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CN-121991192-A - Pentanediamine transporter mutant and application thereof in production of pentanediamine

CN121991192ACN 121991192 ACN121991192 ACN 121991192ACN-121991192-A

Abstract

The invention discloses a pentamethylene diamine transporter mutant and application thereof in pentamethylene diamine production, and belongs to the technical field of protein engineering. The invention provides a high-activity mutant M284A, M D of a pentamethylene diamine transporter CgmA, and the mutant is expressed in recombinant corynebacterium glutamicum, so that the accumulation amount of intracellular pentamethylene diamine is reduced by 47.7% and 46.2%, and compared with wild type CgmA, the production amount of pentamethylene diamine is increased by 34.7% and 37.8%, respectively.

Inventors

  • LIU LIMING
  • SONG LONGFEI
  • GAO CONG
  • LIU JIA
  • HU GUIPENG
  • LI XIAOMIN

Assignees

  • 江南大学

Dates

Publication Date
20260508
Application Date
20260129

Claims (10)

  1. 1. The pentanediamine efflux protein mutant is characterized by having a mutation of M284A, M284D or M382A on the basis of the parent shown in SEQ ID NO. 1.
  2. 2. The mutant of the pentylene diamine exoprotein according to claim 1, wherein the mutant of the pentylene diamine exoprotein is obtained by mutating methionine at 284 to alanine or aspartic acid or by mutating methionine at 382 to alanine based on the parent shown in SEQ ID No. 1.
  3. 3. A gene encoding the pentanediamine efflux protein mutant of claim 1 or 2.
  4. 4. A recombinant plasmid carrying the gene of claim 3.
  5. 5. The recombinant plasmid according to claim 4, wherein said plasmid includes, but is not limited to pCES208,208, said recombinant plasmid further comprising a promoter Ptrc thereon, and wherein expression of said pentanediamine efflux protein mutant is regulated by said promoter Ptrc.
  6. 6. Recombinant corynebacterium glutamicum expressing the pentanediamine efflux protein mutant of claim 1 or 2.
  7. 7. The recombinant corynebacterium glutamicum according to claim 6, wherein said recombinant corynebacterium glutamicum further expresses lysine decarboxylase gene HaLdcC as set forth in SEQ ID No. 2.
  8. 8. A process for preparing pentamethylenediamine by fermentation, comprising culturing the recombinant corynebacterium glutamicum of claim 6 or 7 in a medium for a period of time and collecting the pentamethylenediamine in the fermentation broth.
  9. 9. The method according to claim 8, wherein glucose and ammonium sulfate are fed during the fermentation, and the concentration of glucose and ammonium sulfate in the fermentation system is less than or equal to 1 g/L, respectively.
  10. 10. The use of the pentanediamine efflux protein mutant of claim 1 or 2 in constructing genetically engineered bacteria.

Description

Pentanediamine transporter mutant and application thereof in production of pentanediamine Technical Field The invention relates to a pentamethylene diamine transporter mutant and application thereof in pentamethylene diamine production, belonging to the technical field of protein engineering. Background Pentanediamine is a key high-value chemical product, and is widely applied to the fields of industrial manufacture, agricultural production, medical appliances and the like, and the bio-based plastic produced by the pentalene diamine gradually replaces petroleum-based plastic, so that the problem of petroleum resource exhaustion is relieved, and the environmental pollution and the emission of greenhouse gases are reduced. Worldwide polyamide products are in constant demand and consume more than 3000 ten thousand tons per year. At present, the pentanediamine is mainly synthesized by a chemical method of petroleum fossil raw materials and a whole-cell catalytic bioconversion method. However, accumulation of high concentration of pentanediamine in cells causes severe cytotoxicity, and causes cell lysis and death and metabolic flow disturbance. The biological catalysis or fermentation production of the 1, 5-pentanediamine can reduce fossil energy consumption, save cost and realize low-carbon production. Current research mainly uses glucose fermentation or catalytic production with lysine as a substrate. However, in the catalytic or fermentation production of pentanediamine, the toxic effect on cells cannot be avoided, and particularly, the toxic phenomenon caused by accumulation of intracellular pentanediamine is serious, so that the productivity is low and the industrialization cannot be realized. Therefore, there is a need to develop transporter mutants with higher external transport activity of pentamethylenediamine in order to further increase the yield and production efficiency of pentamethylenediamine. Disclosure of Invention Aiming at the problem of low transport efficiency of CgmA pentamethylenediamine in the prior art, the invention provides a CgmA efficient mutant and application thereof. The invention provides a pentanediamine efflux protein mutant which has a mutation of M284A, M284D or M382A on the basis of a parent shown in SEQ ID NO. 1. In one embodiment, the pentylene diamine exoprotein mutant is based on the parent shown in SEQ ID NO.1, mutating methionine at position 284 to alanine or aspartic acid. In one embodiment, the pentylene diamine exoprotein mutant is based on the parent shown in SEQ ID NO.1, mutating methionine at position 382 to alanine. The invention also provides a gene for encoding the pentanediamine efflux protein mutant. The invention also provides a recombinant plasmid carrying the gene. In one embodiment, the plasmid includes, but is not limited to pCES208,208. In one embodiment, the recombinant plasmid further comprises a promoter Ptrc, and the expression of the pentanediamine efflux protein mutant is regulated by the promoter Ptrc. The invention also provides recombinant corynebacterium glutamicum expressing the pentanediamine efflux protein mutant. In one embodiment, the recombinant corynebacterium glutamicum uses corynebacterium glutamicum FMME-L007 as an initial strain, and expresses a lysine decarboxylase gene HaLdcC shown in SEQ ID NO. 2. In one embodiment, the corynebacterium glutamicum FMME-L007 and methods of constructing the same are disclosed in the patent application publication number CN116656566 a. The invention also provides a method for promoting extracellular secretion of pentamethylene diamine by corynebacterium glutamicum, which is to express the mutant in corynebacterium glutamicum. In one embodiment, the corynebacterium glutamicum includes, but is not limited to, corynebacterium glutamicum expressing lysine decarboxylase gene HaLdcC. The invention also provides a method for preparing the pentanediamine by applying the recombinant corynebacterium glutamicum to ferment. In one embodiment, the method is to culture the recombinant corynebacterium glutamicum in a medium for a period of time and collect pentamethylenediamine in the fermentation broth. In one embodiment, the medium contains glucose, ammonium sulfate, corn steep liquor dry powder, betaine, potassium chloride. In one embodiment, glucose and ammonium sulfate are also fed during the fermentation process, and the concentration of glucose and ammonium sulfate in the fermentation system is respectively less than or equal to 1 g/L. The invention also provides application of the pentanediamine efflux protein mutant in construction of genetically engineered bacteria. The beneficial effects are that: The invention provides a high-activity mutant M284A or M284D of a pentamethylene diamine transporter CgmA, and the mutant is expressed in recombinant corynebacterium glutamicum, so that the accumulation amount of intracellular pentamethylene diamine is respectively reduced by 47.7% and 46.2%, and compared with