KR-20260065481-A - Variant Microorganism for Production 1,3-Propandiol and Method for Producing 1,3-Propandiol Using the Same

Abstract

The present invention relates to a mutant microorganism that produces 1,3-propanediol and a method for producing 1,3-propanediol using the same. Specifically, the present invention recombined a strain in which the PP pathway (pentose phosphate pathway) and the ED pathway (Entner-Doudoroff pathway) were enhanced by genetically modifying the microorganism to enhance the citric acid cycle (TCA cycle) and the glycoxylate pathway, thereby enabling the production of 1,3-propanediol. Therefore, using the mutant microorganism according to the present invention, high concentrations of 1,3-propanediol can be produced.

Inventors

• 이준학
• 타이얍 이슬람
• 조승현
• 정성원
• 박성훈
• 조윤기

Assignees

• 주식회사 엑티브온

Dates

Publication Date

20260508

Application Date

20250626

Priority Date

20241030

Claims (4)

1. Includes the dhaB gene, gdrAB gene, and yqhD gene, and The ldhA , poxB , adhE , pta, ackA , yciA , pdhR , and pgi genes are deleted, and The expression of the zwf gene and gnd gene increases, and One or more of the iclR gene or arcA gene are deleted, Escherichia coli mutant microorganism capable of producing 1,3-propanediol.
2. In paragraph 1, The above-mentioned Escherichia coli mutant microorganism is one in which the glpK gene is additionally deleted, Escherichia coli mutant microorganism capable of producing 1,3-propanediol.
3. In paragraph 2, The above-mentioned Escherichia coli mutant microorganism is one in which glpF gene expression is increased, Escherichia coli mutant microorganism capable of producing 1,3-propanediol.
4. A method for producing 1,3-propanediol comprising: a step of culturing the Escherichia coli mutant microorganism of claims 1 to 3 in a medium containing glucose; and a step of recovering 1,3-propanediol from the medium in which the Escherichia coli mutant microorganism is cultured.

Description

Variant Microorganism for Production 1,3-Propandiol and Method for Producing 1,3-Propandiol Using the Same The present invention relates to a mutant microorganism that produces 1,3-propanediol and a method for producing 1,3-propanediol using the same. 1,3-Propanediol is a polyhydric alcohol with the chemical formula C₃H₅O₂,generally obtained by fermenting corn starch liquid or sugar. 1,3-Propanediol is used in cosmetics as a solvent, skin emollient, preservative, to improve product texture and homogeneity, and to enhance absorption. Compared to 1,2-Propanediol, known as propylene glycol (PG), its manufacturing process is more environmentally friendly and it has superior safety in cosmetics, playing an important role in the modern beauty industry. Traditional production methods for 1,3-propanediol, which has a wide range of uses, rely mainly on chemical synthesis methods, such as the hydration of acrolein and the hydroformylation of ethylene oxide in the presence of phosphine. However, these chemical production methods have limitations due to high costs and the inclusion of environmentally harmful production processes. To overcome these limitations, methods for producing 1,3-propanediol using microorganisms are being developed, typically using glycerol or sugars. However, the production of 1,3-propanediol using microorganisms has the disadvantage of producing various byproducts, such as lactic acid, acetic acid, ethanol, and 2,3-butanediol, through glycerol oxidation metabolism while simultaneously generating 1,3-propanediol as a glycerol reduction metabolite. Although various attempts are being made to increase the production of 1,3-propanediol while reducing these byproducts, much research is still needed to increase the production of 1,3-propanediol while suppressing the generation of byproducts. Figure 1 is a schematic diagram showing the plasmid used to construct a strain that produces 1,3-propanediol at a high concentration. Figure 2 illustrates the process of culturing a strain and producing 1,3-propanediol, showing a flask test method to confirm strain characteristics and a fermentation test method to confirm the effect by culturing in a fed-batch culture system. Figure 3 is a graph showing A) 1,3-propanediol concentration and growth amount of the strain, and B) specific production rate over time when the E. coli PK19-D1Q1 strain was cultured in a fed-batch system. One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments. Example 1: Materials and Method All restriction enzymes, DNA polymerases, and other DNA modifying enzymes for gene cloning were purchased from New England Bio-Labs (Beverly, MA, USA). Plasmid isolation, DNA extraction, and isolation were performed using mini-plasmid isolation kits, DNA gel extraction kits, and genomic DNA isolation kits purchased from Cosmotech Co. Ltd. (Seoul) and Promega (Madison, Wisconsin, USA), respectively. RNA isolation kits were purchased from Qiagen (Mannheim, Germany). The iScript cDNA Synthesis Kit and SyBr green RT-PCR master mix were purchased from BioRad (Seoul, Korea). Oligonucleotide synthesis and DNA sequencing were conducted through Macrogen (Macrogen Co. Ltd., Seoul, Korea). Tryptone and yeast extract were purchased from Difco (Becton Dickinson, Franklin Lakes, NJ, USA). Glycerol, glucose, and all other chemicals and enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified in this specification. Example 2: Production of recombinant strain The 1,3-propanediol production process is shown in Figure 1. A recombinant strain was constructed considering the 1,3-propanediol production pathway. Recombinant strains were prepared and used, including existing strains and plasmids, as listed in Table 1 below. Genetic recombination was performed as follows. Escherichia coli DH5α was used for the development of overexpression and deletion plasmids. Chromosomal genetic manipulation was performed using the in-frame tagged deletion/insertion method. Briefly, 500 bp of the upstream (upstream fragment, A) and downstream (downstream fragment, B) regions of the target gene were PCR amplified and ligated using the nested PCR method. Subsequently, fragments AB were cloned into the pKOV vector at the Not I and Xba I or Bam HI restriction sites. Using the resulting plasmids, the target gene was deleted from the chromosomal DNA via homologous recombination. Finally, deletion mutant strains were screened using PCR and verified by sequencing. For the replacement of all target genes, the target gene was first removed, and then the gene of interest was inserted using a similar in-frame tagged method. To construct a recombinant strain that produces high levels of 1,3-propanediol, a plasmid was used as shown in Figure 1. The recombined strains using the above pla