C7 Building Block Production via BioI Monooxygenase Oxidative Cleavage
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Solution Overview
Problem
There is a need for sustainable and economically viable methods to produce the C7 building blocks pimelic acid, 7-hydroxyheptanoic acid, 7-aminoheptanoic acid, heptamethylenediamine, and 1,7-heptanediol, as existing petrochemical routes are not economically feasible, and biotechnology offers an alternative through biocatalysis, but natural microorganisms do not efficiently produce these compounds.
Innovation Solution
Construction of biochemical pathways using fatty acid synthesis enzymes and cytochrome P450 encoded by BioI from microorganisms like Bacillus subtilis, combined with metabolic engineering and cultivation strategies, to enzymatically synthesize seven carbon chain aliphatic backbones and form terminal functional groups such as carboxyl, amine, or hydroxyl groups, in recombinant host cells like Escherichia coli or Bacillus subtilis, using enzymes like ω-transaminases, monooxygenases, and thioesterases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If biocatalysis is used to produce C7 building blocks, then environmental sustainability is improved, but production efficiency is insufficient because no wild-type microorganism naturally overproduces these compounds
Solution Approach 1:
The patent divides the synthesis pathway into modular enzymatic steps, each catalyzed by a specific enzyme (e.g., monooxygenase for oxidative cleavage, synthetases for carbon-carbon bond formation). This segmentation allows independent optimization of each reaction step and enables the construction of efficient heterologous pathways in host organisms, thereby improving productivity while maintaining the sustainability of biocatalysis.
Solution Approach 2:
The patent employs engineered intermediary metabolites and enzyme complexes to bridge the gap between natural metabolic pathways and the desired C7 building block production. By introducing heterologous enzymes and optimizing metabolic flux through intermediary compounds, the system achieves high productivity while preserving the environmentally benign nature of biocatalytic processes.
2Productivity
If metabolic engineering strategies are applied to direct carbon flux toward C7 building blocks, then production yield is improved, but host biomass growth is reduced due to the optimality principle
Solution Approach 1:
The patent employs dynamic metabolic engineering strategies where enzyme expression levels, substrate feed rates, and cultivation conditions are optimized over time to balance biomass growth and product formation. This dynamic approach allows the system to achieve high production yields without completely sacrificing host growth, as the metabolic flux is continuously adjusted to meet both objectives.
Solution Approach 2:
The patent utilizes parameter changes in cultivation conditions (e.g., pH, temperature, dissolved oxygen, nutrient composition) and genetic expression levels to optimize the trade-off between biomass growth and C7 building block production. By carefully tuning these parameters, the system can shift metabolic flux toward product formation when needed while maintaining sufficient growth for sustained production.
3Productivity
If high concentrations of C7 building blocks are produced, then economic viability is improved, but host cell tolerance is exceeded leading to reduced productivity
Solution Approach 1:
The patent employs efflux pump systems and product extraction strategies to remove C7 building blocks from the host cell interior and transfer them to the extracellular environment. This extraction mechanism prevents toxic accumulation within the cell, maintaining host cell tolerance while enabling high product concentrations in the culture medium, thereby improving economic viability.
Solution Approach 2:
The patent introduces intermediary transport proteins and periplasmic binding proteins that facilitate the controlled export of C7 building blocks. These intermediary systems act as buffers, allowing high production rates while protecting the host cell from toxic effects, thus maintaining both high concentration and host cell reliability.
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 allows for efficient production of C7 building blocks by directing carbon flux and overcoming the optimality principle, achieving high yields and improving host tolerance to high concentrations of these compounds through selective cultivation and efficient efflux strategies.
Implementation Method 1
oxidative cleavage of a long chain acyl-[acp] intermediate by a monooxgenase encoded by BioI
Implementation Method 2
Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds
Data Source
AI summary
This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the fatty acid synthesis pathway and oxidative cleavage of long chain acyl-[acp] intermediates by a monooxgenase (e.g., cytochrome P450) such as that encoded by BioI from microorganisms such as Bacillus subtillis.