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CN-121975627-A - Engineering algae strain capable of synthesizing alpha-farnesene and preparation method thereof

CN121975627ACN 121975627 ACN121975627 ACN 121975627ACN-121975627-A

Abstract

The invention relates to the field of genetic engineering and biological engineering, in particular to engineering algae capable of synthesizing alpha-farnesene and a preparation method thereof. According to the invention, the MEP metabolic network of the synechocystis PCC 6803 is modified, and through single copy tandem recombination and multi-copy combination of dxs, ispA, idi and AFS genes, the optimization of the farnesene MEP metabolic pathway is realized, the carbon flux of the direct synthesis farnesene is accurately regulated and controlled, and under the optimal effect, the yield of alpha-farnesene within 9 days is 83.07 mg/L (25.82 mg/g DCW), so that the method has economic application value.

Inventors

  • WANG BILI
  • ZHANG QIQI
  • JIANG HUI
  • YAO GE
  • HAN PENGGANG
  • Wan Xiukun
  • BAO SHAOHENG

Assignees

  • 中国人民解放军军事科学院防化研究院

Dates

Publication Date
20260505
Application Date
20251228

Claims (10)

  1. 1. An engineering algae strain capable of synthesizing alpha-farnesene is characterized in that the engineering algae strain takes synechocystis as chassis algae, and the algae strain comprises modified plasmids of a plurality of key enzyme gene expression frames, so that the metabolic flux of MEP (methyl ethyl acetate) channels is enhanced, and the synthesis and accumulation of the alpha-farnesene are promoted; The key enzyme genes comprise dxs (1-deoxy-D-xylulose 5-phosphate synthase), idi (isopentenyl pyrophosphate isomerase), ispA (farnesyl pyrophosphate synthase) and AFS (alpha-farnesene synthase), and are shown as Seq No. 1-4; The key enzyme gene expression frame is constructed by the key enzyme genes singly or in combination; the enzyme gene expression frame is integrated in series with one or more than two of neutral sites Slr0168, slr9394 and Slr 1556; the enzyme gene expression frame drives expression through one or more of a promoter PcpcB, a promoter PrbcL and a promoter 6803.
  2. 2. The engineered strain of algae capable of synthesizing α -farnesene according to claim 1, wherein the engineered strain employs a multicopy tandem strategy, and wherein the key enzyme genes are repeated at different neutral sites.
  3. 3. The engineered strain of algae capable of synthesizing α -farnesene according to claim 1, wherein the enzyme gene is inserted between the upstream and downstream homology arms of the neutral site.
  4. 4. The engineered strain of algae capable of synthesizing α -farnesene of claim 1, wherein the combination of key enzyme gene expression cassettes comprises idi-ispA、idi-ispA-dxs、AFS-ispA、AFS-idi、AFS-dxs、AFS-ispA-idi、AFS-ispA-dxs、AFS-idi-dxs、AFS-ispA-idi-dxs.
  5. 5. The engineered algae strain capable of synthesizing alpha-farnesene according to claim 1, wherein PrbcL6803 drives dxs and combinations containing dxs in the key enzyme gene expression frame, pcpcB560 drives the rest of genes.
  6. 6. The engineering alga strain capable of synthesizing alpha-farnesene according to claim 1, wherein the engineering alga strain is characterized in that Slr0168 site is inserted into AFS-ispA, slr1556 site is inserted into AFS-ispA, and Slr9394 site is inserted into AFS-ispA-idi-dxs.
  7. 7. A method for preparing the engineering algae strain according to any one of claims 1 to 6, comprising the following steps: s1, synthesizing a key enzyme gene AFS, ispA, idi, dxs; s2, constructing an expression frame containing the genes, inserting one or more than two of neutral sites Slr0168, slr9394 and Slr1556, and connecting with a plasmid vector to form a modified plasmid; S3, transforming the transformed plasmid, screening resistance and verifying by PCR to obtain the engineering algae strain with stable integration.
  8. 8. The method for producing an engineered strain of algae according to claim 7, wherein the plasmid vector is pUC19.
  9. 9. An application of the engineering algae strain according to any one of claims 1-6 in synthesizing alpha-farnesene.
  10. 10. The use of an engineered strain of algae according to claim 9 for the synthesis of α -farnesene, wherein the strain of algae performs the photosynthesis of α -farnesene under light conditions using CO 2 as a carbon source.

Description

Engineering algae strain capable of synthesizing alpha-farnesene and preparation method thereof Technical Field The invention relates to the field of genetic engineering and biological engineering, in particular to engineering algae capable of synthesizing alpha-farnesene and a preparation method thereof. Background Farnesene (3, 7, 11-trimethyl-1, 3,6, 10-dodecatetraene; FARNESENE; C 15H24) is also known as farnesene, is an acyclic volatile sesquiterpene, is found in apple peel at the earliest, is mainly found in essential oils such as sweet orange oil, rose oil, ylang-ylang oil and orange oil, and has important value in the fields of medicines (nutritional health products), cosmetics, bioenergy and the like. For example, farnesene can be used to prepare high heating value, efficient, clean and renewable fuels, which are ideal alternatives to blended aviation fuels and thus have attracted considerable attention. Farnesene is an important raw material in the field of medicine, and can be used for synthesizing precursor isophytol of vitamin E, wherein the vitamin E is one of the largest vitamin products in the world, and the carbon emission in the production process is reduced by 60%, so that the farnesene is more environment-friendly than the traditional chemical synthesis technology. In the chemical industry, farnesene can also be used as an additive to give better plasticity to rubber, and these new farnesene based materials have different properties such as reduced rolling resistance, improved compression and permanent set, and higher softness. In addition, farnesene is useful in the production of lubricants, surfactants and cosmetics. Extraction of farnesene from plants for mass market use is challenging because of the relatively low content of farnesene in plants and the significant effects of plant growth due to seasonal, regional climates, etc. With respect to studies on chemical synthesis of farnesene, availability of materials, high production cost, low production efficiency and environmental pollution are unavoidable problems in chemical synthesis. The microbial growth cycle is short, the microbial growth cycle is less affected by the environment and does not occupy cultivated land, and the microbial synthesis overcomes the defects of natural extraction and chemical synthesis and is an ideal choice for producing farnesene. Alpha-farnesene has been synthesized by metabolic engineering strategies in different microorganisms, including yeast, E.coli and cyanobacteria. Among them, yeasts and escherichia coli as heterotrophs rely on expensive organic carbon sources, which impair the economic and environmental benefits of the process. Blue algae have great advantages over plants in photosynthetic efficiency (above 10%) and growth rate. In addition, the biosynthesis of the biofuel and the chemical is realized by utilizing the photosynthetic microorganism cell factory, and compared with the traditional chemical synthesis, the method has the advantages of good stereoselectivity, mild reaction process and less process toxicity. To date, some progress has been made in the preparation of farnesenes using cyanobacteria as host cells. The application of blue algae can effectively utilize carbon dioxide, realize 'light-driven cell factory' for green synthetic chemical substance production, and simultaneously relieve urgent environment and energy pressure, thereby having important significance. Blue algae is an ideal cell factory for producing chemical substances by carbon negative, and has great potential to directly utilize light and carbon dioxide as the only energy and carbon source. The terpene compounds have long and complex metabolic pathways, poor synthetase specificity, low selectivity and low efficiency, and are troublesome for the synthesis of terpenes in heterologous cell factories. At present, the efficient directional synthesis of various terpenes is realized in different blue algae chassis, and the feasibility of the terpenes synthesis by taking blue algae as the chassis is fully verified. Synechocystis PCC 6803, synechococcus PCC 7942 and synechococcus PCC 7002 have become very promising terpene production hosts. However, none of the reported engineering algae was ideal in terms of farnesene yield. In cyanobacteria, farnesene is synthesized by the methylerythritol-phosphate (MEP) pathway, and glyceraldehyde-3-phosphate and pyruvic acid produced by the action of light are used as substrates to convert into the common precursors of all terpenes, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). For farnesene synthesis, two molecules of IPP and one molecule of DMAPP can generate farnesyl pyrophosphate (FPP), and farnesene can be produced by over-expressing farnesene synthase by taking FPP as a substrate. Although metabolic strategies have produced progressive improvements, the titres obtained in engineered cyanobacteria strains (e.g., ,~2.22 mg/L/d,Sun, J., Xu, X., Wu, Y., Sun,