KR-102962642-B1 - ISOPRENOL HIGH PRODUCING STRAIN AND PRODUCING METHOD OF ISOPRENOL USING THE SAME

Abstract

The present invention relates to a method for producing isoprenol in a minimal medium by precisely controlling the isoprenol biosynthetic metabolic pathway and the growth of a strain. Specifically, since recombinant E. coli with maximized isoprenol productivity can be manufactured and cultured in a minimal medium, superior isoprenol can be produced economically, and this can be utilized in various ways in the aviation fuel industry, where isoprenol is used as a precursor, or in the biofuel industry, where it is used directly.

Inventors

• 정규열
• 정금영
• 김승진

Assignees

• 포항공과대학교 산학협력단

Dates

Publication Date

20260508

Application Date

20231229

Claims (9)

1. Recombinant strain into which the following recombinant vector was introduced: (1) A recombinant vector comprising the mvaE gene represented by SEQ ID NO. 1 connected to the Tet promoter represented by SEQ ID NO. 7 and the mvaS gene represented by SEQ ID NO. 2 connected to the Nar promoter (P FdeA ) represented by SEQ ID NO. 8; (2) A recombinant vector comprising the MK (mevalonate kinase) gene represented by SEQ ID NO. 3 linked to the T7 promoter represented by SEQ ID NO. 9, the PMK (phosphomevalonate kinase) gene represented by SEQ ID NO. 4 linked to the T7 promoter represented by SEQ ID NO. 9, and the MDD (mevalonate diphosphate decarboxylase) gene represented by SEQ ID NO. 5 linked to the T7 promoter represented by SEQ ID NO. 9; and (3) A recombinant vector containing the nudF gene represented by sequence number 6 linked to the T7 promoter represented by sequence number 9.
2. A recombinant strain according to claim 1, characterized in that the recombinant vector of (1) is pACYC-based.
3. A recombinant strain characterized in that, in claim 1, the recombinant vector of (2) is pET-based.
4. A recombinant strain characterized in that, in claim 1, the recombinant vector of (3) is pCDF-based.
5. A recombinant strain according to claim 1, characterized in that the strain is Escherichia coli.
6. A method for producing isoprenol, obtained by culturing a recombinant strain described in any one of claims 1 to 5 in a minimal medium.
7. A method for producing isoprenol according to claim 6, characterized in that the minimum medium is M9 medium.
8. A method for producing isoprenol according to claim 6, characterized in that the minimum medium includes aTc and naringenin.
9. A method for producing isoprenol according to claim 6, characterized in that the culture is a fed-batch culture.

Description

Isoprenol High-Producing Strain and Producing Method of Isoprenol Using the Same The present invention relates to a method for producing isoprenol in a minimal medium by precisely controlling the isoprenol biosynthetic metabolic pathway and the growth of a strain. More specifically, it relates to a recombinant strain with improved isoprenol productivity by optimizing carbon flow for isoprenol production through the simultaneous precise control of foreign genes with different derivatives using different inducible promoters, and a method for producing isoprenol using the same. The importance of biofuels is becoming increasingly critical to address oil depletion and the accumulation of greenhouse gases caused by climate change. While there is progress in electric vehicles in the transportation market, limitations exist for long-distance vehicles and aircraft, making it necessary to replace oil with sustainable biofuels. As an alternative, there is isoprenol, which is considered a gasoline substitute due to its energy density and combustion efficiency similar to gasoline. In other words, isoprenol is a substance attracting attention in the sharing economy as a biological precursor to DMCO, a future high-performance aviation fuel feedstock. The biological production of isoprenol can be achieved through the introduction of foreign genes to avoid feedback inhibition of endogenous genes, and this process is called isoprenol production following the mevalonic acid production pathway. The mevalonic acid upstream consists of the mvaE and mvaS genes derived from Enterococcus faecalis , and the mevalonic acid downstream isol is produced by introducing the MK, PMK, and MDD genes derived from Saccharomyces cerevisiae and nudF derived from Bacillus subtilis . Since mevalonic acid is an important precursor for isoprenol production, it is crucial to concentrate acetyl-CoA upstream of mevalonic acid into the isoprenol production pathway. However, within the cell, there was a challenge in that acetyl-CoA competed with isoprenol production, cell building block formation, and energy production. Therefore, most prior studies to date have avoided the aforementioned competition by culturing cells in a nutrient-rich medium supplemented with a large amount of nutritional supplements (beef extract, yeast extract, peptone, citrate) to solve the aforementioned difficulties. These media, containing various nutrients, are rich in amino acids and cell building blocks, allowing carbon flow from the substrate to be concentrated on isoprenol rather than being dispersed to other pathways such as the TCA cycle; however, this presents a problem in that it entails significant economic costs. To address the aforementioned issues, research is currently underway to increase yields by balancing precursors through sophisticated gene expression regulation in key metabolic pathways. It is expected that utilizing this research approach will help improve the economic efficiency of biofuel production, as it enables the production of sufficient amounts of isoprenol even in minimal, economical media. Accordingly, while continuing research on the productivity of isoprenol by precisely controlling gene expression levels to balance the precursors, the inventors completed the present invention by identifying a recombinant strain that improves isoprenol productivity by optimizing carbon flow toward mevalonic acid and strongly expressing genes downstream of mevalonic acid. Figure 1 is a schematic diagram showing an overview of the invention for the efficient production of isoprenol in E. coli. Figure 2 is a graph confirming isoprenol production by regulating mvaE and mvaS gene expression in the BI02 strain of the present invention by combining two inducers, naringenin and aTc, at various concentrations. Figure 3 is a graph showing the production of isoprenol by the BI01 strain of the present invention in M9 medium (A), the production of isoprenol by the BI01 strain in M9Y medium (B), and the production of isoprenol by the BI02 strain in M9 medium under optimized induction conditions (C). Figure 4 is a graph showing the results of fed-batch culture of the BI02 strain of the present invention. The present invention will be described in detail below. Most previous studies on isoprenol production have cultured isoprenol-producing strains in nutrient-rich media supplemented with large amounts of nutritional supplements to avoid competition between the flow of intracellular acetyl-CoA into the isoprenol production pathway and the pathways for cell building block formation and energy production. Media containing various nutrients include abundant amounts of amino acids and cell building blocks, which allows carbon flow from the substrate to be concentrated on isoprenol rather than being dispersed to other pathways such as the TCA cycle. However, for economic reasons, it is necessary to increase isoprenol production without the addition of nutritional supplements. In isop