RESEARCH

Curcumin, a natural compound extracted from the ginger plant, is one of the edible natural pigments and a food additive approved by the Codex Alimentarius Commission of the Food and Agriculture Organization of the United Nations.
In addition, it has attracted much attention worldwide for its antioxidant, anticancer and anti-fungal properties. The common method used to extract curcumin from plants often involves the pressing and dissolving method, which employs various organic solvents and likely pollutes the environment.
Also, this method consumes a lot of energy, and the yield is far from meeting the market demand. So we chose to create the engineering E. coli to generate curcumin.

DESIGN

In plants, 4-coumarate coenzyme A ligase (4cl) converts ferulic acid to feruloyl coenzyme A while Acetyl coenzyme A carboxylase (acc) can transform acetyl coenzyme A into malonyl coenzyme A during metabolism. Dcs catalyzes ferulic acid coenzyme A and malonyl CoA to β-ketoyl-CoA, and ferulic acid coenzyme A and β-ketoyl CoA synthesize curcumin by curs.
Therefore, if we insert the genes encoding the four key enzymes 4cl, acc, dcs, and curs into E. coli BL21(DE3) and take the advantage of some Endo diacyl coenzyme A which is produced by E. coli during its own metabolism, we can realize the synthesis of curcumin in E. coli by supplying ferulic acid as a substrate.

BUILD

We extracted RNA from Arabidopsis thaliana and performed reverse transcription to generate cDNA. cDNA was used as a template to clone acc and 4cl genes, which were fused later as a fusion gene in the expression vector pETEXba by a linker, and pETEXba-acc-4cl plasmid was constructed successfully.
In the same way, we cloned the dcs gene from turmeric and curs from fresh ginger, and then fused the two genes into pGEX-MCM vector to construct a pGEX-dcs-curs plasmid.
We finally constructed the engineered Escherichia coli capable of synthesizing curcumin by transforming the above two plasmids into the chassis organism BL21 (DE3).

TEST

We validated the construction of the pGEXMCM-dcs-curs plasmid through antibiotic sensitivity experiments, plasmid size verification, and PCR amplification. Similarly, we validated the construction of the pET28EXba-acc-4cl plasmid through antibiotic sensitivity experiments, plasmid size verification, and PCR amplification. Our next step will be to use engineered bacteria for fermentation to produce curcumin. Prior to this, we explored the experimental conditions for detecting curcumin. Using analytical pure turmeric as the standard, turmeric stem extract was used as the experimental group. We obtained the standard sample with a maximum absorption wavelength of 420 nm, and the experimental group with a maximum absorption wavelength of 417 nm, successfully verifying the presence of curcumin in the extract of turmeric stem block. Subsequently,We investigated the mass spectrum conditions and obtained the mass spectrum information of curcumin.We opened the raw data by the Qualitative Analysis analysis software that comes with the instrument's computer. The molecular weight(M) of curcumin is 368.39, and the molecular formula(Z) is C21H20O6. The mass spectrometry was detected using positive ion mode. The detected mass-to-charge ratio of curcumin is added to the molecular weight of a hydrogen atom, which means that the mass spectrum characteristic of curcumin is mainly [M+H]+.Therefore, the m/z of this curcumin is 369.133. Basing on the molecular weight value of 369.133, we found the detected curcumin in the data and saved the spectrum. The peak graph with this value is the curcumin we detected.

LEARN

At the beginning, we attempted to clone the target genes using primers with restriction endonucleases but it failed. Afterwards, we used primers without restriction endonucleases to succeed to clone four targets' genes acc, 4cl, dcs and curs.
In the process of constructing the vector, we designed the bridging primers and cloned the fused gene fragment using dcs (removing the termination codon) and curs fusion methods.
We also used fusion expression to connect acc (without removing the termination codon) and 4cl to pETEXba at once. However, we found that the cloned fragments had many heterobands and concentrations were too low to be constructed into the vector. Subsequently, the vector was constructed using a separate connection method.
Later, we reviewed the literature and found that curcumin inhibits the growth of Escherichia coli. In order to solve the problem of insufficient growth of bacteria which results in the decrease of curcumin production, we designed a population sensing system: when the number of bacteria reaches a certain level, the curcumin synthesis pathway will be activated; when the number of bacteria decreases to a certain level, the curcumin synthesis pathway will be shut down, so that the number of bacteria will gradually increase. This strategy may realize the autonomous and dynamic regulation of metabolic flux.

REDESIGN

We used the comqxpa quorum sensing system of Bacillus subtilis, which consists of four genes: comx, comq, comp, and coma. Comx encodes a precursor peptide (pre comx) with 55 amino acid residues, which is then modified by the comq gene's encoding product, the isoprene transferase comq, to produce an active isoprene like peptide pheromone comx composed of 10 amino acid residues at its c-terminal.
As the population density of bacteria increases, the pheromone molecule comx released into the extracellular space continues to accumulate. When the pheromone molecule comx reaches a certain threshold, it binds to the transmembrane protein histidine kinase comp and activates the enzyme, promoting itself autophosphorylation.
The phosphorylated comp then transfers the phosphate group to the COMA regulatory protein and activates it. The activated coma protein then binds to the downstream target gene dcs::curs promoter Psrfa and activates the expression of downstream target genes. This receptive system can dynamically regulate the curcumin synthesis pathway and reduce the toxicity of curcumin to chassis organisms.
The curcumin produced by our constructed engineering bacteria can be used for cultural relic restoration, vegetation dyeing, and pigment production.
The engineering bacteria have been systematically optimized to provide a theoretical basis for future generations. Biosynthesis of curcumin is time-saving and efficient, with minimal environmental pollution.