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Self-flocculation halomonas aydingkolgenesis M1 and application thereof

A self-flocculation, halomonas technology, applied in the direction of bacteria, the use of carriers to introduce foreign genetic material, enzymes, etc., can solve the problems of high energy consumption, low yield, and environmental pollution by waste water discharge.

Active Publication Date: 2020-08-28
北京微构工场生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve many problems such as high energy consumption, low yield and environmental pollution caused by waste water discharge in the production process in the middle and downstream processes of the existing industrial large-scale biological fermentation.

Method used

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  • Self-flocculation halomonas aydingkolgenesis M1 and application thereof
  • Self-flocculation halomonas aydingkolgenesis M1 and application thereof
  • Self-flocculation halomonas aydingkolgenesis M1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Isolation and identification of strains

[0083] 1. Separation

[0084] Soil and water samples were obtained from Aiding Lake, Xinjiang Salt Lake, and the isolates were screened. Well-grown colonies were tested for PHA accumulation by Fourier transform infrared (FI-IR). Ten strains of PHA-producing halophilic bacteria were obtained, one of which was named M1.

[0085] 40-LB medium for isolation and purification of strains: sodium chloride 40g / L, peptone 10g / L, yeast powder 1g / L, pH value is about 8.5;

[0086] The medium for judging whether or not PHA can accumulate is solid mineral glucose (MM-G) medium supplemented with 2% agar.

[0087] MM-G medium:

[0088] 1 liter of MM-G medium was prepared as follows: 20 g glucose, 40 g sodium chloride, 0.5 g yeast powder, 2 g ammonium sulfate, 0.2 g magnesium sulfate, 1.5 g potassium dihydrogen phosphate, 9.65 g dodecahydrate dihydrogen phosphate Sodium, 10 ml of trace element I and 1 ml of trace element II were ...

Embodiment 2

[0104] Example 2: Production of PHB (i.e. P3HB) by non-sterile continuous fermentation using Halomonas aydingkolgenesis M1

[0105] (1) Cultivation of seed solution

[0106] Both the primary and secondary seed liquid medium are 40-LB medium, each liter of 40-LB medium is composed of: 40g sodium chloride, 10g peptone, 5g yeast powder, supplemented with distilled water to 1 liter, sterilized at 121 °C 20 minutes.

[0107] Take 20 μl of Halomonas aydingkolgenesis M1 CGMCCNO.19880 stored in a glycerol tube at -80 °C and transfer it to 20 mL of medium, and culture at 37 °C and 200 rpm overnight to obtain a first-class seed culture solution. The first-level seed culture solution was inserted into a 500mL conical flask equipped with 100mL of culture medium according to 5% of the inoculum, and connected 3 bottles (about 300mL in total), and cultivated at 37 ° C and 200 rpm for about 12 hours to obtain a second-level seed culture. liquid.

[0108] (2) Fermentation process and contro...

Embodiment 3

[0126] Example 3: Using Halomonas aydingkolgenesis M1 as a chassis cell to construct a recombinant bacterium capable of producing P3HB4HB, and using the recombinant bacterium to produce P3HB4HB through non-sterile continuous fermentation

[0127] (1) Construction of recombinant bacteria Halomonas aydingkolgenesis M1-orfZ

[0128] orfZ is the gene encoding 4-hydroxybutyryl-CoA transferase from the chromosome of Clostridium krusei. The function of 4-hydroxybutyryl-CoA transferase is to catalyze the conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA. 4-Hydroxybutyric acid can be obtained from the carbon source substrate by hydrolysis of γ-butyrolactone, and 4-Hydroxybutyryl-CoA can be polymerized into a polymer of 4-Hydroxybutyric acid under the catalysis of PHA polymerase, Including P4HB homopolymer, P(3HB-co-4HB) copolymer, etc. Therefore, it is expected that the Halomonas aydingkolgenesis M1 recombinant strain that can normally express the exogenous orfZ gene has the ab...

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Abstract

The present invention discloses a halomonas aydingkolgenesis strain and an application thereof. The strain is halomonas aydingkolgenesis M1 and has a deposit number of CGMCC NO.19880 in the China General Microbiological Culture Collection Center. Studies show that the halomonas aydingkolgenesis M1can be cultured without sterilization under fermentation conditions in an opening way and can efficiently accumulate polyhydroxyalkanoate (PHA) and / or a small molecule compound of tetrahydropyrimidine, etc. A method for continuous culture of the halomonas aydingkolgenesis M1 is established and a method for co-producing the PHA and tetrahydropyrimidine is successfully established. The method by using the halomonas aydingkolgenesis M1 to produce the PHA and / or tetrahydropyrimidine can realize high yield, is low in production cost, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of industrial biotechnology, and relates to a halophilic bacterium with self-flocculation characteristics and its application in industrial biotechnology. Background technique [0002] Industrial biotechnology is widely used in the production of bio-based chemicals, bio-based materials, bio-fuels, bio-environmental protection and enzyme preparations. In recent years, with the continuous deepening of the concept of green development, industrial biotechnology has become the focus of a new round of technological revolution and industrial transformation in China, and its status and importance in my country's technological innovation strategy has steadily increased. [0003] Microbes are the core of industrial biotechnology, one of the three pillars supporting the development of the biomanufacturing industry, and a key factor in determining production costs. The characteristics of microorganisms determine their culture co...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/62C12P17/12C12N1/21C12N15/74C12R1/01
CPCC12N9/13C12N15/74C12P7/625C12P17/12C12Y208/03C12N1/205C12R2001/01
Inventor 陈国强叶健文吴赴清
Owner 北京微构工场生物技术有限公司
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