Mutant IscR Apoprotein Cell Factories for Enhanced Biochemical Yields

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Solution Overview

Problem

Current methods for producing a wide range of compounds such as vitamins, biofuels, and pharmaceuticals rely on costly chemical synthesis, and biosynthetic pathways dependent on iron-sulfur (Fe—S) cluster proteins face limitations due to growth inhibition and toxicity issues when overexpressing these proteins.

Innovation Solution

A genetically modified prokaryotic cell with a mutant IscR gene that exists solely as an apoprotein, allowing for enhanced expression and activity of Fe—S cluster polypeptides without forming toxic holo-proteins, thereby overcoming growth inhibition and increasing production yields of compounds like biotin, heme, and vitamins.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If Fe-S cluster polypeptides are overexpressed to enhance production of biochemical compounds, then productivity increases, but growth inhibition and toxicity occur due to formation of holo-proteins

Engineering Contradiction:

Improveproduction yield of biochemical compoundsVSAvoidcell growth and viability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention extracts and removes the harmful function of Fe-S cluster formation by deleting the iscR gene, which normally regulates Fe-S cluster assembly. This prevents the formation of toxic holo-proteins while allowing overexpression of Fe-S cluster polypeptides, thereby resolving the contradiction between productivity and cell viability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of promoting Fe-S cluster formation to enhance productivity, the invention inverts the approach by preventing Fe-S cluster assembly through iscR deletion. This paradoxical strategy eliminates toxicity while maintaining high levels of Fe-S cluster polypeptide expression, solving the growth inhibition problem

Inventive Principle:

Principle #13The other way round (Inversion)

2Ease of manufacture

If chemical synthesis methods are used to produce vitamins and pharmaceuticals, then production cost is high, but biosynthetic pathways face toxicity limitations

Engineering Contradiction:

Improveproduction costVSAvoidtoxicity and growth inhibition

Core Design Contradiction:

Ease of manufactureVSObject-affected harmful factors

Solution Approach 1:

The invention converts the harmful effect of Fe-S cluster formation into a beneficial outcome by deleting iscR. This prevents toxicity while enabling high-level expression of biosynthetic pathways, thereby making biosynthetic production both cost-effective and viable

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The invention introduces an intermediary genetic modification (iscR deletion) that mediates between the need for high Fe-S cluster polypeptide expression and the need to avoid toxicity. This genetic intermediary enables biosynthetic pathways to function at high levels without the harmful effects that would otherwise limit cost-effective production

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS20220127311A1Cell factories for improved production of compounds and proteins dependent on iron sulfur clusters

Publication Date: 2022.04.28 BIOSYNTIA APS

AI summary

The invention relates to a genetically modified prokaryotic cell capable of improved iron-sulfur cluster delivery, characterized by a modified gene encoding a mutant Iron Sulfur Cluster Regulator (IscR) and one or more transgenes or upregulated endogenous genes encoding iron-sulfur (Fe—S) cluster polypeptides or proteins that catalyze complex radical-mediated molecular rearrangements, electron transfer, radical or non-redox reactions, sulfur donation or perform regulatory functions. The prokaryotic cells are characterized by enhanced activity of these iron-sulfur (Fe—S) cluster polypeptides, enhancing their respective functional capacity, and facilitating enhanced yields of compounds in free and protein-bound forms, including heme, hemoproteins, tetrapyrroles, B vitamins, amino acids, δ-aminolevulinic acid, biofuels, isoprenoids, pyrroloquinoline quinone, ammonia, indigo, or their precursors, whose biosynthesis depends on their activity. The invention further relates to a method for producing said compounds or their precursors using the genetically modified prokaryotic cell of the invention, and the use of the genetically modified prokaryotic cell.