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A kind of β-ketothiolase related to the 3-hv monomer synthesis of phbv, its coding gene and application

A technology for encoding genes and amino acids, applied in the field of beta-ketothiolase

Inactive Publication Date: 2011-12-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, international research on the extreme halophilic archaea PHA is still mainly focused on the optimization of the fermentation process, but there are relatively few studies on the genes related to PHA synthesis in such microorganisms, and there is no PHA of the archaeal archaea in public reports. There is no report on the synthesis of related β-ketothiolase (PhaA), and there is no report similar to the modification of these related genes in bacteria through genetic engineering techniques to seek PHA with better performance and achieve low-cost production of PHA

Method used

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  • A kind of β-ketothiolase related to the 3-hv monomer synthesis of phbv, its coding gene and application
  • A kind of β-ketothiolase related to the 3-hv monomer synthesis of phbv, its coding gene and application
  • A kind of β-ketothiolase related to the 3-hv monomer synthesis of phbv, its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Obtaining of β-ketothiolase and its coding gene

[0038] The bacterial strain was isolated from the bottom mud of the salt pond in Tianjin sea salt field, and the culture medium used for the separation was AS-168 medium (containing: 5.0g casamino acids per liter, 5.0g yeast extract, 1.0 g sodium glutamate (sodium glutamate), 3.0g sodium citrate, 200g NaCl, 20g MgSO 4 ·7H 2 O, 2.0g KCl, 0.36g FeSO 4 4H 2 O and 0.36 mg MnCl 2 4H 2 O, pH 7.2). An extreme halophilic archaeal strain XH1001 was isolated. The strain can form reddish colonies when grown on AS-168 agarose plate (AS-168+1.2% agarose). Colonies are opaque with smooth edges. The bacterium is Gram-negative, and the cells are usually 1 to 2 μm wide and 2 to 3 μm long by electron microscopy. Physical and chemical experiments found that the optimum growth NaCl concentration of XH1001 is 20% to 25%, and it can still grow at 10% NaCl concentration; the growth pH range is 5.2 to 8.0; it is aerobic; it ...

Embodiment 2

[0041] Example 2, functional identification of phaA1

[0042] 1. Transcription of gene phaA1 in fermentation medium

[0043] 1. Extraction of total RNA from Haloferax sp.XH1001 CGMCC No.3822 in the fermentation medium

[0044] 1) Put Haloferax sp.XH1001 CGMCC No.3822 in AS-168 medium (each liter contains: 5.0g acid hydrolyzed casein (casamino acids), 5.0g yeast extract (yeast extract), 1.0g sodium glutamate ( sodiumglutamate), 3.0g sodium citrate, 200g NaCl, 20g MgSO 4 ·7H 2 O, 2.0g KCl, 0.36g FeSO 4 4H 2 O and 0.36 mg MnCl 2 4H 2 O, the rest is water, pH 7.2) and cultivated at 37°C for 3-4 days to make it enter the logarithmic growth phase.

[0045] 2) According to the inoculation amount of 5%, inoculate (Haloferax sp.) XH1001 CGMCC No.3822 in MG (containing: 200g NaCl, 20g MgSO 4 ·7H 2 O, 2.0g KCl, 1g sodium glutamate, 37.5mg KH 2 PO 4 , 50mg FeSO 4 ·7H 2 O, 0.36 mg MnCl 2 4H 2 O, 1g of yeast extract (yeastextract), 20g of glucose (glucose, pH 7.2) in the cult...

Embodiment 3

[0101] Embodiment 3, phaA1 gene application

[0102] The recombinant plasmid pWL102-A constructed in Step 2 of Example 2 was transformed into the wild-type strain Haloarcula hispanica CGMCC No.1.2049 (purchased from CGMCC, The strain number is CGMCC No.1.2049), and the obtained recombinant strain is named Haloarcula hispanica / pWL 102-A.

[0103] Fermentation and analysis were carried out according to the method described in Example 2. Using the same method, the recombinant bacteria obtained by introducing the empty vector pWL102 into Haloarcula hispanica CGMCC No.1.2049 (named Haloarcula hispanica / pWL102) were fermented and analyzed as a control. The wild-type strain Haloarculahispanica CGMCC No.1.2049 without any treatment was also used as a control.

[0104] The experiment was repeated 3 times, and the results were averaged. Experimental results such as Figure 5 A is the result of polyhydroxybutyrate copolyester produced by Haloarculahispanica / pWL102 (or the result of p...

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Abstract

The invention provides a research and an application of beta-ketothiolase in extreme halophilic archaeon, wherein beta-ketothiolase is related to 3-HV monomer synthesis of a biodegradable material PHBV. The protein provided by the invention is a protein showed by the following description (1) or (2): (1) a protein composed of an amino acid sequence represented by a sequence 1 in the sequence table; (2) a protein formed after the amino acid sequence represented by the sequence 1 is processed after substitutions and / or deletions and / or additions of one or more amino acid residues, wherein the obtained protein is derived from the protein represented by (1), and is PHBV and / or 3-HV related. As a result of experiments, when the coding gene of the poly(3-hydrobutyrate-co-3-hydroxyvalerate) and / or 3-hydroxyalkanoate synthesis related protein is introduced into host bacteria, the molar ratio of generated 3-HV monomer for synthesizing PHBV is greater than 3-HV generated from the host bacteria.

Description

technical field [0001] The invention relates to the research and application of a β-ketothiolase related to the synthesis of 3-HV monomer of degradable material PHBV in extreme halophilic archaea. Background technique [0002] Many prokaryotes, including bacteria and some extreme halophilic archaea, can accumulate polyhydroxyalkanoate (PHA) in their cells under the culture conditions of excess carbon source and limited nitrogen, phosphorus, sulfur, oxygen and other elements. PHA is a kind of biological polyester composed of 3-hydroxy fatty acid (3HA), which is mainly used as a storage material for carbon source, energy and reducing power. PHA has physical and chemical properties similar to traditional petrochemical plastics such as polyethylene and polypropylene, and it is completely biodegradable and synthesized from renewable resources. It is considered an environmentally friendly "biodegradable plastic" ", can alleviate the problem of "white pollution" caused by petroleu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P7/62C12P7/42C12R1/01
Inventor 向华冯博韩静
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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