Team:SCU China/Malatonion

01  Background

      Sleeping is one of the most mysterious yet important activity in our life – it costs us 25 years in average lifetime. However, the quality of sleeping and the habit of sleeping are largely ignored in public awareness. According to the surveys, about one third of the adults in the US sleep less than the suggested 7 hours per day1.; in the UK, 32% people admit that they sleep poorly and 14% have used OTC (over-the-counter) remedies2. It seems that young people suffer more from sleeping issues: according to a survey on people under 44 in China, only one in ten interviewees say that they can sleep through the night, and 91.4% say that they do not feel relaxed and energetic after sleep 3.
      A CDC report states that the ratio of suffering depression or heart attack in short sleep group (<7 hours) is about 1.5 times of that of the sufficient sleep group, and insufficient sleep would also lead to the increased risk in having stroke, cancer and diabetes, etc4. Sleep is important, but it is of unaware importance.
      Melatonin is a hormone secreted by the pineal gland and controlled by the suprachiasmatic nuclei (SCN) whose endogenous rhythm is entrained to the light/night period. Melatonin carries the information about daily light/dark cycle and spread that message to the whole body through bloodstream. According to the previous research, melatonin not only plays as a very robust biochemical signal of night to synchronize the circadian rhythms, it may also take part in the regulation of human seasonal rhythms, immune function, antioxidative defenses, hemostasis and glucose regulation etc. The regulating system of melatonin is complex, and the physiological function of melatonin is of great variety.
      Since melatonin is a hormone secreted by our body naturally and has close liaison with sleeping, it becomes an ideal drug of regulating sleep and tackling sleeping disorders. However, nowadays most of the melatonin is chemically synthesized, which employs complex steps yet obtains low yield, bringing the side effects of hazardous co-products, as well as low energy efficiency 5. Biosynthesis of melatonin would avoid the production of toxic compounds and reduce the possible pollution; also, the great compatibility with human body enables long-term effect and easy modulation. Thus, we tried to build the E. coli that could synthesis melatonin, and coupling it with repressilator to achieve periodic production and secretion of melatonin in human body.

02  Design

     The biosynthesis of melatonin from tryptophan needs 4 kinds of reaction, including 5-hydroxylation, decarboxylation, acetylation and transmethylation. However, the sequence of these four kinds of reaction differs in different species. Based on the previous study of serotonin production from tryptophan 6 and melatonin synthesis from serotonin7. we chose to apply the biosynthesis pathway of melatonin in Figure1 in our project. At first, tryptophan decarboxylase (TDC) catalyzes tryptophan into tryptamine. Next, tryptamine is converted into serotonin by tryptamine 5-hydroxylase (T5H). And then, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of caffeic acid O-methyltransferase (COMT), resulting in the synthesis of O-methylated NAS, also known as melatonin

Fig. 1 Biosynthesis pathway of melatonin

      Then, how do we get these four enzymes expressed to produce enough melatonin periodically? Firstly, in order to couple the biosynthesis of melatonin with repressilator, the last enzyme, COMT, is under the regulation of promoter pTetR, a key part in the repressilator. In this case, the expression of COMT only occurs during the period when the inhibition of TetR is lifted, and melatonin can be produced periodically. Secondly, we want to use mathematical modelling to obtain the optimal ratio of the four enzymes with a higher yield of N-acetylserotonin (NAS), and to choose the combination of the constitutive promoters for expressions of the first three enzymes based on this modeling result. This could reduce the extra energy consumption caused by excessive exogenous protein expression as well as the impact on basic growth metabolism of cells, and accumulate enough NAS. (Fig.2)

Fig. 2 Schematic diagram of our design

03  Results

To facilitate purification and expression identification, we assembled the coding sequence of each enzyme with pET15b containing an N-6x His tag, and results of colony PCR and sequencing demonstrated that we successfully constructed these plasmids.(Fig.1)

Fig.3 Electrophoresis result of constructed plasmid of (1) pET15b-TDC, (2)pET15b-T5H, (3)pET15b-SNAT, (4) COMT

      We purified these four enzymes by affinity chromatography with Ni-NTA Resin and concentrated the purified protein with superfiltration tubes to determine whether these four enzymes were expressed in E. coli BL21 (DE3) respectively. And the results showed that TDC, SNAT and COMT has been expressed successfully under the regulation of promoter pT7 in BL21(DE3). (Fig.4) However, T5H seemed not to be expressed successfully. Thus, we used western blot to further confirm the expression of T5H. Sadly, the results of western blot analysis indicated that T5H might not be expressed successfully in E.coli, because the target band (~70kDa) of T5H existed not only in the line1 (20x concentrated T5H purified by affinity chromatography) and line 2(supernatant of cell lysate (E.coli with pET15b-T5H)), but also in the negative control line 3(supernatant of cell lysate (E.coli without pET15b-T5H)).(Fig.5) In addition, there existed a light target band in the line 7(Fig.6), and a obvious band in the line 1 and line 3 respectively. the SDS-PAGE result indicated that pSB1C3-pTet-COMT could be expressed in E,coli, but most expressed protein were insoluble. This may be caused by constitutive overexpression with a high amount, which showed the need of modeling to determine a optimize proportion of enzymes to some extent.(Fig.6)

Fig.4 SDS-PAGE result for the expression of four enzymes in E.coli BL21(DE3).
(1) supernate of cell lysate containing TDC;
(2) 10x concentrated purified TDC (56kDa);
(3) supernate of cell lysate containing T5H;
(4)10x concentrated purified T5H (71kDa) ;
(5)supernate of cell lysate containing SNAT;
(6) 10x concentrated purified SNAT (23kDa);
(7)10x concentrated purified COMT(40kDa);
(8) 10x concentrated purified cell lysate of BL21(DE3);
(9)supernate of cell lysate of BL21(DE3).

Fig.5 immunoblot analysis result for the expression of T5H in E.coli BL21(DE3).
(1) 20x concentrated T5H purified by affinity chromatography;
(2) supernatant of cell lysate (E.coli with pET15b-T5H);
(3) supernatant of cell lysate (E.coli without pET15b-T5H).

Fig.6 SDS-PAGE result for the purification of pSB1C3-pTet-COMT in E.coli BL21(DE3) via affinity chromatography.
(1) cell lysate;
(2) supernatant of cell lysate;
(3) precipitate of cell lysate;
(4) flow through;
(5) wash;
(6) elution;
(7) 10x concentrated elution;
(8) positive control: COMT protein.

      To verify the biological function of these four enzymes expressed in E.coli, we used high performance liquid chromatography (HPLC) to analyze tryptophan, tryptamine, serotonin, NAS and melatonin content in extracellular medium fraction of E.coli containing these plasmids respectively and wild-type E.coli BL21(DE3). Tryptophan, tryptamine, serotonin (5-hydroxyltryptamine, 5-HT), NAS and melatonin were identified on the basis of retention times related to standard sample. Content was determined by integrating peak areas and converted to concentration. According to the retention time of the standard sample mix of tryptophan and tryptamine, tryptamine compound of extracellular medium from culture of E.coli containing pET15b-TDC was confirmed, which meant TDC expressed in E.coli could function properly.(Fig. 7) Likewise, the biological function of SNAT and COMT expressed in E.coli were confirmed as well. However, the function of T5H could not be verified by us again, because the retention time of 5-HT was similar to a peak of something unknown existed in extracellular medium fraction of E.coli’s culture.

Fig.7 HPLC analysis results of TDC.
(Top) Supernatant fraction of overnight culture of WT BL21 in LB medium at 20℃ with 1mM IPTG and 1mM tryptophan;
(Middle) Supernatant fraction of overnight culture of TDC overexpressed BL21 in LB medium at 20℃ with 1mM IPTG and 1mM tryptophan;
(Bottom) Standard sample of the mix of 1mM tryptophan and 1mM tryptamine.

Fig.8 HPLC analysis results of T5H.
(Top) Supernatant fraction of overnight culture of WT BL21 in LB medium at 20℃ with 1mM IPTG and 1mM tryptamine;
(Middle) Supernatant fraction of overnight culture of T5H overexpressed BL21 in LB medium at 20℃ with 1mM IPTG and 1mM tryptamine;
(Bottom) Standard sample of the mix of 1mM tryptamine and 1mM serotonin.

Fig.9 HPLC analysis results of SNAT.
(Top) Supernatant fraction of overnight culture of WT BL21 in LB medium at 20℃ with 1mM IPTG and 1mM serotonin;
(Middle) Supernatant fraction of overnight culture of SNAT overexpressed BL21 in LB medium at 20℃ with 1mM IPTG and 1mM serotonin;
(Bottom) Standard sample of the mix of serotonin and NAS

Fig.10 HPLC analysis results of COMT.
(Top) Supernatant fraction of overnight culture of WT BL21 in LB medium at 20℃ with 1mM IPTG and 1mM NAS;
(Middle) Supernatant fraction of overnight culture of TDC overexpressed BL21 in LB medium at 20℃ with 1mM IPTG and 1mM NAS;
(Bottom) Standard sample of the mix of tryptophan and NAS.

04  Reference

1. Centers for Disease Control and Prevention. 2017. CDC – Data and Statistics – Sleep and Sleep Disorders.
Retrieved from

2.The Sleep Council. 2013. The Great British Bedtime Report.
Retrieved from

3.China Sleep Research Association. 2017. 2017 China Youth Sleeping Quality White Paper [in Chinese].
Retrieved from

4.Claustrat, B., Brun, J., & Chazot, G. The basic physiology and pathophysiology of melatonin. Sleep Medicine Reviews, 9(1), 11–24 (2005).

5.Flaugh, M. E., Crowell, T. A., Clemens, J. A., & Sawyer, B. D. Synthesis and evaluation of the antiovulatory activity of a variety of melatonin analogues. Journal of Medicinal Chemistry, 22(1), 63–69 (1979).

6. Park, S. et al. Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli. Appl. Microbiol. Biotechnol. 89, 1387–1394 (2011).

7. Byeon, Y. & Back, K. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase. Appl. Microbiol. Biotechnol. 100, 6683–6691 (2016).