We selected four coding genes acc (encoding acetyl CoA carboxylase, ACC), 4cl (encoding 4-alginate CoA ligase, 4Cl), dcs (encoding diketone CoA synthase, DCS), and curs (encoding curcumin synthesis, CURS) as candidate genes that play important roles in the curcumin synthesis pathway. In plants, 4Cl converts ferulic acid to ferulic CoA, ACC converts acetyl CoA to malonyl CoA, DCS catalyzes the binding of ferulic CoA and malonyl CoA to produce ferulic diketone CoA, CURS catalyzes the conversion of ferulic diketone CoA to β- Ketonic acid condenses with another molecule of ferulic coenzyme A to form curcumin (as shown in Fig 1). We chose pETEXba and pGEXMCM as carriers and cloned four catalytic enzymes into BL21 (DE3) substrate organisms to achieve the goal of high curcumin production (as shown in Fig 2).
Fig 1: The biosynthetic pathway of curcumin.
Fig 2 The recombinant plasmid and engineering bacteria constructed in this project.
Construction of acc-4cl expression system
Design
Cloned acc into the expression vector pET28EXba containing a constitutive, shown in Fig 3.1.
Fig 3.1 The construction of P-acc.
Build
We extracted RNA from Arabidopsis thaliana (as shown in Fig 3.2A) and obtained the entire genome of Arabidopsis thaliana through reverse transcription. Using the entire Arabidopsis genome as a template and amplified acc by polymerase chain reaction (PCR) as shown in Fig 3.2B.
Fig 3.2 (A). Lane 1-3: RNA extracted from Arabidopsis thaliana. (B) Lane 1: DNA Marker, Lane 2: acc gene fragment, and the band size was as expected.
Extracted pET28EXba carrier using alkaline lysis method (as shown in Fig3.3A). According to the restriction endonuclease sites, the corresponding restriction endonucleases were used to digest acc and pET28EXba. The digested pET28EXba vector is shown in Fig 3.3B. After digestion, we used T4 DNA ligase to connect acc and pET28EXba, and then transferred them to DH5α.
Fig 3.3 (A). Lane 1: DNA Marker, Lane 2: pET28EXba. (B) Lane 1: DNA Marker, Lane 2: pET28EXba vector digested with NdeⅠ and XhoⅠ, and the band size was as expected.
Test
Through antibiotic sensitivity experiments (as shown in Fig 3.4A), plasmid size, and validation of target genes (as shown in Fig 3.4B), pET28EXba-acc was successfully obtained.
Fig 3.4 Validation of pET28EXba-acc. (A). The result of resistance screening experiment, the left: DH5α/ pET28EXba-acc ,the right: DH5α. (B). Validation of the plasmid size and PCR amplification of the candidate genes. Lane 1: DNA Marker, Lane 2: pET28EXba, Lane 3: pET28EXba-acc, Lane 4: acc gene fragment, used pET28EXba-acc, as the template and the band size was as expected.
Construction of pET28EXba-acc-4cl expression system
Design
Cloned 4cl into the expression vector pET28EXba-acc containing a constitutive, shown in Fig 4.1.
Fig 4.1 The construction of P-acc-4cl.
Build
Using the entire Arabidopsis genome as a template and amplified 4cl by PCR as shown in Fig 4.2A. Extracted pET28EXba-acc from DH5α (as shown in Fig4.2B). According to the restriction endonuclease sites, the corresponding restriction endonucleases were used to digest acc and pET28EXba-acc. After digestion, we used T4 DNA ligase to connect 4cl and pET28EXba-acc, and then transferred them to DH5α.
Fig 4.2 Lane 1: DNA Marker, Lane 2: 4cl gene fragment, amplified by PCR, and the band size was as expected. (B) Lane 1: DNA Marker, Lane 2: pET28EXba-acc.
Test
Through antibiotic sensitivity experiments (as shown in Fig 4.3A), plasmid size, and validation of target genes (as shown in Fig 4.3B), pET28EXba-acc-4cl was successfully obtained.
Fig 4.3 Validation of pET28EXba-acc-4cl. (A). The result of resistance screening experiment, the left: DH5α, the right: DH5α/pET28EXba-acc-4cl. (B). Validation of the plasmid size and PCR amplification of the candidate genes. Lane 1: DNA Marker, Lane 2: pET28EXba-acc, Lane 3: pET28EXba-acc-4cl, Lane 4: acc gene fragment, Lane 5: 4cl gene fragment, Lane 4 and Lane 5 all amplified used pET28EXba-acc, as the template and the band size was as expected.
Construction of pGEXMCM-dcs-curs expression system
Design
Cloned dcs-curs into the expression vector pGEXMCM, shown in Fig 5.1.
Fig 5.1 The construction of P-dcs-curs.
Build
We purchase and plant turmeric and ginger, extract their RNA (as shown in Fig5.2A), and obtain the entire genome of turmeric through reverse transcription. Using the turmeric genome as a template, the dcs gene was amplified using primers with linkers added. The curs gene was amplified using primers with linkers added to the ginger genome as a template, and then the dcs and curs fragments were fused into the dcs-curs through linkers to form a connection (as shown in Fig 5.2B). Using corresponding restriction endonucleases to digest dcs-curs and pGEXMCM, the digested pGEXMCM carrier is shown in Fig 5.2C. After digestion, we used T4 DNA ligase to connect dcs-curs and pGEXMCM, and then transferred them to DH5α.
Fig 5.2 (A). Lane 1-3: RNA extracted from turmeric. (B) Lane 1-3: RNA extracted from ginger. (C) Lane 1: DNA Marker, Lane 2: dcs gene fragment, Lane 3: zurs gene fragment, Lane 4: fusion fragment dcs-curs, and the band size was as expected.
Test
Through antibiotic sensitivity experiments (as shown in Fig 5.3A), plasmid size, and validation of target genes (as shown in Fig 5.3B), pGEXMCM dcs curs were successfully obtained.
Fig 5.3 Validation of pGEXMCM -dcs-curs. (A). The result of resistance screening experiment, the left: DH5α, the right: DH5α/pGEXMCM-dcs-curs. (B). Validation of the plasmid size and PCR amplification of the candidate genes. Lane 1: DNA Marker, Lane 2: pGEXMCM, Lane 3: pGEXMCM-dcs-curs, Lane 4: dcs-curs gene fragment, amplified used pGEXMCM-dcs-curs, as the template and the band size was as expected.