Synthetic Gene Circuits Enable Escherichia coli To Use Endogenous

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Synthetic gene circuits enable Escherichia coli to use endogenous H2S as a signaling molecule for quorum sensing Huaiwei Liu, Kaili Fan, Huanjie Li, Qingda Wang, Yunyun Yang, Kai Li, Yongzhen Xia, and Luying Xun ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00210 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Synthetic Biology

Synthetic gene circuits enable Escherichia coli to use endogenous H2S as a signaling molecule for quorum sensing Huaiwei Liu1, Kaili Fan1, Huanjie Li1, Qingda Wang1, Yunyun Yang1, Kai Li1, Yongzhen Xia1*, Luying Xun1,2* 1State

Key Laboratory of Microbial Technology, Shandong University, Qingdao,

266237, People’s Republic of China. 2School

of Molecular Biosciences, Washington State University, Pullman, WA,

99164-7520, USA.

ACS Paragon Plus Environment

ACS Synthetic Biology 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

Abstract Microorganisms often use specific autoinducers other than common metabolites for quorum sensing (QS). Herein, we demonstrated that Escherichia coli produced sulfide (H2S, HS-, and S2-) with the concentrations proportionally correlated to its cell density. We then designed synthetic gene circuits that used H2S as an autoinducer for quorum sensing. A sulfide:quinone oxidoreductase converted diffusible H2S to indiffusible hydrogen polysulfide (HSnH, n≥2), and a gene regulator CstR sensed the latter to turn on the gene expression. We constructed three element libraries, with which 24 different circuits could be assembled for adjustable sensitivity to cell density. The H2S-mediated gene circuits endowed E. coli cells within the same batch or microcolony with highly synchronous behaviors. Using them we successfully constructed cell factories capable of autonomous switch from growth phase to production phase. Thus, these circuits provide a new tool-kit for metabolic engineering and synthetic biology.

Keywords：Hydrogen sulfide; quorum sensing; Escherichia coli; synthetic gene circuit; dynamic metabolic engineering.


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Dynamic metabolic engineering, involving in dynamically controlling of a metabolic pathway with a metabolite sensing unit, can avoid buildup of undesired intermediates and redistribute the metabolic fluxes automatically (1,2). It has become a new strategy for constructing smart, responsive cell factories (3,4). A key factor in dynamic metabolic engineering is finding/constructing appropriate sensor systems. Consequently, various metabolite sensing circuits have been constructed, including the acetyl-phosphate (AcP), acetyl-CoA, and melonyl-CoA sensing circuits (5-7). Growth sensing circuits are also developed by using quorum sensing (QS)-based circuits (6, 8, 9). Naturally, bacteria use QS systems to communicate and collectively modify behaviors in response to changes in the cell density and species composition of the surrounding microbial community (10). The signaling molecular for QS (autoinducer) is usually not a common metabolite, but a specific molecule produced and released by bacteria, such as acyl-homoserine lactones (AHL) in gram negative bacteria. The membrane of gram negative bacteria is usually permeable to AHL, which can enter cell and directly interact with a cognate transcription factor (TF) (11). After binding, the sensor turns on its cognate promoter—the actuator, then QS-dependent genes are expressed11. For gram positive bacteria, the autoinducer usually does not enter cells, which necessitates the existence of a converter, a transmembrane protein that converts the extracellular signal to an intracellular signal detectable by the sensor (12). The most significant advantage of QS is that it can quickly synchronize all cells of one community when the autoinducer accumulates above a certain level. Through



applying this system in metabolic pathway control, one can construct autonomous cell factories that dynamically turn on production pathways at certain growth phases (13). Nature is enriched with an incredible diversity of signal compounds and sensors that are currently underexploited. Hydrogen sulfide (H2S) is a small molecule produced by many bacteria (14,15). It can shuttle freely between bacterial cells. Mammalian cells and some bacteria can oxidize H2S to hydrogen polysulfide (HSnH, n≥2) via sulfide:quinone oxidoreductase (SQR) (16,17). Unlike H2S, HSnH stays within the cell (18,19). Recently, a few HSnH-sensing TFs have been identified from bacteria (20). Herein, using these natural elements, we developed a library of H2Smediated gene circuits in Escherichia coli, which can synchronize E. coli cells via H2S. The circuits showed valuable applications in cell factory construction, providing a new toolkit for dynamic metabolic engineering.

Results H2S is a metabolite proportional to E. coli cell density Although E. coli producing H2S has been reported long time ago (21), the production dynamics and titers are still unclear. We cultured E. coli BL21 in LB medium, and analyzed its H2S production. It produced and excreted H2S into the culture. The production started at the mid-log phase and reached the maximum (~15 μM) at the stationary phase (Fig. 1A). H2S production of other strains including E. coli W3110, E. coli MG1655, and E. coli Nissle 1917 were also tested and no significant difference was observed (data not shown). Since H2S tends to vaporize at


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pH5000 cells was shown.

Discussion Cell-to-cell variability in genetically identical cells is attributed to fluctuations in intracellular components and the stochastic nature of gene expression (25-27). When QS behaviors are required, such as simultaneously expressing virulence factors, cells need a signaling molecule to conquer this variability and synchronize all members (28,29). In this study, we turned H2S into a cell synchronizing signal via synthetic gene circuits. We found that in E. coli, H2S production is cell density dependent. Although disrupting some genes involved in H2S metabolism could affect its



production, the dependency on cell density is not altered. E. coli endogenous catalase may oxidize part of H2S to HSnH (30); however, the amount is too low to cause a dramatic activity increase of synthetic actuator. After introducing an exogenous H2S oxidation enzyme SQR, HSnH amount reaches to a critical level for turning the actuator ON. Through manipulating the host, the converter, and the actuator, synthetic gene circuits with different dynamics can be obtained. Our H2S-mediated gene circuits provide a new tool-kit for cell factory construction. They can be directly used in pathway control for realizing the autonomous two-phase fermentation, also can be combined with other regulating elements. When coupled with the T7 RNA polymerase-T7 promoter system for protein overexpression, it is more efficient, tunable, and cost-efficient than the original system requiring IPTG induction. It is noteworthy that IPTG is toxic to E. coli, whereas the H2S-mediated system is not, evidenced by the growth difference of xylonic acid producing strains harboring each system (Fig. 6A). As an artificial QS system, the H2S-mediated gene circuit may be more advantageous than native QS because it converts the self-produced H2S to HSnH and keeps the latter within the bacteria. The property may be critical for the QS behavior of strains that form microcolonies and/or biofilms in harsh environments.

Methods Strains, plasmids, and chemicals


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Strains, human cells, and plasmids are listed in Table 1 and Supporting Table S2. E. coli strains were grown in Lysogeny broth (LB) or M9 medium. Ampicillin or kanamycin (50 μg/mL) was added when required. SSP4 (3’,6’-Di(Othiosalicyl)fluorecein) was purchased from Dojindo Molecular Technologies. HSnH (n≥2) was prepared by following Kamyshny & Alexey’s method (31). The fisR gene was amplified from Cupriavidus pinatubonensis JMP134. The bigR and cstR genes were synthesized by Genewiz (Shanghai) Company. Plasmid construction was performed by using the TEDA method (32).

Table 1. Strains, cells, and plasmids used in this study Strains E. coli BL21(DE3) E. coli BL21 E. coli W3110 E. coli MG1655 E. coli DH5α E. coli ∆sseA E. coli ∆cysK E. coli ∆cysKM E. coli ∆xylA ∆yagE∆yjhH Plasmidsa pTrcHis2A pTrcHis30a pTrcHis2A-Xdh aPlasmids

Characteristics/Purposes For testing IPTG based T7 promoter For circuits construction For circuits construction For circuits construction For plasmid construction For circuits construction For circuits construction For circuits construction For xylonic acid cell factory construction

References Lab stock Lab stock Lab stock Lab stock Lab stock Lab stock Lab stock Lab stock Lab stock

Backbone of H2S mediated QS like circuits For testing IPTG based T7 promoter For xylonic acid cell factory construction

Lab stock Lab stock Lab stock

constructed for TF testing, sensor-actuators, and circuits, are listed in Supporting Table S2.

H2S and HSnH analysis H2S and HSnH were analyzed by following a reported protocol (33). Briefly, samples were derivatized with mBBr then analyzed by HPLC (LC-20A, Shimadzu) equipped with a fluorescence detector (RF-10AXL, Shimadzu) and a C18 reverse



phase column (VP-ODS, 150×4 mm, Shimadzu). The column was eluted with gradients of solvent and the flow rate was set to 0.75 mL/min. For detection, the excitation wavelength was set to 340 nm and emission wavelength was set to 450 nm. HSnH was also analyzed by cyanolysis (34) and SSP4 methods. For SSP4 method, cells were washed with and resuspended in HEPES buffer (50 mM, pH 7.4), then 10 μM SSP4 and 0.5 mM CTAB were added. After incubating at 37°C for 15 min in the dark with gently shaking (125 rpm), reagents were washed off with the HEPES buffer. Fluorescence of reacted-cells was detected by flow cytometry. Fluorescence analysis Cell fluorescence from mKate was analyzed with Synergy H1 Microplate reader or BD AccuriTM C5 flow cytometer. When microplate reader was used, E. coli samples were first diluted to equal density then subjected to 588 nm excitation. Emission intensity at 633 nm was recorded and standardized to per OD600nm. For flow cytometry analysis, E. coli samples were diluted to OD600nm=0.1 and for each sample, >5000 cells were analyzed in FL3-A channel. The average fluorescence and CV value were recorded. Xylose and xylonic acid analysis Xylose and Xylonic acid were analyzed by using HPLC. The Bio-Rad Aminex HPX-87H column (300×7.8 mm) and RID-20A refractive index detector were used. 5 mM H2SO4 was used as the eluent and the flow rate was set to 0.4 mL/min. The column temperature was maintained at 55oC. Xylonic acid could not be accurately


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determined due to its peak partially overlaps with the xylose peak; therefore, xylonic acid concentration was corrected by using a hydroxamate method (35). Microfluidic cultivation CellASIC® ONIX Microfluidic System (Merck) was used. E. coli cells were cultivated in a B04A-02 microfluidic bacteria plate and imaged with IX83 inverted fluorescence microscope (Olympus). The Cellsens Dimension software was used for image analysis. The hypoxic condition (1% O2, 5% CO2) was set by following manufacturer's instruction. To maintain relatively static condition, the pressure for LB medium inlet was set to 0.25 psi, corresponding to a flow rate of 1–2 μL/h.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxx Supporting Figure S1. The relation between H2S production and growth for E. coli BL21 in LB and M9 media; Supporting Table S1. The sequences of PL and PR promoter libraries; Supporting Table S2. Plasmids newly constructed in this study. Author Information

Corresponding Author Yongzhen Xia: [email protected]; Tel. +86 532 58631572. Luying Xun: [email protected]; Tel. +1-509-335-2787. ORCID Huaiwei Liu: 0000-0002-0483-5318

Yongzhen Xia: 0000-0001-9950-1910 Luying Xun: 0000-0002-5770-9016



Author Contributions Luying Xun, Huaiwei Liu, and Yongzhen Xia conceived and designed the study; Huaiwei Liu, Kaili Fan, Huanjie Li, Kai Li and Qingda Wang performed the experiments; Yunyun Yang performed the computational work; Huaiwei Liu drafted the manuscript. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgments Funding Sources The work was financially supported by grants of National Key R&D Program of China (2018YFA0901200), the National Natural Science Foundation of China (31770093, 91751207). References 1. Anesiadis, N., Cluett, W.R., and Mahadevan, R. (2008) Dynamic metabolic engineering for increasing bioprocess productivity. Metab. Eng. 10, 255-266. 2. Brockman, I.M., and Prather, K.L. (2015) Dynamic metabolic engineering: New strategies for developing responsive cell factories. Biotechnol. J. 10, 1360-1369. 3. Lalwani, M.A., Zhao, E.M., and Avalos, J.L. (2018) Current and future modalities of dynamic control in metabolic engineering. Curr. Opin. Biotechnol. 52, 56-65. 4. Peng, Xu. (2018) Production of chemicals using dynamic control of metabolic fluxes. Curr. Opin. Biotechnol. 53, 12-19. 5. Farmer, W.R., and Liao, J.C. (2018) Improving lycopene production in Escherichia coli by engineering metabolic control. Nat. Biotechnol. 18, 533-537. 6. Soma, Y., Tsuruno, K., Wada, M., Yokota, A., and Hanai, T. (2014) Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch. Metab. Eng. 23, 175-184.


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Synthetic Gene Circuits Enable Escherichia coli To Use Endogenous

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