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Rhodococcus pyridinivorans polyhydroxyalkanoate synthetase, coding gene and application thereof

A technology of polyhydroxyalkanoate and Rhodococcus pyridineophilus, which is applied in the field of genetic engineering, can solve the problem of low content of 3-hydroxyvaleric acid monomer, and achieve the effect of high substrate affinity

Active Publication Date: 2022-02-08
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For this reason, it is necessary to provide Rhodococcus pyridineophilus polyhydroxyalkanoate synthetase and its encoding gene and application, to solve the relatively low content of 3-hydroxyvaleric acid monomer (3-HV) in the PHBV synthesized by existing engineering microorganisms question

Method used

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  • Rhodococcus pyridinivorans polyhydroxyalkanoate synthetase, coding gene and application thereof
  • Rhodococcus pyridinivorans polyhydroxyalkanoate synthetase, coding gene and application thereof
  • Rhodococcus pyridinivorans polyhydroxyalkanoate synthetase, coding gene and application thereof

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Embodiment 1

[0036] Rhodococcus pyridineophilus polyhydroxyalkanoate synthase PhaC Rp Phylogenetic tree construction

[0037] The whole genome of Rhodococcus pyridinivorans P23 has been sequenced, and the polyhydroxyalkanoate synthase PhaC of Rhodococcus pyridinivorans Rp The gene number of the coding gene in the genome is gene3026, and a total of 20 sequences of the published I, II, III and IV polyhydroxyalkanoate synthetases are selected to be related to the polyhydroxyalkanoate of Rhodococcus pyridineophilus described in the present invention. ester synthase PhaC Rp The software MEGA6 was used to construct the phylogenetic tree. From figure 1 Rhodococcus pyridinephila polyhydroxyalkanoate synthase PhaC Rp Between type I and type II polyhydroxyalkanoate synthetases in the phylogenetic tree, it is on a branch alone, indicating that the polyhydroxyalkanoate polymerase PhaC of Rhodococcus pyridineophilus of the present invention Rp The primary sequence has a strong novelty.

Embodiment 2

[0039] Rhodococcus pyridineophilus polyhydroxyalkanoate synthase PhaC Rp Heterologous expression, isolation and purification in Escherichia coli

[0040] A kind of rhodococcus pyridineophilus polyhydroxyalkanoate synthetase PhaC according to the present invention Rp Amino acid residue sequence, add 6×His tag at the C-terminus of the sequence to facilitate the purification of the target protein, and then design the gene sequence according to the codon preference of E. The optimized gene sequence of Rhodococcus pyridine polyhydroxyalkanoate synthetase, the optimized gene sequence after codon optimization and 6×His tag is shown in SEQ ID NO: 3, which is expressed in Escherichia coli with plasmid pET-30a as a vector Source expression of Rhodococcus pyridineophilus polyhydroxyalkanoate synthase PhaC Rp .

[0041] will contain synthetic PhaC Rp Vector pET-30a-phaC encoding gene Rp Transform Escherichia coli BL21(DE3) and grow to OD in LB medium containing kanamycin resistance ...

Embodiment 3

[0043] Heterologous expression of polyhydroxyalkanoate synthase PhaC from Rhodococcus pyridineophilus Rp activity assay

[0044] Mixing 100 μM DL-3-hydroxybutyryl-CoA with 100 nM of heterologously expressed Rhodococcus pyridineophilus polyhydroxyalkanoate synthase PhaC Rp While incubating at room temperature, detect the change of absorbance value at 236nm wavelength, from image 3 OD can be observed 236 Rapid decrease within 2 min, indicating heterologous expression of Rhodococcus pyridineophilus polyhydroxyalkanoate synthase PhaC Rp Has good catalytic activity.

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Abstract

The invention relates to the technical field of gene engineering, and discloses a rhodococcus pyridinophilus polyhydroxyalkanoate synthetase, a coding gene and application thereof. According to the invention, the rhodococcus pyridinophilus polyhydroxyalkanoate synthetase is derived from rhodococcus pyridinophilus, has good sequence novelty, has good short-chain monomer polymerization activity, has better substrate preference on a 3-hydroxyvaleryl coenzyme A monomer, can be applied to microbial chassis cells for synthesis of PHBV, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the rhodococcus pyridineophilus polyhydroxyalkanoate synthetase and its coding gene and application. Background technique [0002] Polyhydroxyalkanoate (PHA) is an aliphatic polyester synthesized by many bacteria as an intracellular carbon source and energy storage substance. PHA is a degradable plastic with good biodegradability. After being discarded into the environment, water and carbon dioxide can be produced by environmental microorganisms. The use of PHA as an environmentally friendly material may solve or reduce a series of environmental problems such as white pollution caused by petroleum-based plastics, and can be used to prepare biodegradable films, containers and other packaging materials. PHA also has the characteristics of tissue compatibility, so it also has great application prospects in the field of tissue engineering materials, and can be used in me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P7/625C12R1/19
CPCC12N9/1029C12N15/70C12P7/625
Inventor 郭文斌
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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