Phosphite-Utilizing Transformant for Contained Detection

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Solution Overview

Problem

Developing transformants that depend on phosphite for proliferation, applicable to various host organisms, while ensuring high containment effects and cost-effectiveness, is challenging due to difficulties in expressing hypophosphite transporter proteins in hosts like cyanobacteria.

Innovation Solution

Creating transformants defective in phosphate and phosphate ester transporter functions and introducing a hypophosphite transporter protein with a signal peptide substituted from a closely related host organism, enabling phosphite utilization and high containment, and using a method to detect reduced phosphorous compounds by culturing these transformants.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If a hypophosphite transporter protein from a different species is introduced into a host organism, then the transformant can utilize phosphite for proliferation, but the protein may not be properly expressed or localized due to signal peptide incompatibility

Engineering Contradiction:

Improvephosphite utilization capabilityVSAvoidprotein expression reliability

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The signal peptide sequence of the hypophosphite transporter protein is modified to match the host organism's signal peptide characteristics. This parameter change in the amino acid sequence enables proper protein localization and expression in the host, resolving the incompatibility issue while maintaining phosphite utilization capability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The signal peptide from the host organism is copied and applied to the hypophosphite transporter protein. This copying of the signal peptide sequence ensures that the foreign protein is recognized and processed by the host's cellular machinery, enabling reliable expression

Inventive Principle:

Principle #26Copying

2Adaptability or versatility

If conventional phosphate transporters are used in the transformant, then the organism can proliferate in various environments, but the containment effect is reduced because the transformant can utilize phosphate instead of being dependent on phosphite

Engineering Contradiction:

Improveenvironmental adaptabilityVSAvoidcontainment effect

Core Design Contradiction:

Adaptability or versatilityVSObject-affected harmful factors

Solution Approach 1:

The phosphate transporter genes are removed or inactivated in the transformant. This extraction of the phosphate utilization capability forces the organism to depend exclusively on phosphite, which is not naturally present in the environment, thereby achieving high containment effect while maintaining the ability to proliferate in controlled settings

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of enabling phosphate utilization to increase adaptability, the invention inverts the approach by removing phosphate transporter function. This inversion creates a strain that is adapted to use phosphite (an uncommon compound) rather than phosphate (a common compound), thereby achieving containment while maintaining controlled environment adaptability

Inventive Principle:

Principle #13The other way round (Inversion)

3Adaptability or versatility

If transformants are developed for various host organisms, then the application scope is expanded, but the difficulty of preparing functional transformants increases due to species-specific expression issues

Engineering Contradiction:

Improvehost organism rangeVSAvoidtransformant preparation ease

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The signal peptide modification approach creates a universal solution that can be applied across different host organisms. By adapting the signal peptide to match each host's characteristics, the same hypophosphite transporter protein can be successfully expressed in various species, expanding application scope while maintaining ease of manufacture

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The signal peptide sequence is locally modified to match each host organism's specific requirements. This localized adaptation of the signal peptide region enables proper protein expression in each host species without requiring complete redesign of the entire protein, thus expanding host range while keeping the preparation process manageable

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS12180533B2Transformant, and method using said transformant to detect presence or absence of reduced phosphorous compound

Publication Date: 2024.12.31 HIROSHIMA UNIVERSITY

AI summary

It is an object of an aspect of the present invention to provide (i) transformants, whose proliferation depends on phosphite, of various species of organism and (ii) a method for detecting the presence of a reduced phosphorous compound with use of such a transformant. Use is made of a transformant which is defective in functions of a gene encoding a phosphate transporter protein and a gene encoding a phosphate ester transporter protein and into which a gene encoding a hypophosphite transporter protein is introduced, a signal peptide of a hypophosphite binding protein being substituted with a signal peptide derived from a host or a species of organism closely related to the host.