A lycopene-producing rhodobacter sphaeroid engineering strain and its construction method

A technology of Rhodobacter sphaeroides and lycopene, which is applied in the field of metabolic engineering, can solve the problems of complex metabolic regulation, increased production cost and safety, and lack of molecular manipulation technology of B. trispora, and achieves the effect of strong strain stability.

Active Publication Date: 2022-04-05
SHAANXI NORMAL UNIV
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  • Claims
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Problems solved by technology

[0004] At present, the only strain used for industrial production of lycopene is Blakesleatrispora, but Blakesleatrispora lacks effective molecular manipulation technology, and its bacteria are divided into positive and negative bacteria, with complex metabolic regulation. Cyclase inhibitors need to be added when producing lycopene, which undoubtedly increases production costs and safety issues

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  • A lycopene-producing rhodobacter sphaeroid engineering strain and its construction method

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Embodiment 1

[0063] according to figure 1 Lycopene biosynthetic pathway in the shown Rhodobacter sphaeroides, construction of Rhodobacter sphaeroids engineering strains producing lycopene, the specific construction method is as follows:

[0064] 1. crtI of Rhodospirillum rubrum 4 Gene replacement of crtI in Rhodobacter sphaeroides 3 Gene

[0065] Using the gene of Rhodospira rubrum ATCC 11170 (NCBI website: https: / / www.ncbi.nlm.nih.gov / nuccore / NC_007643.1) as a template, the primer crtI was designed according to the principles of overlap extension PCR primer design 4 -F and crtI 4 -R, using the fidelity enzyme Pfu PCR to amplify the phytoene four-step dehydrogenase gene crtI of Rhodospirillum rubrum ATCC 11170 4 , the PCR amplification system is: 2×Pfu PCR mix 10μL, crtI 4 -F (10 μM) 1 μL, crtI 4 -R (10 μM) 1 μL, Rhodospirillum rubrum gene (20 μg / μL) 1 μL, ddH 2 O7 μL, the reaction program was: denaturation at 94°C for 3 min, followed by denaturation at 94°C for 30 s, annealing at 6...

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Abstract

The invention discloses a lycopene-producing rhodobacter bacterium engineering strain and a construction method thereof. The method utilizes the phytoene four-step dehydrogenase gene crtI derived from Rhodospirillum rubrum 4 Replacement of endogenous phytoene three-step dehydrogenase gene crtI in Rhodobacter sphaeroides 3 , and knock out the endogenous streptosporine hydroxylase gene crtC of Rhodobacter sphaeroides and knock out the key gene 6-phosphate glucose dehydrogenase gene zwf of the central metabolic pathway of Rhodobacter sphaeroides, and finally integrate at the position where zwf is knocked out By expressing the endogenous MEP pathway rate-limiting enzyme gene 1-deoxyxylulose-5-phosphate synthase gene dxs of Rhodobacter sphaeroides, an engineering strain of Rhodobacter sphaeroides producing lycopene is obtained, which is fermented and cultivated to produce lycopene The content can reach 10.32mg / gDCW.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and in particular relates to a method for constructing a lycopene-producing strain by transforming Rhodobacter sphaeroides through metabolic engineering. Background technique [0002] Lycopene is a linear polyunsaturated aliphatic hydrocarbon containing 11 carbon-carbon conjugated double bonds and 2 non-conjugated double bonds. Its molecular structure characteristics not only give it a bright red color but also make it a super antioxidant At present, it has been widely used in the fields of functional food, medicine and health care and cosmetics, and the demand in the international market is increasing. [0003] Natural lycopene is mainly derived from plants and microorganisms, but the production cost of plant-derived lycopene remains high, while chemically synthesized lycopene has low biological activity and its safety has been questioned. Due to the characteristics of easy large-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/90C12N1/21C12R1/01
CPCC12N9/0006C12N9/001C12N9/1022C12N15/74C12N15/902C12Y101/01049C12Y103/99028C12Y103/99031C12Y202/01007
Inventor 孟永宏苏安平陈芝李颖强珊
Owner SHAANXI NORMAL UNIV
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