Team:Botchan Lab Tokyo/Description




The goal of our project is to eliminate nicotine from being released into the environment. It has been calculated that between 0.6 and 32 μg/L of nicotine is currently released into the environment and as an endocrine disruptor, it affects the reproduction and growth of organisms such as daphnids. Since nicotine is a difficult substance to break down or convert into other substances through chemical pathways, we felt that using synthetic biology would be appropriate. We know that nicotine is released into the environment when we dispose of water from smoking areas into the sewage system, when we incinerate factory waste from tobacco production, when farmers dispose of excess tobacco leaves, and when cigarette butts are littered in the streets. We focused on the water that accumulates in the smokehouse and the tobacco that is littered into the city in this project. (For more information on how we came to this decision, please see Integrated Human and Practice) By contacting companies that produce tobacco and people in the community that produce a lot of tobacco leaf and generate income, we gained a realistic understanding of the sources of nicotine release.

Extraction of nicotine

First of all, nicotine needs to be extracted from the tobacco waste. By using the method by Qayyum et al., the percentage yield was 0.6 % by weight.(Qayyum et al., 2018) Figure 1 shows the procedure. 1 g of tobacco waste is added to 100 mL of 5 % NaOH solution and shake them well for about 15 minutes. More yield might be gained by adding ethanol to 5% NaOH as it was done by Xing et al. (Xing et al., 2009) Using filter paper, filter the mixture. Take the tobacco waste residue and add 30 mL of water. Stir well and filter again. Add this solvent to the first solvent. Transfer this solvent to separatory funnel and add 25 mL of diethyl ether. Extract the organic layer three times and collect them. Add K2CO3 to the extracted layer and filter them. Evaporate the diethyl ether on water bath. Take the liquid nicotine and measure the boiling point. The boiling point should be 247 ℃. (Qayyum et al., 2018) By infrared radiation spectroscopy (IR spectroscopy), peaks should be found at f =2970-2781 cm-1, 1677 cm-1, 16911 cm-1, 717-904 cm-1.

Figure 1. The extraction method of nicotine. Solve the tobacco waste into 5% NaOH and filter them. Add diethyl ether and evaporate the organic layer.
Converting nicotine into 5-aminolevulinic acid

After extracting nicotine from tobacco waste, nicotine will be converted to 5-aminolevulinic acid. There are two phases in this process. The first step will be converting nicotine to 2,5-dihydroxypyridine biologically. The second step will be converting 2,5-dihydroxypyridine to 5-aminolevulinic acid chemically. There are two ways for the first step. One way is using Escherichia coli as a chassis and the other is using Pseudomonas putida S16(P. putida S16) as a chassis.

Using E. coli as a chassis

As shown in figure 2, genes related to cell communication and nicotine degradation will be inserted to E. coli. There are two devices. The first device which consists of constitutive promoter(BBa_J23100), luxI(BBa_C0061), and luxR(BBa_C0062) is responsible for regulating the second device. The second device which consists of nicotine degrading genes form P. putida S16 and a molybdenum cofactor gene(molybdopterin cytidylyltransferase, mocA: BBa_K2768008) is responsible for degrading nicotine and producing 2,5-dihydroxypyridine.

Figure 2. The genes introduced to E. coli. a: The regulating device consists of constitutive promoter, luxI, and luxR. The nicotine degrading device consists of nicotine degrading genes and mocA. b and c: First, the lux promoter is not activated. If the cells reach to certain density, the lux promoter will be activated by quorum sensing. d: By activating of lux promoter, nicotine degrading genes and mocA will be transcribed.

The regulating device uses quorum sensing. Here is the mechanism of quorum sensing.(Goryachev et al., 2006) The luxI gene produces autoinducer 3-oxohexanoyl-homoserine lactone which can freely diffuse through the cell membrane. The autoinducer will be in equilibrated inside and outside the cell membrane. This will rely on passive permeability of the cell membrane. The intracellular autoinducer will bind to the LuxR protein. The luxR protein with the autoinducer will dimerize and bind to the lux promoter. The lux promoter will be activated by the binding and initiate transcription of genes.
The degrading device starts with the activation of the lux promoter. The lux promoter initiates transcription of nicotine degrading genes and a molybdenum cofactor gene. The nicotine degrading genes are nicotine oxidoreductase (nicA2 : BBa_K2569036), pseudooxynicotine amine oxidase (pnao : K2768006 ), 3-succinoylsemialdehyde-pyridne dehydrogenase (sapd), 3-succinoylpyridine monooxygenase (spmABC), and 6-hydroxy-3-succinoylpyridine hydroxylase (hspB). One of the genes, spmABC, has never expressed functional protein. It is said that this is due to molybdenum cofactor.(Tang et al., 2013) In E. coli, molybdopterin guanine dinucleotide(MGD) or molybdopterin cytosine dinucleotide(MCD) is formed from molybdopterin via adding GMP or CMP to the C4’ phosphate of molybdenum cofactor.(Iobbi-Nivol & Leimkühler, 2013) In E. coli, MGD is the organic part of the molybdenum cofactor. (Tang et al., 2013) MCD is crucial for functional SpmABC. (Tang et al., 2013) By expressing mocA which catalyzes adding CMP to molybdopterin and increasing the amount of MCD, functional SpmABC might be formed.

Using Pseudomonas putida S16 as a chassis

The second way for converting nicotine into 2.5-dihydroxypyridine is done using P. putida S16 as a chassis. A gram-negative bacterium P. putida S16 has known to degrade nicotine.(Liu et al., 2015) As Tang et al. did in 2012(figure 3) , by knocking out 2,5-dihydroxypyridine dioxygenase, 2,5-dihydroxypyridine can be obtained.(Tang et al., 2012)

Figure 3. a: Amount of nicotine of wildtype P. putida S16 and hpo knockout mutant. b: Amount of 2,5-dihydroxypyridine of wildtype P. putida S16 and hpo knockout mutant. (Tang et al., 2012)

More yield could be achieved by quorum sensing as shown in figure 4. Two devices, regulating device and hpo complement device, are introduced to hpo knockout mutant of P. putida S16. The regulating device consists of constitutive promoter, luxI , and luxR. The hpo complement device consists of tet promoter, tetR, tet promoter, and hpo. First, lux promoter is not activated and tet promoter is activated, meaning transcription of tetR is suppressed and hpo is promoted. hpo from plasmid is complementing deleted genome hpo. By mechanism explained above, (The regulating device uses quorum sensing.) if the cells reach to certain density, lux promoter is activated and tetR is transcribed. TetR is the repressor of tet promoter and inactivates it, which suppress transcription of hpo. Both plasmid and genome hpo is suppressed.

Figure 4. a: The regulating device consists of constitutive promoter, luxI, and luxR. The hpo complement device consists of lux promoter, tetR, tet promoter, and hpo. b: First, lux promoter is not activated and tet promoter is activated. This means tetR is not transcribed and hpo is transcribed. hpo inserted in plasmid is complementing the deleted hpo of the genome. c: If the cells reach to certain density, the lux promoter will be activated by quorum sensing. d: By activating of lux promoter, TetR will suppress tet promoter and hpo transcription. Hence, both plasmid and genome hpo is suppressed.
Pros and cons of two plans

Both plans have own good points. For plan using E. coli as a chassis, E. coli is more used in industrial field, for example, the production of L-alanine by Ichiro Chibata. (Takamatsu et al., 1986) Compared with P. putida S16, E. coli has better understanding of basic synthetic biology tools, for instance, lux and tet system. Also, by pushing this plan, genes form S16 will be characterized and future iGEMer and synthetic biologist can benefit from them.

For plan using P. putida S16 as a chassis, the metabolic pathway of P. putida S16 is used. Regarding the fact that environment for enzymes is suitable and nicotine might be harmful for E. coli, higher yield and better degradation ability are assumed.

Converting 2,5-hydroxypyridine to 5-aminolevulinic acid

After producing 2,5-dihydroxypyridine, it will be converted to 5-aminolevulinic acid. The chemical characteristics of 2,5-dihydroxypyridine is unknown, so ways to collect it from medium needs work. We plan to identify n-octanol-water partition coefficient and find out whether 2,5-dihydroxypyridine is solvent to organic layer or water layer.

Once 2,5-dihydroxypyridine is collected, 5-aminolevulinic acid is made. As shown in figure 4, it is known that this can be done chemically. (Herdeis and Dimmerling, 1984) 2,5-dihydroxypyridine takes equilibrium between 5-hydroxy-2-pyridone. This is known lactam-lactim tautomer. By heating 30 ℃ for 5 hours with palladium on carbon, it will be converted to 2,5-piperidinedione. React this with HCl for 12 hours, finally, 5-aminolevulinic acid is made.

Figure 5. 2.5-dihydryoxypyridine is converted to 5-aminolevulinic acid. 2,5-dihydroxypyridine is equilibrium with 5-hydroxy-2-pyridine due to lactam-lactim tautomer. With palladium on carbon, 2,5-piperidinedione is made. Adding HCl to the product, 5-aminolevulinic acid is produced.


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