Team:USTC/m/m
Design
Sensing System
Overview
The goal of our sensor is to detect nicotine in liquid phase. In industrial production, it is important to monitor nicotine’s concentration in tobacco waste. However, continuously producing enzymes in a high expression level is a burden for bacteria, and also, present methods to measure nicotine concentration are too inconvenient and expensive, which are not suitable for industrial production. Therefore, so we design a nicotine biosensor to detect nicotine and regulate the expression of enzymes of degradation system. After bacteria uptaking nicotine, it should produce visible signal (GFP) and synthesize signal molecule to activate the next system. Nicotine concentration can be known by measuring fluorescence intensity.
Important components:
HdnoR: transcriptional repressor of 6hdno, from plasmid pAO1
VppA: Nicotine dehydrogenase, catalyzing the reaction nicotine -> 6-Hydroxy nicotine, from genome of Ochrobactrum sp. Strain SJY1, needing molybdenum cofactor MCD.
6-hdno: 6-hydroxy-D-nicotine oxidase, catalyzing the reaction 6-hydroxy-D-nicotine -> 6-Hydroxypseudooxynicotine, from plasmid pAO1.
6-hlno: 6-hydroxy-L-nicotine oxidase, catalyzing the reaction 6-hydroxy-L-nicotine -> 6-Hydroxypseudooxynicotine, from plasmid pAO1.
GFRA: GFP with SsrA tag (AANDENYADAS, see part BBa_M0052) on the C terminus. This tag make GFP degrade very fast in bacteria which only need several minutes.
HdnoR Mechanism
HdnoR binds to the 6-hdno operator with a Kd of 21 nM, which is similar to transcriptional regulators of the TetR family. It binds to 2 fragments named IR1, which covers the 6hdno promoter region, and IR2 on the upstream from the 6-hdno promoter. 6-Hydroxy-D-nicotine and 6-hydroxy-Lnicotine work as inducers of 6hdno expression, which can bind to hdnoR and stop the inhibition.
Detail design of the sensor
a. Sensing nicotine
Promoter 1 is a constitutive promoter to produce enzyme VppA. When nicotine exists, it will be converted to 6-hydroxy nicotine. However, VppA has no enzyme activity without molybdenum cofactor, so we used gene moa, moc, moe and mog from E.coli genome to produce molybdenum cofactor MCD (shown in Figure 1). Promoter 2 is also a constitutive promoter to express hdnoR, and the hdnoR will inhibit expression of pHdno (originally is 6-hdno’s promoter on plasmid pAO1). Therefore, there is nearly no GFP or AHL produced without nicotine. 6-Hydroxy nicotine can bind to hdnoR and release hdnoR’s repression. If there exists nicotine, it would be converted to 6-hydroxy nicotine and activate expression of GFP and LuxI downstream of pHdno (shown in Figure 1). If detected fluorescence, it meant that there existed nicotine, and AHL would be another output signal, acting on the next system.
Figure 1. Biosynthesis of molybdenum cofactor in E. coli
(From Iobbi-Nivol C, Leimkühler S. Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2013, 1827(8-9): 1086-1101.)
Figure 2. The gene circuit we designed in our original sensing system
b. Showing nicotine’s running out
The design above might not be good enough, because it can only show nicotine’s existence and we don’t know when would nicotine be used up. Thus we add 6-hdno and 6-hlno, and SsrA tag on the C term of GFP. When pHdno is activated, 6-hdno and 6-hlno will be produced to consume 6-hydroxy nicotine. After nicotine been used up, 6-hydroxy nicotine will be depleted in a short time. Then, pHdno will be repressed again, so GFRA (GFP with SsrA tag) will be degraded fast (shown in Figure 2). After all, we can see the fluorescence intensity decline quickly till disappeared.
Figure 3. The gene circuit we designed in the improved sensing system
[1] Bernauer H, Mauch L, Brandsch R. Interaction of the regulatory protein NicR1 with the promoter region of the pAO1‐encoded 6‐hydroxy‐D‐nicotine oxidase gene of Arthrobacter oxidans[J]. Molecular microbiology, 1992, 6(13): 1809-1820.
[2] Sandu C, Chiribau C B, Brandsch R. Characterization of HdnoR, the transcriptional repressor of the 6-hydroxy-D-nicotine oxidase gene of Arthrobacter nicotinovorans pAO1, and its DNA-binding activity in response to L-and D-nicotine derivatives[J]. Journal of Biological Chemistry, 2003, 278(51): 51307-51315.
[3] Yu H, Tang H, Li Y, et al. Molybdenum-containing nicotine hydroxylase genes in a nicotine degradation pathway that is a variant of the pyridine and pyrrolidine pathways[J]. Applied and environmental microbiology, 2015, 81(24): 8330-8338.
[4] BRANDSCH R, HINKKANEN A E, MAUCH L, et al. 6‐Hydroxy‐D‐nicotine oxidase of Arthrobacter oxidans: Gene structure of the flavoenzyme and its relationship to 6‐hydroxy‐L‐nicotine oxidase[J]. European journal of biochemistry, 1987, 167(2): 315-320.
[5] Grether‐Beck S, Igloi G L, Pust S, et al. Structural analysis and molybdenum‐dependent expression of the pAO1‐encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans[J]. Molecular microbiology, 1994, 13(5): 929-936.
[6] McGinness K E, Baker T A, Sauer R T. Engineering controllable protein degradation[J]. Molecular cell, 2006, 22(5): 701-707.
[7] Iobbi-Nivol C, Leimkühler S. Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2013, 1827(8-9): 1086-1101.]
Regulation System
Overview
In our sensing system, adding nicotine leads to production of AHL, so we use quorum sensing system to regulate expression of enzymes of degradation system. After nicotine being detected by our sensing system, enzymes will be largely expressed.
Our design
Promoter lux pR has a low transcription level when AHL’s concentration is lower than 0.1nM, but with luxR-AHL complex, the expression of downstream genes will be activated. Thus we use luxR and lux pR to construct the regulation system, using AHL synthetized by sensing system to activate the expression of enzymes (shown in Figure 3). At the same time, we considered that nicotine concentration might be so low that made AHL is not enough to activate expression of lux pR, so we add another LuxI after lux pR to form a positive feedback, aiming at enlarging AHL signal (shown in Figure 4)
Figure 4. luxR-lux pR system
Figure 5. Regulation System we design with positive feedback
Degradation (Harvest) System
Overview
Degradation system contains several enzymes, and it is on the downstream of regulation system. After been activated by sensing system and regulation system, degradation is able to produce nicotine degradation enzymes. We want to make good use of nicotine, so we use several enzymes to convert nicotine to 3-succinylpyridine, a precursor in the production of hypotensive agents. We can degrade nicotine and produce some useful chemicals at the same time!
Our design
We use 3 enzymes to achieve our goal: nicA2 (nicotine oxidoreductase), pnao (pseudooxynicotine oxidase), and sapd (3-succinoylsemialdehyde-pyridine dehydrogenase), which are all from Pseudomonas putida S16. NicA2 can convert nicotine to N-Methylmyosmine; pnao can convert Pseudooxynicotine to 3-Succinoylsemialdehyde-pyridine; sapd can convert 3-Succinoylsemialdehyde-pyridine to 3-succinylpyridine (shown in Figure 5). These 3 enzymes can produce our target product from nicotine.
Figure 6. Reaction pathway to 3-succinylpyridine
[1] Hu H, Wang W, Tang H, et al. Characterization of pseudooxynicotine amine oxidase of Pseudomonas putida S16 that is crucial for nicotine degradation[J]. Scientific reports, 2015, 5: 17770.
[2] Tang H, Wang L, Wang W, et al. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas[J]. PLoS genetics, 2013, 9(10): e1003923.
[3] Wang S N, Xu P, Tang H Z, et al. “Green” route to 6-hydroxy-3-succinoyl-pyridine from (S)-nicotine of tobacco waste by whole cells of a Pseudomonas sp[J]. Environmental science & technology, 2005, 39(17): 6877-6880.
[4] Qiu J, Ma Y, Wen Y, et al. Functional identification of two novel genes from Pseudomonas sp. strain HZN6 involved in the catabolism of nicotine[J]. Applied and environmental microbiology, 2012: AEM. 07025-11.
Summary
When nicotine doesn’t exist, there are nearly no enzyme of degradation system was expressed, which avoid toxicity of too much expression of protein. Sensing system ensure that in comment situation, bacteria can grow in normal state. When bacteria have reached enough quantity, we can add TW to produce 3-succinylpyridine. After bacteria uptaking nicotine, the sensing system can produce detectable GFP and AHL to activate regulation system. Activated regulation system will produce nicotine degradation enzymes to convert nicotine to 3-succinylpyridine. And then, when nicotine’ concentration is too low or even nicotine is used up, expression of GFP will stop, and GFP with SsrA tag is going to degrade quickly so we can see the fluorescence intensity decline rapidly, and we can harvest the production then.