Intelligent Microbial Heat-Regulating Engine (IMHeRE) for Improved

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Intelligent Microbial Heat-Regulating Engine (IMHeRE) for Improved Thermo-Robustness and Efficiency of Bioconversion Haiyang Jia,† Xiangying Sun,† Huan Sun,† Chenyi Li,† Yunqian Wang,† Xudong Feng,† and Chun Li*,†,‡ †

Department of Biological Engineering, School of Life Science, Beijing Institute of Technology, Beijing 100081, P. R. China State Key Laboratory of System Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China

‡

S Supporting Information *

ABSTRACT: The growth and production of microorganisms in bioconversion are often hampered by heat stress. In this study, an intelligent microbial heat-regulating engine (IMHeRE) was developed and customized to improve the thermo-robustness of Escherichia coli via the integration of a thermotolerant system and a quorum-regulating system. At the cell level, the thermotolerant system composed of different heat shock proteins and RNA thermometers hierarchically expands the optimum temperature by sensing heat changes. At the community level, the quorum-regulating system dynamically programs the altruistic sacrifice of individuals to reduce metabolic heat release by sensing the temperature and cell density. Using this hierarchical, dynamical, and multilevel regulation, the IMHeRE is able to significantly improve cell growth and production. In a real application, the production of lysine was increased 5-fold at 40 °C using the IMHeRE. Our work provides new potential for the development of bioconversion by conserving energy and increasing productivity. KEYWORDS: hierarchical thermotolerance, quorum sensing, altruistic cell death, bioconversion, synthetic biology

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ments provide great insights for development of robust hosts. In response to heat stress, thermophilic microorganisms can efficiently activate an ancient signaling pathway that leads to the transient expression of heat shock or heat stress proteins with different functions,6,7 e.g. molecular chaperones,8 ubiquitin proteins,9 and so on. Recently, identification and introduction of such genes from extremophiles have been proven as an effective approach for engineering the cellular robustness of microbes.10,11 In addition to intracellular protection mechanisms, bacterial cells increase the chances of survival using programmed cell death (PCD) when they encounter an extreme stress, which can be viewed as an altruistic trait. One of the best-studied forms of PCD in bacteria is the toxin− antitoxin system, MazEF.12,13 The product of mazF is a stable toxin that cleaves mRNA at a specific site(s).14 The toxic effect obtained by ectopic overproduction of MazF can be reversed by the action of the antitoxin MazE, which is ectopically overexpressed at a later time, and suggests that rather than inducing cell death, MazF induces a state of reversible bacteriostatic conditions.15 Certain cells trigger cell death and release such stress-relieving substances as actors’ skin, which increases the chances of survival of other cells within the population for the “public good”.16 We believe that the “public good” events may not all manifest in the form of substances,

icrobial fermentation and bioconversion play essential roles in the production of pharmaceuticals, biofuels, and chemicals.1 The market competitiveness of these fermented products is primarily determined by their production efficiency and cost, which are often limited by a number of stress conditions due to environmental fluctuations or metabolism imbalances, i.e., high temperature, high salt, strong acids, toxic metabolites, and other factors. More specifically, high temperature is one of the main adverse factors that causes cell morphological abnormalities, inhibits cell division and growth, destroys cytoskeletal integrity, and impacts metabolic activity.2,3 Unfortunately, the commonly used industrial fermentation strains, i.e., bacteria and yeast, are mesophilic and have low robustness to high temperature. Therefore, the development of new methods that can improve the thermotolerance of microorganisms for the fermentation industry could significantly improve productivity, shrink production costs, reduce energy consumption, increase substrate utilization, and mitigate the risk of contamination.4 Previous studies used mutagenesis methods or adaption evolution5 to improve the thermotolerance of microorganisms, but these methods also have side effects on production ability and genetic stability. Heat represents a significant barrier to life. Cells with biological robustness can adapt to unpredictable internal or external stresses through dynamic controllability, modular and hierarchical organization, or functional redundancy in a cellular system.1 In particular, microbes that live in extreme environ© XXXX American Chemical Society

Received: August 24, 2015

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Figure 1. Thermo-robustness mechanism of the intelligent microbial heat-regulating engine.

was assembled with a thermotolerant system and a quorumregulating system. These two systems perform different functions and display their functions in different sections, but they work together, similar to two gears in a machine, to ensure cell survival in a wide range of high temperatures. The thermotolerant system was constructed with different heat shock proteins (HSPs) to extend the optimum temperature using hierarchical thermotolerance. The hierarchy of a thermotolerant system was revealed for the first time by RNA thermometers, which triggered the expression of several HSPs at different temperature in a living cell, and functions via whole integration through complementary interactions. Biological heat is one of the main factors that lead to the temperature rising during the fermentation process. It is because a culture at a specific physiological state produces a constant rate of heat per unit of biomass and the heat per bacterial cell increased, which was explained by an increased cell size and amount during growth.21,22 Therefore, a large amount of biological heat will be released when the cell density reaches a high level in the large-scale vigorous fermentation. This heat would lead to the rise of fermentation temperature exceeding the capability of the thermotolerant system and affecting metabolism, which is found in the literature regarding an explicit coupling between a reduced specific rate of heat production and an increased

and actions that decrease the adverse conditions, i.e., heat stress, represent another type of “public good” for the community. To construct thermo-robustness in a complicated artificial biological system, it is insufficient and unrealistic to use a simple or single strategy, i.e., overexpression of heat shock proteins.17 In addition, cells with properties that overcome stress conditions should leverage multidimensional synergetic effects of different functional genes or even systems.18 Synthetic biology, which is defined as the application of engineering principles to biology, aims to systematize the process of designing genetically encoded biological systems, thus rendering them predictable, robust, scalable, and efficient. 19 Consequently, by supplying cells with devices and components that confer resistance to stress, synthetic biology offers a powerful approach for integrating sophisticated biological functionalities at both intra - and intercellular levels20 and improving the properties of existing microbial biotransformation systems.18 In this study, inspired by the natural thermotolerance mechanisms of microorganisms, we constructed an intelligent microbial heat-regulating engine (IMHeRE) using synthetic biology to improve the thermotolerance of Escherichia coli at both the cell and community levels (Figure 1). The IMHeRE B

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Figure 2. Test of temperature-dependent RFP expression controlled by artificial synthetic RNA thermometers. (a) RNATs structure modeled using the RNAfold web server and fluorescence intensity assay. RNAT0 is the control without RNA thermometer. Raw cytometry data for (b) RNAT0, (c) RNAT3, and (d) RNAT12.

metabolic efficiency.21 In this case, the quorum-regulating system programs altruistic cell death by sensing a certain temperature (T0) and cell density (D0) in a process of dynamical feedback regulation, which may be coupled with the ease of release of biological heat and release of public goods that provide direct or indirect benefits to the survivors. Finally, through the synergistic effect of the two systems at both the cell and community levels, the cells can adapt to fluctuating high-temperature stress. Therefore, if used in industry, the chassis with IMHeRE could provide a fermentation process that is less dependent on cooling systems and reduces the production cost of bioconversion. At the same time, the cells can thrive under the expanded conditions in a manner similar to their behavior at their original optimum

temperature. Therefore, the activity of enzymes in the cell could be increased, and the efficiency of microbial metabolism could be improved as well.

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RESULTS AND DISCUSSION Thermotolerant Parts Customized for the Desired Strain. The HSPs play a critical role in protection from the cellular damage associated with elevated temperatures.23 Although the HSP expression level rises rapidly in the heat shock response,24 the ability of endogenous HSPs to shelter cells is temporarily available7 and rather limited. In addition, overexpression of the native HSPs does not help the cells to thrive under harsh conditions (high temperature).25 Thus, heterologous and more efficient HSPs are desperately needed. C

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Figure 3. Verification of the customized thermotolerant system. (a) Device combined with groES and thiF controlled under RNAT3 and RNAT12, respectively. (b) Verification of heat tolerance by measuring the OD600 at 37, 40, and 43 °C for 24 h. (c) Counted cell viability by dilution and spot titration of control and engineered strains cultured at high temperature for 24 h. Triangles indicate 10-fold serial dilutions of spotted cells. The control is the BL21 (DE3) strain with an empty pSB1A3 plasmid. The wild type is the control without any plasmid.

protein ThiF28 was most effective at 46 °C and second most effective at 43 °C, indicating that cells under different degrees of heat stress might require different forms of aid. Thermotolerant System for Hierarchal Heat Resistance. HSPs are essential for the adaptation of cells to the high temperature, but they are redundant at normal temperature. Under normal condition, the overexpression of HSPs can be a burden for cells and cause the waste of energy and substrate, which will significantly hamper production process and harm cells.29 Therefore, the control will be needed to reduce the side effect. In nature, bacteria use complex strategies to control the expression of these genes in response to ambient temperature changes.30 Many genes that encode heat shock proteins and virulence factors are regulated by temperature-sensing RNA sequences known as RNA thermometers (RNATs) that respond to temperature changes quickly, precisely, and controllably. The RNATs are located in the 5′untranslated region of the mRNA and fold into a secondary structure to influence translational efficiency through the shedding of the ribosome binding site (RBS) at low temperature. Once the temperature increases to a certain value, RNATs will release RBS to express the blocked genes. Most natural RNATs fold into rather complex secondary structures and have been suggested to undergo gradual conformational changes in response to changes in temperature.31 Also there are not enough available RNA thermometers that were sensitized to different high temperatures. Therefore, smaller, more diverse and controllable synthetic RNATs must be designed and constructed for controlling the expression of heat shock proteins at different high temperatures. The design of synthetic RNA thermometers is based on the assumption that the thermometers function by the proposed simple RNA-melting mechanism.38 According to this assumption, a modular design of simple synthetic thermometers was designed with a single stem-loop structure. The structure of the RNATs includes RBS (5′-AAGGAG-3′) sequence, a complementary anti-RBS

Previous efforts to improve the heat resistance of cells only focused on a single heat-resistant gene, i.e., irrE gene26,27 or a single system (GroeSL system10), which can only improve the heat-resistant characteristics for specific strain. In contrast to the previously reported research, in this study, the biocompatibility between the heat-resistant device and the microbial system was considered in constructing the thermotolerant system, with the aim of adaptively customizing the thermotolerance for E. coli. To customize the thermotolerant system for different target hosts, a HSP library was constructed that included many types of HSPs mined from numbers of thermotolerant species. The heat-resistant proteins from Thermoanaerobacter tengcongensis MB4 perform well in E. coli.16 Therefore, the ubiquitous heat-resistant genes, i.e., GroESL system, DnaKJ system, transcription factors, and ubiquitin-like proteins, were mined from the sequenced genome of T. tengcongensis MB4. Homologies of HSPs between T. tengcongensis MB4 and E. coli were compared using bioinformatics. Sixteen heat-resistant genes that exit in both strains and show high homologies were chosen as candidates and expressed under the control of the strong constituent promoter Pgapa (Supplementary Table S1), showing that half of the heterologous proteins were well expressed in E. coli (Supplementary Figure S1). Proteins that did not express normally in E. coli were eliminated. The engineered E. coli strains with the remaining candidates were tested by culturing the cells at selected high temperatures (40 °C, 43 and 46 °C). The biomass of the strains with HSPs was approximately 1- to 2-fold higher than that of the wild type at high temperature (Supplementary Figure S2), and the cell viability of the engineered strains was also higher than that of the control (Supplementary Figure S3). Additionally, heterologous HSPs with different functions benefitted cell growth at different high temperatures. By comparing all the data in Supplementary Figure S3, we can see the GroES conferred the best heat resistance on E. coli at both 40 and 43 °C, and the ubiquitin-like D

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Figure 4. Temperature-sensitive and cell-density-sensitive ANDGATE design and characterization. (a) Design of the ANDGATE module used in the PCD progress. The ANDGATE was constructed with the pluxR promoter and RNAT12, which were used as the two inputs to drive the transcription of mazF LuxI synthesizes AHL (3OC6HSL) as a signal of cell density, which binds to the activator (LuxR) and activates the pluxR promoter. The RNAT12 is an RNA thermometer that senses 43 °C. J23102 is a strong constitutive promoter from iGEM Registry. (b) Truth table for the ANDGATE; D indicates cell density; T denotes temperature. (c,e) Mathematical simulation according to the truth table for the ANDGATE. (d,f) Experimental verification of the ANDGATE according to the truth table. The control is the BL21(DE3) strain with an empty pSB1A3 plasmid as negative control. QS-PCD is a kind of positive control without RNATs, which can only program cell death through quorum-sensing system.

(ARBS) sequence and the circle sequence. The RNATs needed be constructed as BioBrick. Because these constructs would produce a nick between two assembly parts using the 3A assembly method (http://parts.igem.org/), the RNATs that we modeled contained two nicks. Then, through changing stem length, mismatches in the stem and the size of the loop, a series of RNATs with different minimum free energies was designed

through the RNAfold Web Server (http://rna.tbi.univie.ac.at/ cgi-bin/RNAfold.cgi) (Supplementary Table S2). To test a variety of different structures, a reporter gene cassette was constructed formed by a strong constitutive promoter (BBa_J23119, the standard biological part from iGEM Registry), the RNATs, and red fluorescent protein (RFP). All of the engineered strains were grown at four different E

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ACS Synthetic Biology physiological temperatures of 33, 37, 40, and 43 °C followed by measurement via flow cytometry (FCM). The characteristics of the RNATs reflected by the experiments were in accordance with the predicted opening order together with temperature elevation through the structure and minimum free energy. The opening level of the constructs increased with the increase in temperature. The results showed that the RFP controlled with RNATs all showed slight background expression at low temperature. The fluorescence intensity of RFP controlled with RNAT3 and RNAT12 multiplied at 40 and 43 °C, respectively (Figure 2), and the corresponding amount of expressed protein was also investigated via SDS-PAGE, as shown in Supplementary Figure S5. The RNAT8, RNAT9, RNAT10, and RNAT11 display their activities at temperatures greater than 46 °C (Supplementary Figure S4). Next, to achieve hierarchical heat resistance, a more efficient device was constructed through combined expression of groES and thiF controlled under RNAT3 and RNAT12, respectively (Figure 3a). The expression level of genes controlled with the RNATs increased with the increase in temperature. Therefore, when the temperature reached 40 °C, a small amount of ThiF would function at an imminent higher temperature (43 °C), which prepared cells for heat shock earlier and adapted them more effectively for the heat stress. After measuring the OD600 at 37, 40, and 43 °C, we found that the strain with the engineered device grew better at every investigated temperature compared with the control (Figure 3b), thus demonstrating that the optimal growth temperature of the engineered E. coli with this device was extended to 40 °C. The advantage of the engineered strain was reflected more significantly at 43 °C in the aspects of growth (OD600) (Figure 3b) and cell viability (Figure 3c). The GroES is a type of molecular chaperone that aids denatured proteins in refolding. The ThiF is a member of the ubiquitin-like proteins that works to clear misfolded and irreversibly aggregated proteins. RNATs render the entire system seamless between two high temperatures to achieve hierarchical heat resistance, which is the bridge to contact two different heat shock proteins. Thus, the cooperation of these two helpers in a hierarchical manner could lead to synergistic effects and improved behavior as a whole, which would benefit the cells to a greater extent than each HSP alone, and thus, the effect would be more obvious at higher temperature. On the other hand, RNATs were innovatively used to control the expression of HSPs at the right time for an economic system that avoids waste of matter and energy. Quorum Regulating System for Altruistic Cell Death. In a natural system, bacteria control their programmed death according to many types of damage induced by stress, but the elements that are involved in the death pathway are unclear.13 Indeed, under heat stress, two factors are primarily involved in programmed death. The main and most direct factor associated with heat stress is temperature, and thus, temperature-sensitive PCD could be constructed using the RNATs. The other factor is cell density, which is mainly responsible for the heat stress in fermentation because as the cell density increases, more heat will be released. In the literature, use of a quorum-sensing system to program cell death has been well studied.13,32 The quorum-sensing system contains two functional proteins, i.e., LuxI and LuxR, which are derived from the marine bacterium Vibrio fischeri.33,34 The LuxI protein synthesizes a small and diffusible acylhomoserine lactone (AHL) signaling molecule. The AHL accumulates both in the medium and inside the cells as the cell

density increases. At sufficiently high concentrations, AHL binds and activates the LuxR transcriptional regulator, which in turn induces transcription of the gene under the control of a promoter (pluxR35). The pluxR is related to the quantity of AHL-LuxR complex, which makes it convenient for setting the required cell density via regulation of the AHL synthesis.36 Therefore, in this study, the quorum-sensing system and the RNATs were combined together to design a temperaturesensitive and cell-density-sensitive ANDGATE, which is the main component of the quorum-regulating system for regulating cell death (Figure 4a). Cells can grow freely in the fermentation process under normal conditions; however, a high cell density will increase the temperature. When the temperature increases to a certain degree, the PCD acts to decrease the cell density to a safe level as set by the quorum-sensing system. In industry, the PCD regulation process should not be too drastic or it could disturb the fermentation. Therefore, the MazEF system, which is a safe, moderate, and controllable system, was constructed in the quorum-regulating system. The mazF was controlled strictly by pluxR(BBa_R0062) and RNAT12, and the cell used the native MazE37 to inhibit the small and leaky MazF at normal temperatures (37 °C). A mathematical model from the transcription and translation perspective was developed to simulate the quorum-regulating system (the simulation process can be found in Supporting Information). The simulated results that illustrate the working principle of the ANDGATE are shown in Figure 4(c,e). The results showed that the quorum-regulating system can function well as an ANDGATE according to the truth table. The corresponding experimental results are shown in Figure 4(d,f). The trends of the experiment results were generally in accordance with the simulation results. QS-PCD-ANDGATE was the engineered strain with quorum-regulating system that can sense temperature and cell density. QS-PCD was the positive control without RNATs, which can only program cell death through a quorum-sensing system. When the temperature was 43 °C, RNAT was in complete open conformation and had no inhibition effect on the translation of MazF, so QSPCD-ANDGATE and QS-PCD would function in a similar trend. The negative control contained one empty plasmid. In Figure 4d, from 18 h to 21 h, both cell density (D) and the temperature (T) were at the logic lever “1”, so the output of the ANDGATE went to a logic level “1” (effective). The cell density of the engineered strain (QS-PCD-ANDGATE) decreased from the control level to the QS-PCD level. The cell density of control and QS-PCD also showed a slight drop due to the heat stress. However, compared with QS-PCDANDGATE, the changes of cell density caused by heat stress were negligible. When temperature was changed to 37 °C, the temperature was at the logic lever “0”, and cell density was still at logic level “1”, so the ANDGATE returned to a logic level “0” (ineffective). QS-PCD-ANDGATE can recover to the same density with control, since the RNAT12 was off. In Figure 4f, cell density and temperature were both at logic level “0” from 0 h to 8 h, so the output was at logic level “0” (ineffective). The OD600 for all three strains were the same. Along with cell growth, the cell density reached level “1”, so the QS-PCDANDGATE is activated and maintained a similar cell density as QS-PCD. However, when the temperature was down to normal after the long-term heat stress, QS-PCD-ANDGATE can regrow faster than QS-PCD due to the inactivation of ANDGATE, Cells with the quorum-regulating system were able to sensitively respond to the temperature and cell density. F

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Figure 5. (a) Synergistic thermotolerant effect of IMHeRE. The control is the BL21(DE3) strain with two empty plasmids (pSB1A3; pSB3C5) as negative control. Control-QS-PCD-ANDGATE is the strain with pSB1A3 empty plasmid and quorum-regulating system. (b) Lysine production by engineered E. coli with IMHeRE at 40 °C. K-Control are the negative control with two empty plasmids (pSB1A3; pSB3C5).

to valuable chemicals (i.e., amino acid, xylitol, and ethanol)39 through enzymatic hydrolysis or simultaneous saccharification and fermentation (SSF).40 Saccharification enzymes (60 °C) have optimal temperatures that are much higher than that of fermentation of E. coli at 37 °C, and therefore, SSF and the fermentation process should be performed at the maximum possible temperature depending on the applied strain. Therefore, high-temperature fermentation provides a number of potential advantages in terms of cost and yield, which are all based on improving the heat-resistance of E. coli. In this research, the IMHeRE was used to improve the efficiency of the lysine production. The lysine-producing strain used in this study is a temperature-sensitive industrial E. coli strain. It can overproduce lysine at 37 °C (14.5 ± 2.2 g/L, 48 h, Biolector); however, when the strain undergo heat shock, the production will be effected seriously. When introducing IMHeRE into the industrial E. coli strain, although the yield at high temperature was a little lower than the yield of the control at 37 °C, the lysine production ability was surprisingly increased to approximately five times than that of the control within 48 h at 40 °C, as shown in Figure 5b. The hightemperature production capability have been enhanced drastically. In this case, to keep good production condition, we chose 40 °C as fermentation temperature to avoid quorumregulating system switching on all the time to affect cell growth. On the one hand, the production improvement is related to the enhanced growth at high temperature. The biomass of the engineered strain was increased about 10% compared with that of the control (Supplementary Figure S7). On the other hand, heat shock proteins help the lysine producing related metabolic enzymes keeping activity, which may be another important factor for the enhancement of the production at high temperature. In conclusion, when the thermotolerance of E. coli was enhanced via the IMHeRE, the production capacity was improved under high temperature. In this process, high temperature is no longer a stress but can accelerate production. At the same time, high-temperature fermentation may decrease the energy cost used for cooling the raw materials after SSF and sustaining the fermentation temperature. Perspective for Further Applications Using IMHeRE. The IMHeRE provides the cells with biological robustness through dynamic controllability, hierarchical organization, and functional redundancy, similar to those in man-made complex systems, i.e., an automatic flight control system in a modern

If the temperature did not reach the threshold value, the cells grew as well as the control did. In our model, the effect of high temperature on cell growth is not considered, and thus, after the heat stress, the cells grow as well as before the stress. In fact, the growth property of cells changed according to the heat stress time and temperature. After undergoing a long-term exposure under high-temperature stress (43 °C), the growth ability of control cells was disrupted, and they recovered quite slowly even when the temperature was returned to normal. However, the engineered strain controlled by the ANDGATE could recover and grow quite quickly once the temperature returned to normal (Figure 4f). Compared with the only quorum-sensing system that programs cell−cell communication and regulates killing,38 our quorum-regulating system was safer and more suitable for improvement of thermotolerance. The sensing temperature and cell density of the ANDGATE can be regulated conveniently according to practical needs. This work represents the first report of an artificial biological system sensitive to both temperature and cell density that programs cell death for improved heat resistance at the community level. Intelligent Microbial Heat Regulating Engine (IMHeRE). To ensure that cells acquire thermotolerance in multidimensional aspects, the intelligent microbial heatregulating engine (IMHeRE) was constructed by combining the thermotolerant system and the quorum-regulating system. By modeling heat stress with artificial variation of temperature, the IMHeRE achieved the expected performance intended to aid the cell in resisting heat damage. As shown in Figure 5a, with the aid of the thermotolerant system, the strain with IMHeRE grew better than the control when cells suffered heat stress (43 °C) initially (3−8 h) but did not reach the threshold of the set density. However, when the cell density increased to the threshold, the quorum-regulating system would function to program cell death and control cell density (10−22 h). The density of the strain with IMHeRE would keep more or less the same with the strain with only quorum-regulating system. In addition, after undergoing heat stress for 22 h, the recovery abilities of the strains were different in terms of initial recovery time. The density of the wild-type control did not change obviously. The strain with IMHeRE was able to regrow after 4 h when the temperature was shifted from 43 °C to back to 37 °C and at a rate faster than that of the strain with only the quorum-regulating system. Application of IMHeRE to High-Temperature Fermentation. The cellulose-enriched solid phase can be transformed G

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the negative control (RFP-free). Cells were assayed at a low flow rate until 30 000 total events were collected. Lysine Fermentation. Strains (industrial E. coli, K- E. coli) were cultured in 48-well plates by Biolector (m2p-labs, 1000 rpm) at 40 °C for 48 h. The medium was a given medium (glucose 40 g/L, ammonium sulfate 10 g/L, phosphoric acid 0.6 mL/L, sodium chloride 0.8 g/L, lycine 0.4 g/L, magnesium sulfate 1.2 g/L, and corn steep liquor 0.4 g/L). In the fermentation process, 5% aqueous ammonia was added every 2 h to adjust the pH to approximately 7.0 (depending on the pH test strips). Samples were taken every 6 h. The fermentation broth was centrifuged for 2 min at 12 000 rpm. The supernatant was diluted to less than 100 mg mL−1 with water, and the concentrations of lysine were measured using a SBA-40C biosensor (Institute of Biology, Shandong Academy of Sciences, China). All experiments were performed in triplicate, and differences between the mean values were considered.

airplane that contains well-understood robustness control mechanisms that maintain a stable flight path regardless of perturbations in the atmospheric conditions.41 For this reason, we refer to our system as an “engine”, i.e., an intelligent microbial heat-regulating engine. In the future, other system similar to IMHeRE will be constructed for other different chassis. Along with the richness of the HSPs library, the thermotolerace of strains will be extended to a higher level. Moreover, the customized thermotolerant systems will benefit different metabolic pathways according to the properties of the pathway and the needs of the production process. Additionally, microbes can adapt well to the fluctuating temperature, and the engine can thus also be applied in nonisothermic temperature fermentation in order to avoid the negative effects of changing temperature. In summary, this study shed new light on the development of thermotolerant bacteria for industrial application and also will contribute to increased productivity and enhanced energy conservation.

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METHODS Plasmid Construction. Plasmid construction and DNA manipulations were performed following standard molecular biology techniques. The HSPs genes were cloned from the genome of T. tengcongensis MB4. The promoters used in this study were taken from two resources: one from our lab and the other from the iGEM Distribution Kit. All of the genes in this study are listed in Supplementary Table S3. The groES, thiF, and RNATs were constructed in BioBrick following the BioBrick standard (http://partsregistry.org). Plasmids pSB1C3, pSB1K3, and pSC1A3 were used in the 3A assembly. In the HSP screening and verification experiments, the HSPs were controlled with the Pgapa promoter and constructed in the pET28a (+). Strains and Growth Conditions. Plasmid cloning work was performed in E. coli (TOP10). The characterization work of the circuit constructs was performed in E. coli BL21(DE3) unless otherwise indicated. All characterization experiments were performed in standard LB medium. The antibiotic concentrations used were 50 μg mL−1 for kanamycin, 25 μg mL−1 for chloramphenicol and 100 μg mL−1 for ampicillin. Normally, cells inoculated from single colonies on freshly streaked plates were grown overnight in 50 mL of LB in a 100 mL flask at 37 °C with shaking (170 rpm.). For hightemperature fermentation, the overnight cultures were diluted into 300 mL of LB medium at a 1:100 ratio at 37 °C. When the OD600 of all bottles reached approximately 1.0, 300 mL of cell culture was dispensed into three 250 mL flasks, which were separately placed at a constant temperature of 37, 40, and 43 °C. After 20 h, bacterial growth reached a stable state, and samples were collected to determine the OD600 values for comparison of the growth of different strains at high temperature. All experiments were performed in triplicate, and differences between the mean values were considered significant at p < 0.05. Fluorescence Assay. To measure the RNATs function, an overnight culture was diluted 100-fold into fresh LB culture. Next, the cell culture was dispensed into several 12 mL culture tubes that were separately placed at constant temperatures of 33, 37, 40, and 43 °C to grow for 12 h. The FCM assays were performed using a Becton−Dickinson FACSCalibur flow cytometer with 584 nm excitation and 607 nm emission. Culture cells were pelleted and resuspended in PBS filtered with a 0.22-μm membrane. The flow cytometer was tuned with

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.5b00158. Supplementary Figures S1−S7, supplementary Tables S1−S4, and supplementary mathematical modeling (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: +86 10 68913171. Author Contributions

H.J. and C.L. designed the experiments. H.J., X.S., and H.S. performed experiments and analyzed data. H.J., C.L., Y.Q., and X.F. built the mathematical model. H.J., X.F., and C.L. wrote the paper, and all authors reviewed the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge funding support from the National Natural Science Foundation of China (No. 21376028, No. 21425624, No. 21476026) and the Major State Basic Research Development Program of China (973 Program) (No. 2013CB733900).

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ABBREVIATIONS IMHeRE, intelligent microbial heat-regulating engine; PCD, programmed cell death; HSPs, heat shock proteins; RNATs, RNA thermometers; RBS, ribosome binding site; FCM, flow cytometry; AHL, acyl-homoserine lactone; SSF, simultaneous saccharification and fermentation

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REFERENCES

(1) Zhu, L., Zhu, Y., Zhang, Y., and Li, Y. (2012) Engineering the robustness of industrial microbes through synthetic biology. Trends Microbiol. 20, 94−101. (2) Torija, M. J., Rozes, N., Poblet, M., Guillamón, J. M., and Mas, A. (2003) Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae. Int. J. Food Microbiol. 80, 47−53. (3) Davidson, J. F., Whyte, B., Bissinger, P. H., and Schiestl, R. H. (1996) Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 93, 5116−5121.

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DOI: 10.1021/acssynbio.5b00158 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acssynbio.5b00158 ACS Synth. Biol. XXXX, XXX, XXX−XXX