Autoinduced AND Gate Controls Metabolic Pathway Dynamically in

Dec 8, 2016 - Quorum sensing (QS) systems have been widely applied in biotechnology and synthetic biology that require coordinated, community-level ...
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Autoinduced AND Gate Controls Metabolic Pathway Dynamically in Response to Microbial Communities and Cell Physiological State Xinyuan He, Yan Chen, Quanfeng Liang,* and Qingsheng Qi State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China S Supporting Information *

ABSTRACT: Quorum sensing (QS) systems have been widely applied in biotechnology and synthetic biology that require coordinated, community-level behaviors. Meanwhile, the cell physiological state is another key parameter that affects metabolic pathway regulation. Here, we designed an autoinduced AND gate that responds to both microbial communities and the cell physiological state. A series of tunable QS systems in response to different cell densities were obtained through random mutagenesis of LuxR and optimization of the luxRI promoter; the corresponding suitable stationary phase sensing system was selected after monitoring the fluorescence process during cell growth. The application of the final synthetic device was demonstrated using the polyhydroxybutyrate (PHB) production system. The AND gate system increased PHB production by 1−2-fold in Escherichia coli. This synthetic logic gate is a tool for developing a general dynamic regulation system in metabolic engineering in response to complex signals, without using a specific sensor. KEYWORDS: dynamic control, AND gate, quorum sensing system, PrpoS, polyhydroxybutyrate

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appropriate time.14 A dynamic control system for a biosynthetic pathway requires a sensor that detects the pathway flux or pathway intermediate levels and a regulator that responds to the sensor and accordingly regulates enzyme expression. Several dynamic sensor−regulator systems have been developed on the basis of natural metabolite-sensing proteins.15,16 However, limited sensors can sense metabolites, making it particularly difficult to regulate heterologous metabolic pathways where no natural sensor is available.17 Quorum sensing (QS) systems offer an alternative approach that can sense the cell density and subsequently, by feedback, control the expression of desired pathways. This activity enables the cells to delay the induction of various genetic programs, which are more effective if coordinated at the population level.18 QS systems have been used in biotechnology and synthetic biology applications that require coordinated, community-level behaviors.19 Recently, Soma et al. constructed a synthetic lux system as a tunable cell density sensor−regulator for isopropanol production.20 Liu et al. developed an autonomous controller module through QS and successfully increased the production of 1,4-butanediol from a de novo designed pathway.21 Meanwhile, the cell physiological state (exponential growth phase and stationary phase) is another key parameter that affects metabolic pathway regulation. Using the sensors to regulate genes’ expression in response to the physiology state

icroorganisms have been used as cell factories for producing homologous and heterologous metabolic products, including central metabolic products and numerous secondary metabolites.1−3 However, constitutive expression or overexpression of pathway enzymes during cell growth competes with the biomass for substrates or directly inhibits cell growth, which reduces production efficiency.4 On the other hand, expression of pathway enzymes at too low a level also results in bottlenecks in the reaction pathway, limiting the product yield and titers. Strategies for optimizing engineered pathways include static controls, such as tuning the promoter strength,5,6 ribosome binding sites,7 or gene copy number,8,9 to enable a balanced pathway reaction flux and eliminate bottlenecks. Recently, novel optimization strategies, including the dynamic control of metabolic pathways where gene expression can be triggered on demand, were developed.10,11 Separation of the cell growth stage from the production stage, allowing cells to accumulate biomass before channeling resources for forming the desired chemicals (two-stage fermentation).12 Such a system would minimize the accumulation of pathway intermediates, thus reducing excess proteins.13,14 One common implementation of two-stage fermentation is growing cells (growth stage) to production stage (e.g., stationary phase, anaerobic phase, etc.) in which biosynthetic genes were induced. However, use of inducer adds cost and potential regulatory hurdles; thus, alternative approaches are required. Dynamic control strategies without inducer have been proposed to allow for two-stage fermentation and dynamic control of gene expression at the © XXXX American Chemical Society

Received: June 23, 2016 Published: December 8, 2016 A

DOI: 10.1021/acssynbio.6b00177 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Table 1. Characterizations of LuxR Mutantsa

creates an association between the cell cellular state and induction of the metabolic pathway. Synthetic stationary-phase and stress-induced promoters have been used as tools for finetuning the expression of recombinant proteins 22 and production of chemicals.17,23 It is worth noting that cell density does not always correspond to the cell physiological state in different environments. For example, cells grown in large bioreactors are markedly different from those under which the strains were originally constructed and optimized (i.e., in the uniform environment of a shaker flask or small fermenter).24,25 On the other hand, diverse target compounds’ fermentation may require the optimal induction of target metabolic pathways at different appropriate cell densities.20 Therefore, it is critical to develop a strategy that enables not only an autoregulated two-stage fermentation but also biomass sensing. Such a system can be achieved using an AND gate that controls dynamic metabolic pathways in response to microbial communities and the cell physiological state. To initiate the expression of a target gene, an inducible AND gate must simultaneously meet a combination of at least two intra- or intercellular conditions, which improves the accuracy and specificity of conditional gene expression.26,27 In this study, we developed and characterized an autoinduced AND gate controlled dynamic metabolic pathway in response to the cell density and stationary phase. To demonstrate the biotechnological potential of our synthetic device, it was applied to polyhydroxybutyrate (PHB) production.

LuxR mutant

nucleotides substitution

amino acids substitution

[3OC6HSL]50 (nM)

LuxR wt R1 R2 R3 R4

NA C149T A206T T111A, A342T C316T

NA S50F Y69F H37Q, K114N S106P

13.76 807.4 79.93 475.1 163.2

a

Overnight seeds were diluted 1:100 in M9 minimal medium supplemented with 0, 0.1, 1, 10, 100, 1000, and 10 000 nM 3OC6HSL. Optical cell densities and green florescence were detected after shaking in 96-well microassay plates for 12 h at 37 °C. Three replicates were performed, and data were fitted to Hill equations, with different dissociation constant or [3OC6HSL]50.

medium, whereas the mutants R1, R2, R3, and R4 required 807.4, 79.93, 475.1, and 163.2 nM 3OC6HSL, respectively. Considering the structure of the homology protein TraR,32,33 all mutation sites were supposed to be located at 3OC6HSLbinding regions rather than at DNA-binding regions, which guaranteed a decreased 3OC6HSL-binding ability without compromising DNA affinity. Moreover, these mutations were not directly engaged in 3OC6HSL binding and were less likely to completely disable the binding ability of LuxRs.32 We subsequently constructed variant promoters of LuxRI to reduce LuxR protein and N-acyl homoserine lactone (AHL) production. Soma et al. previously designed a synthetic promoter, PluxlacO, by adding various concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG)20 to control the LuxI expression level, whereas Wang et al. have attempted to regulate the LuxR expression level by employing wellcharacterized constitutive promoters.34 In this study, relatively weaker promoters, namely Plac uv5, PJ23101, PJ23104, and PJ23108 (http://parts.igem.org/Main_Page), were employed to substitute Ptet and reduce the LuxR expression level and AHL production. The resulting strains QSL0, QS10, QS40, QS80, and QST0 were characterized in 96-well microassay plates using a Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) (Figure 1).

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RESULTS AND DISCUSSION Construction of a Series of Tunable QS Systems in Response to Different Cell Densities. The cell density thresholds or cell densities for QS of almost all synthetic or native QS systems were fixed at a rather low value.28 Microbial fermentation requires the optimal induction of target metabolic pathways at appropriate cell densities, which may differ among diverse target compounds. Rewiring of the existing QS circuits19 enables modifications of the cellular response to autoinducers, with the final response being graded: thresholdlike or bistable. In a series of studies, directed evolution has been used to enhance the activities of the LuxR transcriptional activator29 and alter the specificity of LuxR for its cognate signal molecule to allow the creation of unique LuxR proteins that can be used for constructing synthetic circuits with minimal cross talk.30,31 Here, we engineered the LuxI-LuxR QS system of Vibrio f ischeri toward the low sensitivity to cell density by using two strategies: directed evolution for LuxR optimization and variant promoter construction for LuxRI expression. First, error-prone polymerase chain reaction (PCR)-based directed evolution was used to reduce the activities of the LuxR transcriptional activator. Positive mutants were identified through fluorescence screening of cells containing the mutant LuxR proteins. Screening of 3000 colonies identified eight putative positive mutants. Subsequent sequencing and fluorescence quantification of the mutant responses confirmed decreased responses of four of the eight putative positive mutants compared with wild-type LuxR. The characterization of LuxR mutants is shown in Supplementary Figure 1. The mutants showed one or two amino acid substitution and lower sensitivities to N-(β-ketocaproyl)-L-homoserine lactone (3OC6HSL), with their [3OC6HSL]50 values varying between 58- and 5-fold more than those of the respective wild-type proteins (Table 1). To achieve a half-saturated expression of Plux, wild-type LuxR required 13.74 nM 3OC6HSL in M9

Figure 1. In vivo characterization of luxRI promoters. Strains were characterized in M9 minimal medium by using 96-well microassay plates. The strains were inoculated at OD600 = 0.05. OD600 was normalized, and GFP was standardized using OD. Dotted lines represent GFP expression at 2000 and 8000 au. Each dot indicates at least four replicates. B

DOI: 10.1021/acssynbio.6b00177 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Table 2. Plasmids Used in This Study plasmid

relevant characteristics

pLuxRI pQST00 pQST01 pQST02 pQST03 pQST04 pQST0 pQSL0 pQS10 pQS40 pQS80 pBW213ara-hrpS pBW313 lux-hrpR pBW400 hpL-gfp p3T0 p3T1 p3T2 p3T4 p3L0 p310 p2B p2A p2C p4PHB pQKZ103

CmR Plac/ara‑1-luxR-luxI ori ColE1 CmR, pMB1, sfGFP under expression of Plux, LuxR under expression of Ptet pQST00 derivative, LuxR-S50F pQST00 derivative, LuxR-Y69F pQST00 derivative, LuxR-H37Q, K114N pQST00 derivative, LuxR-S106P pQST00, luxI Ptet substituted by Plac uv5 in pQST0 Ptet substituted by PJ23101 in pQST0 PJ23101 substituted by PJ23104 in pQS10 PJ23101 substituted by PJ23108 in pQS10 CmR, araC, hrpS under expression of PBAD KanR, LuxR under expression of Ptet, hrpR under expression of Plux AmpR, GFP under expression of PhrpL pBW313 lux-hrpR derivative, LuxRI under expression of Ptet p3T0 derivative, LuxR-S50F p3T0 derivative, LuxR-Y71F p3T0 derivative, LuxR-S106P Ptet substituted by Plac uv5 in p3T0 Ptet substituted by PJ23101 in p3T0 Ptet+RBS33 replaced by rpoSP1+RBS+codon 8 in pBW213ara-hrpS Ptet+RBS33 replaced by rpoSP1+RBS in pBW213ara-hrpS Ptet+RBS33 replaced by Plac uv5+RBS+codon 8 in pBW213ara-hrpS pBW400 hpL-gfp derivative, gf pmut2b substituted by phbCAB AmpR, phbCAB from R. eutropha

ref 49 this this this this this this this this this this 26 26 26 this this this this this this this this this this 52

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study study study study study study study study study study

As a control, G00 was supplemented with 1.3 mM Ara and 100 nM 3OC6HSL [G00 (+)], and its growth was largely affected (Supplementary Figure 3). To fully activate the expression of the AND gate, these inducers were added at the beginning of inoculation (Supplementary Figure 4). As shown in Figure 2, marked fluorescence (GFP/OD600 = 200) was observed in G00 (+) at OD600 = 0.51, followed by G01, G06, G03, G07, and G05 at OD600 = 1.58, 2.11, 2.27, 2.68, and 3.63, respectively. G02 failed to reach the expression level; moreover, G01, G00 (+), and G03 reached GFP/OD600 = 600 au at OD600 = 3.89, 4.1, and 4.3, respectively. However, G06 and G07 could only reach GFP/OD600 = 400 au at OD600 = 3.46 and 4.16, respectively (Figure 2). These results indicated that a series of QS systems in the AND gate exhibited decreased sensitivity to cell density at different levels. Selection of a Suitable Stationary Phase Sensing System in Response to the Cell Physiological State. Stationary phase sensing systems is composed mainly of the stress-induced promoters which can only be markedly activated at the stationary phase.35,36 A previous study reported that physiological stress promotes both reduced growth and PrpoS activation, and thus, PrpoS can increase gene transcription under unfavorable conditions and maximize gene expression in the stationary phase.37−39 For selecting a suitable stress-induced promoter in the AND gate, we employed 3 forms of PrpoS, namely rpoSp1 + RBS + codon 8, rpoSp1 + RBS, and Plac uv5 + RBS + codon 8, considering their expression level and stressinduced ability at the stationary phase.35 These DNA fragments were assembled into pBW213ara-hrpS, and the successfully constructed plasmids were subsequently transformed into Escherichia coli Top10 containing pBW313lux-hrpR and pBW400hrpL-gfp. The resulting strains, G10, G20, and G30, were characterized in the M9 medium supplemented with 100 nM 3OC6HSL

As expected, all strains with weaker promoters showed decreased fluorescent responses than did the control strain QST0. All strains showed a similar growth curve (Supplementary Figure 2). QST0 with the strongest promoter exhibited the highest green fluorescent protein (GFP) expression, followed by QS40 and QSl0, which contained the relatively weaker promoters PJ23104 and Plac uv5, respectively. The GFP expression of QS10 and QS80 was lower. On inoculating the cells at OD600 = 0.05, the GFP expression of QST0, QSL0, and QS40 was more than 2000 au, whereas QS10 and QS80 required OD600 = 0.48 and 1.23, respectively. Moreover, QST0 was the first to reach the expression level of GFP/OD600 = 8000 au at OD600 = 1.5, followed by QS40 and QSL0 at OD600 = 1.76 and 1.89, respectively. However, QS80 and QS10 failed to reach GFP/OD600 = 8000 au before OD600 = 3.0 (Figure 1). These results show a series of autoinduced sensing systems in response to different cell densities were obtained by using two strategies. Characterization of Different Cell Density Sensing Systems in the AND Gate. We constructed a series of sensing systems with different sensitivities and introduced them to the AND gate. The autoinduced AND gate is based on an inducible AND gate that includes 2 transcriptional coactivators, HrpR and HrpS, as the inputs and their controlled promoter, PhrpL, as the output.26 We cloned the AHL-synthesizing gene luxI from pQST0 and integrated it into the plasmids pBW313lux-hrpR and p3T0 (Table 2). Furthermore, the LuxR variants (R1, R2, and R4) and promoters (Plac uv5 and PJ23101) were cloned into pBW313lux-hrpR, generating the strains G01, G02, G03, G05, G06, and G07 (Supplementary Table 1). To examine the expression level of the autoinduced QS systems in the AND gate, these strains were half autoinduced AND gates that output GFP following excess Larabinose (Ara) addition (Figure 2). C

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Figure 3. Characterization of suitable stationary phase sensing systems in the AND gate. (a) Schematic illustration of a variety of stationary phase sensing systems in the AND gate. HrpR is produced by the excess addition of 3OC6HSL, and PBAD was substituted by PrpoS. (b) Characterization of three forms of stationary phase sensing systems in the AND gate. Optimized PrpoS fragments, namely rpoSp1 + RBS + codon 8, rpoSp1 + RBS, and Plac uv5 + RBS + codon 8, were employed. Furthermore, 100 nM 3OC6HSL was added as an inducer of the QS system. Error bars indicate three replicates.

Figure 2. Characterization of autoinduced QS systems in the AND gate. (a) Circuit diagram of QS in the AND gate. Input 1 is the autoinduced QS system that induces HrpR expression. HrpS is expressed upon the activation of PBAD by excess addition of Ara. HrpR and HrpS coactivate PhrpL and output GFP. (b) Introduction of the characterized autoinduced QS system into the AND gate. G00 (+) was induced by both 1.3 mM Ara and 100 nM 3OC6HSL as the positive control, and the remaining strains were induced with only 1.3 mM Ara. Error bars indicate three replicates.

(Figure 3b). As a negative control, G00 induced by 100 nM 3OC6HSL alone exhibited no GFP expression. Although G10 stably expressed GFP at the early exponential phase, the expression level was very low (GFP/OD600 = 100). The expression of G20 and G30 rapidly increased at the early exponential phase (0−6 h) because of the stress-induced activation. However, the expression level remained constant from the early to mid-exponential phases (8−16 h). Subsequently, the expression markedly increased at the late exponential phase and later peaked at the early stationary phase. In conclusion, G20 and G30 exhibited considerable stressinduced characteristics compared with G00, and G20 is an ideal alternative in our AND gate construction because of its more desirable growth and prompt GFP expression (Figure 3b and Supplementary Figure 5). Construction of an Autoinduced AND Gate To Dynamically Control Metabolic Pathways. The QS system is a cell density-dependent device, whereas PrpoS activation is dependent on the cell physiological state. Hengge-Aronis stated that the QS system and PrpoS have different mechanisms.40 To further examine their characteristics in our constructed AND gate, we compared them with different inoculation cell densities (Figure 4). Initially, the GFP expression of the AND gate containing PrpoS was weak at low cell densities. Subsequently, the expression markedly increased, possibly because of the low

Figure 4. Comparison between a QS system and a stationary phase sensing system. G01 induced by 1.3 mM Ara was employed to investigate the expression characteristics of the QS system in the AND gate. G20 induced by 100 nM 3OC6HSL was employed to characterize PrpoS in the AND gate. Both strains were inoculated at OD600 = 0.1 and 0.01. G00 induced by 1.3 mM Ara, and 100 nM 3OC6HSL was set as a control. Error bars represent three replicates.

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the early cell growth phase. By contrast, the autoinduced AND gate showed similar GFP expression with the control strain G00. The results revealed that the completely autoinduced AND gate has advantages of regulating the target gene and maintaining cell growth compared with the original AND gate. An Example for Application of the Autoinduced AND Gate. As proof of concept for the practical application of our synthetic device, AND gates with various cell density-sensing QS systems and rpoSP1 + RBS were employed for PHB production (Figure 6a). The autoinduced AND gate exhibited marked advantages in PHB accumulation compared with the inducible AND gate (Figure 6b). P00 induced by 1.3 mM Ara

growth rate at the early exponential phase. From the early to mid-exponential phases (OD 600 = 1.0−2.5), the GFP expression remained constant (Figure 4 and Supplementary Figure 6). Furthermore, the GFP expression increased steadily until the stationary phase, when an unfavorable growth condition occurred. By contrast, the GFP expression of the AND gate-harboring QS system was weak at low cell densities and even lower than that of the AND gate containing PrpoS. However, the expression increased steadily during the entire growth period (Figure 4). Moreover, the GFP expression of PrpoS was considerably affected by the inoculation cell densities, whereas no clear effect of inoculation cell densities on the GFP expression of the QS system. G20 inoculated at OD600 = 0.1 and 0.01 shared the expression level of 1000 au at OD600 = 5.0 (Figure 4). These results indicated different expression characteristics of the QS system and PrpoS in our constructed AND gate. After individually constructing and characterizing the autoinduced QS system and a stress-induced system in the AND gate, we built a completely autoinduced AND gate by combining both systems (Figure 5a). p3T0 in addition to p2A was co-transformed into E. coli Top10 containing pBW400hrpL-gfp, and the resulting strain G21 was characterized in M9 medium without inducers (Figure 5b). Compared with G00 supplemented with 100 nM 3OC6HSL and 0.33 mM Ara, G21 showed favorable growth, particularly at

Figure 6. Demonstration of AND gate in PHB production. (a) Graphic illustration of applying the autoinduced AND gate to PHB production. (b) Time profile of PHB production at 35, 60, and 72 h fermentation. PHB production was normalized by cell dry weight. P00 (−) was the negative control. P00-1 was induced by 1.3 mM Ara and 100 nM 3OC6HSL. P00-2 was induced by 0.33 mM Ara and 100 nM 3OC6HSL. P01 (single QS) was induced by 1.3 mM Ara and P20 (single PrpoS) was induced by 100 nM 3OC6HSL. (c) Growth conditions. Error bars indicate three replicates.

Figure 5. Construction and characterization of an autoinduced AND gate by employing the QS system and the stationary phase sensing system. (a) Genetic graph of the autoinduced AND gate. (b) Characterization of the autoinduced AND gate. G00 supplemented with 100 nM 3OC6HSL and 0.33 mM Ara was set as the control. Filled squares and circles represent the GFP expression level; empty squares and circles represent the cell density. Error bars indicate three replicates. E

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with 15 g/L agar powder) was used for plasmid construction and LuxR mutant screening. For GFP characterization and fermentation experiments, we used M9 medium (15.3 g/L Na2HPO4·12H2O, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 0.24 g/L MgSO4, 0.015 g/L CaCl2, 2 g/L Amicase, 0.1 g/L VB1) supplemented with 0.5% or 2% glycerol as the carbon source. To maintain plasmids, antibiotics, namely chloramphenicol (25 μg/mL), kanamycin (25 μg/mL), and ampicillin (100 μg/mL), were used. 3OC6HSL (No. 10011207; Cayman) and Ara (Code 101253904; Sigma) were added as inducers, if required. Plasmid Construction. pQST00 was the basic plasmid in the QS system in our study. By using pMB1 (http://parts.igem. org/Part:pSB1A7) as the replication origin, we assembled Ptet and Plux fragments from pBW313lux-hrpR26 to upstream of LuxR49 and sf GFP, respectively; the assembled plasmid was designated as pQST00. luxI was assembled from pLuxRI49 to pQST00, and pQST0 was thus generated. Subsequently, 4 constitutive promoters, PJ23101, PJ23104, PJ23108 (http://parts. igem.org/Main_Page), and Plac uv5 were used to substitute Ptet in pQST0. These promoters were designed within primers and were assembled into the corresponding pQST0 backbone of the DNA fragment digested using DpnI; therefore, pQS10, pQS40, pQS80, and pQSL0 were constructed. An inducible AND gate comprising two input plasmids (pBW213ara-hrpS and pBW313lux-hrpR) and one output plasmid (pBW400hrpL-gfp) was provided by Prof. Martin Buck from Imperial College London. We constructed an autoinduced AND gate on the basis of these three plasmids. First, to autoactivate the QS system at a certain cell density, wild-type LuxR in pBW313lux-hrpR was substituted by LuxRI from pQST0, and subsequently, p3T0 was obtained. Similarly, p3T1, p3T2, p3T4, p3L0, and p310 were constructed. Second, PBAD was substituted by a stress-induced promoter PrpoS from E. coli. DNA fragments, namely rpoSP1 + RBS + codon 8, rpoSP1 + RBS, and Plac uv5 + RBS + codon 8 were cloned from E. coli or synthesized and assembled to substitute PBAD in pBW213arahrpS, resulting in p2B, p2A, and p2C. Furthermore, p4PHB was constructed by introducing phbCAB genes 5 0 into pBW400hrpL-gfp. Construction and Selection of LuxR Mutant Library. To improve the QS system with a higher cell-density threshold, directed evolution on LuxR or promoter substitution for Ptet were conducted in pQST00. The wild-type luxR DNA fragment and relative backbone were separately cloned from pQST00. After two cycles of high-rate mutation (http://www.agilent. com/cs/library/usermanuals/Public/200550.pdf) on luxR (Agilent Catalog 200550), the purified luxR DNA fragment was assembled into the backbone and transformed into E. coli DH5α. Compared with the colonies of wild-type LuxR, those of less sensitive LuxR mutants on the LB plate supplemented with 100 nM 3OC6HSL tended to be weak or showed delayed green fluorescence under an ultraviolet detector. Plasmids containing potential LuxR mutants were retransformed into E. coli DH5α and sequenced. In this method, four LuxR mutants with different sensitivities, namely LuxR-S50F, LuxR-Y71F, LuxR-H39Q/K116N, and LuxR-S106P, were obtained; the plasmids pQST01, pQST02, pQST03, and pQST04 were constructed accordingly. Quorum Sensing System Characterization. Plasmids containing various LuxR mutants were retransformed into E. coli Top10 and cultured on LB agar with 25 μg/mL chloramphenicol. Single colonies were subsequently transferred

and 100 nM 3OC6HSL (P00-1) produced 8.22% (w/w) PHB content of the cell dry weight. Because of the excess addition of inducers, the growth of P00-1 was markedly affected and delayed (Figure 6c).41 However, all fermentation growth profiles reach steady state in 72 h, even cell density of P00-1 strain. Although P00 induced by 0.33 Ara and 100 nM 3OC6HSL (P00-2) did not show an apparent growth delay, PHB content was only 8.31% (w/w). P01 produced about 5.16% (w/w) PHB at 36 h, and the production increased to 10.29% (w/w) at 72 h. And the corresponding figures for PrpoS were 6.17% (w/w) and 9.79% (w/w). Both QS and PrpoS showed increased production in PHB production at higher cell density or late cell state. By contrast, the autoinduced AND gate showed desirable growth and produced 1−2 times higher PHB content than did P00-2. P21 containing the wild-type QS system yielded 13.79% (w/w) PHB. Moreover, P23, P25, P26, and P27 that harbored the optimized QS system produced more PHB than did P21, with a PHB content of the cell dry weight of 16.37% (w/w), 14.83% (w/w), 24.24% (w/w), and 19.51% (w/w), respectively (Figure 6b). Although efforts have be made for constructing AND gates,26,42−44 only a few studies have discussed its potential application in biotechnology. Moser et al. tested the induced AND logic gate at the microreactor to bioreactor levels by using different selection media, strains, and growth rates.45 Shong et al. constructed and characterized two synthetic AND gate promoters that require both a QS signal and an exogenously added inducer (IPTG or aTc) to induce report gene expression.46 In this study, we constructed an autoinduced AND gate controlled dynamic metabolic pathway in response to microbial communities and the cell physiological state and applied it for PHB production. The application of the autoinducible and self-monitored genetic circuit may reduce the cost of fermentation processes because the addition of external inducers and external monitoring of the reactor is not required.16,47 On the other hand, this strategy may be useful in a range of metabolic engineering applications as it can respond to both cell density and a second physiological state signal, such as aerobicanaerobic conditions and the redox state.

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CONCLUSION

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METHODS AND MATERIALS

We described the development and characterization of AND gate dynamic controllers, which combine two dynamic control strategies: microbial communities and the cell physiological state. As proof of concept, these dynamic controllers were applied for PHB production, revealing a 1−2-fold increased production in E. coli. This logic gate provides a tool for developing a general dynamic regulation system in metabolic engineering in response to complex signals, without using a specific sensor.

Strains, Plasmids, Primers, and Culture Medium. All plasmids in this article are shown in Table 2, and all strains and primers are listed in Supplementary Tables 1 and 2, respectively. Primers were synthesized for plasmid construction through PCR and Gibson assembly.48 E. coli DH5α was used for plasmid construction, whereas E. coli Top10 was used for GFP characterization and PHB fermentation. Luria−Bertani (LB) broth (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl) was used for plasmid cloning; LB agar (LB broth supplemented F

DOI: 10.1021/acssynbio.6b00177 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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to 5 mL of LB broth with appropriate antibiotics. Following culturing at 250 rpm/min and 37 °C for 12 h, 1% seeds were washed and inoculated into a 96-well microassay plate, with a total volume of 200 μL M9 medium supplemented with 0, 0.1, 1, 10, 100, 1000, and 10 000 nM 3OC6HSL. The 96-well microassay plate was cultured at 37 °C under vigorous shaking. The cell density at OD600 and green fluorescence (excitation at 485 nm and emission at 528 nm) were detected after a 12 h inoculation using a Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.). The data were fitted to Hill equations, and [3OC6HSL]50 was accordingly decided. For promoter characterization, no inducer was required; OD600 and GFP were collected every 20 min. AND Gate Characterization. AND gate plasmids were transformed into E. coli Top10 and grown on LB agar supplemented with 25 μg/mL chloramphenicol, 25 μg/mL kanamycin, and 100 μg/mL ampicillin. Single colonies were grown in 5 mL of LB broth at 250 rpm/min and 37 °C for 12 h. Furthermore, 1 mL of culture was transferred into a 50 mL LB flask and grown at 250 rpm/min and 37 °C for another 12 h. The cells were collected, washed, and resuspended in M9 medium, with inoculation at OD600 = 0.1 or 0.01. Cultured at 250 rpm/min and 37 °C, samples were collected every 4 h, and OD600 and green fluorescence (excitation at 485 nm and emission at 528 nm) were detected after washing. Polyhydroxybutyrate Fermentation Analysis. Seed preparation is described in AND gate characterization. However, the seeds were inoculated in M9 medium supplemented with 20 g/L glycerol, except for 5 g/L glycerol in GFP characterization. Samples were collected approximately every 8 h, and the pH was adjusted to 7.0 with 4 M NaOH. The cell density was detected at OD600 by using a spectrophotometer. Glycerol was analyzed through high-performance liquid chromatography.1 At 60 h, cells were collected, and approximately 15−20 mg portions of lyophilized cells were subsequently treated with 150 μL of H2SO4, 850 μL of CH3OH, and 1 mL of CHCl3 at 100 °C for 1 h. Furthermore, 1 mL of H2O was added. The mixture was allowed to stand and layered, and the CHCl3 layer was analyzed through gas chromatography.51

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X.H. and Y.C. performed experiments and analyzed data. Q.L. and X.H. designed research and drafted the manuscript. Q.L. and Q.Q. conceived and supervised the study. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Professor Martin Buck from Imperial College London for kindly providing the plasmids of original AND gate. This work was financially supported by a grant from the National Basic Research Program of China (2012CB725202), grant from the National Natural Science Foundation of China (31370085, 31170097), a grant from National Undergraduate Training Program for Innovation and Entrepreneurship (201610422074) and Shandong Science and Technology Development Plan (2015GSF121042).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00177. Supplementary Tables 1−3, listing strains, primers, and features of constitutive promoters used in this study; Supplementary Figures 1−6, showing graphic characterization of LuxR mutants, growth condition of promoter selection in QS system, growth condition of AND gate harboring various QS systems, G00 induced by 1.3 mM Ara and 100 nM 3OC6HSL at 0, 6, and 12 h, growth condition of AND gate containing three forms of PrpoS, and growth comparison between QS system and PrpoS (PDF)

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DOI: 10.1021/acssynbio.6b00177 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acssynbio.6b00177 ACS Synth. Biol. XXXX, XXX, XXX−XXX