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Engineering cyanobacteria for biosynthesis of p-coumaric acid and preparation method of engineering cyanobacteria

A technology for biosynthesis and stearic acid, applied in the field of bioengineering, can solve the problems of long growth cycle, low yield, low yield, etc., and achieve the effects of fast growth, mature genetic manipulation technology, and cost reduction.

Active Publication Date: 2021-04-16
江苏甬泽生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Through the above analysis, the existing problems and defects of the prior art are: the main sources of the existing p-coumaric acid are plant extraction, chemical synthesis and biosynthesis, and plant extraction faces problems such as long growth cycle, low yield and great impact on the environment. , while chemical synthesis faces problems such as high energy consumption, low yield and environmental pollution

Method used

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  • Engineering cyanobacteria for biosynthesis of p-coumaric acid and preparation method of engineering cyanobacteria
  • Engineering cyanobacteria for biosynthesis of p-coumaric acid and preparation method of engineering cyanobacteria
  • Engineering cyanobacteria for biosynthesis of p-coumaric acid and preparation method of engineering cyanobacteria

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Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1: The carrier construction that is used to produce p-myrisin engineering cyanobacteria

[0048] In order to confirm the role of alaninase and cinnamic acid-4-hydroxylase in the synthesis of p-myrisin in cyanobacteria, and to increase the production of p-myrisin in cyanobacteria by increasing the expression of the enzymes, a The vector pCA0168 used to integrate the phenylalaninase gene (PAL) and the cinnamic acid-4-hydroxylase gene (C4H) driven by the Pcpc560 promoter into the cyanobacterial genome at the slr0168 site. In order to further increase the expression level of alaninase and cinnamic acid-4-hydroxylase so as to improve the output of p-maric acid synthesized by cyanobacteria, a phenylalanase gene (PAL) for driving by the Pcpc560 promoter was constructed. and the cinnamic acid-4-hydroxylase gene (C4H) were integrated into the vector pCA2081 at the slr2081 site of the cyanobacteria genome.

[0049] Above-mentioned carrier transforms cyanobacteria PCC6...

Embodiment 2

[0064] Embodiment 2: Transformation of cyanobacteria and screening of transformants

[0065] (1) Cyanobacterium PCC6803 was cultured statically in BG11 medium under continuous light conditions with a temperature of 30°C and a light intensity of 30 μE m-2s-1. Glucose at a final concentration of 5 mM was added to BG11 during mixed culture. If passaging or screening on a solid plate, add agar powder to a final concentration of 1.4%, TES-NaOH (pH8.0) to 8mM, Na 2 S 2 o 3 to 0.3% and glucose to 5mM.

[0066] (2) Take 10 mL of cyanobacterial cells in the logarithmic growth phase (OD730 is about 0.5-1.0), and collect the cells by centrifugation; wash the cells twice with fresh BG11 medium, and then resuspend the cells in 1 mL of BG11 medium (1.5 g / L NaNO 3 , 40mg / L K 2 HPO 4 ·3H 2 O, 36mg / L CaCl 2 2H 2 O, 6mg / L citric acid, 6mg / L ferric ammonium citrate, 1mg / L EDTA disodium salt, 20mg / L NaCO 3 , 2.9mg / LH 3 BO 3 , 1.8mg / LMnCl 2 4H 2 O,0.22mg / LZnSO 4 ·7H 2 O, 0.39mg / LN...

Embodiment 3

[0075] Embodiment 3: the output of cyanobacteria of genetic engineering transformation

[0076] 1. Experimental procedure: use a transparent conical cell culture flask, fill with 150mL liquid BG11 medium (including the corresponding resistance mentioned above), the initial inoculation concentration is OD730 = 0.1, at 30°C, 30μEm-2s-1 light conditions After culturing under continuous light for one week, when the OD730 reached 1.5, the cell pellet and supernatant were collected by centrifugation, and the cells were broken with a cell disruptor, and used for detection by high performance liquid chromatography.

[0077] 2. Experimental results

[0078] The present inventors respectively detected the production of p-amyric acid in cyanobacteria PCC6803 and SYN005, the production of p-amyric acid was not detected in the wild-type cyanobacteria PCC6803, and the production of p-amyric acid was detected in SYN005. Figure 4 and Figure 5 show the growth of cyanobacteria PCC6803 and SYN0...

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Abstract

The invention belongs to the technical field of bioengineering, and discloses engineering cyanobacteria for biosynthesis of p-coumaric acid and a preparation method of the engineering cyanobacteria. A carrier for biosynthesis of p-coumaric acid comprises a phenylalaninase gene and a cinnamate- 4-hydroxylase gene which are operably connected by a promoter and have activity in cyanobacteria; and the phenylalanase gene encodes a protein having phenylalanase activity, and the cinnamate -4-hydroxylase gene encodes a protein having cinnamate- 4-hydroxylase activity. According to the method, carbon dioxide is fixed in the photosynthetic microorganism cyanobacteria through solar energy to synthesize the p-coumaric acid, energy for synthesizing the p-coumaric acid comes from solar energy, a carbon source comes from the carbon dioxide, and compared with plant extraction or chemical synthesis, the cost can be effectively reduced; and the plant natural product prepared by the technology is not limited by insufficient raw materials, the growth speed of microorganisms is high, the genetic manipulation technology is mature, and the fermentation process does not pollute and damage the environment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an engineering cyanobacteria for biosynthesizing p-myrisic acid and a preparation method thereof. Background technique [0002] At present, p-coumaric acid, also known as "4-hydroxycinnamic acid", is one of the natural products widely present in plants in nature. P-maric acid mainly exists in fruits, vegetables, grains and fungi, especially in Chinese herbal medicines. As an important precursor compound of phenylpropanoids, stilbenes and flavonoids, p-maric acid has anti-oxidation, anti-inflammatory, anti-tumor, anti-platelet aggregation, cardiovascular protection, prevention and improvement of diabetes and neuroprotective effects And other biological activities beneficial to the human body have a wide range of application values ​​in biomedicine, cosmetics and food industries. In addition, p-coumaric acid is an intermediate product of the secondary metabolic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/60C12N15/53C12N9/88C12N9/02C12P7/42C12R1/01
CPCY02P20/59Y02P20/133
Inventor 高恶斌詹诸明
Owner 江苏甬泽生物科技有限公司
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