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CN-115820523-B - Method for synthesizing propionic acid by using 1, 2-propylene glycol and recombinant bacterium used in method

CN115820523BCN 115820523 BCN115820523 BCN 115820523BCN-115820523-B

Abstract

The invention discloses a method for synthesizing propionic acid by using 1, 2-propylene glycol and recombinant bacteria used by the method. The invention belongs to the technical field of biology, and particularly relates to a method for synthesizing propionic acid by using 1, 2-propylene glycol and recombinant bacteria used by the method. The pseudomonas putida contains a glycerol dehydratase gene and an activator protein gene thereof, and can also contain an arabinose inducible promoter P BAD expression cassette, and the coding genes of the glycerol dehydratase and the activator protein thereof and the coding genes of the promoter P BAD are introduced into the pseudomonas putida, and the lacI gene is knocked out, so that the propionic acid yield of the obtained recombinant pseudomonas putida is obviously improved, and the research provides a new idea for synthesizing high-purity bio-based propionic acid.

Inventors

  • SHI YANAN
  • TAO YONG
  • YU BO

Assignees

  • 中国科学院微生物研究所

Dates

Publication Date
20260508
Application Date
20221129

Claims (8)

  1. 1. The recombinant pseudomonas putida is characterized in that the recombinant pseudomonas putida contains KPpdu gene clusters, and the KPpdu gene clusters code for glycerol dehydratase and glycerol dehydratase activation proteins; The KPpdu gene cluster is derived from klebsiella pneumoniae; the glycerol dehydratase comprises three proteins of a large subunit pduC of the glycerol dehydratase, a subunit pdu D in the glycerol dehydratase and a small subunit pduE of the glycerol dehydratase: 1) The large subunit pduC of the glycerol dehydratase is a protein of which the amino acid sequence is a sequence 2 in a sequence table; 2) Subunit pdu D in the glycerol dehydratase is protein with an amino acid sequence of sequence 3 in a sequence table; 3) The small subunit pduE of the glycerol dehydratase is a protein with an amino acid sequence of a sequence 4 in a sequence table; The glycerol dehydratase activator protein comprises the following proteins of a glycerol dehydratase activator protein alpha subunit pduG and a glycerol dehydratase activator protein beta subunit pduH: 1) The alpha subunit pduG of the glycerol dehydratase activating protein is a protein with an amino acid sequence of a sequence 5 in a sequence table; 2) The beta subunit pduH of the glycerol dehydratase activating protein is a protein with an amino acid sequence of a sequence 6 in a sequence table; the recombinant pseudomonas putida is obtained by introducing the KPpdu gene cluster into a receptor pseudomonas putida; The receptor pseudomonas putida is pseudomonas putida KT2440.
  2. 2. The recombinant pseudomonas putida of claim 1, further comprising a promoter for initiating transcription of the KPpdu gene cluster, wherein the promoter is designated as P BAD promoter and the P BAD promoter is a DNA molecule having a nucleotide sequence from nucleotide 880 to nucleotide 1229 of sequence 1 in the sequence listing.
  3. 3. The recombinant pseudomonas putida of claim 1 or 2, wherein the recombinant pseudomonas putida does not contain a lacI gene encoding a transcriptional regulator protein.
  4. 4. A method for constructing the recombinant pseudomonas putida according to any one of claims 1 to 3, comprising introducing the KPpdu gene cluster according to any one of claims 1 to 3 into a recipient pseudomonas putida, so as to obtain the recombinant pseudomonas putida, wherein the recipient pseudomonas putida is pseudomonas putida KT2440.
  5. 5. The method of claim 4, wherein said KPpdu gene cluster is introduced into said recipient pseudomonas putida by a DNA fragment comprising said KPpdu gene cluster and a promoter that initiates transcription of said KPpdu gene cluster, said promoter having the nucleotide sequence of SEQ ID No.1 at nucleotide numbers 880-1229.
  6. 6. The method of claim 5, wherein integration of the KPpdu gene cluster into the position of the lacI gene of the receptor pseudomonas putida knocks out the lacI gene of the receptor pseudomonas putida, the lacI gene encoding a transcriptional regulator protein.
  7. 7. Use of a recombinant pseudomonas putida according to any one of claims 1-3 for the preparation of propionic acid.
  8. 8. A process for the preparation of propionic acid, characterized in that it comprises culturing the recombinant pseudomonas putida according to any one of claims 1 to 3 to obtain a fermentation product from which propionic acid is obtained.

Description

Method for synthesizing propionic acid by using 1, 2-propylene glycol and recombinant bacterium used in method Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for synthesizing propionic acid by using 1, 2-propylene glycol and recombinant bacteria used by the method. Background Propionic Acid (PA) is an important intermediary metabolite in organisms. Propionic acid and calcium propionate are one of the most widely used food additives, artificial flavors and preservatives, which are also certified by the Food and Drug Administration (FDA) as "GENERALLY RECOGNIZED AS SAFE" (GRAS), and are highly safe. In addition, propionic acid is an important intermediate for the production of a variety of industrial compounds, and has wide application in paints, cosmetics, pharmaceuticals and pesticides. At present, the chemical synthesis of propionic acid mainly adopts an ethylene hydrogenation method, and the final raw material is petroleum. Although chemical synthesis methods have high yields and high yields, this often results in serious environmental pollution problems, and raw material petroleum is a non-renewable resource, exacerbating the resource shortage problem. Therefore, a method for producing propionic acid by fermentation using renewable resources as a substrate in a microbial cell factory has been attracting attention. Pseudomonas putida KT2440 (Pseudomonas Putida KT 2440) is a soil microorganism, can degrade various pollutants, and has biological control function on plant diseases. KT2440 is the most well-characterized strain of the pseudomonas putida population and is becoming the laboratory model strain of great interest in the fields of synthetic biology and metabolic engineering. KT2440 was certified by the recombinant DNA consultation Committee (Recombinant DNA Advisory Committee) as a biosafety strain that can be used to produce a variety of industrial chemicals, including products for direct human use (Nelson 2002). And the pseudomonas putida KT2440 has a complex aldehyde dehydrogenase system in the cell, so that the pseudomonas putida has extremely strong oxidizing capability, and is an excellent industrial acid-producing strain. Ma Chaodeng designed the path of synthesizing propionic acid from L-threonine, and realized efficient catalytic synthesis of propionic acid in Pseudomonas putida KT2440 (patent number: ZL 201811381133.2; application number: 201910572968.4; application number: 202111253877.8), but the theoretical yield from L-threonine to propionic acid was only 0.62g/g, and the atomic economy was not high. Disclosure of Invention The technical problem to be solved by the invention is to improve the propionic acid yield of microorganisms. In order to solve the technical problems, the invention provides a recombinant pseudomonas putida. The recombinant pseudomonas putida provided by the invention contains KPpdu gene clusters, and the KPpdu gene clusters code for glycerol dehydratase and glycerol dehydratase activation proteins. The recombinant pseudomonas putida also contains a promoter for promoting the KPpdu gene cluster to transcribe, the name of the promoter is P BAD promoter, and the P BAD promoter is any one of the following DNA molecules: 1) A DNA molecule with the nucleotide sequence of one chain being 880 th to 1229 th nucleotides of the sequence 1 in the sequence table; 2) A DNA molecule having 80% or more identity with the DNA molecule of 1) and having a promoter function. The recombinant pseudomonas putida does not contain a lacI gene, and the lacI gene codes for a transcription regulatory factor protein. The KPpdu gene cluster is derived from klebsiella pneumoniae. In the recombinant pseudomonas putida, the glycerol dehydratase comprises three proteins of a glycerol dehydratase large subunit pduC, a glycerol dehydratase subunit pdu D and a glycerol dehydratase small subunit pduE: The large subunit pduC of the glycerol dehydratase is a protein of which the amino acid sequence is a sequence 2 in a sequence table; subunit pdu D in the glycerol dehydratase is protein with an amino acid sequence of sequence 3 in a sequence table; the small subunit pduE of the glycerol dehydratase is a protein with an amino acid sequence of a sequence 4 in a sequence table. The glycerol dehydratase activator protein comprises the following proteins of a glycerol dehydratase activator protein alpha subunit pduG and a glycerol dehydratase activator protein beta subunit pduH: the alpha subunit pduG of the glycerol dehydratase activating protein is a protein with an amino acid sequence of a sequence 5 in a sequence table; The beta subunit pduH of the glycerol dehydratase activating protein is a protein with an amino acid sequence of a sequence 6 in a sequence table; the pduC gene is any one of the following DNA molecules: C1 A DNA molecule whose coding sequence is indicated at positions 1230 to 2894 of SEQ ID NO. 1; c2 A DNA molecule having 90%