HcCYP87D19-Engineered Yeast for Cucurbitacin Intermediate Production
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The limited understanding of cucurbitacin biosynthetic pathways and the complexity of extracting and purifying cucurbitacins from plants hinder their clinical application and biological research, while existing synthetic methods face challenges in producing high-yield cucurbitacin precursors and intermediates.
Innovation Solution
The application of Hemsleya chinensis cytochrome oxidase HcCYP87D19 and its transgenic engineering bacteria, co-transfected with HcCYP81Q58, in Saccharomyces cerevisiae to produce cucurbitacin intermediates, utilizing a customized expression vector and genetic engineering to optimize the production process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If cucurbitacins are extracted and purified from plants, then cucurbitacin compounds can be obtained, but the extraction and purification process is complex and time-consuming
Solution Approach 1:
The patent introduces heterologous expression systems (bacterial and yeast hosts) as intermediaries to produce cucurbitacin precursors and intermediates. Instead of directly extracting from plants, the biosynthetic pathway enzymes are expressed in microbial hosts that serve as factories to generate the desired compounds, thereby avoiding complex plant extraction processes
Solution Approach 2:
The patent replaces the mechanical extraction and purification processes with biological synthesis systems. By expressing biosynthetic enzymes in heterologous hosts, the system uses biological mechanisms (enzymatic catalysis) to substitute for mechanical extraction methods, achieving more efficient production
2Productivity
If chemical synthesis methods are used to produce cucurbitacin precursors and intermediates, then compounds can be synthesized, but high-yield production is difficult to achieve
Solution Approach 1:
The patent changes the fundamental parameter of synthesis methodology from chemical to biological. By using heterologous expression of biosynthetic enzymes in living systems, the patent exploits the catalytic efficiency and specificity of enzymatic reactions to achieve high yields that are difficult to obtain through chemical synthesis
Solution Approach 2:
The patent segments the cucurbitacin biosynthetic pathway into individual enzymatic steps and expresses them in heterologous hosts. This allows for modular optimization of each step and enables the reconstruction of the complete pathway to achieve high-yield production of final products
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 approach enables the synthesis of cucurbitacin intermediates, laying a foundation for the regulation of dihydrocucurbitacin biosynthesis and providing a basis for future bio-industrial production of cucurbitacins.
Implementation Method 1
CYP450 has a wide range of catalytic activity, it can insert an oxygen atom into a hydrophobic molecule in a biochemical reaction to obtain higher activity or hydrophilicity
Implementation Method 2
Cucurbitadienol is catalyzed by cytochrome P450 and is oxidized at specific sites by introducing hydroxyl, carboxyl, or epoxy groups
Implementation Method 3
The use of synthetic biology to achieve a heterologous synthesis of cucurbitacin monomers is one of the most important strategies
Implementation Method 4
there is no report on the production of cucurbitacin monomers and intermediates by metabolic engineering
Data Source
AI summary
The invention provides a Hemsleya chinensis cytochrome oxidase HcCYP87D19 gene, and the sequence of the gene is shown in SEQ NO: 1. This invention constructs a transgenic Saccharomyces cerevisiae engineering bacteria co-transfected with Hemsleya cathayensis cytochrome oxidase HcCYP87D19 and HcCYP81Q58, the bacteria can produce a variety of cucurbitacin intermediates including 11-Carbonyl-cucurbita-5,23-diene-3β,16a,20,25-tetrol (6), 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,23-tetrol (6b), 11-Carbonyl-cucurbita-5,23-diene-16α,20,25-triol-3-one (6a), 11-Carbonyl-cucurbita-5,24-diene-16α,20,23-triol-3-one (6c), it provides a source for cucurbitacin production.