PfuS Protease Expression Without Signal Peptide in Bacillus

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Solution Overview

Problem

The production of thermostable secreted protease PfuS from Pyrococcus furiosus has low yields when using expression DNA constructs with a secretion signal, making industrial production economically challenging.

Innovation Solution

Expressing PfuS protease in Bacillus host cells without a translationally fused signal peptide, resulting in higher yields and direct recovery from the culture broth without the need for costly cell lysis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If a signal peptide is used in the expression construct, then the protease is directed into the secretory machinery and secreted into the culture broth, but the expression yield is low

Engineering Contradiction:

Improveease of recoveryVSAvoidexpression yield

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The patent inverts the conventional approach by removing the signal peptide from the expression construct instead of adding it. This inversion leads to surprisingly high expression yields, contradicting the traditional belief that signal peptides are necessary for secretion and recovery in Bacillus host cells.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The patent extracts and removes the signal peptide component from the expression construct. By taking out this traditionally essential element, the invention achieves higher productivity while maintaining the ability to recover the protease from the culture broth, suggesting the signal peptide is not actually required for the desired outcome.

Inventive Principle:

Principle #2Taking out (Extraction)

2Productivity

If a signal peptide is used, then the protease is secreted into the surrounding culture broth, but cell lysis is required for recovery which increases process complexity and cost

Engineering Contradiction:

Improveexpression yieldVSAvoidrecovery process complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent removes the signal peptide from the expression construct, which eliminates the need for cell lysis in the recovery process. This simplification maintains high expression yields while reducing process complexity and operational costs.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The protease expression system serves itself by naturally secreting the protease into the culture broth without requiring external intervention for cell lysis. This self-service mechanism simplifies the recovery process and reduces operational complexity.

Inventive Principle:

Principle #25Self-service

3Ease of manufacture

If a signal peptide is used, then the fused polypeptide is directed into the secretory machinery, but the signal peptide is retained in the cell and degraded while the matured polypeptide is secreted

Engineering Contradiction:

Improveease of recoveryVSAvoidsignal peptide degradation

Core Design Contradiction:

Ease of manufactureVSLoss of substance

Solution Approach 1:

The patent extracts and eliminates the signal peptide from the expression construct, thereby preventing its retention in the cell and subsequent degradation. This removes the source of substance loss while maintaining the ability to recover the mature protease from the culture broth.

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentEP3013962B1Expression of natively secreted polypeptides without signal peptide

Publication Date: 2018.10.10 NOVOZYMES AS

AI summary

The present invention relates to methods of recombinantly producing a natively secreted polypeptide, the method comprising the steps of providing a microorganism host cell comprising an exogenous polynucleotide encoding a natively secreted polypeptide without a translationally fused signal peptide; cultivating the microorganism host cell under conditions conducive to the expression of the polypeptide and, optionally, recovering the polypeptide, as well as microorganisms, certain polynucleotides, expression constructs and protease substitution variants.