Codon-Optimized Phytase Expression in Pichia pastoris
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Solution Overview
Problem
Current methods for producing phytase enzymes in host cells face challenges such as low expression levels, instability, and limited pH and temperature tolerance, which hinder large-scale industrial production and efficiency in animal feed applications.
Innovation Solution
A novel nucleic acid sequence encoding a phytase protein from Serratia odorifera, optimized for expression in Pichia pastoris, is developed, featuring codon optimization and the removal of the N-terminal signal peptide, resulting in enhanced stability and activity over a broad pH range and temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional phytase production methods are used, then production process is simple, but expression levels are low and stability is poor
Solution Approach 1:
The patent applies parameter changes by optimizing the nucleic acid sequence through codon optimization for the host organism and modifying expression control parameters (promoter strength, induction conditions) to achieve high expression levels while maintaining process feasibility
Solution Approach 2:
The patent implements preliminary action by pre-optimizing the gene sequence before expression, including removing signal peptides and adjusting codon usage, so that when the optimized sequence is introduced into the host cell, high expression levels are achieved without requiring complex post-production modifications
2Reliability
If conventional phytase sequences are used, then gene structure is simple, but protein stability and activity range are limited
Solution Approach 1:
The patent changes the nucleotide sequence parameters through codon optimization and removes the N-terminal signal peptide sequence, resulting in a more stable protein with extended pH and temperature activity ranges while keeping the overall gene structure manageable
Solution Approach 2:
The patent extracts and removes the N-terminal signal peptide sequence from the phytase gene, which improves protein stability and extends functional activity ranges without introducing additional complexity to the core coding sequence
3Adaptability or versatility
If wild-type phytase is used, then gene is naturally occurring, but pH and temperature tolerance are limited
Solution Approach 1:
The patent modifies the gene sequence parameters through codon optimization and signal peptide removal to achieve broad pH and temperature tolerance (activity from pH 2-11 and -70°C to +100°C) without requiring extensive gene synthesis or multiple cloning steps
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 optimized phytase protein exhibits significantly higher expression levels and stability, with activity maintained at various temperatures and pH levels, effectively improving the efficiency and cost-effectiveness of phytase production for animal feed applications.
Implementation Method 1
The phosphorous present in the phytate form cannot be made bioavailable to non-ruminant animals, because they lack the digestive enzyme phytase that is required to remove phosphate from the inositol in the phytate molecule
Data Source
AI summary
The invention generally relates to novel and improved nucleic acid sequences encoding a polypeptide or protein having the enzymatic activity of a phytase and methods for effectively expressing such enzymes in a host cell. In a first aspect, the present invention relates an isolated nucleic acid molecule i) comprising the nucleotide sequence according to SEQ ID NO: 1 encoding a protein with phytase activity or ii) having a sequence identity of at least 83% to a nucleotide sequence according to SEQ ID NO: 1 encoding a protein with phytase activity. The present invention also relates to an expression construct or a vector comprising the above mentioned isolated nucleic acid molecule. The present invention further relates to a method of producing a protein having the enzymatic activity of a phytase comprising the steps of: a) introducing into a host cell a vector comprising: i) elements regulating the expression that are functional in the host cell; and ii) operatively linked thereto a nucleic acid molecule according to the invention; b) cultivating the host cells obtained in step a) under conditions suitable for expression of the protein, and optionally c) recovering the protein produced in step b) from the cell culture. The present invention further relates to proteins obtained by the method according to the invention and the use thereof for the manufacture of additives for animal feed.