Engineered Microbial Cells for Histamine Production

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Solution Overview

Problem

Current methods for producing histamine through microbial fermentation from simple carbon and nitrogen sources are inefficient, as they rely on native metabolic pathways that are subject to feedback inhibition and precursor consumption, limiting the production titers of histamine.

Innovation Solution

Engineered microbial cells are developed to express non-native histidine decarboxylase and enhance upstream pathway enzymes, while reducing the activity of enzymes that consume histamine precursors, using techniques such as promoter replacement and feedback-deregulation to improve histamine production titers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If native metabolic pathways are used for histamine production, then the process is simple, but the production titer is limited due to feedback inhibition and precursor consumption

Engineering Contradiction:

Improvehistamine production titerVSAvoidmetabolic pathway complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent segments the histamine production pathway into distinct modular components: (1) upstream histidine biosynthesis genes, (2) the histidine decarboxylase gene from Lactobacillus plantarum, and (3) downstream pathway elements. This modular segmentation allows independent optimization of each module, enabling high-level histamine production while managing pathway complexity through organized genetic constructs

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent performs preliminary action by pre-assembling the complete histamine biosynthetic pathway into engineered microbial cells before fermentation. The pathway is constructed in advance with optimized gene sequences, promoters, and regulatory elements, allowing the cells to immediately produce high titers of histamine upon fermentation without requiring gradual pathway development

Inventive Principle:

Principle #10Preliminary action

2Productivity

If feedback inhibition is maintained in native pathways, then energy consumption is low, but histamine production is limited

Engineering Contradiction:

Improvehistamine production titerVSAvoidmetabolic energy consumption

Core Design Contradiction:

ProductivityVSUse of energy by moving object

Solution Approach 1:

The patent extracts and removes the feedback inhibition mechanism from the engineered histamine production pathway. By introducing heterologous genes with constitutive promoters and optimizing pathway architecture, the system operates without the natural feedback regulation that would otherwise limit histamine accumulation, thereby achieving high production titers

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent applies parameter changes by modifying key metabolic parameters including: (1) increasing enzyme activity through optimized gene sequences and promoters, (2) adjusting pathway flux distribution to favor histamine production, and (3) modifying cellular metabolism to reduce precursor consumption by competing pathways, thereby maximizing histamine titer

Inventive Principle:

Principle #35Parameter changes

3Productivity

If precursor consumption by competing pathways is maintained, then metabolic balance is preserved, but histamine production efficiency decreases

Engineering Contradiction:

Improvehistamine production efficiencyVSAvoidhistidine precursor loss

Core Design Contradiction:

ProductivityVSLoss of substance

Solution Approach 1:

The patent converts the harmful effect of precursor consumption by competing pathways into a benefit by strategically redirecting metabolic flux. Through pathway optimization and enzyme engineering, the system channels precursors that would otherwise be lost into the histamine production pathway, transforming potential waste into productive output and improving overall efficiency

Inventive Principle:

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

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 engineered microbial cells achieve significantly higher histamine production titers, up to 505 mg/L, by optimizing the metabolic pathway and reducing precursor consumption, thereby overcoming the limitations of native pathways.

Implementation Method 1

Histamine is the decarboxylation product of histidine that is catalyzed specifically by the enzyme histidine decarboxylase (EC 4.1.1.22)

Methodology Applied

Scientific EffectDecarboxylation:

Implementation Method 2

the engineered microbial cell expresses a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 3

Production of histamine in an industrial fermentation from simple, non-protein, carbon and nitrogen sources requires assembly of a pathway

Methodology Applied

Scientific EffectFermentation: Fermentation

Data Source

PatentUS11739355B2Engineered biosynthetic pathways for production of histamine by fermentation

Publication Date: 2023.08.29 ZYMERGEN INC

AI summary

The present disclosure describes the engineering of microbial cells for fermentative production of histamine and provides novel engineered microbial cells and cultures, as well as related histamine production methods.