Sustainable production of cannabinoids from simple precursor feedstocks using Saccharomyces cerevisiae
A technology of cannabinoids and precursors, applied in the field of cannabinoid production, can solve the problems of high energy consumption, unsustainability and high cost
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] Example 1: Modification of the cannabinoid biosynthetic pathway
[0042] In order to reconstitute the cannabinoid biosynthetic pathway in microorganisms, it is necessary to first understand the enzymes present in cannabis that catalyze each step in the pathway. See figure 1 .
[0043] The upstream biosynthetic pathway can be divided into two functional parts, the polyketide pathway produces dihydroxyamyl benzoic acid (OLA) as the final product and the isoprenoid pathway produces geranyl pyrophosphate (GPP).
[0044] Polyketone production OLA
[0045] polyketide pathway (see figure 1 , red) starting with the substrates caproic acid and malonyl CoA. An acyl-activating enzyme named CsAAE1 (SEQ ID NO:4) is responsible for the addition of the CoA moiety to caproic acid in cannabis leaf trichomes (Stout et al., Plant J., 2012, 71(3): 353-365). Then, a type III polyketide synthase named olivetol synthase (OLS; SEQ ID NO: 6) catalyzes the tetraketothioester (3, 5,7-Triox...
Embodiment 2
[0055] Example 2: Construction of the assembly in Saccharomyces cerevisiae
[0056] The molecular cloning strategy for constructing constructs in Saccharomyces cerevisiae is a set of plasmids based on Golden-Gate assembly called the YeastFab system (Guo et al.). The assembly system allows the modular assembly of transcriptional units such as promoters and terminators into open reading frames (ORFs), followed by the assembly of expression cassettes of up to six different transcriptional units.
[0057] The suite of YeastFab plasmids has been expanded to assemble up to eight transcription units together. The modular nature of the assembly facilitates downstream optimization, as it is relatively easy to alter transcriptional regulatory units such as promoters and terminators, enabling differential regulation of the expression of each gene in a biosynthetic pathway. In this pathway assembly method, type II restriction enzymes, such as BsaI and BsmBI, cleave near the enzyme recogn...
Embodiment 3
[0064] Example 3: Cannabinoid Bioproduction
[0065]After the expressed gene is assembled into the desired plasmid, it is transferred into a single organism (Saccharomyces cerevisiae) for biological production of the desired product. Yeast strain BY4741 is an auxotrophic strain for the amino acids methionine, leucine, histidine and uracil (Brachmann et al., Yeast, 1998, 14(2): 115-132) for cannabinoid biogenesis Production. The step-by-step approach can be used as a precaution to ensure that each plasmid transformed yeast subsequently expresses a functional enzyme. The products of each step (eg OLA, CBGA and CBDA) are then detected using liquid chromatography-mass spectrometry (LC-MS) before proceeding to the next step in the biosynthetic pathway.
[0066] Saccharomyces cerevisiae cultures were grown to stationary phase to express the enzymes. Substrates (ie hexanoic acid and malonic acid) were added to the cultures and incubated overnight at 25°C. The culture was spindown...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More