Lactococcus Natural Competence via ComX-Modulated Transformation

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Solution Overview

Problem

There is a lack of effective methods for naturally transforming Lactococcus strains using natural competence, and there is a need to identify strains within the genus that can be transformed by this process.

Innovation Solution

A method involving the modulation of ComX protein production in Lactococcus strains, either through expressing an exogenous or endogenous comX gene, followed by contacting the strain with an exogenous DNA polynucleotide and incubating to integrate it into the genome, with selection for successful integration.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If classical selection methods are used to improve Lactococcus strains, then growth and fermentation properties can be gradually improved, but the process is time-consuming and lacks efficiency

Engineering Contradiction:

Improvestrain improvement efficiencyVSAvoidtime for strain improvement

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent applies preliminary action by pre-modulating ComX protein production in Lactococcus strains before transformation. This involves expressing the comX gene constitutively or inducing it under specific conditions (such as carbon starvation or using inducers like glucose, galactose, or lactose) to prepare the strain in advance for efficient DNA uptake and integration, thereby accelerating the overall strain improvement process

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent utilizes parameter changes by manipulating environmental conditions (carbon source availability, temperature, pH) and genetic parameters (ComX protein expression levels) to optimize natural competence. By changing these parameters, the transformation efficiency is significantly improved, allowing rapid strain modification without time-consuming classical selection methods

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If natural transformation methods are developed for Lactococcus, then new non-GMO strains with useful industrial properties can be generated, but the transformability of different Lactococcus strains varies and needs to be identified

Engineering Contradiction:

Improvestrain transformabilityVSAvoidstrain identification complexity

Core Design Contradiction:

Adaptability or versatilityVSDifficulty of detecting and measuring

Solution Approach 1:

The patent implements feedback mechanisms to assess and identify transformable Lactococcus strains. This involves testing different strains for their natural competence through controlled transformation assays, using markers (such as antibiotic resistance genes or fluorescent proteins) to detect successful transformation events, and adjusting ComX modulation strategies based on strain-specific responses to optimize transformation efficiency

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The patent replaces complex multi-parameter strain screening with a more straightforward approach focused on ComX protein modulation. By substituting the need to evaluate multiple genetic and phenotypic traits with a single key regulator (ComX) that controls natural competence, the strain identification process is simplified and made more scalable

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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

This approach successfully transforms Lactococcus strains by natural competence, demonstrating the first evidence of transformation in L. lactis strains and confirming natural competence in Lactococcus raffinolactis and Lactococcus lactis species.

Implementation Method 1

ComX binds to a specific DNA sequence named Com-box or Cin-box, which is located at least in the vicinity of promoters of late competence (com) genes/operons responsible for DNA uptake (e.g.; comG, comF and comE operons), DNA protection (e.g. ssb) and DNA recombination (e.g. recA, dprA, coiA), and positively controls their expression.

Methodology Applied

Scientific EffectTranscriptional regulation:

Implementation Method 2

DNA acquisition by natural transformation is widespread among prokaryotes and has been identified in over 80 species. Various functions are attributed to competence for natural transformation: genome plasticity, DNA repair, and/or nutrition.

Methodology Applied

Scientific EffectNatural transformation:

Implementation Method 3

Above a threshold concentration, competence pheromones activate the master regulator ComX (alternative sigma factor x), which ultimately leads to a transcriptional reprogramming of cells (globally known as late competence phase) including the induction of genes strictly required for DNA transformation.

Methodology Applied

Scientific EffectTranscriptional reprogramming:

Data Source

PatentUS20250388933A1Methods and strain

Publication Date: 2025.12.25 INT N&H DENMARK APS

AI summary

The present invention relates to a method for transforming a strain of the Lactococcus genus through natural competence. The present invention further relates to strains obtained or obtainable by said method. The present invention also relates to a method for identifying a strain of the Lactococcus genus which is transformable through natural competence.