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Synechocystis-6803 genetically engineered bacterium capable of producing 3-hydroxypropionic acid, and construction method and application thereof

A technology of genetically engineered bacteria and trihydroxypropionic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of complex separation and purification of cyanobacteria that have not yet been reported on genetically engineered bacteria that produce trihydroxypropionic acid , Large-scale industrial application gap and other issues

Inactive Publication Date: 2015-07-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 3-HP is traditionally produced mainly by chemical synthesis, but the separation and purification of the product is more complicated, and the production cost is correspondingly higher. It can only be synthesized in a small amount for laboratory use, and there is a big gap from large-scale industrial application.
Therefore, it is of great significance to use Synechocystis sp. 6803 as the host to produce 3-HP through genetic modification, but there is no report on the genetic engineering of trihydroxypropionate-producing cyanobacteria.

Method used

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  • Synechocystis-6803 genetically engineered bacterium capable of producing 3-hydroxypropionic acid, and construction method and application thereof
  • Synechocystis-6803 genetically engineered bacterium capable of producing 3-hydroxypropionic acid, and construction method and application thereof
  • Synechocystis-6803 genetically engineered bacterium capable of producing 3-hydroxypropionic acid, and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Construction of Synechocystis 6803 Genetically Engineered Strain SMPA Producing Trihydroxypropionic Acid:

[0028] (1) In vitro amplification of the target gene:

[0029] Extraction of the genome of C. aurantiacus using bacterial genome extraction kit

[0030] Using SEQ ID No.1 and SEQ ID No.9 in the sequence list as the upstream and downstream primers, and the genome of C. aurantiacus as a template, the malonyl-CoA reductase gene mcr was amplified by PCR. The sequence of the gene mcr was used SEQ ID No. 17, shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8. The sequences shown are the upstream primers, respectively, with SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16. The sequences shown are the downstream primers. The Bacterial Genome Extraction Kit was used to extract the Synechocystis 6803 genome, and the Synechocystis 6803 genome was used as a templat...

Embodiment 2

[0042] Conditions for 3-HP production in the constructed strain SMPA

[0043] The constructed strain SMPA was cultured in normal BG 11, and the setting parameters of the shaker were the light intensity of 2000 Lux, the rotating speed of 130 rpm, and the temperature of 30°C. Take OD during vaccination 730nm Add 5 mL of 0.2 fresh cells to 20 mL of culture medium. Make 3 parallel samples in each group. Use a UV-1750 spectrophotometer to measure the absorbance at a wavelength of 730 nm. 730nm When it is 1.0, centrifuge 25mL bacterial solution at 900xg for 15min, resuspend with 10mL fresh BG11 medium, cultivate and produce 3-HP, and add 1M NaHCO every day 3 0.5mL, the 3-HP production was measured after 6 days.

Embodiment 3

[0045] Expression and identification of malonyl-CoA reductase

[0046] The cells were collected, centrifuged at 7600 rpm for 10 minutes, and the cells were kept in a boiling water bath at 100°C for 10 minutes, and then subjected to SDS-PAGE electrophoresis ( image 3 ).

[0047] From image 3 Comparing the total protein of the constructed strain and the wild-type strain, it can be observed that the malonyl-CoA reductase is expressed and its size is 132KD.

[0048] 3-HP production detection

[0049] Cells were collected, centrifuged at 7600rpm at 4°C for 10min, the supernatant was left for sample derivatization, 100μl of supernatant was taken, and after drying, 10μl of methoxyamino hydrochloride pyridine solution (4mg / ml) was added with 30°C 750rpm constant temperature shaking for 90min. Add 90μl MSTFA 37°C 450rpm constant temperature shaking for 30min. Centrifuge at 13000rpm for 10min at room temperature, take 70~75μl and load the sample, taking care not to absorb the white floccules...

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Abstract

The invention discloses a synechocystis-6803 genetically engineered bacterium capable of producing 3-hydroxypropionic acid, and a construction method and application thereof. The construction method is as follows: a malonyl coenzyme A reductase gene in orange chloroflexus is cloned into synechocystis 6803; and acetylcoenzyme A carboxylase, biotin acylase and an NAD(P) transhydrogenase gene in the synechocystis 6803 are expressed. The synechocystis 6803 is transformed through a synthetic biology method to obtain the synechocystis-6803 genetically engineered bacterium capable of producing the 3-hydroxypropionic acid. The experiments prove that the final yield of the 3-HP (3-hydroxypropionic acid) of the genetically engineered bacterium is up to 837.18mg / L, which is of great theoretical and practical significances for the production of the 3-HP through photosynthetic microorganisms.

Description

Technical field [0001] The invention belongs to the field of industrial microorganisms, and specifically relates to a genetically engineered bacteria producing trihydroxypropionic acid and a construction method and application thereof. Background technique [0002] 3-hydroxypropionic acid (3-HP), also known as β-hydroxypropionic acid, is a colorless, odorless, oily liquid that is miscible with water, alcohol, ether and other organic solvents. Both ends of the molecule have a hydroxyl group and a carboxyl group respectively, which are more active molecules. The Office of Energy Efficiency and Renewable Resources of the U.S. Department of Energy put forward six projects on biomass research and energy, renewable resources, and economic resource production in April 2002. Among them, the United States Cargill and Codexis jointly researched grains. The new fermentation process for carbohydrate production of 3-HP is one of the six projects. The US Department of Energy report in August...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/42C12R1/89
Inventor 王云鹏陈磊张卫文
Owner TIANJIN UNIV
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