Part:BBa_K4955001
Enzyme that converts 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) to Picrocrocin
Part confirmed to convert 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) to piclocrocin in E.coli.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 253
Illegal PstI site found at 520
Illegal PstI site found at 604 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 253
Illegal PstI site found at 520
Illegal PstI site found at 604 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1206
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 253
Illegal PstI site found at 520
Illegal PstI site found at 604 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 253
Illegal PstI site found at 520
Illegal PstI site found at 604
Illegal NgoMIV site found at 1389 - 1000COMPATIBLE WITH RFC[1000]
Profile
name: UGT709G1
Base Pairs: 1548 bp
Function(summary): Enzyme that converts 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) to picrocrocin
Introduction
Picrocrocin is a secondary metabolite of saffron (Crocus Sativus) and has been shown to have positive effects on depression, cancer, thrombosis, oxidation, myocardial ischemia, atherosclerosis [1], and cerebral ischemia [2]. Thus, it is attracting attention as a high-value-added glycoside terpenoid.
Current sources of picrocrocin rely on complex extraction and purification from saffron [3]. The purified product is of low purity and requires extensive processing. In addition, approximately 150,000 to 200,000 flowers must be harvested by hand to produce 1 kg of saffron’s floral stigma. This is why saffron is known as “red gold” due to the increased cost of saffron-based ingredients. They are also responsible for serious child labor in Iran and other countries where saffron is mainly produced [4].
Saffron is also vulnerable to negative environmental factors such as climate, and its triploid nature makes gene editing difficult. Due to its complex structure and abundant chiral centers, saffron-derived components can easily form inactive or toxic optical isomers during chemical synthesis. For these reasons, the cost of saffron and saffron-derived components remains high [4]. When considering saffron-derived components as food additives, picrocrocin is especially difficult to substitute because it is a flavor-related component [5].
We aimed to achieve low-cost, stable picrocrocin production by microorganisms. Two previous iGEM teams have tried this, but both have failed.
| 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. |
Table 1: Previous teams that have tried to produce picrocrocin and their results [6][7]
We assembled BBa_K4955001 downstream of BBa_K4955000 assembled in pRK404 [8] and introduced it into the BL21(DE3) strain. After culturing in TB 50 ml culture, the culture medium was analyzed by HP-LC-MS/MS, and picrocrocin production was confirmed.
This was the world’s first success in the biotransformation of picrocrocin from HTCC by heterologous expression and the establishment of a metabolic pathway to synthesize picrocrocin under conditions without the special substrate addition in a heterologous manner.
Biology & Function
UGT709G1: Crocus sativus L.
Measurement
Plasmid
We have assembled BBa_K4955001 downstream of BBa_K4955000 assembled on pRK404 [8] using Gibson assembly.
When zeaxanthin was degraded by the CCD2 enzyme, crocetin dialdehyde and HTCC, the precursor of crocetin, were formed. Our goal was to produce picrocrocin under conditions without specific substrate addition. Since we had confirmed the production of crocetin in BBa_K4955000, we assumed that HTCC was produced and decided to assemble it in the same plasmid.
Engineering success
This plasmids we designed is created through the engineering cycle, which is the core philosophy of iGEM.
Design 1:
From the beginning, our goal was to biosynthesize picrocrocin in E. coli under culture conditions that did not include any special substrates. Therefore, we designed a new metabolic pathway (enzyme group A) that was known to biosynthesize 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) (precursor of picrocrocin) in E. coli under the conditions described above and a new metabolic pathway (enzyme group B) that was known to biosynthesize picrocrocin in vitro from HTCC. We decided to biosynthesize picrocrocin by introducing the metabolic pathway (enzyme group A), which was known to biosynthesize HTCC (precursor of picrocrocin) in E. coli, and the enzyme group B, which was known to convert HTCC to picrocrocin in vitro, into BL21(DE3) strain.
Build 1:
We designed Parts (BBa_K4955000) as enzyme group A and received a plasmid from RIKEN BRC (https://web.brc.riken.jp/en/) cloned into the Duet-1 vector, pRK404. Next, Parts (BBa_K4955001) was designed as enzyme B, synthesized by Teist Bioscience, and cloned into the pET21a vector. The two plasmids were then introduced into the BL21(DE3) strain, which was constructed to have both enzyme group A and enzyme B.
Test 1:
Lycopene, β-Carotene, and Zeaxanthin, which are intermediates in the biosynthetic pathway to HTCC, are carotenoids. If the colonies of the BL21(DE3) strain produced in Build 1 are yellow, the production of the intermediates has been successful, but if the colonies are white, even the production of the intermediates has not been successful.
Learn 1:
When we checked the colonies of the strain produced in Build 1, we found that they were clearly white in color. The color did not change even after several days, indicating that not even carotenoids, which are intermediates in the biosynthetic pathway of picrocrocin, were being produced.
~Design 2:
In Round 1, enzyme group A was designed downstream of the tac promoter, whereas enzyme group B was designed downstream of the T7 promoter; since the T7 promoter is a very strong promoter, only enzyme B was expressed in the cells, possibly suppressing the expression of enzyme group A. The T7 promoter is a very strong promoter.
Build 2:
The plasmid was redesigned so that enzyme group A and enzyme group B were downstream of the same promoter. Then, transformation of BL21(DE3) strain was performed.
Test 2:
As in Round 1, we first checked the production of intermediates in color.
Learn 2:
The color of the colony was confirmed to be yellow. Therefore, we conducted a culture for substance production and analyzed by HPLC-MS, which confirmed the production of picrocrocin.
E. coli strain
BL21(DE3) strain was selected as the host for biosynthesis. The above plasmids were transfected by the heat shock method.
Cultivation Conditions
Transformed E. coli were cultured under the following conditions:
Preculture:
Inoculation: 5 mL of LB, supplemented with the appropriate antibiotic, was inoculated with E. coli and cultured in test tubes.
Conditions: Shaking culture at 140 rpm, 37°C.
Duration: 18-24 hours, after which E. coli were transferred to the main culture.
Main Culture:
Inoculation: 1 mL of the preculture solution was added to 49 mL of TB, supplemented with the appropriate antibiotics.
Conditions: Cultured in a 300 mL Erlenmeyer flask, shaking at 150 rpm, 25°C.
Induction: 0.1 mM IPTG was added when the culture reached an OD600 of 8-12.
Duration: Subsequent incubation for 72 hours.
Identification by HP-LC-MS/MS
Extraction
500 µl of culture medium was suspended in an equal volume of methanol and vortexed for 1 hour.
HP-LC-MS/MS analysis & MS Spectrum
Liquid chromatography was performed on a SHIMADZU Ultra-Fast Liquid Chromatography (UFLC) Nexera system (Shimadzu, Kyoto). The mobile phase conditions were A 0.1% formic acid-H2O, B 0.1% formic acid-acetonitrile (10-100% B for 5 min, 100% B for 2.5 min, and then 10% B for 2.5 min).
A SCIEX Triple TOF X500R system (Sciex, Tokyo) was used as the mass spectrometer.
Picrocrocin [M (C16H26O7)] was analyzed in negative ion mode using the LC-MS analyzer described above and generated an extracted ion chromatogram (EIC) of m/z 329.16058 [M-H]-. A single peak was identified, and the production of picrocrocin was confirmed.
References
[1]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.
[2]Anastasia Kyriakoudi, Stella A Ordoudi, Marta Roldán-Medina and Maria Z Tsimidou.(2015)Saffron, A Functional Spice.Austin Journal of Nutrition and Food Sciences
[3]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.
[4]Majid Shokrpour. (2019).Saffron (Crocus sativus L.) Breeding: Opportunities and Challenges. Advances in Plant Breeding Strategies: Industrial and Food Crops Volume 6 675-706.
[5]Rezaee R, Hosseinzadeh H. 2013. Safranal: from an aromatic natural product to a rewarding pharmacological agent. Iranian Journal of Basic Medical Sciences 16: 12–26.
[6]Team:WashU - 2012.igem.org
https://2012.igem.org/Team:WashU[7]Team:Uppsala - 2013.igem.org
https://2013.igem.org/Team:Uppsala[8] 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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