Bifunctional Terpenoid Biosynthesis via Metabolic Flux Redirection
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Solution Overview
Problem
The terpenoid and polyketide chemical space in organisms such as Yarrowia lipolytica has not been fully explored due to high flux towards lipid accumulation, limiting the production of novel terpenoids and polyketides.
Innovation Solution
Overexpressing β-hydroxy β-methylglutaryl-CoA reductase (HMGR) and farnesyl pyrophosphate synthase (FPPS) in Yarrowia lipolytica, combined with additional cytochrome P450 enzymes, increases the production of bifunctional terpenoid compounds by redirecting metabolic flux through the mevalonate pathway.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional genetic manipulation to overexpress heterologous biosynthetic enzymes is performed, then production of target compounds is attempted, but results are unpredictable and high flux towards lipid accumulation limits terpenoid production
Solution Approach 1:
The invention changes key metabolic parameters by overexpressing specific enzymes (HMGR, FPPS, and P450s) to alter flux distribution in the mevalonate pathway. This redirects carbon flow from dominant lipid pathways to non-dominant terpenoid pathways, achieving predictable and enhanced terpenoid production with up to 60-fold increase in diacid compounds.
2Productivity
If carbon flux is directed toward non-dominant terpenoid pathways, then novel terpenoid molecules are produced, but this requires overcoming high flux towards lipid accumulation
Solution Approach 1:
The invention segments the mevalonate pathway into distinct functional modules by overexpressing specific enzymes at different stages. HMGR overexpression increases precursor supply at the beginning of the pathway, while FPPS and P450 overexpression enhances downstream terpenoid synthesis, effectively dividing the metabolic flux redirection into manageable enzymatic steps.
Solution Approach 2:
The invention changes metabolic parameters by simultaneously overexpressing multiple enzymes (HMGR, FPPS, and CYP52 family P450s) to alter flux distribution. This coordinated parameter change redirects carbon flow from dominant lipid pathways to non-dominant terpenoid pathways, achieving substantial increases in terpenoid production.
3Productivity
If oxidative capacity is increased through P450 overexpression, then bifunctional terpenoid compounds are produced, but this requires coordinated increase in precursor supply
Solution Approach 1:
The invention merges multiple enzymatic functions by co-overexpressing HMGR, FPPS, and CYP52 family P450s in a single engineered system. This combination creates a synergistic effect where increased precursor supply from HMGR/FPPS works together with enhanced oxidative capacity from P450s to produce bifunctional terpenoids, with the greatest effect observed when all three are overexpressed simultaneously.
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 results in a 60-fold increase in terpenoid diacid production, uncovering a novel terpenoid chemical space and demonstrating the potential of genetic engineering to enhance precursor supply and oxidative capacity in microorganisms.
Implementation Method 1
overexpressing β-hydroxy β-methylglutaryl-CoA reductase (HMGR)
Implementation Method 2
overexpressing β-hydroxy β-methylglutaryl-CoA reductase (HMGR) and farnesyl pyrophosphate synthase (FPPS)
Implementation Method 3
additional overexpression of cytochrome P450 enzymes in the CYP52 family increased the production of the various chain length bifunctional terpenoid compounds
Implementation Method 4
overexpression of cytochrome P450 enzymes in the CYP52 family increased the production
Data Source
AI summary
A strain of Yarrowia lipolytica was engineered to overexpress β-hydroxy β-methylglutaryl-CoA reductase and farnesyl pyrophosphate synthase, as rate-limiting enzymes in the mevalonate and sesquiterpenoid synthesis pathways respectively. Metabolite extracts from this strain were run on LC-MS and showed a number of novel compounds being produced, including terpenoids varying in lengths and oxidation states. Upon NMR and MS/MS structure validation as well as biochemical assays, these compounds were determined as a new class of non-natural compounds, bifunctional terpenoids. Studies on the overexpression of P450 enzymes, alcohol oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase showed that expression of these enzymes in addition to β-hydroxy β-methylglutaryl-CoA reductase and farnesyl pyrophosphate synthase increase the production of bifunctional terpenoids. Bioactivity assays demonstrate the application of bifunctional terpenoids.