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CN-122012252-A - Monascus purpureus capable of producing essential amino acids at high yield, screening method and application

CN122012252ACN 122012252 ACN122012252 ACN 122012252ACN-122012252-A

Abstract

The invention belongs to the field of microorganisms, and particularly relates to monascus purpureus capable of producing essential amino acids in high yield, a screening method and application thereof. The invention provides a novel transmembrane differentiation ARTP mutagenesis method for filamentous fungi, and optimizes protoplast pretreatment and repairing liquid components before mutagenesis aiming at the characteristics of multi-core and thick wall of monascus, so that the mortality and positive mutation rate are optimally balanced, and the problem of high genetic instability of conventional ARTP on monascus is solved. Based on the new mutagenesis method, the invention successfully mutagenizes and screens to obtain the mutant strain AZA5. The essential amino acid content of AZA5 is up to 58%. The invention not only provides excellent strain resources for the industrial production of high-quality microbial proteins, but also has important scientific value and wide industrialization application potential.

Inventors

  • LI MU
  • SUO YI
  • Mu Diehua
  • HAN XINJIE
  • SHANG ZHIHUI
  • HOU DAO

Assignees

  • 华中农业大学

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. 1. A novel monascus purpureus (Monascus purpureus) AZA5 is characterized in that the monascus purpureus AZA5 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.42055 at 2025.7.14.
  2. 2. The monascus purpureus AZA5 according to claim 1, wherein the ITS sequence of the monascus purpureus AZA5 is shown as SEQ ID NO. 1.
  3. 3. A bacterial preparation prepared by using the monascus purpureus AZA5 according to claim 1 or 2, or a bacterial preparation containing the monascus purpureus AZA5 according to claim 1 or 2.
  4. 4. Use of aspergillus purpureus AZA5 as claimed in claim 1 or 2 or of a bacterial preparation as claimed in claim 3 for the preparation of microbial proteins.
  5. 5. A monascus mutagenesis method is characterized by comprising the following steps: (1) Synchronous pretreatment of spores of an original strain, namely placing spore liquid of the original strain in a 4 ℃ environment for 18-36 hours to enable all spores to be in a dormancy stage; (2) ARTP mutagenesis, namely uniformly coating synchronized spore liquid on a metal slide for ARTP mutagenesis, wherein the mutagenesis treatment time is longer mutagenesis time with the mortality rate of 90% +/-2%; (3) And (3) repairing the specific membrane and genetically fixing, namely after mutagenesis is finished, putting the slide into a repairing liquid within 30 seconds, immersing the slide in the repairing liquid, and then carrying out light-shielding and standing repairing for 2-4 hours at 20-25 ℃, wherein the repairing liquid contains calcium ions and sorbitol.
  6. 6. The method of claim 5, wherein the repair liquid formulation of step (3) comprises a buffer, sorbitol, calcium ions, and an antioxidant.
  7. 7. The method according to claim 5 or 6, wherein the method further comprises: (4) Pressure screening, namely inoculating the strain subjected to the first round of mutagenesis to a screening culture medium, culturing for 2-4 days at 20-30 ℃, and selecting 2-4 colonies with the largest colony diameter; preparing spores from the selected bacterial colonies, carrying out ARTP mutagenesis again according to the method of the step (2), and then inoculating the strain subjected to the mutagenesis again to a new screening culture medium, and repeating the pressure screening process for more than 5 times to obtain the required strain; The screening culture medium is a culture medium taking sodium glutamate and aspartic acid as the only carbon sources.
  8. 8. The method of claim 7, wherein the selection medium comprises glucose 、KH 2 PO 4 、Na 2 HPO 4 、MgSO 4 、CaCl 2 、ZnSO 4 •7H 2 O、FeSO 4 •7H 2 O、CoSO 4 •7H 2 O、CuSO 4 •5H 2 O、MnSO 4 •H 2 O、 sodium glutamate and aspartic acid.
  9. 9. Monascus obtained by the method of any one of claims 5-8.
  10. 10. Use of monascus as claimed in claim 9 for the preparation of microbial proteins.

Description

Monascus purpureus capable of producing essential amino acids at high yield, screening method and application Technical Field The invention belongs to the field of microorganisms, and particularly relates to monascus purpureus capable of producing essential amino acids in high yield, a screening method and application thereof. Background As the global population grows and the level of living increases, so does the demand for high quality proteins. The traditional animal protein production mode faces the problems of large resource consumption, serious environmental pollution, long production period and the like, so that the development of alternative protein sources becomes an important research direction in the current food science and biotechnology fields. Microbial proteins are an emerging protein source, and have the advantages of short production period, no limitation of seasons and regions, high nutritive value and the like, and have been paid attention in recent years. Fungal proteins are considered to be one of the most promising sources of microbial proteins because of their balanced amino acid composition and abundant essential amino acid content. Essential amino acids are amino acids which cannot be synthesized by humans and animals themselves and must be taken up from the outside. The content of essential amino acids and their proportion to total amino acids directly determine the nutritional and biological value of the protein. The higher the proportion of essential amino acids in the protein, the more can the nutritional requirements of human and animals be met. One of the key challenges facing the current microbial protein industry is increasing the amount of essential amino acids to produce a high quality protein product that truly has high nutritional value. Disclosure of Invention The invention aims to provide monascus purpureus capable of producing essential amino acids at high yield, a screening method and application thereof. In order to achieve the aim of the invention, the technical scheme adopted by the invention is that a new monascus purpureus (Monascus purpureus) AZA5 is preserved in the China general microbiological culture collection center (CGMCC) with the preservation number of 42055 at 2025.7.14. The ITS sequence of the monascus purpureus AZA5 is shown as SEQ ID NO. 1. Correspondingly, the monascus purpureus AZA5 is utilized to prepare a bacterial preparation, or the monascus purpureus AZA5 is contained. Correspondingly, the monascus purpureus AZA5 or the bacterial preparation is applied to the preparation of microbial proteins. Correspondingly, the monascus mutagenesis method comprises the following steps of: (1) Synchronous pretreatment of spores of an original strain, namely placing spore liquid of the original strain in a 4 ℃ environment for 18-36 hours to enable all spores to be in a dormancy stage; (2) ARTP mutagenesis, namely uniformly coating synchronized spore liquid on a metal slide for ARTP mutagenesis, wherein the mutagenesis treatment time is longer mutagenesis time with the mortality rate of 90% +/-2%; (3) And (3) repairing the specific membrane and genetically fixing, namely after mutagenesis is finished, putting the slide into a repairing liquid within 30 seconds, immersing the slide in the repairing liquid, and then carrying out light-shielding and standing repairing for 2-4 hours at 20-25 ℃, wherein the repairing liquid contains calcium ions and sorbitol. Preferably, the repair liquid formulation includes a buffer (e.g., MES buffer), sorbitol, calcium ions (e.g., caCl 2), and an antioxidant (e.g., glutathione). (4) The pressure screening is carried out by inoculating the strain subjected to the first round of mutagenesis to a screening culture medium, culturing for 2-4 days at 20-30 ℃, selecting 2-4 colonies with largest colony diameters, preparing spores from the selected colonies, carrying out ARTP mutagenesis again according to the method of the step (2), then inoculating the strain subjected to the mutagenesis again to a new screening culture medium, and repeating the pressure screening process for more than 5 times to obtain the required strain, wherein the screening culture medium is a culture medium with sodium glutamate and aspartic acid as unique carbon sources. Preferably, the formulation of the screening medium comprises glucose 、KH2PO4、Na2HPO4、MgSO4、CaCl2、ZnSO4•7H2O、FeSO4•7H2O、CoSO4•7H2O、CuSO4•5H2O、MnSO4•H2O、 sodium glutamate and aspartic acid. Correspondingly, the monascus obtained by the method. Correspondingly, the monascus is applied to the preparation of microbial proteins. The invention has the following beneficial effects: Aiming at the pain point of monascus multi-core genetic instability, the invention provides a novel transmembrane differentiation ARTP mutagenesis method aiming at filamentous fungi. Aiming at the characteristics of multi-core and thick wall of monascus, the invention optimizes the protoplast pretreatment and repair liqui