Search

CN-121975875-A - Method for regulating efficient conversion of methanol based on (p) ppGpp signal molecule

CN121975875ACN 121975875 ACN121975875 ACN 121975875ACN-121975875-A

Abstract

The invention discloses a method for regulating efficient conversion of methanol based on a (p) ppGpp signal molecule. Specifically, the synthesis and hydrolysis balance of a microorganism intracellular (p) ppGpp signal molecule are regulated, and the expression of key genes and the amino acid metabolism flux of a methanol metabolic pathway are enhanced by combining promoter engineering and nutrition condition optimization, so that the utilization efficiency of the microorganism on methanol and the synthesis capability of a target product are obviously improved. The invention defines the regulation mechanism of (p) ppGpp on the conversion of methanol under the stress of low nitrogen source for the first time, realizes the efficient conversion of methanol to organic acids such as acetic acid, butyric acid and the like, provides a brand-new strategy for preparing high-added value chemicals through biological conversion of methanol, has the advantages of economy, environmental protection, high conversion efficiency and wide application prospect.

Inventors

  • WANG XIN
  • QIN RUIRUI
  • CHEN KEQUAN
  • GUO YUANKE
  • Zhuang Feiyi

Assignees

  • 南京工业大学

Dates

Publication Date
20260505
Application Date
20260209

Claims (7)

  1. 1. A method for regulating efficient conversion of methanol based on (p) ppGpp signal molecules, which is characterized by comprising the following steps: Step1, constructing a (p) ppGpp metabolic regulation system: Cloning drosophila-derived (P) ppGpp hydrolase Mesh1 gene, constructing a recombinant expression plasmid PXY3-P thl -Mesh1 based on a PXY3 vector, and electrically transferring into methylbutyrate bacillus to obtain a recombinant strain B. methylotrophicum/PXY3-P thl -Mesh1; step2, precise regulation and optimization of a promoter: P th1 from Clostridium acetobutylicum ATCC 824 is used as a promoter for subsequent regulation of Mesh1 gene expression, random mutation is carried out on the promoter to obtain a promoter with the strength of P th13 (58% -20), and then the promoter P thl in the recombinant expression plasmid PXY3-P thl -Mesh1 constructed in the step 1 is replaced by the promoter P thl3 to obtain the recombinant strain B. methylotrophicum/PXY3-P thl3 -Mesh1 after the promoter is optimized; step 3, nutrient condition optimization, recombinant strain culture and metabolite analysis: Selecting PB fermentation medium with yeast powder content of 0.5 g/L, culturing the recombinant strain B. methylotrophicum/PXY3-P thl3 -Mesh1 after optimizing the promoter, culturing in a 37 ℃ anaerobic workstation, controlling the initial OD 600 to be 0.1 and the volume ratio of N 2 :CO 2 :H 2 to be 8:1:1, sampling and detecting every 48 h during the culturing process, and performing real-time fluorescence quantitative PCR (RT-PCR) detection and transcriptome sequencing analysis on methanol metabolic pathway related genes of the recombinant strain B. methylotrophicum/PXY3-P thl3 -Mesh 1.
  2. 2. The method of claim 1, wherein the amino acid sequence of the gene Mesh1 used in the recombinant plasmid PXY3-P thl -Mesh1 in step 1 is shown in SEQ ID NO.57, and the nucleic acid sequence is shown in SEQ ID NO. 61.
  3. 3. The method of claim 1, wherein the recombinant plasmid PXY3-P thl -Mesh1 in step 1 is constructed and synthesized by the Anhui general company and codon optimized.
  4. 4. The method of claim 1, wherein the optimized promoter mutant PXY3-P thl3 -Mesh1 in step 2 has the nucleotide sequence of the upstream primer P thl3 -F shown in SEQ ID NO.24, the nucleotide sequence of the downstream primer P thl3 -R shown in SEQ ID NO.25, and the nucleotide sequence of the promoter P thl3 shown in SEQ ID NO. 19.
  5. 5. The method of claim 1, wherein the optimized culture medium formulation in step 3 is potassium dihydrogen phosphate 4 g/L, dipotassium hydrogen phosphate 6 g/L, ammonium chloride 1 g/L, magnesium chloride hexahydrate 0.1 g/L, calcium chloride dihydrate 0.1 g/L, 0.5 g/L yeast powder, 2% (V/V) microelement solution, 2% (V/V) vitamin solution, 50 mM methanol, 20mM NaHCO 3 .
  6. 6. The method of claims 1-5, wherein the target products of high efficiency methanol conversion comprise acetic acid and butyric acid.
  7. 7. The method of claim 6, wherein in the efficient conversion of methanol, methanol consumption is 7.0 g/L, methanol consumption is increased by 22.8%, acetic acid yield is up to 2.3 g/L, butyric acid yield is up to 1.7 g/L, and carbon conversion is increased to 70.4%.

Description

Method for regulating efficient conversion of methanol based on (p) ppGpp signal molecule Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for regulating and controlling efficient conversion of methanol based on (p) ppGpp signal molecules. Background Methanol is a non-food-grade C1 raw material, and is a potential application raw material due to the characteristics of easy storage, easy transportation and the like. With the increasing interest in green sustainable economy, single carbon such as methanol compounds as alternative raw materials for saccharides have attracted strong attention. However, the existing methanol conversion microorganisms generally have the problems of low methanol utilization rate, slow cell growth and high dependence on organic nitrogen sources, and limit industrial application. (P) ppGpp is a key signal molecule for bacteria to cope with nutritional stress, and can help microorganisms adapt to adverse environments by regulating amino acid metabolism, ribosome synthesis and carbon nitrogen metabolism balance. Existing studies indicate that (p) ppGpp can alleviate the problem of amino acid starvation during methanol metabolism, but a systematic (p) ppGpp regulation strategy has not yet been formed, and its strengthening mechanism for methanol conversion under low nitrogen source conditions is not fully exploited. Therefore, the development of a multidimensional regulation strategy based on (p) ppGpp has important significance for breaking through the bottleneck of methanol bioconversion efficiency. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a method for regulating and controlling the efficient conversion of methanol based on (p) ppGpp signal molecules, which constructs an efficient methanol conversion regulating and controlling system by integrating gene editing, promoter regulation and nutrition optimization, remarkably improves the utilization efficiency of microorganisms on methanol and the synthesis capability of products, and is particularly suitable for the anaerobic microorganism-mediated conversion process of methanol to organic acid. In order to solve the problems in the prior art, the invention adopts the following technical scheme: A method for regulating and controlling efficient conversion of methanol based on (p) ppGpp signal molecules comprises the following steps: Step1, constructing a (p) ppGpp metabolic regulation system: Cloning drosophila-derived (P) ppGpp hydrolase Mesh1 gene, constructing a recombinant expression plasmid PXY3-P thl -Mesh1 based on a PXY3 vector, and electrically transferring into methylbutyrate bacillus to obtain a recombinant strain B. methylotrophicum/PXY3-P thl -Mesh1; step2, precise regulation and optimization of a promoter: P th1 from Clostridium acetobutylicum ATCC 824 is used as a promoter for subsequent regulation of Mesh1 gene expression, random mutation is carried out on the promoter to obtain a promoter with the strength of P th13 (58% -20), and then the promoter P thl in the recombinant expression plasmid PXY3-P thl -Mesh1 constructed in the step 1 is replaced by the promoter P thl3 to obtain the recombinant strain B. methylotrophicum/PXY3-P thl3 -Mesh1 after the promoter is optimized; step 3, nutrient condition optimization, recombinant strain culture and metabolite analysis: Selecting PB fermentation medium with yeast powder content of 0.5 g/L, culturing the recombinant strain B. methylotrophicum/PXY3-P thl3 -Mesh1 after optimizing the promoter, culturing in a 37 ℃ anaerobic workstation, controlling the initial OD 600 to be 0.1 and the volume ratio of N 2:CO2:H2 to be 8:1:1, sampling and detecting every 48 h during the culturing process, and performing real-time fluorescence quantitative PCR (RT-PCR) detection and transcriptome sequencing analysis on methanol metabolic pathway related genes of the recombinant strain B. methylotrophicum/PXY3-P thl3 -Mesh 1. As an improvement, the amino acid sequence of the gene Mesh1 used by the recombinant plasmid PXY3-P thl -Mesh1 in the step 1 is shown as SEQ ID NO.57, and the nucleic acid sequence is shown as SEQ ID NO. 61. Preferably, the recombinant plasmid PXY3-P thl -Mesh1 in the step 1 is constructed and synthesized by Anhui general company and subjected to codon optimization. As an improvement, the nucleic acid sequence of the upstream primer P thl3 -F used in the optimized promoter mutant PXY3-P thl3 -Mesh1 in the step 2 is shown as SEQ ID NO.24, the nucleic acid sequence of the downstream primer P thl3 -R is shown as SEQ ID NO.25, and the nucleic acid sequence of the promoter P thl3 is shown as SEQ ID NO. 19. As improvement, the culture medium formula optimized in the step 3 is potassium dihydrogen phosphate 4 g/L, dipotassium hydrogen phosphate 6 g/L, ammonium chloride 1 g/L, magnesium chloride hexahydrate 0.1 g/L, calcium chloride dihydrate 0.1 g/L, 0.5 g/L yeast powder, 2%