Recombinant Yeast Fermentation for Olivetol and Olivetolic Acid Production
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Solution Overview
Problem
There is a lack of scalable and commercially viable methods for producing olivetol, olivetolic acid, divarin, and divarinic acid through fermentation, which are crucial for synthesizing cannabinoids.
Innovation Solution
The use of recombinant heterologous host microorganisms, such as Saccharomyces cerevisiae, engineered with Cannabis sativa olivetol synthase, olivetolic acid cyclase, and acyl activating enzyme, to ferment glucose or galactose, producing these compounds in significant quantities through processes involving alkaline extraction and decarboxylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional fermentation methods are used, then production simplicity is maintained, but productivity and yield of olivetol and olivetolic acid are insufficient for commercial scale
Solution Approach 1:
The patent divides the complex biosynthetic pathway into separate functional modules: introducing individual genes (OLS, OAC, AAE) encoding specific enzymes into the yeast genome. Each gene is expressed independently to produce the required enzymatic activities for converting substrates into olivetol and olivetolic acid, thereby achieving high productivity through modular genetic construction
Solution Approach 2:
The patent uses yeast (Saccharomyces cerevisiae) as a heterologous host microorganism to mediate the production of olivetol and olivetolic acid. The yeast serves as an intermediary system that takes in simple substrates (glucose, galactose, fatty acids) and transforms them through engineered metabolic pathways into the desired complex cannabinoid precursors, enabling scalable commercial production
2Manufacturing precision
If traditional synthesis methods are used, then process simplicity is maintained, but manufacturing precision and purity of gateway molecules are insufficient
Solution Approach 1:
The patent employs alkaline extraction to separate and purify olivetol and olivetolic acid from the fermentation broth. The alkaline conditions selectively extract the acidic compounds (olivetolic acid and divarinic acid) into the aqueous phase, leaving neutral compounds and impurities in the organic phase. Subsequent acidification and filtration yield high-purity products, demonstrating effective separation based on chemical properties
Solution Approach 2:
The patent utilizes pH parameter changes to control the extraction and purification process. By adjusting the pH to alkaline conditions (pH 8-10), the carboxylic acid groups of olivetolic acid and divarinic acid are deprotonated, increasing their water solubility and enabling selective extraction. After extraction, acidification (pH 2-4) reprotonates the acids, causing precipitation of pure products. This parameter-based separation achieves high manufacturing precision
3Productivity
If fermentation is used to produce gateway molecules, then scalability is improved, but the complexity of genetic engineering and process optimization increases
Solution Approach 1:
The patent uses a universal yeast-based fermentation platform that can produce multiple cannabinoid gateway molecules (olivetol, olivetolic acid, divarin, divarinic acid) through a single engineered strain. The yeast system serves multiple functions: substrate conversion, pathway catalysis, and product secretion. This multi-functional platform enables scalable production of various cannabinoids from a single fermentation process
Solution Approach 2:
The patent performs preliminary genetic engineering of the yeast strain before fermentation, introducing and optimizing the expression of OLS, OAC, and AAE genes. This preliminary action establishes the complete biosynthetic pathway in advance, allowing the fermentation process to directly produce high yields of target compounds without requiring complex process modifications during production. The pre-configured genetic system enables immediate scalable manufacturing
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the commercial-scale production of olivetol and olivetolic acid, and divarin and divarinic acid, facilitating the synthesis of various cannabinoids with high yields and efficiency.
Implementation Method 1
employing a recombinant heterologous host microorganism to ferment glucose or galactose, producing olivetol, olivetolic acid, divarin, and divarinic acid
Implementation Method 2
engineered with Cannabis sativa olivetol synthase, olivetolic acid cyclase, and acyl activating enzyme
Implementation Method 3
processes involving alkaline extraction and decarboxylation
Implementation Method 4
processes involving alkaline extraction and decarboxylation
Data Source
AI summary
Provided herein are processes, such as commercially viable processes, of producing alkyl resorcinols, such as olivetol and olivetolic acid, and analogs of each thereof. Certain of these processes utilize a recombinant, heterologous host microorganism. Certain of the heterologous microorganisms include a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS). Certain of the heterologous microorganisms include a Cannabis sativa olivetolic acid cyclase (csOAC). Certain of the heterologous microorganisms include a Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAE1. In certain of these processes, glucose is fermented. In certain of these processes, the fermentation further comprises a carboxylic acid, RCO2H where R is defined as herein, or a salt thereof. Certain of these processes provide olivetol and olivetolic acid in a combined amount of at least 3 g/liter.