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CN-121975750-A - Novel KstD enzyme nucleic acid, recombinant vector, saccharomyces cerevisiae and application

CN121975750ACN 121975750 ACN121975750 ACN 121975750ACN-121975750-A

Abstract

The invention relates to the field of genetic engineering, and belongs to the field of applied microorganism and enzyme engineering. The invention discloses a novel KstD enzyme nucleic acid, a recombinant vector, saccharomyces cerevisiae and application thereof. The invention discloses a protein shown in SEQ ID No.6 and a nucleic acid molecule shown in SEQ ID No.5 as a coding gene thereof, wherein the 3-sterone-delta 1 -dehydrogenase (KstD enzyme) is derived from Fusarium solani. The invention heterologously expresses 3-sterone-delta 1 -dehydrogenase (KstD enzyme) protein in saccharomyces cerevisiae, and shows that the invention has the capability of catalyzing substrates such as 4AD, 9 alpha-OH-4 AD, progesterone, testosterone and the like, and has industrial application value.

Inventors

  • LIAO GUOJIAN
  • Long Mengfei
  • ZHANG YING
  • ZHOU ZIYI

Assignees

  • 西南大学

Dates

Publication Date
20260505
Application Date
20260311

Claims (9)

  1. 1. A3-sterone-delta 1 -dehydrogenase, the amino acid sequence of which is shown in SEQ ID No.6, is derived from Fusarium solani (Fusarium solani).
  2. 2. A nucleic acid molecule encoding the 3-sterone- Δ 1 -dehydrogenase, the nucleic acid molecule being a gene encoding the 3-sterone- Δ 1 -dehydrogenase of claim 1, the nucleotide sequence of the nucleic acid molecule being shown in SEQ ID No. 5.
  3. 3. A recombinant expression vector, which is a recombinant plasmid pJFE-FsKstD containing the dehydrogenase encoding gene of claim 2.
  4. 4. A recombinant saccharomyces cerevisiae strain SC-FsKstD of 3-sterone-delta 1 -dehydrogenase expresses the protein as set forth in claim 1, and the nucleotide for encoding the recombinant strain SC-FsKstD is shown as SEQ ID No.5 in a sequence table.
  5. 5. The method for constructing a recombinant saccharomyces cerevisiae strain of 3-sterone-delta 1 -dehydrogenase according to claim 1, comprising the following steps: 1) The empty plasmid pJFE is linearized with the restriction enzyme PstI. 2) The gene fragment encoding 3-sterone-delta 1 -dehydrogenase is obtained by PCR amplification technology, and the template is cDNA of Fusarium solani. 3) And constructing the gene fragment on pJFE vectors to obtain recombinant plasmids with the target enzyme genes. 4) The recombinant plasmid is introduced into Saccharomyces cerevisiae through chemical transformation to obtain recombinant Saccharomyces cerevisiae strain with steroid C1,2 dehydrogenation, named SC-FsKstD. There is no chronological limitation between steps 1) and 2).
  6. 6. The construction method according to claim 5, wherein the Saccharomyces cerevisiae in step 3) is BY4741 strain knocked out of the ayr1 gene and designated SC-ayr1.
  7. 7. Use of the recombinant s.cerevisiae according to claim 4 for the synthesis of C1, 2-dehydro-steroids from substrate steroids, comprising the steps of: s1, inoculating the recombinant saccharomyces cerevisiae into a seed culture medium, culturing at 220 rpm and 28-30 ℃ until the OD600 is 1.3-1.5, and obtaining seed liquid; S2, inoculating the seed solution to a fermentation medium YPD, and culturing for 24 hours at 220 rpm and 28-30 ℃; S3, adding a pre-dissolved substrate (1 g/L) when fermenting and culturing for 24 hours, and reacting at 28-30 ℃ until the substrate is completely converted; the substrate pre-dissolution in step S3 is performed by using N, N-Dimethylformamide (DMF) as a solvent.
  8. 8. The use according to claim 7, wherein the seed medium in step S1 is SD-Ura broth.
  9. 9. The use according to claim 7, wherein the substrate in step S3 comprises 4AD, 9α -OH-4AD, progesterone, testosterone, etc.

Description

Novel KstD enzyme nucleic acid, recombinant vector, saccharomyces cerevisiae and application Technical Field The invention relates to the field of genetic engineering, and belongs to the field of applied microorganism and enzyme engineering. In particular to 3-sterone-delta 1 -dehydrogenase (KstD enzyme) in Fusarium solani, and a coding gene and application thereof. Recombinant vectors or yeast strains containing the nucleic acid and the application thereof in catalyzing substrates such as 4AD, 9 alpha-OH-4 AD, progesterone, testosterone and the like have industrial values. Background The steroid is a kind of organic micromolecule taking cyclopentane polyhydrophenanthrene as a mother nucleus, is widely distributed in the nature, has various kinds, complex structure, exquisite configuration and wide biological activity, and is a very important natural organic compound. The steroid medicine is mainly used for treating （Fernandes P, Cruz A, Angelova B, et al. Microbial conversion of steroid compounds: recent developments [J].Enzyme Microb Technol, 2003, 32(6): 688-705）. of endocrine disturbance, cardiovascular, collagenous diseases, lymphoblastic leukemia, human organ transplantation, anti-inflammatory, anti-tumor, bacterial encephalitis, skin diseases, rheumatism, senile diseases and the like in clinic, and currently, the world approval of the marketed steroid medicine is more than 400, the production value is more than trillion dollars, and the product is only second to antibiotics. The 3-sterone-delta 1 -dehydrogenase is responsible for catalyzing the dehydrogenation reaction of C1 and 2 sites on the A ring of the 3-ketosteroid, and the physiological activity of the steroid is obviously improved through the introduction of double bonds on the C1 and 2 sites on the A ring. At present, the dehydrogenation reaction of steroid C1 and 2 of microorganisms is involved in the production process of a plurality of important steroid compounds clinically, and most of adrenocortical hormone with anti-inflammatory capability, such as prednisone, dexamethasone, palatinose, betamethasone, triamcinolone, methylprednisolone and the like. At present, only the biological and physiological effects of 3-sterone-Delta 1 -dehydrogenase, a minority of the strains of Rhodococcus (Rhodococcus sp.), mycobacterium (Mycobacterium sp.), arthrobacter (Arthrobacter), have been systematically reported and show remarkable species and strain specificity （JIA Hong-Chen, LI Fang, ZHENG Xin-Ling,et al. Research progress of 3-ketosteroid-Δ1-dehydrogenase, a key enzyme for steroid microbial conversion[J].Microbiology China, 2020, 47(7): 2218-2235.）. The C1,2 double bond steroid medicine and its derivative, including 1, 4-Androstenedione (ADD), prednisolone (prednisolone), baodanone (boldenon), etc., the clinical application is wide, the market value is high, the present synthesis mostly relies on chemical synthesis, there are disadvantages such as the complicated synthetic route, use of a large amount of organic reagents, production of by-products and organic reagent residue, on the contrary, the microbial transformation has advantages such as few synthetic steps, short production cycle, few by-products and mild reaction conditions, etc., and meanwhile the microbial transformation has high regioselectivity, some reactions such as hydroxylation, C1,2 dehydrogenation, etc. that are difficult to realize can be carried out by means of microbial transformation. Microbial transformation methods are now increasingly being applied to the production of steroid intermediates and some steroid drugs. The invention aims to dig out the enzyme with the activity of C1 and 2 dehydrogenation reaction, and introduce the coding genes into a heterogeneous host such as yeast for expression, so as to obtain the bacterial strain with the capacity of catalyzing the steroid C1 and 2 dehydrogenation, expand a KstD enzyme resource library and provide a practical application foundation for efficiently synthesizing steroid drugs or natural products. Disclosure of Invention One of the purposes of the invention is to provide a 3-sterone-delta 1 -dehydrogenase (KstD enzyme) in Fusarium putrescens, the other of the invention is to provide a nucleic acid encoding the 3-sterone-delta 1 -dehydrogenase, the other of the invention is to provide a recombinant vector or yeast strain containing the nucleic acid of the 3-sterone-delta 1 -dehydrogenase, the fourth of the invention is to provide the application of the recombinant vector or yeast strain containing the nucleic acid of the 3-sterone-delta 1 -dehydrogenase in preparing androstenedione, and the fifth of the invention is to provide the application of the recombinant vector or yeast strain containing the nucleic acid of the 3-sterone-delta 1 -dehydrogenase in preparing substrates such as 9 alpha-OH-4 AD, progesterone, testosterone and the like. The product androstenedione is a precursor substance for synthesizing various sex hormon