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Phytoene dehydrogenase mutant gene and application thereof in carotenoid synthesis

A technology of phytoene and lycopene, applied in the field of genetic engineering, can solve problems such as structure and binding mechanism that have not yet been resolved

Inactive Publication Date: 2018-05-15
ZHEJIANG OCEAN UNIV
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Problems solved by technology

[0003] However, the structure and binding mechanism of phytoene dehydrogenase CrtI to the substrate have not yet been resolved

Method used

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Experimental program
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Embodiment

[0012] Embodiment: at first will be derived from aquatic paracoccus Agrobacterium aurantiacum phytoene dehydrogenase encoding gene crtI Insert into Yarrowia lipolytica integrated expression single-copy vector pINA1269, and then use error-prone PCR technology to crtI Carry out mutations, verify and screen forward mutants by observing the primary screening method and HPLC method of colony color changes of lycopene-producing strains, and sequence the target forward mutants.

[0013] Through the above method, the A178S mutant was obtained in the first round of directed evolution, and the yield of lycopene was increased by 27.3%. Then, based on the directed evolution of this mutant, two mutants, A178S / N280T and A178S / M234I, were obtained. The yield of lycopene was further increased by 51.4% and 18.3%, but the combination of A178S, N280T and M234I failed to further increase the yield of lycopene; continued to perform directed evolution on the A178S / N280T mutant, and obtained the ...

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Abstract

The invention discloses a phytoene dehydrogenase mutant gene and application thereof in carotenoid synthesis. The mutant gene is obtained by performing multiple rounds of mutation on a phytoene dehydrogenase coding gene by means of iterative orthogenesis and through a high-throughput screening method based on bacterial colony color change. When the phytoene dehydrogenase mutant gene is used to synthesize lycopene, A178S mutant is obtained in the first round of orthogenesis, and lycopene yield is increased by 27.3%; A178S / N280T mutant and A178S / M234I mutant are obtained by using the A178S mutant as a base for orthogenesis, and lycopene yield is further increased by 51.4% and 18.3% while the lycopene yield is not further increased by combining A178S, N280T and M234I; A178S / N280T / E239P mutantis obtained by performing orthogenesis on the A178S / N280T mutant, and A178S / N280T / E239P mutant is finally obtained through three rounds of orthogenesis and screening.

Description

technical field [0001] The invention relates to a phytoene dehydrogenase mutant gene and its application in carotenoid synthesis, belonging to the technical field of genetic engineering. Background technique [0002] In the process of carotenoid biosynthesis, phytoene dehydrogenase catalyzes the continuous four-step dehydrogenation reaction from phytoene to lycopene, and is the key rate-limiting enzyme of the pathway. Therefore, improving the catalytic activity of phytoene dehydrogenase to obtain a preferred phytoene dehydrogenase is of vital significance for improving the productivity of carotenoid biosynthesis pathway. [0003] However, the structure and binding mechanism of phytoene dehydrogenase CrtI binding to the substrate have not been resolved yet. In the absence of enzyme structure or catalytic mechanism information, directed evolution technology is a powerful tool to improve enzyme catalytic performance. Therefore, obtaining a preferred phytoene dehydrogenase by ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12P5/02
CPCC12N9/001C12P5/007C12Y103/99031
Inventor 陈艳丁国芳杨最素余方苗唐云平黄芳芳
Owner ZHEJIANG OCEAN UNIV
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