Team:FAFU-CHINA/Design
Overview
In order to really make the microorganisms produce floral substances, we began our investigation with the
screening of individual scented substances. We found that only compound aromas can simulate the fragrance of
flowers, and dynamic
changes of aroma components can give the flavor verve.
In order to achieve these goals, we designed a photoinduced co-culture system to produce incense.
Fragrance survey
Referring to the aroma description of aromatic substances and the difficulty of synthesis, we targeted neroli
and linalool as the target products.
Neroli: A steady and balanced fragrance that brings light, sweet and pleasant sensations
Linalool: With sweet wood smell, like rose wood.Bouquet of lilac, lily of the Valley and rose, woody, fruity
notes.The aroma is soft, light and long lasting.
This can be achieved by combining nerolol with linalool and allowing the ratio of the two components to change
dynamically over time.
Photoinduced Electron Transfer Systems
Our goal is to achieve open-loop regulation and automatic rhythm.
Photoperiod gave us the idea that we could use the dynamics of light intensity throughout the day as input to
the system. And we can do that by making the substrate organisms that produce linalool and neroli respond in
reverse to the light
signal.
Coculture system
Neroli and linalol inhibit the growth of E.coli, so we wanted to reduce the burden of E.coli, and assigned the
task of producing neroli to S. cerevisiae. In the co-culture system of E.coli and yeast, the competition
between E.coli and yeast could be eliminated by adding xylose. They can do their job in the medium without
interfering with each other.
Construction of strain
The construction of E.coli is relatively simple. The traditional idea is that the production of different
aroma substances can be achieved by regulating the endogenous MVA pathway and cutting into different foreign
enzyme genes[1]
MVA pathway exists in yeast, and IPP can be enriched by strain optimization. We got in touch with Mr. Sun Jie
from Zhejiang University of Technology, who agreed to provide ipP-enriched saccharomyces cerevisiae
strain[2] for our
IGEM
experiment. Then all we have to do is transfer the genes that produce the aroma into yeast.
Culture medium design
The medium needs to eliminate competition from E. coli and yeast for carbon sources and use inorganic nitrogen sources. Czapek - Dox Medium was redesigned and its performance was verified.A synthetic medium [3]composed of :
| Culture medium design | ||
|---|---|---|
| 20g/L glucose | 8g/L xylose | 1.00 g /LMgSO4·7H2O |
| 1.10 g/L KCl | 0.15 g/L CaCl2·2H2O | 1.00 g /L(NH4)2HPO4 |
| 8.75 g/L(NH4)2SO4 | 60.3 mg/L myo-inositol | 30.0 mg/L Capantothe-nate |
| 6.0 mg/L thiamine HCl | 1.5 mg/L pyridoxine HCl | 20 mg/L Leucine |
| 20 mg/L Histidine | 20 mg/L Uracil | 15.0 mg/L FeCl3·6H2O |
| 10.6 mg/L MnCl2, | 9.0 mg/L ZnSO4·7H2O | 20mg/L Leucine |
| 20mg/L histidine | 20mg/L Uracil | 2.4mg/L CuSO4 ·2H2O per liter of water |
Reference
[1] Xia Y, Li K, Li J, Wang T, Gu L, Xun L. T5 exonuclease-dependent assembly offers a low-cost method for
efficient cloning and site-directed mutagenesis. Nucleic Acids Res. 2019 Feb 20;47(3):e15.
[2] Qu Z, Zhang L, Zhu S, Yuan W, Hang J, Yin D, Tang X, Zheng J, Wang Z, Sun J. Overexpression of the
transcription factor HAC1 improves nerolidol production in engineered yeast. Enzyme Microb Technol. 2020
Mar; 134:109485.
[3] Hanly TJ, Urello M, Henson MA. Dynamic flux balance modeling of S. cerevisiae and E.coli co-cultures for
efficient consumption of glucose/xylose mixtures. Appl Microbiol Biotechnol. 2012 Mar;93(6):2529-41.