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CN-122012421-A - Flavin dependent oxidase SavA and application thereof in asymmetric synthesis of 4-alkyl butenolide

CN122012421ACN 122012421 ACN122012421 ACN 122012421ACN-122012421-A

Abstract

The invention discloses a flavin dependent oxidase SavA and application thereof in asymmetric synthesis of 4-alkyl butenolide. The SavA enzyme provided by the invention is a novel multifunctional enzyme with the functions of desaturation, hydroxylation and lactonization, and can catalyze fatty acyl thioester to be converted into (4S) -4-alkyl butenolide with a single configuration. The SavA enzyme provided by the invention is a flavin dependent enzyme which naturally exists in a form of combining FAD, an external enzyme reaction does not need to add exogenous FAD, any other coenzyme and cofactor are not involved, only O 2 is used as an oxidant, the reaction process is green and efficient, and the enzyme has the potential of developing into an efficient tool enzyme for synthesizing 4-substituted butenolide by asymmetric biocatalysis.

Inventors

  • ZHOU SHANSHAN
  • LI DONGHUI
  • LI WENRUI
  • ZHAO JINLIAN

Assignees

  • 中国科学院微生物研究所

Dates

Publication Date
20260512
Application Date
20251218

Claims (10)

  1. 1. The flavin-dependent oxidase is characterized by having an amino acid sequence shown as SEQ ID NO.1 or having an amino acid sequence in which residues 453-504 of the amino acid sequence shown as SEQ ID NO.1 are truncated or any region within residues 453-504 are truncated, or having an amino acid sequence which is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown as SEQ ID NO.1 and has the same function, or having an amino acid sequence obtained by conservative substitution of the amino acid sequence shown as SEQ ID NO.1 or having the same function by substitution, deletion or addition of amino acids.
  2. 2. A kit, characterized in that it comprises a flavin-dependent oxidase according to claim 1.
  3. 3. Fusion protein, characterized in that it comprises a flavin dependent oxidase according to claim 1 and a tag for detection and/or purification, optionally selected from one or more of His6, GST, MBP and SUMO.
  4. 4. A nucleic acid molecule encoding a flavin dependent oxidase according to claim 1 or a fusion protein according to claim 3.
  5. 5. An expression vector comprising the nucleic acid molecule of claim 4.
  6. 6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.
  7. 7. A method for producing a flavin-dependent oxidase, characterized in that the method comprises the steps of culturing the host cell according to claim 6 and purifying the obtained flavin-dependent oxidase.
  8. 8. A process for the preparation of (4S) -4-alkyl butenolide, characterized in that the process is carried out using a flavin dependent oxidase according to claim 1.
  9. 9. The method according to claim 8, comprising the step of reacting a fatty acyl thioester with the flavin dependent oxidase, optionally the fatty acyl thioester is a 10-methyldodecanoyl thioester, optionally the 10-methyldodecanoyl thioester is selected from the group consisting of 10-methyldodecanoyl pantetheine thioester and 10-methyldodecanoyl mercaptoethylamine thioester.
  10. 10. Use of a flavin dependent oxidase according to claim 1, a kit according to claim 2, a fusion protein according to claim 3, a nucleic acid molecule according to claim 4, an expression vector according to claim 5 or a host cell according to claim 6 in the preparation of (4S) -4-alkylbutenolide, optionally (4S) -4-alkylbutenolide being (4S) -4- (6-methyloctyl) butenolide.

Description

Flavin dependent oxidase SavA and application thereof in asymmetric synthesis of 4-alkyl butenolide Technical Field The invention belongs to the field of genetic engineering, in particular to a flavin-dependent oxidase SavA and application thereof in asymmetric synthesis of 4-alkyl butenolide, and more particularly relates to a new flavin-dependent oxidase SavA in a biological synthesis path of streptomycete signal molecule abamectin, a nucleic acid molecule thereof, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector, a preparation method of the flavin-dependent oxidase, and application of the flavin-dependent oxidase or the host cell in asymmetric synthesis of 4-alkyl butenolide. Background The signal molecules containing the gamma-butenolide structure or derived from the gamma-butenolide are commonly existed in streptomycete and related actinomycetes, and the molecular structure of the streptomycete is regulated and controlled or the biosynthesis of secondary metabolites is regulated by combining with specific transcription inhibitor, so that the molecular structure has important application value for the fermentation production of industrial antibiotics. Avermectin (avenolide) is a signal molecule with a 4-alkyl butenolide skeleton, and is structurally characterized in that a long-chain alkyl chain is introduced into the C4 position of gamma-butenolide, and literature reports that the avermectin has specific regulation and control on the generation of （Kitani S., Miyamoto K. T., Takamatsu S., et al. Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis[J]. Proceedings of the National Academy of Sciences, 2011, 108(39): 16410-16415）, of avermectin in the middle path of Streptomyces AVERMITILIS MA-4680, and avermectin and semisynthetic derivatives (such as ivermectin) thereof are used as broad-spectrum efficient antibiotic biopesticides and antiparasitic drugs, and are widely used for controlling agricultural pests, controlling parasites in animal husbandry and treating certain parasitic infections of human beings. In addition, gamma-butenolide is widely used in the fields of medicine, agriculture, perfume, materials and the like as a structural unit which has various functions and is ubiquitous. It can function as a key active pharmacophore in many active natural products (e.g., bullatacin with anticancer activity) and important synthetic drugs (e.g., anti-inflammatory drugs firocoxib). Agricultural pesticides such as spirodiclofen, flupirfuranone and the like also have gamma-butenolide structural fragments. In addition, gamma-butyrolactone (such as gamma-undecalactone, also called peach aldehyde) which is a carbon-carbon double bond reduction product has fruit fragrance and peach-like fragrance, and is widely used in spices and food additives. Currently, the chemical synthesis of gamma-butenolide ring generally involves multiple synthesis steps and multiple reagents, and the synthesis is cumbersome and has low efficiency. From a sustainability point of view, these methods are laborious and expensive and are serious to environmental pollution. Biocatalysis is a hot spot of research in recent years due to its green, environmental protection and sustainable characteristics. However, despite the large number of natural products containing gamma-butenolide structures in nature, the current knowledge of the biosynthetic mechanisms is very limited, and the reported biocatalytic gamma-butenolide synthesis pathways are very complex, usually involving several enzymes with different cofactor requirements, and difficult to directly apply to industrial production. Therefore, how to develop a more concise and efficient biocatalytic preparation method of gamma-butenolide has become one of the focuses of attention of a plurality of prospective researchers in the field, and has important scientific significance and application value. Disclosure of Invention The invention aims to provide a streptomycete-derived flavin-dependent enzyme oxidase SavA, an effect thereof in biosynthesis of abamectin and an application of an enzymatic reaction thereof in asymmetric synthesis of (4S) -4-alkyl butenolide. In one aspect, the invention provides a flavin-dependent oxidase which has an amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence obtained by truncating 453 th to 504 th residues of the amino acid sequence shown as SEQ ID NO.1 or truncating 453 th to 504 th residues in any region, or an amino acid sequence which has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown as SEQ ID NO.1 and has the same function, or an amino acid sequence obtained by conservative substitution of the amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence with the same function formed by substitution, deleti