Recombinant Yeast Synthesizing Andrographolide Intermediate via CYP450 Enzymes
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Solution Overview
Problem
There is currently no eukaryotic microbial recombinant strain for the de novo synthesis of 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid, an important intermediate compound for andrographolide synthesis, which lacks a clear catalytic pathway involving CYP450 enzymes.
Innovation Solution
The heterologous expression of CYP71A8 and CYP71D10 enzymes from Andrographis paniculata, along with the knockout of ROX1 and GAL80 genes and integrative expression of GGPP synthase and CPS diterpene synthase in Saccharomyces cerevisiae, is used to create a recombinant strain capable of synthesizing 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid through microbial fermentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If eukaryotic expression hosts are used to express CYP450 enzymes, then the expressed protein is closer to its natural conformation with complete post-translational modifications, but the device complexity and construction difficulty increase
Solution Approach 1:
The patent uses eukaryotic expression hosts (Pichia pastoris and Saccharomyces cerevisiae) as intermediary systems that provide the necessary cellular machinery for proper CYP450 enzyme folding and post-translational modifications. These hosts act as mediators between the heterologous CYP450 genes and the desired functional enzyme product, ensuring proper conformation through endogenous eukaryotic modification systems.
2Reliability
If endogenous CPR of eukaryotic microorganisms is used to mediate electron transfer, then the CYP450 enzymes can exert their catalytic function effectively, but the specificity and control over catalytic reactions decrease
Solution Approach 1:
The patent employs the endogenous CPR (cytochrome P450 reductase) system of the eukaryotic expression hosts to automatically provide electron transfer to the heterologously expressed CYP450 enzymes. The host cell's native redox partner proteins self-service the foreign CYP450 enzymes, eliminating the need for additional engineering of electron transfer components and enabling straightforward expression of catalytic function.
3Loss of information
If heterologous expression of CYP71A8 and CYP71D10 is performed, then their catalytic functions are discovered and applied, but the lack of existing eukaryotic microbial recombinant strains for de novo synthesis of 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid increases the difficulty of strain construction
Solution Approach 1:
The patent performs preliminary heterologous expression of CYP71A8 and CYP71D10 in eukaryotic hosts to discover and characterize their catalytic functions before constructing the complete biosynthetic pathway. This preliminary action establishes the functional basis and optimizes expression conditions, which then facilitates the subsequent construction of the full de novo synthesis pathway by providing known working components and protocols.
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 successfully achieves the de novo synthesis of 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid, providing a reference for analyzing the biosynthesis pathway of andrographolide and enabling its metabolic engineering for related derivatives.
Implementation Method 1
CYP71A8 and CYP71D10 derived from Andrographis paniculata belong to the Class I CYP71 family and require electron transport protein to catalyze their activity
Implementation Method 2
Cytochrome P450 (P450) belongs to the heme oxygenase superfamily, named after the maximum absorption peak near 450 nm when the reduced heme oxygenase forms a complex with CO
Implementation Method 3
Class I requires additional electron transport proteins to transfer electrons NAD(P)H to P450 enzymes active center to complete the entire catalytic process
Implementation Method 4
The heterologous expression of CYP71A8 and CYP71D10 enzymes from Andrographis paniculata, along with the knockout of ROX1 and GAL80 genes and integrative expression of GGPP synthase and CPS diterpene synthase in Saccharomyces cerevisiae, is used to create a recombinant strain capable of synthesizing 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid through microbial fermentation
Data Source
AI summary
The present disclosure provides a P450 cytochrome enzyme for andrographolide synthesis and its application, belonging to the field of bioengineering. The present disclosure uses Saccharomyces cerevisiae CEN.PK2-1D as a host, and implements knockout of ROX1 and GAL80 genes on the genome, and integrative expression of GGPP synthase encoding gene and CPS diterpene synthase encoding gene at ROX1 site; and implements free expression of ApCPR and CYP71A8 and CYP71D10 both with truncated signal peptides, successfully constructing recombinant S. cerevisiae, and achieving de novo synthesis of 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid. Compared with the blank, a response value of a product peak reaches 1.9*106, and this strategy provides necessary reference for analyzing biosynthetic pathway of andrographolide and using metabolic engineering to synthesize andrographolide and related derivatives thereof.