Programming Integrative Extracellular and ... - ACS Publications

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg

Letter

Programming Integrative Extracellular and Intracellular Biocatalysis for Rapid, Robust and Recyclable Synthesis of Trehalose Ling Jiang, Xiaogang Song, Yingfeng Li, Qing Xu, Jiahua Pu, He Huang, and Chao Zhong ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03445 • Publication Date (Web): 26 Jan 2018 Downloaded from http://pubs.acs.org on January 27, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

1

Programming Integrative Extracellular and Intracellular Biocatalysis for

2

Rapid, Robust and Recyclable Synthesis of Trehalose

3

Ling Jiang1, Xiaogang Song1, Yingfeng Li2, Qing Xu3, Jiahua Pu2, He Huang3,*, Chao Zhong2,*

4 5 6 7 8 9 10 11 12 13

1

College of Food Science and Light Industry, Nanjing Tech University, Nanjing 210009, PR China;

2

School of Physical Science and Technology, Shanghai Tech University, Shanghai 201210, PR China;

3

College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, PR China

*Corresponding authors. Emails: [email protected] (He Huang) and [email protected] (Chao Zhong)

1

ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

ABSTRACT We herein introduce a strategy that leverages and integrates the attributes of whole-cell catalysis with enhanced stability of extracellular immobilized enzymes for rapid, robust, recyclable enzyme cascade reactions in a scalable fashion. We demonstrated the utility of the integrative strategy for catalytic synthesis of trehalose from soluble starch with two-step sequential bioconversion enzymatic reactions, implemented by coupling the enzymatic immobilization of β-amylase (BA), based upon E. coli biofilm curli display technique, with intracellular expression of trehalose synthase (TreS) within the same cells. This integrative strategy, compared with a strategy based on cells coupled with isolated BA, enabled a 103.5 ± 18.7% increase in the maximum trehalose formation rate by efficiently reducing the average distance of BA to intracellluar TreS enzyme. In addition, the maximum yield of starch into trehalose reached as high as 59.0 ± 1.3% at a relatively high starch concentration (10% w/v) with 15 g/L of engineered cells. We further showed that the productivity of trehalose and the percentages of cell viability remained 89.1 ± 4.4% and 85.2 ± 3.6% respectively, even after 8 continuous rounds of biocatalysis. In addition, this strategy exhibited superb operational stability even under harsh conditions, for example, solutions rich in high amount of organic solvents. The strategy demonstrated here opens up research opportunities of combining extracellular catalysis with intracellular reactions for rapid and robust production of various value-based products. KEYWORDS: Biocatalysis; Capsule-Like Particles (CLPs); Biofilms; Trehalose; Amyloid

2


Page 2 of 13

Page 3 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

ACS Catalysis

Biocatalysis covers a broad spectrum of applications in industrial chemical synthesis, and is an environmentally friendly and scalable alternative to traditional chemical synthesis for complex chemical transformations.1-3 In biotransformations, enzymes can be used as biocatalysts in purified or immobilized form as well as within whole cells.4, 5 Isolated enzymes or whole cells are attractive versatile biocatalysts because of their inherent regio- and stereo-selectivity, high-efficiency and substrate specificity, but both have their own limitations.6 For example, catalysis based on isolated enzymes has several disadvantages such as high costs associated with enzyme purification, lack of long-term stability and low catalyst recovery. Whole-cell biocatalysis as functional biocatalyst module unit presents a multistep approach by easily coupling both artificial and native reaction pathways, but it is mainly confined to batch processes, often with low transformation efficiency due to poor intracellular mass transport.7 A new approach that integrates enhanced enzyme stability, recyclable catalysis and multistep enzymatic modules with fast catalysis rate is highly desirable but remains challenging. Here, we introduce a strategy that leverages and integrates the attributes of whole-cell catalysis with the enhanced stability of immobilized enzymes for fast, robust and recyclable enzyme cascade reactions in a scalable fashion. Specifically we demonstrated the utility of the strategy for catalytic synthesis of trehalose from soluble starch via two-step sequential bioconversion enzymatic reactions. To such ends, β-amylase (BA) enzymes were first docked onto engineered E. coli biofilms for the first-step catalysis reaction, while trehalose synthase (TreS), intracellularly produced by the same cells, catalyzed the second-step enzyme reaction. In this way, we harnessed the cells containing multiple enzymes as programmable living factories to implement both intracellular catalysis and extracellular immobilized enzyme catalysis simultaneously (Figure 1). We first leveraged a genetically programmable biofilm curli display platform to anchor the BA enzyme onto biofilms.8, 9 Relevantly, curli fibers, formed through self-assembly of an extracellular protein CsgA, are robust functional amyloid structures of Escherichia coli biofilms.10, 11 Previous studies have shown that curli fibers could be endowed with additional functionalities without affecting its self-assembling properties, providing an excellent platform to display various functional proteins including enzymes onto nanofibers.12, 13 As such, we constructed a strain harboring a plasmid with the targeting gene that can express CsgA-SpyTag proteins. Upon secretion, the CsgA-SpyTag proteins can extracellularly self-assemble into amyloid fibers displaying functional SpyTag, which can covalently capture the SpyCatcher-tagged β-amylase through SpyTag/SpyCatcher protein partner interaction under ambient conditions (Figure S1).14, 15 In addition, we transformed the same E. coli strain with another plasmid containing the targeting gene that can express the TreS enzyme inside the cells. We termed this engineered BA-displaying cells that simultaneously express TreS upon induction as Capsule-Like Particles (CLPs) for biosynthesis of trehalose (Figure S4). To implement the outlined strategy, we first designed two constructs (Figure S1, Table S1, S2). The first one, referred to as PET28a/β-amylase-SpyCatcher (abr. PET28a/BA-SpyCatcher), can express BA-SpyCatcher under IPTG induction. Another one, a bicistronic expression vector (PET28a/TreS + CsgA-SpyTag), can co-express TreS and CsgA-SpyTag by adding a ribosomal binding site between these two fragments.

3


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

Figure 1. An integrative cell-based catalysis strategy for synthesis of trehalose from soluble starch based upon two-step sequential bioconversion enzymatic reactions. The strategy was achieved by coupling immobilized enzymes on extracellular amyloid curli nanofibers with independent TreS enzymes expressed inside E. coli cells. Schematic showing the whole processes (A) and the corresponding two-step sequential enzymatic reactions (B) for synthesis of trehalose starting from soluble starch: (a) BA displayed on CsgA-SpyTag biofilm hydrolyzes starch into maltose; (b) The resulting maltose enters the cells; (c) Intracellular TreS hydrolyzes the maltose into trehalose and glucose simultaneously (Figure S4); (d) Extracellular release of trehalose and glucose. Glucose as side product provides additional supply for cell growth. Note: the catalytic biofilms displaying BA were successfully constructed by covalent reactions between CsgA-SpyTag nanofiber subunits and BA-SpyCatcher fusion proteins, achieved via SpyTag/SpyCatcher protein partner interaction. We next turned to morphologically characterize the secreted biofilms with both SEM and TEM. Fibrous structures could be observed wrapped around E. coli cells when the expression of CsgA-SpyTag was induced with IPTG (Figure 2b). In contrast, such fibrous structures were not present in the control strain (the csgA deletion mutant PHL628) expressing no curli fibers, as revealed by SEM image (Figure 2a). These results were consistent with TEM observations, which showed fibrous networks containing curli nanofibers with a diameter of approximately 4-7 nm adhering to the engineered cells but not in the control strain (Figure 2c-d). To confirm if the nanofibers still bear amyloid features, we carried out Congo red (CR) assay, a dye assay frequently used to detect amyloid fibers (Figure 2e). The absorption spectra indicated that mixing CR with 4


Page 4 of 13

Page 5 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

101 102 103 104 105 106 107 108 109 110 111 112

ACS Catalysis

CsgA-SpyTag amyloid fibrils caused a blue shift of λmax from 495 to 490 nm, which is consistent with previous reports for amyloid fibrils.16 In addition, Congo red (CR) bound strongly to the cell pellets after centrifugation (Figure 2e insert). The absorbance of the CR supernatant became progressively lower, suggesting that the amount of CsgA-SpyTag amyloid fibrils produced by the cells increased after 48 h inducible expression (Figure 2f). To verify that extracellular amyloid nanofiber materials were indeed formed by CsgA-SpyTag protein, we isolated the extracellular materials from the induced recombinant cells and disassembled the curli fibers into their monomeric components (details in supporting information). SDS-PAGE analysis of the resulting samples confirmed the presence of both TreS (69 kDa) (Figure 2g) and CsgA-SpyTag fusion protein (17 kDa) (Figure 2h) in all samples except for the control without curli fibers (data not shown). Collectively, these results suggested that CsgA-SpyTag proteins had been successfully secreted and would assemble into fibrous structures with “amyloid” features.

113 5


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Characterization of E. coli biofilms composed of functional amyloid nanofibers. FE-SEM and TEM images of the control strain expressing no curli fibers (a, c) and the recombinant E. coli expressing the functionalized curli fibers comprising CsgA-SpyTag subunits (b, d); (e) CR binding assay of CsgA-SpyTag biofilms. The inserted image shows the Congo red solution alone and Congo red solution mixed with cell fibrils at different inducing time (10–20 h). (f) The correlation between the CR binding and the amount of amyloid CsgA-SpyTag produced by the cells at different induced time (10–48 h). SDS-PAGE with Coomassie staining for TreS (g), CsgA-SpyTag (h) and BA-SpyCacther (i). Note: In (g-i), the word “marker”, “crude” and “purified” from left to right refers to the standard marker lane, proteins from crude extract and purified proteins, respectively.

114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137

In order to ensure conjugation efficiency between BA-SpyCatcher and the fiber-bound SpyTag on E. coli cells, we used an excess of BA-SpyCatcher fusion proteins with the mass ratio of BA-SpyCatcher fusion proteins to cells setting in 0.3–0.6 mg/g cells (supporting information, formula 3). To optimize the immobilization efficiency, we varied the conjugation reaction time from 6 to 72 hours. We purposely extended the conjugation time for this system as the SpyTag/SpyCatcher protein partner interaction became a heterogeneous reaction due to the insolubility of CsgA-Spycatcher curli fibers. As incubation time increased, the efficiency gradually increased and almost reached a plaque after 48 h, with 61.2 ± 5.4% immobilization efficiency (Figure 3). The mass ratio of immobilized BA-SpyCatcher fusion proteins (on curli fibers) to cells could reach 0.22 mg/g cells. These results thus suggested that BA-SpyCatcher could be effectively immobilized on the CsgA-SpyTag biofilms, even though the immobilization efficiency would saturate to a certain degree possibly due to the fact that most of SpyTag residues displayed onto the biofilms had been consumed.

138 139 140 141

Figure 3. Effects of incubation time on immobilization percentage of BA-SpyCatcher proteins on CsgA-SpyTag biofilms. The immobilization percentage is determined by measuring the difference of enzyme protein content between residual BA-SpyCatcher and initial BA-SpyCatcher 6


Page 6 of 13

Page 7 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

ACS Catalysis

enzyme using the Bradford method (supporting information, formula 4). CLPs were thus applied to catalyze soluble starch conversion into trehalose through two-step sequential biocatalysis reactions. The expression of BA-SpyCatcher was confirmed by SDS/PAGE (Figure 2i). The enzymatic activity of purified BA-SpyCatcher fusion proteins in CLPs reached 92.7 U/mg (Table S3), which is in good agreement with the activity of ~90 U/mg reported for Saccharomycopsis fibuligera.17 The result suggested that fusion of SpyCatcher onto BA did not affect the enzyme activity of BA. With TreS expressed within the cells, the activity of TreS enzyme in the CLPs reached 326.3 U/g [wet weight] of cells (Table S3), which was of the same order of magnitude compared with the TreS activity in crude cell lysates (139.7 U/g [wet weight] of cells) (Table S3).18, 19 The values thus suggested that the activity of TreS was not affected by the co-expression of TreS and CsgA-SpyTag in our CLPs. Importantly, the BA-displaying engineered cells were able to produce trehalose directly from soluble starch (Figure S5). In contrast, control cells expressing no curli fibers produced no trehalose and maltose (Figure S5). These results thus validated the feasibility of integrating intracellular and extracellular catalysis simultaneously to convert soluble starch into trehalose, reminiscent of previous enzymatic systems based on surface display of hydrolytic enzymes coupled to specific intracellular conversion.20 Notably, the maximum maltose formation rate in the CLPs (17.9 ± 0.5 mmol/L/h) was much higher than that of free BA mixed with cells (14.7 ± 0.6 mmol/L/h) and free BA coupled with free TreS (13.9 ± 0.7 mmol/L/h) (Figure 4a). Furthermore, with the same amount of starch (10% w/v) applied in the three systems, the total amount of trehalose produced in the CLPs reached as high as 59.0 ± 1.3 g/L, significantly higher than that of free BA mixed with cells (36.3 ± 2.9 g/L) and free BA coupled with free TreS (22.3 ± 2.1 g/L), respectively (Figure 4b). A similar trend was found with the maximum trehalose formation rate among the three systems, with 11.6 ± 0.4, 5.7 ± 0.3 and 4.4 ± 0.4 mmol/L/h for CPLs, free BA coupled with cells and free BA combined with free TreS, respectively (Figure 4b). The higher formation rates of maltose and trehalose thus implied that faster mass conversion of starch to maltose and faster mass transfer of maltose into the cells occurred in the case of CLPs compared with free BA mixed with cells.21 It is very likely that the extracellularly immobilized BA provides a more stable and favorable environment for capturing the substrate and thus implements more efficient enzymatic reaction compared with free BA in solution. In addition, the reduced average distance between BA and TreS enzymes in the two systems, with an average distance of 1.85 µm in CLPs vs. 4.27 µm in the system of free BA coupled with cells based on simplified calculation (Figure S6), could facilitate the mass transfer of maltose to the catalytic sites of the TreS enzymes expressed inside the cells.

7


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206

Figure 4. Comparison of the maltose formation rate (a) and trehalose formation rate (b) over a reaction period of 40 hours based on three different approaches, that is, in the case of CLPs, free BA coupled with cells, and free BA combined with free TreS, respectively. Note: the concentration of cells in both CLPs and regular cell system used was fixed at 15 g/L. The amount of free BA used was 2.7 mg/L based on the mass ratio of BA to cells fixing with 0.18 mg/g cells (supporting information, formula 3). In addition, the amount of free TreS used was 7.5 mg/L, which was equal to that of TreS expressed in CLPs and cells (Table S4). The error bars refer to standard errors of the average rate calculated based on three replicates. The incubation time for conjugation of BA-SpyCatcher with CsgA-SpyTag influenced the final yield of trehalose, as the incubation time directly affected the enzyme immobilization efficiency. As expected, the maximum yield of trehalose was obtained with 48 h of immobilization time (Figure 5a), in consistence with the relationship between enzyme immobilization efficiency and incubation time, as revealed in Figure 2. Specifically, the maximum yield of trehalose from starch was 59.0 ± 1.3% in our CLPs system, in stark contrast with the other cases, that is, with 36.3 ± 2.9% in the system of free BA coupled with cells and 22.3 ± 2.1% in the system of free BA coupled with free TreS (Figure 4b, 5b). As further shown in Figrue 5b, 7.0 ± 0.5 g/L of glucose was concurrently produced as by-product during the intramolecular transglucosylation reactions (Figure S4).19 The time courses for the concentrations of maltose and trehalose revealed a dynamic relationship of the two-enzyme catalytic cascade reactions (Figure 5b). The concentration of trehalose increased steadily for the first 20 hours and gradually reached a plaque after 24 hours. In contrast, maltose concentration also increased steadily during the first 12 hours but fell dramatically after 18 hours. Collectively, the data indicated that the ratio of generation rate/consumption rate of maltose changed over time. We thus inferred that hydrolysis of starch to maltose by extracellular BA should be a rate-limiting step during the whole catalytic process. In the repeated utilization of CLPs，the complex biocatalyst yielded a high productivity of trehalose (Figure 5c). In addition, CLPs exhibited robust and continuous operational stability by reaching a total reaction time of 145 h (6 batches) and maintaining more than 89.1 ± 4.4% of the 8


Page 8 of 13

Page 9 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231

ACS Catalysis

initial trehalose productivity even after 8 rounds of reaction at 40 °C. Intriguingly, we found that even after repeated utilization for 90-hour biocatalysis, the viability percentage of BA-displaying engineered cells remained above 85.2 ± 3.6%. The value is even higher compared with the control cells that didn’t perform biocatalysis at all (Figure 5d). We proposed that glucose, produced as a by-product, supply nutrient for cell growth and therefore maintain the high viability of cells. Previous studies had reported trehalose formation based on recombinant β-amylase-TreS bifunctional fusion protein and cellular catalysis coupled with free BA,22-24 However, none of the systems had been aimed for continuous industrial production of trehalose from commercially available and cheap starch, as only one-time catalytic reaction had been demonstrated and relatively a low amount of substrate had been applied in their systems.22, 23 Compared with the ellular catalysis coupled with free BA approach, with similar concentration of starch applied in both systems (10% w/v), our CLPs system exhibited higher yield of starch into trehalose (59% vs 45%).24 In addition, compared with methods based on free BA coupled with free TreS enzyme, the CLPs described in this study showed enhanced temperature stability (from 60 °C to 70 °C) and could be operated over more broad pH range (from pH 5–9 to 4–10) (Figure S7). The enzyme stability in solution over a wide pH range (pH, 4–10) also outperformed the case of recombinant β-amylase-TreS bifunctional fusion protein, with a pH operational range of 5–7.22 Notably, the CLPs can even normally operate in the presence of common organic solvents (e.g., methanol, toluene and acetone) at high concentrations (40%) (Figure S7). Collectively, the CLPs system thus outperforms the previously reported strategies as well as the control systems in our studies in terms of catalytic rate, efficiency and operational stability.22-24 It therefore provides a possible solution for continuous industrial production of trehalose from commercially available and cheap starch even under relatively harsh conditions, which are quite common in the industrial process of enzymatic reactions.3

9


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252

Figure 5. (a) Effects of incubation time for BA binding to the CsgA-SpyTag biofilm on trehalose production with 10% w/v of soluble starch after 24-h sequential catalytic reactions. The cells expressing CsgA-SpyTag biofilms were incubated with BA-SpyCatcher fusion protein at 18 °C for variable time ranging from 6 to 72 h. (b) Time courses of the intermediate, final and side products for the production of trehalose via two-step sequential biocatalysis reactions under different incubation time for conjugation of BA-SpyCatcher with CsgA-SpyTag. The reaction conditions were based on 10% w/v of soluble starch at pH 7.0, 40 °C with 15 g/L of BA-displaying engineered cells expressing CsgA-SpyTag biofilms. BA-SpyCatcher fusion protein was incubated for 48 h. (c) Repeated utilization of CLPs for biocatalysis was operated at 40 °C with 24 h of each batch for trehalose production. (d) Time courses of cell viability measured by resazurin assay. The BA-displaying engineered cell was measured during continuous catalytic reaction, and control cell (the csgA deletion mutant PHL628) for continuous catalytic reaction. Data are averages from three independent experiments. In summary, we demonstrated a new cell-based biocatalysis strategy coupling the intracelluar catalysis with extracelluarly immobilized enzyme catalysis. The usefulness of this integrative strategy was demonstrated based on the immobilization of BA- SpyCatcher onto functional curli nanofibers of E. coli biofilms (for the first catalysis reaction) coupled with simultaneous expression of TreS inside the same E. coli cells (for second catalysis reaction), yielding efficient and robust enzymatic synthesis of trehalose from soluble starch. We envision that our strategy, merging 10


Page 10 of 13

Page 11 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276

ACS Catalysis

extracellular enzyme immobilization with intracellular enzyme catalysis, provides a new and feasible avenue towards biotransformations with complex enzymatic cascade reactions and expands a wide spectrum of applications ranging from microbial fuel cells to biosensors.

AUTHOR INFORMATION Corresponding authors: *Email: [email protected] *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Shanghai Science and Technology Committee (Shanghai Science and Technology Innovation Action, Fundamental Research Project Grant No.17JC1403900), the National Natural Science Foundation of China (No. U1603112, No. 31570972), “Dawn” Program of Shanghai Education Commission, China (No. 14SG56), Joint Funds of the National Natural Science Foundation of China (Seed Grant No. U1532127), and the Six Talent Peaks Project in Jiangsu Province (No. 2015-JY-009). ASSOCIATED CONTENT Supporting Information The Supporting Information for this paper is available free of charge in the online version of the paper. REFERENCES (1) Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K. Nature 2012, 485, 185–194. (2) Choi, J. M.; Han, S. S.; Kim, H. S. Biotechnol. Adv. 2015, 33, 1443–1454. (3) Reetz, M. T. J. Am. Chem. Soc. 2013, 135, 12480–12496. (4) Halan, B.; Buehler, K.; Schmid, A. Trends Biotechnol. 2012, 30, 453–465. (5) Sheldon, R. A.; van Pelt, S. Chem. Soc. Rev. 2013, 42, 6223–6235. (6) Nestl, B. M.; Nebel, B. A.; Hauer, B. Curr. Opin. Chem. Biol. 2011, 15, 187–193. (7) Ladkau, N.; Schmid, A.; Bühler, B. Curr. Opin. Biotech. 2014, 30, 178–189. (8) Nguyen, P. Q.; Botyanszki, Z.; Tay, P. K. R.; Joshi, N. S. Nat. Commun. 2014, 5, 4945–4952. (9) Botyanszki, Z.; Tay, P. K. R.; Nguyen, P. Q.; Nussbaumer, M. G.; Joshi, N. S. Biotechnol. Bioeng. 2015, 112, 2016–2024. (10) Chapman, M. R.; Robinson, L. S.; Pinkner, J. S.; Roth, R.; Heuser, J.; Hammar, M.; Hultgren, S. J. Science 2002, 295, 851–855. (11) Barnhart, M. M.; Chapman, M. R. Annu. Rev. Microbiol. 2006, 60, 131–147. (12) Gilbert, C.; Howarth, M.; Harwood, C.; Ellis, T. bioRxiv. 2016, 087593. (13) Chen, A. Y.; Deng, Z.; Billings, A. N.; Seker, U. O.; Lu, M. Y.; Citorik, R. J.; Zakeri, B.; Lu, T. 11


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

K. Nat. Mater. 2014, 13(5), 515–523. (14) Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M. P. Nat. Acad. Sci. USA 2012, 109, E690–E697. (15) Reddington, S. C.; Howarth, M. Curr. Opin. Chem. Biol. 2015, 29, 94–99. (16) Nilsson, M. R. Methods 2004, 34, 151–160. (17) Chi, Z.; Chi, Z.; Liu, G.; Wang, F.; Ju, L.; Zhang, T. Biotechnol. Adv. 2009, 27, 423–431. (18) Jiang, L.; Lin, M.; Zhang, Y.; Li, Y.; Xu, X.; Li, S.; Huang, H. Plos One 2013, 8, e77437. (19) Schiraldi, C.; Di Lernia, I.; De Rosa, M. Trends Biotechnol. 2002, 20, 420–425. (20) den Haan, R.; van Rensburg, E.; Rose, S. H.; Görgens, J. F.; van Zyl, W. H. Curr. Opin Biotech. 2015, 33, 32–38. (21) Gibney, P. A.; Schieler, A.; Chen, J. C.; Rabinowitz, J. D.; Botstein, D. P. Nat. Acad. Sci. USA 2015, 112(19), 6116–6121. (22) Wang, J. H.; Tsai, M. Y.; Lee, G. C.; Shaw, J. F. J. Agr. Food Chem. 2007, 55(4), 1256–1263. (23) Chang, S. W.; Liu, P. T.; Hsu, L. C.; Chen, C. S.; Shaw, J. F. Food Chem. 2012, 134(4), 1745–1753. (24) Chang, S. W.; Chang, W. H.; Lee, M. R.; Yang, T. J.; Yu, N. Y.; Chen, C. S.; Shaw, J. F. J. Agr. Food Chem. 2010, 58(5), 2908–2914.

12


Page 12 of 13

Page 13 of 13

ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Programming Integrative Extracellular and ... - ACS Publications

Recommend Documents