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CN-121991867-A - Recombinant streptomyces capable of producing terpenes at high yield and application thereof

CN121991867ACN 121991867 ACN121991867 ACN 121991867ACN-121991867-A

Abstract

The invention discloses recombinant streptomyces with high terpene yield and application thereof, and belongs to the technical field of microorganisms. The invention firstly introduces the complete MVA path in streptomycete in a heterologous way, and constructs the kasOp-containing streptomycete through PCR amplification of MVK/DPMD/PMK/IDI/HMG-CoA/HMGR/AACS key enzyme genes The expression vector of the promoter is subjected to joint transfer to obtain the recombinant strain. Lycopene is used as a visual marker, and fermentation verification shows that the terpenoid yield of the recombinant strain is improved by about 50% compared with that of a control. The invention combines promoter activation and metabolic pathway optimization, breaks single regulation and control limitation, and provides a high-efficiency chassis for synthesizing terpenoid derivatives.

Inventors

  • HOU ANWEI
  • LI YUANYUAN
  • XU MIN
  • DENG ZIXIN

Assignees

  • 中国科学院天津工业生物技术研究所
  • 合成生物学海河实验室

Dates

Publication Date
20260508
Application Date
20251231

Claims (10)

  1. 1. A recombinant strain for producing terpenoid with high yield is characterized in that a complete MVA pathway is introduced into a starting strain in a heterologous manner to obtain the recombinant strain.
  2. 2. Recombinant bacterium according to claim 1, characterized in that the introduction of the complete MVA pathway is a key gene introduced into the MVA pathway, comprising MVK, DPMD, PMK, IDI, HMG-CoA, HMGR and AACS genes, preferably derived from streptomyces Streptomyces durocortorensis, the nucleotide sequence of which is shown in SEQ ID nos. 1-7 or the degenerate sequence thereof, respectively.
  3. 3. Recombinant bacterium according to claim 2, characterized in that the starting bacterium comprises a wild-type or genetically engineered prokaryote, preferably comprising streptomyces, escherichia coli and actinomycetes, more preferably the streptomyces comprises a wild-type streptomyces, or a streptomyces obtained by modification, mutation, mutagenesis or genetic recombination, preferably the streptomyces is streptomyces coelicolor.
  4. 4. An expression vector for constructing the recombinant bacterium of claim 1, wherein the vector comprises a promoter and a key gene of the MVA pathway, the key gene of the MVA pathway comprising MVK, DPMD, PMK, IDI, HMG-CoA, HMGR and AACS genes, preferably the promoter is kasOp Promoter, said kasOp The nucleotide sequence of the promoter is shown as SEQ ID No. 8.
  5. 5. A method for constructing a recombinant bacterium according to claim 1, comprising the step of transferring the expression vector according to claim 4 to Streptomyces coelicolor M1154 via E.coli conjugation.
  6. 6. The method according to claim 5, wherein the conjugation transfer uses MS solid medium containing MgCl 2 , and the selection uses nalidixic acid and hygromycin double resistance selection.
  7. 7. Use of a recombinant bacterium according to any one of claims 1-3 or obtained by a construction method according to any one of claims 5-6 for the production of terpenoids.
  8. 8. The use according to claim 7 wherein the terpenoid comprises lycopene.
  9. 9. A production method of a terpenoid is characterized by comprising the steps of fermenting and culturing the recombinant bacterium according to any one of claims 1-3 or the recombinant bacterium obtained by the construction method according to any one of claims 5-6, centrifuging, ultrasonicating and organic extraction treatment of a fermentation product.
  10. 10. The production method according to claim 9, wherein the fermentation culture is performed in SM medium under fermentation conditions of 25-33 ℃ for 4-10 days at 150-300rpm, preferably 30 ℃ for 5-8 days at 200-250 rpm.

Description

Recombinant streptomyces capable of producing terpenes at high yield and application thereof Technical Field The invention belongs to the technical field of microorganisms, and particularly relates to recombinant streptomyces with high terpene yield and application thereof. Background Terpenoids are a generic term for compounds containing isoprene units. It is widely found in nature, and to date, more than 12 tens of thousands of terpenoids have been found in animals, plants and microorganisms. The compounds have a plurality of physiological activities and are widely applied to the industries of foods, cosmetics and medicines. Terpenoid is mainly extracted from natural products, chemically synthesized, fermented by microorganism, etc. The natural product is extracted mainly by extracting, purifying and separating specific plants to obtain corresponding terpenoid, however, the production mode is influenced by climate, variety, geographical position, maturity and other uncontrollable factors, has obvious seasonality and content instability, and has higher cost of large-area planting and cultivation and lower content. In addition, purification and isolation of specific terpenoids in extracts containing multiple components is technically very difficult, which together make many compounds with good physiological activity expensive. The chemical synthesis method has the characteristics of numerous steps, high synthesis difficulty, pollution and the like in the synthesis process, and the quality, safety and application range of the product are limited. The microbial fermentation method mainly utilizes the biological metabolism of microorganisms to convert basic metabolites such as glucose, starch, soybean cake powder and other cheap raw materials into corresponding terpenoid compounds, and the method is not influenced by factors such as seasons, regions, climate and the like, and has the advantages of easily obtained raw materials, short production period, simple process operation, low cost, controllable product quality, easily purified product, high safety and less environmental pollution, thereby not only solving the problem of occupying a large amount of cultivated land due to plant cultivation and the like, but also solving the defect of non-environmental protection of chemical synthesis. Most importantly, terpenoids produced by fermentation methods are natural products, the activity of which is consistent with that of active ingredients extracted from natural plants, and are considered as the most promising methods for the production of terpenoids. CN119120337a (2024.12.13) discloses a method for realizing high yield of terpenoid by modifying streptomycete chassis, the core idea is to enhance the expression of key rate-limiting enzymes in methylerythrose phosphate (MEP) pathway, such as DXS, DXR and GGPS, at specific sites of genome by utilizing homologous recombination technology, thereby remarkably improving the synthesis efficiency of sesquiterpene and diterpene precursors, finally realizing the improvement of the overall yield of terpenoid, and excessively strengthening a single module (MEP pathway) possibly breaks the inherent metabolic balance of cells, resulting in accumulation of toxic intermediates and growth inhibition. CN105176899a (2015.12.23) proposes a strategy for constructing high-yielding recombinant strains, focusing on the efficient enhancement of target gene expression by introducing insulator sequences in the genome and artificially synthesized strong promoters. The method is also applied to streptomycete chassis, and particularly aims at regulating and controlling key genes such as farnesyl diphosphate synthase (FPS) and the like, so that the synthesis of terpenoid is promoted. The example of the patent focuses mainly on the production of lycopene, and achieves remarkable yield increase of target products by optimizing metabolic pathways, but the constitutive strong promoter is continuously expressed with high energy consumption, so that huge metabolic burden can be caused, and strain degradation or productivity reduction is easily caused by long-term culture. In contrast, the research object of CN119875866A (2025.04.25) is recombinant saccharomycetes, which is mainly used for producing the sesquiterpene compound beta-elemene by efficient fermentation. The technical lines include heterologous expression of a variety of key enzyme genes in yeast, such as ERG20 (farnesyl diphosphate synthase), lsLTC (sesquiterpene synthase) and RrFPS (bifunctional enzyme), while downregulating the expression of squalene synthase (ERG 9) to reduce competition of metabolic flux to sterol pathways. Through the metabolic flow optimization design, the synthesis level of beta-elemene is improved obviously, the catalytic efficiency, cofactor requirement and subcellular localization of potential heterologous enzymes (such as RrFPS, lsLTC 2) in industrial application are revealed, and the potential heterologous enz