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Methods and materials for recombinant production of saffron compounds

A technology of saffron and saffron acid, applied in the field of recombinant production of compounds from saffron, a saffron plant, can solve problems such as low efficiency

Inactive Publication Date: 2014-04-30
EVOLVA SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Often, production involves manual removal of stigmas from flowers, which is also an inefficient process

Method used

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  • Methods and materials for recombinant production of saffron compounds
  • Methods and materials for recombinant production of saffron compounds
  • Methods and materials for recombinant production of saffron compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1: Production of beta-carotene in yeast

[0123] Construction of β-carotene-producing yeast reporter strains for use in eYAC experiments designed to discover optimal combinations of saffron biosynthetic genes. Neurospora crassa phytoene desaturase (also known as phytoene desaturase) (accession number XP_964713) and Phaffia GGDP synthase (also known as geranylgeranyl Pyrophosphate synthase or CrtE) (accession number DQ012943) and Phaffia phytoene-β-carotene synthase CrtYB (accession number AY177204) genes were inserted into expression cassettes, and these expression cassettes were integrated into the experimental In the genome of the chamber yeast strain Saccharomyces cerevisiae CEN.PK113-11. Phytoene desaturase and CrtYB were overexpressed under the control of the strong constitutive GPD1 promoter, and CrtE was overexpressed using the strong constitutive TPI1 promoter. Chromosomal integration of the Phaffia CrtE and Neurospora crassa phytoene desaturase expres...

Embodiment 2

[0125] Example 2: Yeast production of optimized HBC and crocetin dialdehyde

[0126] Crocetin is known to be formed from crocetin dialdehyde, and crocetin dialdehyde and hydroxy-β-cyclic citral (HBC) are produced after cleavage of zeaxanthin with the enzyme zeaxanthin-cleaving dioxygenase (ZCD) . Using the eYAC described in Example 1 and the β-carotene-producing yeast strain, a batch of genes was assembled into the eYAC to establish the optimal pathway for the biosynthesis of crocindialdehyde and HBC.

[0127] A panel of genetic analogs of the enzyme for the conversion of β-carotene to crocindialdehyde was generated by yeast codon-optimized synthesis (DNA2.0) and inserted under the promoters of various methionine-repressible genes eYAC enters the vector (Entry Vector). Naesby et al., Microb Cell Fact. 8:45 (2009) describes the use of eYAC technology. The expression cassettes for the 37 saffron biosynthetic genes shown in Table 1 were ligated (with or without UGT genes) ...

Embodiment 3

[0134] Example 3: Discovery of UGTs that form crocinpicrin

[0135] Glucosyltransferases are required to form saffron from hydroxy-β-cyclocitral (HBC). This reaction is aglycone glucosylation rather than glucose-glucose bond formation, and there are families of glycosyltransferases that utilize UDP-glucose to screen for this type of activity.

[0136] Source of HBC substrate

[0137] HBC was synthesized and the desired compound was purified by chiral column chromatography (GVK, Hyderabad).

[0138] Screening for UGT enzymes

[0139] Crocinopicroside formation was assayed for the following UGTs: Stevia 88B1, 76G1, 74G1, 91D2e, 85C2, 73EV12; Vinca UGT2; and Arabidopsis UGT75B1 and Arabidopsis hybridases UGT353 and UGT354 ( image 3 sequence provided).

[0140] Genes encoding these UGTs were cloned into plasmids utilizing the T7 promoter and transformed into E. coli BL21 cells for expression studies. Strains with these UGTs were induced with 0.1 mM IPTG and the induced cultur...

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Abstract

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express a zeaxanthin cleavage dioxygenase alone or in combination with recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce compounds from saffron such as crocetin, crocetin dialdehyde, crocin, or picrocrocin.

Description

[0001] This application claims U.S. Provisional Application No. 61 / 521171 filed August 8, 2011, U.S. Provisional Application No. 61 / 576460 filed December 16, 2011, and U.S. Provisional Application No. 61 / 2012. Priority of 61 / 595450. technical field [0002] The present invention relates to methods and materials for the recombinant production of compounds from the saffron plant Crocus sativus, and more particularly to methods and materials for the recombinant production in recombinant hosts of flavorants, fragrances ( aromatant) and colorant compounds. Background technique [0003] Saffron is a dried spice prepared by extraction from the stigma of Crocus sativus L. flowers and is thought to have been used for over 3500 years. The fragrance has historically been used for a variety of medicinal purposes, but more recently has been exploited primarily for its colorant properties. Crocetin, one of the main constituents of saffron, has antioxidant properties similar to related c...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N9/10
CPCC12N9/1048C12N9/0006C12N9/1051C12P19/46A01H6/00C12N9/10C12N9/88C12N15/80C12N15/81C12R2001/19C12N1/205C12R2001/865C12N1/185
Inventor 沙拉姆·拉加万约恩·汉森赛伦德拉·松卡尔萨西什·库马尔卡利安·K·库马尔穆拉利·潘沙佩奇萨埃斯本·哈尔克耶尔·汉森克拉夫斯·里谢德·汉森
Owner EVOLVA SA
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