Recombinant TrSub Peptide Fermentation for Stable Feed Additives
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Solution Overview
Problem
Existing methods for producing antibacterial peptides like Sublancin168 are costly and inefficient, and their use as feed additives is hindered by stability and safety concerns, necessitating a more efficient and safe production method.
Innovation Solution
A recombinant antimicrobial peptide TrSub is produced in Trichoderma reesei using error-prone PCR and homologous recombination, ensuring thermal stability, acid resistance, and low hemolytic activity, suitable for use in feed additives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibacterial peptides are produced through biological extraction or chemical synthesis, then antibacterial activity is achieved, but production cost increases and efficiency decreases
Solution Approach 1:
The patent uses heterologous expression to copy the Sublancin168 gene into Trichoderma reesei, producing recombinant TrSub peptide. This genetic copying approach enables microbial factories to produce the peptide through natural metabolic processes, replacing costly chemical synthesis and biological extraction while significantly improving production efficiency and enabling mass production
Solution Approach 2:
The patent replaces mechanical extraction processes and chemical synthesis methods with biological expression systems. By using Trichoderma reesei's protein synthesis machinery to produce TrSub, the system substitutes expensive and inefficient mechanical/chemical processes with efficient biological production, achieving high-yield manufacturing
2Reliability
If antibiotics are used to treat bacterial diseases, then treatment effectiveness is improved, but drug resistance emerges threatening human health
Solution Approach 1:
The patent changes the fundamental parameter of antimicrobial agents from traditional antibiotics to antimicrobial peptides (TrSub). This parameter change exploits a different mechanism of action - TrSub peptides disrupt bacterial cell membranes through physical-chemical interactions rather than targeting specific bacterial enzymes, making it difficult for bacteria to develop resistance while maintaining effective antibacterial activity
3Productivity
If foreign proteins are produced in Escherichia coli or Pichia pastoris, then production capability is achieved, but safety requirements for feed additives are not met
Solution Approach 1:
The patent uses Trichoderma reesei as an intermediary host organism that bridges the gap between production capability and safety requirements. As a GRAS (Generally Recognized As Safe) organism, Trichoderma reesei produces TrSub peptide that meets feed additive safety standards, unlike E. coli or Pichia pastoris which require additional purification and cannot be directly applied to feed
Solution Approach 2:
The patent copies the Sublancin168 gene into the Trichoderma reesei expression system, creating a safe production platform. The recombinant TrSub produced in this GRAS host can be directly used in feed applications without the extensive safety validation required for proteins produced in non-GRAS organisms
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 recombinant TrSub demonstrates effective antibacterial activity against Escherichia coli, Salmonella, and Staphylococcus aureus, with good thermal stability and low hemolytic activity, making it suitable for industrial applications.
Implementation Method 1
amplifying a gene fragment of antibacterial peptide Sublancin168 through error-prone PCR reaction
Implementation Method 2
inserting Trichoderma expression vector by homologous recombination in vitro to construct the recombinant expression vector of recombinant antibacterial peptide Sublancin168
Implementation Method 3
transforming a recombinant expression vector into Trichoderma Tu6 by protoplast transformation mediated by polyethylene glycol
Implementation Method 4
the recombinant antibacterial peptide TrSub is obtained by fermentation expression
Data Source
AI summary
A recombinant antibacterial peptide TrSub, preparation method and application for the recombinant antibacterial peptide TrSub is disclosed, and belongs to the field of genetic engineering and biotechnology. The amino acid sequence of the recombinant antimicrobial peptide TrSub is shown in SEQ ID No. 1. The nucleotide sequence encoding the amino acid shown in SEQ ID NO.1 is shown in SEQ ID NO.2. The disclosure also provides a method for preparing the recombinant antimicrobial peptide TrSub, wherein the recombinant antimicrobial peptide TrSub has an inhibitory effect on Escherichia coli, Salmonella, Staphylococcus aureus and Clostridium perfringens. The disclosure has good thermal stability, acid resistance, pepsin resistance and low hemolytic activity, and is beneficial to the application of the disclosure in the preparation of fee and feed additive.