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A mutant of cytochrome p450s

A technology of cell lines and precursors, applied in the field of bioengineering, to achieve broad industrial practical prospects and high-efficiency catalytic activity

Active Publication Date: 2022-02-25
SHANXI AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method may produce cytotoxicity, so balancing the expression ratio of P450s and its chaperones is also critical for electron chain transmission (Renault et al., 2014b)

Method used

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  • A mutant of cytochrome p450s
  • A mutant of cytochrome p450s
  • A mutant of cytochrome p450s

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, protein expression and detection

[0045] Mutated P450 to obtain several mutants, such as V1 (L48F-S49A-I61F-L120T-T352K-L356P), V2 (L48F-S49A-I61F-L120T-K352I-L356P), etc., and further obtained The mutant V2-W119I-L125D that can efficiently produce the mogroside precursor 11H-Cuol.

[0046] These mutants were transformed into yeast expression strain INVSc1, induced to express for 12 hours, the total yeast protein was extracted by trichloroacetic acid-acetone method, and the expression of mutant protein was detected by western blotting. The detection method of trichloroacetic acid-acetone method is as follows: take 5OD yeast. Resuspend with 1ml alkaline lysate (0.25M NaOH+1% β-mercaptoethanol), place on ice for 10min; add 160ul 50% TCA, mix well, place on ice for 10min, centrifuge at 14000g for 10min; fully discard the supernatant, add 1ml Resuspend the pellet in pre-cooled acetone, place it on ice for 10 minutes, and then centrifuge at 14,000g for 10 mi...

Embodiment 2

[0047] Embodiment 2, metabolite detection

[0048] First, in order to ensure sufficient substrate cucurbitadienol, the yeast strain INVSc1 was modified. These four key enzymes, HMGR, ERG20, ERG9, and ERG1, are crucial in the biosynthetic pathway of 2,3-oxidized squalene. These four genes were integrated into the yeast strain chromosome and overexpressed (enhancing the production of cucurbitadienol content). On this basis, transfer into epoxy squalene synthase and P450 redox chaperone CPR (Csa1G423150).

[0049] Then, the enzyme mutant was transformed into the EY10-Bi chassis yeast containing the redox partner CPR, induced for 48 hours, the same quality yeast was taken, and the total yeast metabolites were extracted by alkaline lysis method, and the compounds in the synthetic pathway were detected by HPLC-qTOF (the yield of target product 11H-Cuol and by-products 11C-Cuol, 11C-20H-Cuol). The important detection parameters of metabolites are as follows: Q-TOF system and APCI ...

Embodiment 3

[0052] Embodiment 3, the effect of other mutants

[0053] The P450s mutant of the present invention is based on the amino acid sequence of CPY87D20 of cucumber (cucumis sativus), as shown in SEQ ID No.1, and multiple mutants have been obtained successively, for example: mutant V1, the amino acid sequence of which is shown in SEQ ID No.2 Shown; Mutant V2, the amino acid sequence is shown in SEQ ID No.3; Mutant V3, the amino acid sequence is shown in SEQ ID No.4.

[0054] For another example, mutants further mutated on the basis of the above mutants: V2-I46L-A49L, V2-W119I-L125D, V2-R385Y, V2-W399K, V2-I439H, V2-E463P, V2-I46L-A49L- C343Y, V3-C343Y, V3-C343Y-S49L, V3-C343Y-I46L, V3-K73Y, V3-F89D, V3-Y432E, V3-L125D, V3-R383T, V3-W399D, etc., in the embodiment 1-2 Batch detection under the same conditions found that both the yield of the target product 11H-Cuol and the substrate selection specificity of the enzyme or the protein expression level were significantly higher than th...

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Abstract

The present invention relates to a mutant of cytochrome P450s. The mutant can not only be highly expressed in heterologous hosts, but also can significantly improve the substrate selection specificity and the yield of the target product 11H-Cuol, which strongly promotes the development of mogroside V industrial synthesis technology.

Description

technical field [0001] The invention relates to a mutant of cytochrome P450s, which belongs to the field of bioengineering. Background technique [0002] A diet high in sugar, whether it is natural or synthetic sugar, will cause a series of modern health problems, such as obesity, diabetes, and cardiovascular disease (Sievenpiper, 2014). Therefore, natural non-sugar substances derived from plants have become a new generation of sweeteners to meet the sweetness demand due to their advantages of high sweetness, low calorie, and high safety. Among them, the main active substance of Luo Han Guo, mogroside V, is 250-300 times sweeter than sucrose, low in calories, and has pharmacological activities such as antitussive and expectorant, hypoglycemic, anticancer, and antioxidative (Kasai et al., 1989). Therefore, mogroside V can be widely used in various foods instead of sucrose as a sweetener, and as a sugar substitute for obesity or diabetes patients. However, the growth cycle o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N5/10C12P33/00
CPCC12N9/0081C12P33/00C12Y114/15006C12N15/81Y02A50/30
Inventor 梅超张晓鹏宋倩娜王慧杰霍利光
Owner SHANXI AGRI UNIV