Part:BBa_K4955000
Enzymes involved in Crocetin biosynthesis
Parts confirmed to produce Crocetin in E. coli without the addition of special substrates
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1643
Illegal PstI site found at 5570
Illegal PstI site found at 5981
Illegal PstI site found at 8833
Illegal PstI site found at 8951 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1643
Illegal PstI site found at 5570
Illegal PstI site found at 5981
Illegal PstI site found at 8833
Illegal PstI site found at 8951 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 6388
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1643
Illegal PstI site found at 5570
Illegal PstI site found at 5981
Illegal PstI site found at 8833
Illegal PstI site found at 8951 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1643
Illegal PstI site found at 5570
Illegal PstI site found at 5981
Illegal PstI site found at 8833
Illegal PstI site found at 8951
Illegal NgoMIV site found at 6712
Illegal NgoMIV site found at 6842
Illegal NgoMIV site found at 8638
Illegal NgoMIV site found at 8695
Illegal AgeI site found at 991
Illegal AgeI site found at 2015
Illegal AgeI site found at 2148
Illegal AgeI site found at 2555
Illegal AgeI site found at 7616 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2493
Illegal BsaI.rc site found at 2927
Illegal SapI site found at 1648
Profile
name：HpIDI,crtE,crtB,crtI,crtY,crtZ,fhCCD2,NcALD8
Base Pairs：9233 bp
Function(summary)：Enzymes that produce crocetin in E. coli without the addition of special substrates
Introduction
Crocetin is a secondary metabolite of saffron (Crocus sativus) and is a precursor of crocin, which has been shown to have therapeutic effects on a variety of disorders including psychiatric disorders. In addition, crocetin itself has been shown to have therapeutic effects on depression, oxidative stress [1], cancer, myocardial ischemia, multiple sclerosis [2], blood clot, dementia, cerebral ischemia, Parkinson's disease [3], and fatigue [4]. For this reason, it is attracting attention as a high value-added carotenoid. Current sources of crocetin depend on complicated extraction and purification from the saffron stigma. [5] However, the purity of the refined product is low and the production process requires extensive processing. In addition, approximately 150,000 to 200,000 flowers must be harvested by hand to obtain 1 kg of Saffron columnar heads. These are some of the reasons why Saffron is referred to as Red Gold, driving up the cost of Saffron-derived components. Furthermore, they cause severe child labor in Iran and other countries where Saffron is primarily produced. [6] Saffron is also susceptible to negative environmental factors like climate, and its triploid nature makes gene editing difficult. Saffron-derived molecules' complex structures and abundant chiral centers make it susceptible to formation of inactive or toxic optical isomers in chemical synthesis. These are the reasons why the cost of Saffron and Saffron-derived components remains high. [6]
Our goal was to achieve low cost and stable crocetin production by microorganisms. This has been attempted by four iGEM teams over the period 2012-2022, with no successes.
| Team | Goal | Result |
|---|---|---|
| WashU 2012 | Production of Saffron secondary metabolites in E.coli,Synechocystis | Successful gene cloning but failed to demonstrate function in vivo. |
| Uppsala 2013 | Production of Saffron secondary metabolites in lactic acid bacteria | Successful Plasmid design, but enzyme folding did not work. |
| Upssala 2017 | Production of Saffron secondary metabolites in E. coli | Although the biosynthetic pathway up to Zeaxanthin was
successfully integrated into the chromosome, the later pathways could not be functionally demonstrated in vivo. |
| Latvia-Riga 2022 | Production of Saffron secondary metabolites in Rhodotorula toruloides | Transformants could not be obtained. |
Table.1 Teams that have directly or indirectly aimed to produce crocetin in the past, and their results [7][8][9][10]
We introduced BBa_K4955000 into BL21(DE3) and DH5α strains. After culturing both strains in TB 50 ml culture, the culture medium was analyzed by HP-LC-MS/MS, and the production of crocetin was confirmed.
This part is essential for the production of crocin and picrocrocin, which are secondary metabolites of saffron, without the addition of any special substrates.
Biology & Function
HpIDI：Derived from Haematococcus pluvialis[11]
crtE,crtB, crtI, crtY, crtZ：Likely to E. coli, belongs to γ-Proteobacteria, derived from Pantoea ananatis (reclassified from Erwinia uredovora) [12]
fhCCD2：Derived from Freesia hybrida[11]
NcALD8：Derived from Neurospora crassa[11]
HpIDI：Estimated to make carotenoid production 2~3 times higher [13]
The plasmid includes the illegal sequence in BioBrick. When we received the plasmid, there was a discrepancy with the other research organization, and we did not become aware of the discrepancy until just before the deadline for registration of parts, so we could not demonstrate the functionality of the modified plasmid in time.
Measurement
Plasmid
BBa_K4955000 is deposited at RIKEN BRC (https://web.brc.riken.jp/en/) in its assembled form in the Duet-1 vector, pRK404 [14], and was used.
E. coli strain
The BL21(DE3) and DH5α strains were selected as the host for biosynthesis. The above plasmids were transfected using the heat shock method.
Generally, the BL21(DE3) strain is used for protein expression and material production. However, when we compared E. coli colonies containing the above plasmids on LB agar medium, the DH5α strain had a more distinct yellow color than the BL21(DE3) strain (yellow colonies are produced when carotenoids such as crocetin are produced). Therefore, we cultured the DH5α strain as well as the BL21(DE3) strain to see if crocetin was produced.
Culture Conditions
Transformed E. coli were cultured under the following conditions.
Pre-culture: Inoculated into 5 mL of LB containing appropriate antibiotics and cultured in a test tube with shaking (140 rpm, 37°C). 18-24 hrs later, the culture was transferred to the main culture.
Main culture: 1mL of pre-culture solution and 49mL of TB with appropriate antibiotics were cultured in a 300mL triangular flask with shaking (150rpm, 25°C). 0.1mM IPTG was added at the final OD600=8~12. Then incubated for 72 hours.
Identification by HP-LC-MS/MS
Extraction
Extraction of crocetin from the culture medium was performed according to the following protocol.
Step.1 Centrifuge 500 μl of the E. coli culture medium (6000g, 5 minutes) and discard the supernatant.
Step.2 After suspending the cell pellets in 2ml of methanol (MeOH), extract them with an ultrasonic disruption machine.
Step.3 Add Tris-HCl (50mM, pH 7.5) and vortex.
Step 4 Add 1M NaCl and vortex to confirm that it is dissolved.
Step.5 Centrifugal separation (15000rpm, 5min)
Step.6 Add 2ml of chloroform and vortex for 5 minutes
Step.7 Centrifugation (room temperature, 15,000 rpm, 15 min)
Step.8 Collect 1500 μl of the chloroform layer.
Step.9 Dry by centrifugal evaporation.
HP-LC-MS/MS analysis & MS spectrum
Liquid chromatography was performed on a SHIMADZU Ultra-Fast Liquid Chromatography (UFLC) Nexera system (Shimadzu, Kyoto). Mobile phase conditions were A 0.1% formic acid H2O, B 0.1% formic acid acetonitrile (10-100% B over 5 min, 100% B for 2.5 min, and then 10% B for 2.5 min).
The mass spectrometer was a SCIEX Triple TOF X500R system (Sciex, Tokyo).
Crocetin [M (C₂₀H₂₄O₄)] was analyzed in positive ion mode using the above LC-MS analyzer, and an extracted ion chromatogram (EIC) of m/z 329.17474 [M+H]+ was prepared, showing a single peak. This analysis confirmed the production of crocetin.
The single peak was confirmed by specifying the act-ms of crocetin (329.17474) at the time of positive polarity. (Top and bottom are extracted from DH5α and BL21(DE3) cultures, respectively.)
References
[1]Deepu Pandita.(2021).Saffron (Crocus sativus L.): phytochemistry, therapeutic significance and omics-based biology.Medicinal and Aromatic Plants: Expanding their Horizons through Omics.
[2]Zahra Maqbool, Muhammad Sajid Arshad, Anwar Ali, Afifa Aziz, Waseem Khalid, Muhammad Faizan Afzal, Sneh Punia Bangar, Mohamed Addi, Christophe Hano and Jose Manuel Lorenzo.(2022).Potential Role of Phytochemical Extract from Saffron in Development of Functional Foods and Protection of BrainRelated Disorders.Oxidative Medicine and Cellular Longevity.
[3]Zeliha Selamoglu, Senay Ozgen.(2016).Therapeutic Potential of Saffron Crocus (Crocus sativus L.).Turkish Science and Technology.
[4]María José Bagur, Gonzalo Luis Alonso Salinas, Antonia M. Jiménez-Monreal, Soukaina Chaouqi, Silvia Llorens, Magdalena Martínez-Tomé and Gonzalo L. Alonso.(2017).Saffron: An Old Medicinal Plant and a Potential Novel Functional Food.Molecules.
[5]Wen Wang, Ping He, Dongdong Zhao, Lijun Ye, Longhai Dai, Xueli Zhang, Yuanxia Sun, Jing Zheng and Changhao.(2019).Construction of Escherichia coli cell factories for crocin biosynthesis. Microbial Cell Factories.
[6]Majid Shokrpour. (2019).Saffron (Crocus sativus L.) Breeding: Opportunities and Challenges. Advances in Plant Breeding Strategies: Industrial and Food Crops Volume 6 675-706.
[7]Team:WashU - 2012.igem.org
https://2012.igem.org/Team:WashU[8]Team:Uppsala - 2013.igem.org
https://2013.igem.org/Team:Uppsala[9]Team:Uppsala - 2017.igem.org
https://2017.igem.org/Team:Uppsala#[10]| Latvia-Riga - iGEM 2022
https://2022.igem.wiki/latvia-riga/[11]Norihiko Misawaa, Takashi Maokab and Miho Takemura. (2022). Carotenoids: Carotenoid and apocarotenoid analysis—Use of E. coli to produce carotenoid standards. Methods in Enzymology.
[12]Misawa, N. (Ed.). (2021). Carotenoids: Biosynthetic and biofunctional approaches. (advances in experimental medicine and biology 1261). Singapore: Springer.
[13]Kajiwara, S., Fraser, P. D., Kondo, K., & Misawa, N. (1997). Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochemical Journal, 324, 421–426.
[14] Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X.-W., Finlay, D. R., et al. (1985). Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid, 13, 149–153.
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