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Methods for terpenoid production

A terpene synthase and terpene technology, applied in biochemical equipment and methods, oxidoreductase, lyase, etc., can solve the problems of insufficient activity and limitation of terpene synthase

Pending Publication Date: 2021-06-22
AGENCY FOR SCI TECH & RES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some cases, terpenoid production is limited by insufficient activity of terpene synthase

Method used

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  • Methods for terpenoid production
  • Methods for terpenoid production
  • Methods for terpenoid production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Engineering E. coli strains for TPS characterization

[0149] Wild-type E. coli BL21 produces small amounts of terpene precursors (GPP and FPP), therefore, it is not suitable as a TPS characterization platform. In order to improve the sensitivity and accuracy of detection, DXS and IDI were overexpressed to increase the intracellular precursor ( image 3 ). Unlike existing methods, two DXS mutants (SL3 and SL5) were identified based on random mutagenesis and screening. As shown in Figure 2, SL3 and SL5 have higher solubility and thus higher activity than wild-type DXS compared to wild-type DXS. More importantly, SL5 had higher specific activity than wild-type DXS ( Figure 2B ). Therefore, the lycopene production of strains overexpressing SL3 or SL5 was higher than that of wild-type DXS. Here, lycopene was used as an indicator to demonstrate that GPP and FPP were higher in the strains (SL3 and SL5). Using DXS mutants, the detection sensitivity of the cell platform ...

Embodiment 2

[0151] Analysis of terpenes produced in Amanita cylindrica

[0152] To obtain an assessment of the terpenes produced in Amanita cylindrosum, the volatile compounds produced by its liquid cultures were analyzed. The illudin precursor Δ(6)protoilurene 1 is the main metabolite produced in A. cylindrica ( figure 1 ). In addition, after 26 days of culture, a small amount of α-iocomene was observed # , α-, β-piperene, β-cubene, γ-ylangolene 2, δ-junipene 4, β-celerene # 9. Piper cubeba oleinol, epipiperumba oleenol 10 and Piper cubeba oleinol ( Figure 4 and Figure 5 chemical structures and mass spectra of all terpenes identified in the study, respectively). The results indicated that the mushroom Acromanus cylindrica produced structurally diverse terpenes.

Embodiment 3

[0154] Sesquiterpene Alcohols from Amanita cylindrica

[0155] Twenty putative terpenoids were detected by GC / MS analysis during fruiting of Amanita cylindrica, among which the experimentally identified Δ6-protoilurene # is the most prominent compound ( figure 1 ). Other major compounds are α-piperene, α-isocomene # , β-piperene and δ-juniperene ( Figure 4 The compound structure in Figure 5 mass spectrometry in ). A blastp search of putative STSs present in the A. cylindrica genome revealed 11 genes (Table 2 and Figure 6 ). Seven TPS clustered into at least 3 distinct groups with known Basidiomycete TPS. Four putative TPSs (AAE3_09008, AAE3_06743, AAE3_04444, and AAE3_05024) compile into a cluster ( Figure 6 ). Four of the STS genes (AAE3_10454, AAE3_12839, AAE3_04120 and AAE3_13291) were part of a cluster that included two to five P450 monooxygenases.

[0156] Table 2 Details of the STS gene of Amanitula cylindrica

[0157] protein ID bracket gene...

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Abstract

The present invention relates to a bacteria strain comprising one or more vectors encoding a) one or more enzymes to produce one or more terpene precursors; and b) a fungal terpene synthase (FTPS) and a method of producing terpenoids by a) culturing said bacteria strain in an expression medium; and b) isolating the terpenoid from said expression medium. Also claimed are genetically modified 1-deoxyxylulose-5-phosphate synthase (DXS) enzymes and modified FTPS isolated from agrocybe aegerita or agrocybe pediades.

Description

[0001] Cross References to Related Applications [0002] This application claims priority from Singapore Application No. 10201807514P filed on 31 August 2018, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The invention belongs to the field of biotechnology. In particular, the present invention relates to methods for the discovery of fungal terpene synthases and the use of fungal terpene synthases in the production of terpenoids. Background technique [0004] Terpenoids constitute one of the most structurally diverse classes of natural products and have a wide range of applications: as pharmaceuticals (e.g. paclitaxel and artemisinin), as food colorings (e.g. carotenoids), flavorings and fragrances (e.g. nokadone and sclareol) and biofuels (such as farnesene). Terpene diversity is largely attributed to terpene synthases (TPS), which convert acyclic prenyl diphosphate precursors into diverse cyclic and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P5/00C12N15/09
CPCC12P5/007C12Y402/03C12Y202/01007C12N15/52C12N9/88C12Y402/03046C12Y402/03048C12N9/1022C12N9/90C12P5/002C12Y503/03002
Inventor 张聪强陈茜娴R·马丁朱兴奋
Owner AGENCY FOR SCI TECH & RES
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