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Tropane alkaloid (TA) producing non-plant host cells, and methods of making and using the same

A technology for alkaloids and tropane, which is applied to non-plant host cells producing tropane alkaloids (TA) and the fields of their preparation and use, can solve the problems of long generation time, poor economies of scale, unproven and the like

Pending Publication Date: 2021-12-10
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulties posed by TA stereochemistry, the total chemical synthesis of TA from simple starting materials has not proven sufficiently economical for industrial use
Furthermore, to date, poor economies of scale and long generation times have made engineering transgenic plants or plant cultures with increased TA production an unfeasible strategy for obtaining these compounds

Method used

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  • Tropane alkaloid (TA) producing non-plant host cells, and methods of making and using the same
  • Tropane alkaloid (TA) producing non-plant host cells, and methods of making and using the same
  • Tropane alkaloid (TA) producing non-plant host cells, and methods of making and using the same

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0269] For high-purity, small-scale preparations, TA precursors or TA can be purified in a single step by liquid chromatography.

[0270] Yeast-derived alkaloid APIs versus plant-derived APIs

[0271] Clarified yeast medium (CYCM) may contain various impurities. The clarified yeast medium can be dehydrated by vacuum and / or heat to produce an alkaloid-rich powder. This product is similar to a concentrate of nightshade leaves (CNL), which are used by active pharmaceutical ingredient (API) manufacturers to extract tropane alkaloids subject to further chemical treatment and purification. For purposes of the present invention, CNL is a representative example of any type of purified plant extract from which one or more desired alkaloid products may ultimately be further purified. Table 5 highlights impurities in these two products that may be specific to CYCM or CNL, or may be present in both. By analyzing products of unknown origin for a subset of these impurities, one skilled i...

Embodiment 1

[0311] Example 1. Platform yeast strains engineered for high-level putrescine production

[0312] The tropin portion of TA is derived from the amino acid arginine via the polyamine molecule putrescine. With the aim of increasing intracellular concentrations of TA precursor molecules including putrescine, NMP, 4MAB and NMPy, S. cerevisiae strains with increased flux through the arginine and polyamine biosynthetic pathways were developed. These strains combined genetic modifications for increasing carbon and nitrogen flux from central metabolism to arginine and polyamine biosynthesis in general, and included the introduction of key heterologous enzymes to additionally produce the TA precursor putrescine. Employing genetic modifications, including feedback inhibition of genes encoding native biosynthetic enzymes and regulatory proteins, introduction of mitigation mutations, fine-tuning of transcriptional regulation of native biosynthetic enzymes, deletion or disruption of genes e...

Embodiment 2

[0326] Example 2. Engineering Yeast Strains for the Production of NMPy

[0327] A strain of S. cerevisiae was developed by modifying the putrescine-overproducing strain developed in Example 1 for the production of the TA precursor NMPy. These strains combined genetic modifications aimed at increasing carbon and nitrogen flux from putrescine to NMPy biosynthesis and included the introduction of key heterologous enzymes to produce the TA precursors NMP, 4MAB, and NMPy. Genetic modifications were used, including modification of the N- and / or C-terminal domains of the enzyme of interest to improve activity in a heterologous host, and deletion or disruption of the gene encoding the enzyme that drives the precursor molecule away from the intended pathway .

[0328] 2.1) The biosynthetic pathway in the engineered strain is capable of producing NMPy from endogenous putrescine. Putrescine is first converted to N-methylputrescine (NMP) by SAM-dependent N-methyltransferase (PMT), which...

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Abstract

Provided herein, among other things, is an engineered non-plant cell that produces a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product. A method for producing a tropane alkaloid, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product that makes use of the cell is also described.

Description

[0001] cross reference [0002] This application claims the benefit of U.S. Provisional Application Serial Nos. 62 / 815,709, filed March 8, 2019, 62 / 848,419, filed May 15, 2019, and 62 / 891,771, filed August 26, 2019, which are adopted by incorporated herein by reference. [0003] government rights [0004] This invention was made with government support under contracts GM110699 and AT007886 awarded by the National Institutes of Health. The US Government has certain rights in this invention. Background technique [0005] Tropane alkaloids (TA) are a class of anticholinergic secondary metabolites produced by plants of the family Solanaceae (Solanaceae). Several TAs, including atropine, hyoscyamine, and scopolamine, are listed by the World Health Organization as essential medicines for the treatment of various neurological conditions, such as organophosphate and nerve agent poisoning, gastrointestinal cramps, and heart rhythm disorders, and control of Parkinson's disease sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P43/00C12P17/12C12N15/52C12P7/24C12N9/90
CPCA61P43/00C12P17/12C12N15/52C12Y203/01035C12Y203/01001C12Y305/03001C12Y401/01017C12Y105/03017C12Y401/01019C12Y305/03012C12Y305/01053C12Y305/03011C12Y204/02028C12Y205/01022C12Y207/11001C12Y201/01053C12Y104/03021C12Y101/01206C12Y101/01236C12Y102/01003C12Y104/03022C12Y106/02004C12Y402/01051C12Y206/01057C12Y101/01237C12Y101/01027C12Y207/07009C12Y114/11011C12Y403/01024C12Y403/01025C12N9/00C12P17/188C12N15/81C12N15/80C12N9/88C12N9/78C12N9/0032C12N9/1007C12N9/0022C12N9/0006C12N9/0071C12Y401/01001C12Y104/0301
Inventor 克里斯蒂娜·D·斯默克普拉桑特·斯里尼瓦桑
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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