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Octahydrolycopene dehydrogenase mutant and application thereof

A technology of phytoene and dehydrogenase, which is applied in the field of biosynthesis and can solve problems such as structural analysis

Active Publication Date: 2020-10-30
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The current research on the functional diversity of CrtI usually screens out mutants with different dehydrogenation steps from the original dehydrogenase through random mutations, without in-depth structural analysis of these mutation sites

Method used

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  • Octahydrolycopene dehydrogenase mutant and application thereof
  • Octahydrolycopene dehydrogenase mutant and application thereof
  • Octahydrolycopene dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Determines the mutation site that affects the catalytic function of BtCrtI

[0031] In the previous study (CN201910308068), the BtCrtI mutants in the two mutant strains yCC08 and yCC09 we screened contained two point mutations, namely H136R&Y160F; H453R&N576S. In order to determine the mutation sites that affect the catalytic function of BtCrtI, and to explore the synergy between different mutation points, we constructed four single-point mutant strains and nine combined mutant strains according to the method in the patent (CN201910308068). The bacterial numbers and corresponding CrtI mutant types are listed in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] Shake flask fermentation according to the method in the patent (CN201910308068), the result is as follows figure 1 , figure 2 , as shown in Table 2:

[0036] in conclusion:

[0037] 1. H136R and H453R are the key mutation sites affecting the dehydrogenation function of BtCrtI mutants in...

Embodiment 2

[0043] Example 2 The second round of error-prone PCR mutation screening for H136R and H453R

[0044] In order to obtain CrtI mutants with better performance, we performed the second round of error-prone PCR on the basis of H136R and H453R determined by the first round of error-prone PCR. The mutant strains screened are shown in Table 3:

[0045] table 3

[0046]

[0047]

[0048] Shake flask fermentation results are as follows image 3 Shown:

[0049] The second round of error-prone PCR did not screen out mutants with better performance. However, in the single-point verification test, it was found that the mutation points T545A, A289V, A355V, N148D, A289V&A355V can significantly improve the catalytic activity of CrtI, and the yield of lycopene has also been greatly improved. possible. The strains and fermentation results constructed by the single-point verification test are shown in Table 4, Figure 4 and table 5:

[0050] Table 4

[0051] strain descr...

Embodiment 3

[0057] Example 3 Exploring the Structural Features of Regulating CrtI Function

[0058] In order to explore the evolution rules of phytoene dehydrogenases from different sources at the H136 and H453 sites. We performed phylogenetic tree analysis and sequence alignment of phytoene dehydrogenases from bacteria, fungi, cyanobacteria, and plants. We found that phytoene dehydrogenases in RsCrtI, RcCrtI, and RaCrtI can catalyze a three-step dehydrogenation reaction, and the amino acid corresponding to the H136 position of BtCrtI is arginine; phytoene in cyanobacteria and plants The dehydrogenase can catalyze the two-step dehydrogenation reaction, and the amino acid corresponding to the H453 site of BtCrtI is also arginine, which is consistent with our experimental results, so we speculate that the two sites H136 and H453 are phyto Key structural sites of erythrin dehydrogenases that cause functional differentiation in natural evolution.

[0059] Then we analyzed the structural fea...

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Abstract

The invention relates to the field of biosynthesis, in particular to an octahydrolycopene dehydrogenase mutant and application thereof. Based on a BtCrtI mutant strain screened through error-prone PCRin the earlier stage, single-point and multi-point recombination verification is conducted on contained mutation sites, and key sites influencing BtCrtI catalytic specificity are determined; saturation mutation is performed on the screened BtCrtI key sites so as to increase the functional diversity of the BtCrtI mutant; principal component analysis (PCA) and K-means clustering analysis are performed on a BtCrtI single-point saturated mutation result, the single-point mutants are classified, and different types of representative single-point mutations are combined to further analyze the diversity of a BtCrtI catalytic function.

Description

technical field [0001] The invention relates to the field of biosynthesis, in particular to phytoene dehydrogenase mutants and applications thereof. Background technique [0002] Carotenoids are a large family of pigments widely distributed in nature, and more than 750 carotenoid molecular structures have been determined so far. In recent years, carotenoids have important commercial potential in medicine, food coloring, animal feed, etc. due to their excellent dyeing and antioxidant abilities. In the biosynthetic pathway of carotenoids, phytoene is the initial C40 skeleton compound with 9 double bonds. The subsequent sequential multi-step dehydrogenation catalyzed by phytoene dehydrogenase provides abundant precursors for the synthesis of diverse physiologically active carotenoids. Therefore, it is of great significance to explore the structural sites that affect the catalytic specificity of phytoene dehydrogenase for the customized synthesis of dehydrogenation products an...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12P5/02
CPCC12N9/001C12P5/007C12Y103/99
Inventor 元英进梁楠姚明东肖文海王颖
Owner TIANJIN UNIV
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