Metabolically Engineered E. coli for Extracellular Heme Secretion
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Solution Overview
Problem
Current methods for producing heme are inefficient, with low yields and environmental concerns, and existing recombinant microorganisms accumulate heme inside cells, making extracellular production challenging.
Innovation Solution
A metabolically engineered E. coli variant is developed that overexpresses the heme biosynthetic pathway and the C5 pathway for 5-aminolevulinate production, along with the ccmABC genes to facilitate extracellular heme secretion, optimizing culture conditions and fermentation processes for high-yield heme production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional chemical synthesis or organic extraction methods are used to produce heme, then heme can be obtained, but the process becomes complicated, time-consuming, and environmentally unfriendly with low yield
Solution Approach 1:
The microorganism is engineered to autonomously produce heme through its own metabolic pathways. The engineered E. coli uses its endogenous C5 pathway and heme biosynthetic pathway to synthesize heme de novo, eliminating the need for complex external chemical synthesis processes or extraction procedures. The cell serves itself by converting glucose to heme through its metabolic network.
Solution Approach 2:
The patent replaces mechanical/chemical extraction systems with a biological production system. Instead of using organic solvents, enzymes, or mechanical separation equipment to extract heme from plant or animal sources, the system uses genetically engineered microorganisms that secrete heme directly into the culture medium, simplifying the production process to mere fermentation and filtration.
2Loss of time
If recombinant microorganisms are used to produce heme biosynthetically, then production time is reduced, but heme accumulates inside cells requiring extraction
Solution Approach 1:
The invention extracts the heme product from the intracellular compartment and directs it to the extracellular space. By engineering the CcmABC heme exporter system, heme is actively transported out of the cell into the culture medium, allowing direct recovery without cell lysis or extraction steps. The product is 'taken out' of the cellular factory and deposited where it can be easily harvested.
Solution Approach 2:
The CcmABC transporter system acts as an intermediary mechanism between intracellular heme synthesis and extracellular heme accumulation. This mediator system enables the transfer of heme across the cell membrane, resolving the contradiction by providing a controlled export pathway that maintains intracellular synthesis while delivering product to the extracellular environment for easy recovery.
3Quantity of substance
If heme is produced at high concentrations intracellularly, then production yield increases, but toxicity to microorganisms occurs
Solution Approach 1:
The invention converts the harmful effect of intracellular heme accumulation (toxicity) into a beneficial driving force for extracellular secretion. The cell's natural response to heme toxicity is activated and harnessed: the CcmABC exporter system, normally used for physiological heme management, is upregulated by toxicity and actively pumps heme out of the cell. Thus, the harmful accumulation triggers the very mechanism needed to resolve the problem and deliver product externally.
Solution Approach 2:
The system employs feedback control where intracellular heme concentration regulates its own export. As heme accumulates intracellularly and reaches toxic levels, this feedback signal activates the CcmABC exporter system, which then reduces intracellular concentration by exporting heme extracellularly. This self-regulating feedback loop maintains productive synthesis while preventing toxic accumulation.
4Productivity
If the C5 pathway and heme biosynthetic pathway are overexpressed to increase heme production, then extracellular heme secretion increases, but intracellular heme accumulation causes toxicity
Solution Approach 1:
The system dynamically balances intracellular heme synthesis and export rates. By co-overexpressing both the C5 pathway (for ALA production) and the heme biosynthetic pathway along with CcmABC exporter, the system creates a dynamic flow where heme is continuously synthesized and immediately exported. This dynamic equilibrium prevents static accumulation and maintains toxic levels below harmful thresholds while maximizing extracellular productivity.
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
The E. coli variant achieves significant increases in heme production and secretion, with up to 228.46 mg/L of heme produced extracellularly, surpassing conventional methods by several folds, and reducing intracellular toxicity.
Implementation Method 1
the ccmABC genes are known to encode a heme exporter that directly delivers intracellularly synthesized free heme to proteins involved in cytochrome c biosynthesis in the periplasm
Implementation Method 2
Archaea, plants and most bacteria have the C5 pathway that converts L-glutamate to ALA through a series of reactions that are catalyzed by glutamyl-tRNA synthase (GluRS), glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde 2,1-aminomutase (GSAM)
Implementation Method 3
enhancement of the biosynthesis of free heme can promote the extracellular secretion of heme by the heme exporter expressed from the ccmABC genes, thereby enabling extracellular production of free heme
Data Source
AI summary
The present invention relates to a microorganism variant having the ability to extracellularly produce heme, and more particularly to a metabolically engineered microorganism variant having the ability to extracellularly produce heme and a method of producing heme using the same. According to the present invention, heme, an organometallic compound which is increasingly used as a health food or food supplement for the treatment of porphyria, can be extracellularly secreted and produced in high yield using the microorganism variant, but not conventional chemical synthesis or enzymatic synthesis.