Codon-Optimized Phytase Expression in Aspergillus niger

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Solution Overview

Problem

The challenge lies in efficiently expressing Escherichia coli phytase in filamentous fungi like Aspergillus niger, as existing methods face difficulties in achieving high expression levels and are hampered by the need for methanol as a carbon source in yeast-based systems, which poses safety hazards and increases production costs.

Innovation Solution

The use of a codon-optimized gene encoding Escherichia coli phytase combined with the Aspergillus oryzae TAKA amylase signal peptide, linked into an expression cassette and introduced into Aspergillus niger for enhanced secretory expression, overcoming the limitations of previous methods by improving protein secretion and reducing production risks.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If Escherichia coli phytase is expressed in yeast (Pichia pastoris), then phytase production is achieved, but methanol safety hazards and increased production costs occur

Engineering Contradiction:

Improvephytase productionVSAvoidmethanol safety hazards

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent uses Aspergillus niger as an intermediary host system to express E. coli phytase, replacing the yeast-Pichia pastoris system that requires methanol. This intermediary approach allows the phytase gene to be expressed in a GRAS organism that uses safe, non-flammable carbon sources like glucose or starch, thereby eliminating methanol-related safety hazards while maintaining high phytase production capability

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the expression host parameter from yeast to filamentous fungus (Aspergillus niger), and modifies the carbon source parameter from methanol to safe alternatives like glucose or starch. This parameter change resolves the contradiction by maintaining productivity while eliminating the harmful effects associated with methanol usage

Inventive Principle:

Principle #35Parameter changes

2Object-affected harmful factors

If Escherichia coli phytase is expressed in Aspergillus niger, then safety and cost issues are resolved, but expression levels are initially low

Engineering Contradiction:

Improveproduction safetyVSAvoidphytase expression level

Core Design Contradiction:

Object-affected harmful factorsVSProductivity

Solution Approach 1:

The patent performs preliminary optimization of the phytase gene through codon optimization before introducing it into Aspergillus niger. The gene sequence is pre-modified to use codons preferred by A. niger, and an A. oryzae signal peptide is pre-linked to facilitate secretion. These preliminary actions ensure high expression levels and efficient secretion from the outset, resolving the contradiction between safety and productivity

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent changes the genetic parameter of the phytase gene through codon optimization to match the host organism's preferences, and modifies the expression parameter by using strong A. niger promoters and optimized signal peptides. These parameter changes enable the safe host A. niger to achieve high phytase expression and secretion levels

Inventive Principle:

Principle #35Parameter changes

3Reliability

If Gram-negative bacteria (E. coli) phytase is expressed in filamentous fungi, then GRAS status is achieved, but expression difficulty arises due to species differences

Engineering Contradiction:

ImproveGRAS statusVSAvoidexpression difficulty

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent applies local quality optimization by specifically modifying the phytase gene sequence to match the codon preferences of Aspergillus niger, and by locally optimizing the N-terminus with an A. oryzae signal peptide sequence. These localized modifications address the species-specific barriers while maintaining the overall structure and function of the phytase protein, enabling successful expression in the GRAS host

Inventive Principle:

Principle #3Local quality

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

This approach results in high levels of secreted Escherichia coli phytase in the culture supernatant, enhancing the expression efficiency and safety of the production process while minimizing environmental and operational hazards.

Implementation Method 1

enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus

Methodology Applied

Scientific EffectSecretory expression:

Implementation Method 2

The use of a codon-optimized gene encoding Escherichia coli phytase combined with the Aspergillus oryzae TAKA amylase signal peptide

Methodology Applied

Scientific EffectCodon optimization:

Implementation Method 3

which catalyzes the hydrolysis of phytic acid to produce lower inositol phosphate derivatives and inorganic phosphoric acid

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Data Source

PatentUS11339397B2Expression of phytase in <i>Aspergillus niger</i>

Publication Date: 2022.05.24 NANJING BESTZYME BIO ENG CO LTD

AI summary

Disclosed herein is a method for expressing phytase in a filamentous fungus by using an optimized Escherichia coli phytase gene having a nucleotide sequence as shown in SEQ ID NO. 7 and a signal peptide having a nucleotide sequence as shown in SEQ ID NO. 12.