Phosphorylated Recombinant Casein Production via Microbial Kinases

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Solution Overview

Problem

Current methods for producing recombinant caseins suffer from poor yield and lack functional attributes, limiting their industrial application.

Innovation Solution

A system comprising microbial host cells expressing recombinant caseins and microbial kinases is developed to produce phosphorylated and phosphomimetic caseins, mimicking native phosphorylation sites and enhancing functional properties.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional recombinant casein production methods are used, then casein can be produced, but the yield is poor and functional attributes are lacking

Engineering Contradiction:

Improvecasein production yieldVSAvoidfunctional attributes of casein

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The casein precursor is engineered with pre-designed phosphorylation sites incorporated into the amino acid sequence before expression. This preliminary configuration of phosphorylation motifs enables subsequent phosphorylation by microbial kinases to occur efficiently, achieving both high yield and proper functional attributes simultaneously

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

Microbial kinases are introduced as intermediary enzymes to catalyze the phosphorylation of recombinant casein at specific sites. These kinases act as mediators that transfer phosphate groups from ATP to serine and threonine residues on casein, enabling the acquisition of functional attributes necessary for calcium binding and micelle formation

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If recombinant casein is produced without phosphorylation, then production is simpler, but functional properties are lost

Engineering Contradiction:

Improveproduction simplicityVSAvoidfunctional properties

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The system utilizes the microbial host's own endogenous kinase enzymes to phosphorylate the recombinant casein after expression. This self-service approach eliminates the need for separate phosphorylation steps or external kinase additions, maintaining production simplicity while ensuring proper phosphorylation and functional properties are achieved

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

Microbial kinases serve as intermediary catalysts that enable phosphorylation to occur under simple fermentation conditions. These enzymes mediate the transfer of phosphate groups during the expression process, allowing functional properties to be acquired without complicating the manufacturing procedure

Inventive Principle:

Principle #24Intermediary (Mediator)

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 system achieves efficient production of recombinant caseins with functional properties comparable to native caseins, enabling their use in dairy products and food additives.

Implementation Method 1

a microbial host cell expressing: a. at least one recombinant casein selected from the group consisting of alpha-casein S1 (αS1), alpha-casein S2 (αS2), beta-casein and kappa-casein, and b. at least one microbial kinase; whereby said system is capable of producing one or more phosphorylated recombinant caseins

Methodology Applied

Scientific EffectPhosphorylation: Chemical Bonding

Data Source

PatentUS20250206792A1Method to produce phosphorylated milk proteins in microbes

Publication Date: 2025.06.26 DANMARKS TEKNISKE UNIV

AI summary

The present disclosure relates to methods for production of phosphorylated casein, phosphorylated phosphomimetic casein and phosphomimetic casein, as well as the use of such caseins.