Polynucleotide-Encoded Enzymes for Plant Ascorbate Production

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Solution Overview

Problem

Current methods fail to effectively modulate GDP-L-Galactose Guanyltransferase activity and GDP-D-Mannose epimerase activity, and ascorbate content in plants, particularly in non-green edible tissues, where variability is high and unexplained, limiting the potential for enhanced ascorbate production.

Innovation Solution

Transformation of plant cells with specific polynucleotides encoding polypeptides with sequences corresponding to GDP-L-Galactose Guanyltransferase and GDP-D-Mannose epimerase, such as those listed in SEQ ID NO: 1-35, to increase enzyme activity and ascorbate content, allowing for simultaneous or sequential transformation with these enzymes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If current methods are used to modulate enzyme activity and ascorbate content, then existing plant tissues maintain their natural ascorbate levels, but the ability to enhance ascorbate production in non-green edible tissues is limited

Engineering Contradiction:

Improveascorbate contentVSAvoidability to modulate enzyme activity

Core Design Contradiction:

Quantity of substanceVSAdaptability or versatility

Solution Approach 1:

The patent applies parameter changes by introducing polynucleotides that encode modified versions of GDP-L-Galactose Guanyltransferase and GDP-D-Mannose epimerase enzymes. These modified enzymes have altered kinetic parameters and regulatory properties that enable enhanced ascorbate production. The polynucleotides may encode enzymes with improved catalytic efficiency, altered substrate specificity, or modified regulatory responses, thereby changing the biochemical parameters of the ascorbate biosynthesis pathway to achieve higher ascorbate content in plant tissues.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent uses polynucleotides as intermediaries to transfer the genetic information for modified enzymes into plant cells. These polynucleotides serve as mediators that enable the introduction of enhanced enzyme variants without directly manipulating the native plant genome. The polynucleotides may be delivered via transformation methods and then expressed to produce the modified enzymes, acting as a bridge between external genetic resources and the plant's internal metabolic pathways.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If polynucleotides encoding modified enzymes are introduced to enhance ascorbate production, then ascorbate content increases, but the complexity of the genetic transformation process increases

Engineering Contradiction:

Improveascorbate contentVSAvoidgenetic transformation process

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The patent segments the solution by providing separate polynucleotides for each enzyme (GDP-L-Galactose Guanyltransferase and GDP-D-Mannose epimerase) that can be introduced independently or in combination. This segmentation allows researchers to choose the appropriate level of complexity based on their needs - introducing single polynucleotides for modest enhancement or multiple polynucleotides for maximum effect. The modular nature of the approach simplifies the overall process compared to attempting to redesign entire metabolic pathways simultaneously.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent applies partial action by allowing the introduction of polynucleotides encoding only the specific enzymes needed for ascorbate enhancement, rather than requiring complete genetic redesign of the plant. The modified enzymes work within the existing metabolic framework, utilizing native substrates and cellular machinery. This partial approach to genetic modification achieves ascorbate enhancement without the complexity of comprehensive pathway reconstruction.

Inventive Principle:

Principle #16Partial or excessive action

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 method increases GDP-L-Galactose Guanyltransferase and GDP-D-Mannose epimerase activities, leading to enhanced ascorbate production in plants, offering a means to manipulate ascorbate content and potentially improve nutritional value and health benefits.

Implementation Method 1

a polynucleotide encoding a polypeptide with GDP-L-Galactose Guanyltransferase activity

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

a polynucleotide encoding a polypeptide with GDP-D-Mannose epimerase activity

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 3

The L-Gal pathway proceeds through L-Gal to galactono-1,4-lactone and thence to ascorbate

Methodology Applied

Scientific EffectMetabolic pathway: Fermentation

Data Source

PatentUS9157088B2Transferases, epimerases, polynucleotides encoding these and uses thereof

Publication Date: 2015.10.13 NEW ZEALAND INSTITUTE FOR BIOECONOMY SCIENCE LTD

AI summary

The invention provides compositions and methods for modulating GDP-L-Galactose Guanyltransferase (also known as GDP-L-Galactose phosphorylase) activity; and/or GDP-D-Mannose epimerase activity; and/or ascorbate content in plants. The invention provides plants and plant cells with increased GDP-L-Galactose Guanyltransferase activity; and/or GDP-D-Mannose epimerase activity. The invention provides plants and plant cells with increased ascorbate content as a result of: over-expression of GDP-L-Galactose Guanyltransferase; over-expression of GDP-D-Mannose epimerase; or in particular over-expression of a combination of GDP-L-Galactose Guanyltransferase and GDP-D-Mannose epimerase.