Naringenin product addiction device and application thereof
A naringenin and addiction technology, applied in the field of naringenin product addiction device, can solve the problems of degradation of production traits, unstable production strains, stress and the like, and achieve the effect of stable production traits
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[0022] The preparation method of the naringenin product addiction device of the present invention is not particularly limited, and preferably includes: placing the FdeR binding site FdeO upstream of a constitutive promoter, constructing a naringenin-inducible chimeric promoter, and incorporating the The inducible chimeric promoter was cloned between the AvrII / XbaI restriction sites of the vector pYLXP'2 to replace the original TEF promoter in the vector pYLXP'2; the LEU2 gene was inserted into the SnaBI site of the vector plasmid obtained above , resulting in plasmid pOTEF(136)-LEU2. The genes encoding transcription factors were cloned into the SnaBI sites of plasmids pYaliJ1, pYaliL1 and pOTEF(136), respectively, to obtain plasmids pYaliJ1-FdeR, pYaliL1-FdeR and pOTEF(136)-FdeR. Use AvrII / SalI to digest pYaliJ1-FdeR, pYaliL1-FdeR, and pOTEF(136)-FdeR, respectively, use NheI / SalI to digest pOTEF(136)-LEU2, and finally digest the AvrII / SalI-digested donor plasmid with NheI / Th...
Embodiment 1
[0026] Raw materials: plasmids pYLXP' and pYLXP'2, Yarrowia lipolytica Po1f was purchased commercially, and NarPro / ASC_Rep was a Yarrowia lipolytica strain that synthesized naringenin.
[0027] Yarrowia lipolytica Po1f and NarPro / ASC_Rep were used as the chassis cells to verify the reliability of the product addiction device. Placement of the FdeR-binding sequence FdeO upstream of TEF(136) resulted in a chimeric promoter P OTEF(136), these sequences were synthesized by Genewiz Company. Use the primer pair OTEF(136)F / OTEF(136)R to clone the artificially synthesized DNA sequence, and use Gibson assembly to clone it between the AvrII / XbaI sites of the vector pYLXP'2, replace the TEF promoter therein, The plasmid pOTEF(136) was obtained. The LEU2 gene was cloned using the primer pair LEU2 F / LEU2 R and inserted into the SnaBI site of the plasmid using Gibson assembly to obtain plasmid pOTEF(136)-LEU2. The transcription factor encoding gene FdeR was synthesized by Genewiz Company...
Embodiment 2
[0034] Different strength promoters control the expression of FdeR gene and analysis of its addiction effect
[0035] In order to obtain higher addiction and lower background expression, thereby reducing the escape of low-yielding bacteria, promoters of different strengths were used to control the expression of the transcription factor FdeR ( figure 2 middle a-c). These naringenin-addicted devices were respectively transformed into Yarrowia lipolytica Po1f, and the reliability of the product-addicted devices was analyzed on a leucine-deficient CSM-Leu plate. The formulation of the leucine-deficient CSM-Leu plate is shown in Table 3. The results showed that when the constitutive promoter POX4 and IDP2 were used to control the expression of FdeR, the bacteria could grow normally on the plate containing naringenin ( figure 2 Middle g and h); while on the plate without naringenin, no single colony was obtained ( figure 2 middle d and e). When the inducible promoter OTEF (13...
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