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CN-121975821-A - 13-Hydroxyglucopiericidin A heterologous expression engineering strain, construction method and application thereof

CN121975821ACN 121975821 ACN121975821 ACN 121975821ACN-121975821-A

Abstract

The invention discloses a 13-hydroxyglucopiericidin A heterologous expression engineering strain, a construction method and application thereof. The engineering strain takes Streptomyces avermitilis SUKA as a host, realizes the heterologous synthesis of the pteromycin glycoside, especially 13OH-GPA, by introducing a pteromycin glycoside biosynthesis gene cluster, and is classified and named as Streptomyces sp.SK-SA 23E, and the preservation number is GDMCC NO.67271. The invention realizes the efficient heterologous expression of the Pinctada glycoside biosynthesis gene cluster for the first time, the yield of 13OH-GPA in the engineering strain is improved by 2.8 times compared with that of the original strain, the relative content in the Pinctada glycoside is improved from 23.6% to 48.1%, and the method is suitable for large-scale preparation of 13 OH-GPA.

Inventors

  • ZHOU XUEFENG
  • LIANG JIAQI
  • CAI JIAN
  • XIE YUHUI
  • TANG LAN

Assignees

  • 广州示康药业有限公司

Dates

Publication Date
20260505
Application Date
20260128

Claims (10)

  1. 1. A Pincerlike glycoside biosynthesis gene cluster is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.
  2. 2. A recombinant vector comprising the Pincerlike glycoside biosynthesis gene cluster according to claim 1, preferably, pHZAUBACFXJ-23E.
  3. 3. A heterologous expression engineering strain of a pteromycin glycoside, characterized in that it comprises the pteromycin glycoside biosynthesis gene cluster of claim 1 or comprises the recombinant vector of claim 2.
  4. 4. The heterologous expression engineering strain of a pteromycin glycoside of claim 3, wherein the heterologous expression engineering strain of a pteromycin glycoside is a Streptomycesavermitilis host strain.
  5. 5. The heterologous expression engineering strain of a pteromycin glycoside of claim 4, wherein the heterologous expression engineering strain of a pteromycin glycoside is a Streptomyces avermitilis SUKA host strain.
  6. 6. The heterologous expression engineering strain of a pteromycin glycoside of claim 5, wherein the heterologous expression engineering strain of a pteromycin glycoside is Streptomyces sp.sk-SA 23E with accession No. GDMCC No. 67271.
  7. 7. A method for constructing a Pincerlike glycoside heterologous expression engineering strain according to any one of claims 3 to 6, comprising the steps of: 1) Constructing a DNA library containing the pteromycin glycoside biosynthetic gene cluster of claim 1; 2) Screening and positioning recombinant vectors containing the Pincerlike mycin glycoside biosynthesis gene cluster; 3) Transferring the recombinant vector screened in the step 2) into a host strain through conjugal transfer.
  8. 8. A process for preparing a pteromycin glycoside, characterized by comprising the steps of culturing the pteromycin glycoside heterologous expression engineering strain according to any one of claims 3-6 with a fermentation medium, obtaining a fermentation product and isolating the pteromycin glycoside, preferably the pteromycin glycoside comprises 13-hydroxyglucopiericidin A.
  9. 9. The method according to claim 8, wherein the fermentation medium is an H9 fermentation medium, the composition of which is soluble starch 20g/L, cotton seed meal 10 g/L, yeast extract meal 5g/L, maltodextrin 20g/L, malt extract meal 5g/L, mgSO 4 ·7H 2 O 2 g/L,NaCl 2 g/L,CaCO 3 20g/L, water as a solvent and pH 7.0-7.2, and the culture condition is 28 ℃.
  10. 10. Use of the triptycepin glycoside biosynthesis gene cluster of claim 1, the recombinant vector of claim 2, the triptycepin glycoside heterologous expression engineering strain of any one of claims 3-6 in the preparation of a triptycepin glycoside, preferably the triptycepin glycoside comprises 13-hydroxyglucopiericidin A.

Description

13-Hydroxyglucopiericidin A heterologous expression engineering strain, construction method and application thereof Technical Field The invention belongs to the technical field of pharmaceutical biology, and relates to a 13-hydroxyglucopiericidin A heterologous expression engineering strain, a construction method and application thereof. Background Marine actinomycetes are widely concerned by being capable of producing metabolites with novel structures and unique activities, and become an important source of new drug candidates. Pincerlike glycosides are a class of glycoside natural products of alpha-hydroxypyridine produced mainly by Streptomyces oceanicus, and have been studied in recent years to show good pharmaceutical development potential (Journal of MEDICINAL CHEMISTRY, 2019, 62:7058). In particular to 13-hydroxyglucopiericidin A (13 OH-GPA), has remarkable effect on resisting acute kidney injury (Acta Pharmaceutica Sinica B, 2024, 14, 3232), and has potential of resisting renal fibrosis activity and treating chronic kidney disease (Theranostics, 2022, 12:7158), and the 13OH-GPA also has potential treatment effect on liver, lung and other organ fibrosis when being used as liver kinase B1 (LKB 1) activator. At present, no chemical total synthesis report of the pteromycin glycoside including 13OH-GPA exists. Among the wild Streptomyces species found 13OH-GPA, the yield was extremely low. Even through the optimization and improvement of fermentation conditions, the yield of the 13OH-GPA reported in the prior art is only 2.70 mg/L (patent ZL 202410857442.1), and the homologs of the Pincerlike glycoside are more, so that the separation difficulty of the 13OH-GPA is relatively high. Therefore, the traditional fermentation mode of the wild drug source strain is difficult to meet the requirements of intensive research and new drug development. Therefore, establishing a biosynthesis method capable of efficiently and stably producing the pteromycin glycoside, especially 13OH-GPA, becomes a key for promoting the pharmaceutical application of the compounds. The heterologous expression technology provides an effective way for improving the yield of natural products, and is expected to realize the optimization of metabolic pathways and the remarkable improvement of the yield of the products by introducing target biosynthesis gene clusters into a host easy to genetically operate. Disclosure of Invention The application provides a 13-hydroxyglucopiericidin A heterologous expression engineering strain, a construction method and application thereof, which are suitable for large-scale preparation by integrating a complete Pincerlike glycoside biosynthesis gene cluster into streptomycete Streptomyces avermitilisSUKA (the strain is disclosed in reference ：Komatsu, Mamoru et al. "Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism." Proceedings of the National Academy of Sciences of the United States of America vol. 107,6 (2010): 2646-51. doi:10.1073/pnas.0914833107,, the inventor also holds the strain and ensures that the strain is released to the public within 20 years from the filing date), realizing the efficient heterologous synthesis of Pincerlike glycoside, especially 13OH-GPA, improving the yield and relative content, reducing homologous impurities. The invention adopts the following technical scheme: The first object of the invention is to provide a Pincerlike glycoside biosynthesis gene cluster, the nucleotide sequence of which is shown as SEQ ID NO. 1. Preferably, the triptycene glycoside comprises 13OH-GPA. Preferably, the Pincerlike glycoside biosynthesis gene cluster is derived from Streptomyces psammoticusSCSIO NS126, and the strain is deposited in the Guangdong province microorganism strain collection center (GDMCC) with the deposition number of GDMCC NO. 64524, the deposition date of 2024, 4 and 19 days, and the deposition address of No. 5 building of No. 59, university 100 in Guangzhou City martyr, guangdong province, and the post code of 510070. A second object of the present invention is to provide a recombinant vector comprising the above-mentioned Pincerlike glycoside biosynthesis gene cluster. Preferably, the recombinant vector is pHZAUBACFXJ1-23E. The third object of the present invention is to provide a heterologous expression engineering strain of the pteromycin glycoside, wherein the engineering strain contains the pteromycin glycoside biosynthesis gene cluster or the recombinant vector. Preferably, the heterologous expression engineering strain takes Streptomyces avermitilis as a host strain to express the Pincerlike mycin glycoside biosynthesis gene cluster. Further preferably Streptomyces avermitilisSUKA is used as host strain. Preferably, the Pincerlike glycoside heterologous expression engineering strain is Streptomyces sp.SK-SA 23E, and is deposited in the Guangdong province microorganism strain collection (GDMCC), the deposition number is GDMCC NO. 6