Endogenous signal peptide of komagataella pastoris and screening method and application thereof

By constructing a "zero-background" screening system in *Mamyloidosis motilityis* and using α-amylase as a reporter protein, the extracellular secretion function of endogenous signal peptides can be accurately identified, solving the problem of inaccurate screening in existing technologies, screening out highly efficient signal peptides, and simplifying the production process of recombinant proteins.

CN122234154APending Publication Date: 2026-06-19WUHAN RUIJIAKANG BIOTECHNOLOGY CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
WUHAN RUIJIAKANG BIOTECHNOLOGY CO LTD
Filing Date
2026-03-24
Publication Date
2026-06-19

AI Technical Summary

Technical Problem

In existing technologies, methods for screening endogenous signal peptides in *Fermentomonas motilityis* cannot accurately identify their extracellular secretory function, resulting in unstable secretion efficiency of recombinant proteins and increasing the difficulty and cost of downstream separation and purification.

Method used

A functional reporter system based on substrate impermeability was adopted, using α-amylase as the reporter protein. By designing that starch macromolecules cannot penetrate the cell membrane, a "zero background" screening system was constructed. The positive signal is generated only when the reporter protein is successfully secreted into the extracellular space, eliminating interference from periplasmic secretion and reporter protein leakage.

Benefits of technology

This method enables efficient screening of endogenous signal peptides in *Fermentomonas motilityis*, identifying 12 peptides with secretion efficiency superior to the original AmyE signal peptide. This simplifies the downstream separation and purification process, reduces production costs, and is particularly suitable for the production of industrial enzymes and high-value proteins.

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Abstract

This application discloses an endogenous signal peptide of *Fermentomonas motilityis*, its screening method, and its application. This application constructs a "zero-background" screening system using an α-amylase with a truncated signal peptide as a reporter gene and starch as an extracellular impermeable substrate. Twelve natural endogenous signal peptides with secretion efficiencies superior to the original AmyE signal peptide were successfully screened from the *Fermentomonas motilityis* genome. Their amino acid sequences are shown in SEQ ID NO: 1, 7, 20, 28, 31, 33, 35, 40, 49, 54, 55, and 58. The optimal signal peptide guides the extracellular secretion of amylase at 232.21% of the ability of the original AmyE. This application also provides recombinant nucleic acids encoding the signal peptides, recombinant expression vectors, recombinant bacteria, and their application in the secretory expression of target proteins. This application overcomes the limitation of bioinformatics predictions in distinguishing between periplasmic secretion and extracellular secretion, providing *Fermentomonas motilityis* as a key, highly efficient secretory element for recombinant protein expression.
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