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CN-121975649-A - Engineering microorganism expressing heterologous squalene synthase and construction method and application thereof

CN121975649ACN 121975649 ACN121975649 ACN 121975649ACN-121975649-A

Abstract

The invention discloses an engineering microorganism expressing heterologous squalene synthase, and a construction method and application thereof. The engineered microorganism is a squalene-producing recombinant microorganism obtained by introducing and expressing at least one heterologous squalene synthase in a host microorganism. By introducing and expressing the heterologous squalene synthase, the invention constructs a squalene synthesis channel which is parallel and independent to the endogenous sterol synthesis path, efficiently accumulates squalene, improves the synthesis capacity of squalene, and improves the yield by more than 29% compared with a control.

Inventors

  • ZHU QIAOYUN
  • CHEN CHENG
  • CHEN SHILIN
  • JIANG HANXIAO

Assignees

  • 柯泰亚生物科技(上海)有限公司

Dates

Publication Date
20260505
Application Date
20260409

Claims (9)

  1. 1. The engineered microorganism is a recombinant microorganism for producing squalene, wherein the recombinant microorganism is obtained by introducing and expressing at least one heterologous squalene synthase in saccharomyces cerevisiae, the heterologous squalene synthase is from any one of human, rat or methyl coccus capsulatus, and the amino acid sequence of the heterologous squalene synthase is shown as SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 11.
  2. 2. The engineered microorganism of claim 1, wherein the nucleic acid sequence obtained by codon optimization of the gene encoding the heterologous squalene synthase is the sequences shown in SEQ ID No.12, SEQ ID No.13, and SEQ ID No. 17.
  3. 3. The engineered microorganism of claim 1, wherein the engineered microorganism further overexpresses any one or a combination of at least two of an acetyl-coa transferase gene, a 3-methyl-3-hydroxyglutaryl-coa synthase gene, a 3-hydroxy-3-methylglutaryl-coa reductase gene, a mevalonate kinase gene, a mevalonate-5-phosphate kinase gene, a mevalonate-5-pyrophosphate decarboxylase gene, an isopentenyl pyrophosphate isomerase gene, or a farnesyl pyrophosphate synthase gene.
  4. 4. A method for preparing an engineered microorganism according to any one of claims 1 to 3, comprising introducing into said host microorganism a nucleic acid molecule encoding a heterologous squalene synthase, whereby a recombinant microorganism expressing said heterologous squalene synthase is obtained, said nucleic acid molecule being shown in SEQ ID No.12, SEQ ID No.13 and SEQ ID No. 17.
  5. 5. The method of claim 4, wherein introducing a nucleic acid molecule encoding a heterologous squalene synthase into the host microorganism comprises site-directed integration of the nucleic acid molecule encoding a heterologous squalene synthase into a specific genomic locus of the host microorganism, wherein the specific genomic locus comprises any one of abm, lpp1, dpp1, ho or gal80 loci.
  6. 6. A process for producing squalene, comprising culturing the engineered microorganism of any one of claims 1 to 3, collecting the culture, and purifying to obtain squalene.
  7. 7. The method of claim 6, wherein the culturing is performed in a medium comprising a carbon source comprising any one or a combination of at least two of glucose, fructose, sucrose, glycerol, maltose, acetic acid, lactic acid, or succinic acid.
  8. 8. The method according to claim 6, wherein the temperature of the cultivation is 25-35 ℃ for 1-10 days.
  9. 9. Use of an engineered microorganism according to any one of claims 1 to 3 or an engineered microorganism constructed by a method according to claim 4 or 5 for the production of squalene or for the preparation of a product containing squalene.

Description

Engineering microorganism expressing heterologous squalene synthase and construction method and application thereof Technical Field The invention belongs to the technical fields of bioengineering and microbial fermentation, and relates to an engineering microorganism for expressing heterologous squalene synthase, and a construction method and application thereof. Background Squalene is an important triterpene compound precursor and has wide application in the fields of medicines (such as vaccine adjuvants), cosmetics (such as moisturizers) and high-value health products. Traditional extraction from shark liver oil has problems of resource destruction, poor sustainability and purity, and biosynthesis using microbial cell factories is an ideal alternative route. Saccharomyces cerevisiae, a well-established model microorganism, possesses its own complete mevalonate pathway, and is capable of synthesizing squalene, and is therefore considered an ideal cell factory for achieving green bio-production of squalene. In the sterol synthesis pathway inherent to Saccharomyces cerevisiae, squalene synthase, encoded by the ERG9 gene, catalyzes the condensation of two molecules of farnesyl pyrophosphate to form squalene, a key node reaction leading to sterols and triterpenes. The expression and activity of ERG9 is regulated by multi-level, precise feedback of intracellular sterol levels, potentially making it difficult for carbon flow to be effectively directed to excessive accumulation of squalene. The art of metabolic engineering has attempted to engineer ERG9 with various strategies to increase the ability to produce squalene. The most common thinking is through promoter engineering, or directed evolution attempts on the ERG9 enzyme molecule itself, etc. The invention introduces heterogeneous squalene synthase to construct a squalene synthesis channel which is parallel and independent to the endogenous sterol synthesis path, thereby improving the squalene synthesis capacity. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the invention provides an engineering microorganism for expressing heterologous squalene synthase, and a construction method and application thereof. In order to achieve the aim of the invention, the invention adopts the following technical scheme: In a first aspect, the invention provides an engineered microorganism which is a recombinant microorganism obtained by introducing and expressing at least one heterologous squalene synthase in a host microorganism, wherein the heterologous squalene synthase is derived from any one of human, rat or methyl coccus capsular, and the amino acid sequence of the heterologous squalene synthase is shown as SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 11. By introducing and expressing the heterologous squalene synthase which is not controlled by a host cell endogenous sterol feedback regulation network, the invention constructs an independent squalene synthesis bypass which is decoupled from an endogenous essential sterol synthesis path in regulation. The strategy fundamentally avoids the defect of cell growth caused by disturbance of endogenous ERG9, and realizes separation of squalene synthesis and normal cell growth, thereby breaking through the bottleneck of traditional metabolic engineering on the node. The squalene synthase according to the present invention refers to an enzyme capable of catalyzing the condensation of two molecules of farnesyl pyrophosphate (FPP) to form squalene, which may comprise an enzyme as defined by EC 2.5.1.21, which may be referred to in different databases or literature as squalene synthase, pre-squalene synthase or related to cyclase, but whose core "synthase" activity is well defined and uniform. Squalene synthase Hs.ERG9 amino acid sequence SEQ ID NO.6 from human (homosapiens): MEFVKCLGHPEEFYNLVRFRIGGKRKVMPKMDQDSLSSSLKTCYKYLNQTSRSFAAVIQALDGEMRNAVCIFYLVLRALDTLEDDMTISVEKKVPLLHNFHSFLYQPDWRFMESKEKDRQVLEDFPTISLEFRNLAEKYQTVIADICRRMGIGMAEFLDKHVTSEQEWDKYCHYVAGLVGIGLSRLFSASEFEDPLVGEDTERANSMGLFLQKTNIIRDYLEDQQGGREFWPQEVWSRYVKKLGDFAKPENIDLAVQCLNELITNALHHIPDVITYLSRLRNQSVFNFCAIPQVMAIATLAACYNNQQVFKGAVKIRKGQAVTLMMDATNMPAVKAIIYQYMEEIYHRIPDSDPSSSKTRQIISTIRTQNLPNCQLISRSHYSPIYLSFVMLLAALSWQYLTTLSQVTEDYVQTGEH. Squalene synthase Rn.ERG9 amino acid sequence SEQ ID NO.7 from rat (Rattus norvegicus): MEFVKCLGHPEEFYNLLRFRMGGRRNFIPKMDRNSLSNSLKTCYKYLDQTSRSFAAVIQALDGDIRHAVCVFYLILRAMDTVEDDMAISVEKKIPLLRNFHTFLYEPEWRFTESKEKHRVVLEDFPTISLEFRNLAEKYQTVIADICHRMGCGMAEFLNKDVTSKQDWDKYCHYVAGLVGIGLSRLFSASEFEDPIVGEDTECANSMGLFLQKTNIIRDYLEDQQEGRQFWPQEVWGKYVKKLEDFVKPENVDVAVKCLNELITNALQHIPDVITYLSRLRNQSVFNFCAIPQVMAIATLAACYNNHQVFKGVVKIRKGQAVTLMMDATNMPAVKAIIYQYIEEIYHRVPNSDPSASKAKQLISNIRTQSLPNCQLISRSHYSPIYLSFIMLLAALSWQYLSTLSQVTEDYVQREH. Squalene synthase Ld.ERG9 amino acid sequence SEQ ID NO.8 from Leishmania donovani (LEISHMANIA DONOVANI): MGFFSDSVAMMRVKWQMRSVKIQVPPEETDLRFCYDIMNDVSRSFAVVVAQLADQQLRDAICIFYLVLRALDTLEDDMSVPVDVKLKELPKFHTHTSDMSWCMSGVGEGRERELL