different sources, with or without
333
C-terminal of trehalose synthase from Thermus thermophilus were showed. The data
334
were generated from three independent experiments.
335
19
336 337
Fig. 5. Trehalose production in recombinant E.coli Rosetta (DE3) harboring
338
pET-22b-PpTreS, pET-22b-PpTreS565-linker-TtTreS-C (a), pET-22b-CgTreS,
339
pET-22b-CgTreS-linker-TtTreS-C (b), pET-22b-ScTreS,
340
pET-22b-ScTreS-linker-TtTreS-C (c), pET-22b-TmTreS, and
341
pET-22b-TmTreS-linker–TtTreS-C (d) respectively by varying reaction temperatures.
342
The amount of trehalose under different temperatures were measured by high
343
performance liquid chromatography.
344
20
345
Table 1
346
The details of strains and plasmids. Plasmid or strain Strains E coli DH5α
E coli Rosetta (DE3)
E coli Rosetta (DE3) -1 E coli Rosetta (DE3) -2 E coli Rosetta (DE3) -3 E coli Rosetta (DE3) -4 E coli Rosetta (DE3) -5 E coli Rosetta (DE3) -6 E coli Rosetta (DE3) -7 E coli Rosetta (DE3) -8 E coli Rosetta (DE3) -9
E coli Rosetta (DE3) -10 E coli Rosetta (DE3) -11 E coli Rosetta (DE3) -12 Plasmids pET-22b pET-22b-PpTreS
Descriptiona
Reference or sources
F-ψ80dlacZ△ (lacZYA-argF) U169 endA1 recA1 hsdR17 (rk-, mk+) supE44λ-thi-1 gyrA96 relA1 phoA F-ompT hsdSB(rB-, mB-) galdcmlacY1 (DE3) pRARE (argU, argW, ilex, glyT, leuW, proL) (Cmr) E coli Rosetta (DE3) harboring pET-22b-PpTreS E coli Rosetta (DE3) harboring pET-22b-CgTreS E coli Rosetta (DE3) harboring pET-22b-ScTreS E coli Rosetta (DE3) harboring pET-22b-TmTreS E coli Rosetta (DE3) harboring pET-22b-PpTreS565-TtTtreS-C E coli Rosetta (DE3) harboring pET-22b-CgTreS-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-ScTreS-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-TmTreS-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-PpTreS565-linker-TtTtre S-C E coli Rosetta (DE3) harboring pET-22b-CgTreS-linker-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-ScTreS-linker-TtTreS-C E coli Rosetta (DE3) harboring pET-22b-TmTreS-linker-TtTreS-C
TransGen
TransGen
This study This study This study This study This study This study This study This study This study
This study This study This study
pT7 expression vector, Novagen PBR322origin, Ampr pET-22b vector containing PpTreS This study 21
from pseudomonas putida pET-22b-CgTreS pET-22b vector containing CgTreS from Corynebacterium glutamicum pET-22b-ScTreS pET-22b vector containing ScTreS from Streptomyces coelicolor pET-22b-TmTreS pET-22b vector containing TmTreS from Thermotoga maritime pET-22b-PpTreS565-TtTre pET-22b vector containing PpTreS S-C from pseudomonas putida and TtTreS-C from Thermus thermophilus pET-22b-CgTreS-TtTreS-C pET-22b vector containing CgTreS from Corynebacterium glutamicum and TtTreS-C from Thermus thermophilus pET-22b-ScTreS-TtTreS-C pET-22b vector containing ScTreS from Streptomyces coelicolor and TtTreS-C from Thermus thermophilus pET-22b-TmTreS-TtTreS-C pET-22b vector containing TmTreS from Thermotoga maritime and TtTreS-C from Thermus thermophilus pET-22b-PpTreS565-linker- pET-22b vector containing PpTreS TtTreS-C from pseudomonas putida and TtTreS-C from Thermus thermophilus with linker pET-22b-CgTreS-linker-TtT pET-22b vector containing CgTreS reS-C from Corynebacterium glutamicum and TtTreS-C from Thermus thermophilus with linker pET-22b-ScTreS-linker-TtT pET-22b vector containing ScTreS reS-C from Streptomyces coelicolor and TtTreS-C from Thermus thermophilus with linker pET-22b-TmTreS-linker-Tt pET-22b vector containing TmTreS TreS-C from Thermotoga maritime and TtTreS-C from Thermus thermophilus with linker 347
a
Amp r, ampicillin resistant.
348
22
This study This study This study This study
This study
This study
This study
This study
This study
This study
This study
349
Table 2
350
The details of primer sequences. Name of Primers
Primer sequences
PpTreS-f PpTreS-r CgTreS-f
5’-GGGAAAGGATCCGGCCAAGCGTTCCCGCC-3’ 5’-GGGAAACTCGAGTGACTCTCCCCAGGTACTGATC-3’ 5’-GGGAAACCATGGGCACTGATACCTCTCCGTTGAATTCTCA3’ 5’-GGGAAAAAGCTTTTCCATATCGTCCTTTTCATCGGC-3’ 5’-GGGAAAGGATCCGCACCGTCAACGAGCCCGTAC-3’ 5’-GGGAAAAAGCTTAGCGCGGCGGCCG-3’ 5’-GGGAAAGAATTCGGATGTTGTGTTGAGAGAGCGAAGC-3’ 5’-GGGAAAGTCGACCCTCAACAGATCTAAGAAGAGTTTCAG ATAGTT-3’ 5’-GGGAAAGAATTCGGCCAAGCGTTCCCGCCCGGC-3’
CgTreS-r ScTreS-f ScTreS-r TmTreS-f TmTreS-r PpTreS565(TtTreSC)-f PpTreS565(TtTreSC)-r TtTreS-C(PpTreS56 5)-f TtTreS-C(PpTreS56 5)-r CgTreS(TtTreS-C)-f CgTreS(TtTreS-C)-r TtTreS-C(CgTreS)-f TtTreS-C(CgTreS)-r ScTreS(TtTreS-C)-f ScTreS(TtTreS-C)-r TtTreS-C(ScTreS)-f TtTreS-C(ScTreS)-r TmTreS(TtTreS-C)-f TmTreS(TtTreS-C)-r TtTreS-C(TmTreS)-f TtTreS-C(TmTreS)-r PpTreS565L(TtTreS -C)-f PpTreS565L(TtTreS -C)-r LTtTreS-C(PpTreS5
5’-CGGGGGCGGGCTCCTCGGCGGGCATGCGGTCATGGGCG-3 ’ 5’-CGCCCATGACCGCATGCCCGCCGAGGAGCCCGCCCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGTCGACGACTGATACCTCTCCGTTGAA-3’ 5’-CGGGGGCGGGCTCCTCGGCTTCCATATCGTCCTTTTCA-3’ 5’-TGAAAAGGACGATATGGAAGCCGAGGAGCCCGCCCCCG-3 ’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGAATTCGACCGTCAACGAGCCCGTACC-3’ 5’-CGGGGGCGGGCTCCTCGGCGGATGCGACTCGGGTGAGC-3 ’ 5’-GCTCACCCGAGTCGCATCCGCCGAGGAGCCCGCCCCCG-3 ’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGAATTCGGATGTTGTGTTGAGAGAGCG-3’ 5’-CGGGGGCGGGCTCCTCGGCCCTCAACAGATCTAAGAAG-3 ’ 5’-CTTCTTAGATCTGTTGAGGGCCGAGGAGCCCGCCCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCT-3’ 5’-GGGAAAGGATCCGGCCAAGCGTTCCCGCCCGGCAGCC-3’ 5’-GGCGGCGGCTCCGGCTCCGGGCATGCGGTCATGG-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCG-3’ 23
65)-f LTtTreS-C(PpTreS5 65)-r CgTreSL(TtTreS-C)f CgTreSL(TtTreS-C)r LTtTreS-C(CgTreS)f LTtTreS-C(CgTreS)r ScTreSL(TtTreS-C)f ScTreSL(TtTreS-C)r LTtTreS-C(ScTreS)f LTtTreS-C(ScTreS)r TmTreSL(TtTreS-C) -f TmTreSL(TtTreS-C) -r LTtTreS-C(TmTreS) -f LTtTreS-C(TmTreS) -r
5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’ 5’-GGGAAACCATGGGACTGATACCTCTCCGTTGAATTCTCAG CCG-3’ 5’-GGCGGCGGCTCCGGCTCCTTCCATATCGTCCTTT-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’ 5’-GGGAAAGGATCCGACCGTCAACGAGCCCGTACCTGACAC CTTCGAG-3’ 5’-GGCGGCGGCTCCGGCTCCAGCGCGGCGGCCGATG-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’ 5’-GGGAAAGGATCCGGATGTTGTGTTGAGAGAGCGAAGCAT AGAGG-3’ 5’-GGCGGCGGCTCCGGCTCCCCTCAACAGATCTAAG-3’ 5’-GGAGCCGGAGCCGCCGCCTGGGCCGAGGAGCCCGCCCCC GAG-3’ 5’-GGGAAAAAGCTTGGCTTTTCCGGCCTTGGCCTGCCAGCG CTCGT-3’
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We tested the heterologous expression of TreS genes with or without TtTreS-C in Escherichia coli. Linking TtTreS-C to the C-termini of TreS enzymes could all increase the enzymatic activities. A linker peptide between TreS and TtTreS-C is essential for the activity enhancement. The specific activities of TreS enzymes were also improved by TtTreS-C.
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