Wickerhamomyces Ciferrii Strain Engineering for Markerless TAPS Production
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Solution Overview
Problem
Conventional methods for genetically engineering Wickerhamomyces ciferrii to enhance tetraacetylphytosphingosine (TAPS) production are labor-intensive and inefficient, with challenges in applying expression plasmids and leaving behind selection markers.
Innovation Solution
Development of an expression plasmid with a CEN/ARS origin of replication and uracil selection marker for Wickerhamomyces ciferrii, enhancing serine palmitoyl-transferase and sphinganine C4-hydroxylase activities while weakening long-chain base kinase activity, using methods like electroporation for transformation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional genetic engineering methods (promoter replacement or gene insertion) are used to overexpress specific genes in Wickerhamomyces ciferrii, then TAPS production is improved, but the process requires much time and labor to remove selection markers and leaves traces in the gene
Solution Approach 1:
The invention extracts and eliminates the selection marker from the genetic engineering process by using a markerless gene deletion system. The cre-loxP system allows for the removal of the URA3 selection marker after it has served its purpose in selecting transformants, leaving no trace in the final engineered strain. This resolves the contradiction by maintaining TAPS production improvement while eliminating the time-consuming marker removal process and genetic traces.
Solution Approach 2:
The invention changes the genetic engineering approach from traditional promoter replacement or gene insertion to a markerless deletion system using cre-loxP recombination. This parameter change in the engineering method enables efficient gene deletion without selection markers, significantly reducing time and labor while maintaining the ability to improve TAPS production through metabolic pathway optimization.
2Productivity
If conventional genetic engineering methods are used, then specific genes can be overexpressed to improve TAPS production, but the process is labor-intensive and inefficient
Solution Approach 1:
The selection marker is extracted and removed from the engineering process using the cre-loxP system. The URA3 marker is temporarily introduced for transformation selection, then efficiently removed by cre recombinase acting on loxP sites, leaving a clean gene deletion without markers. This makes the manufacturing process much easier and more efficient while still achieving the desired TAPS production improvement.
Solution Approach 2:
The cre-loxP system acts as an intermediary mechanism that facilitates efficient gene deletion without selection markers. The loxP sites serve as recognition sequences for cre recombinase, enabling precise and clean gene deletion. This intermediary system resolves the contradiction by providing an efficient, markerless method for genetic modification that improves both ease of manufacture and TAPS production.
3Reliability
If selection markers are used in genetic engineering, then transformation efficiency is improved, but traces remain in the engineered gene
Solution Approach 1:
The selection marker is temporarily introduced to ensure high transformation efficiency, then completely removed using the cre-loxP recombination system. The URA3 marker flanked by loxP sites is efficiently excised by cre recombinase, leaving no trace in the final strain. This resolves the contradiction by maintaining high transformation reliability during the process while achieving complete gene purity in the final product.
Solution Approach 2:
The selection marker is preliminarily introduced to ensure efficient transformation and selection of correct transformants. After transformation is complete and correct integrants are selected, the marker is then removed in a subsequent step using cre-loxP recombination. This preliminary use of the marker ensures reliable transformation while the subsequent removal ensures complete gene purity, resolving the contradiction between the two requirements.
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 engineered Wickerhamomyces ciferrii strain achieves significantly higher TAPS production, with titers up to 20 g/L, improving productivity and efficiency in biosynthesis.
Implementation Method 1
an expression plasmid with a CEN/ARS origin of replication
Implementation Method 2
using methods like electroporation for transformation
Implementation Method 3
The condensation of L-serine and palmitoyl-CoA by catalyzed by serine palmitoyl-transferase (SPT)
Implementation Method 4
sphinganine C4-hydroxylase activities
Data Source
AI summary
The present invention relates to a Wickerhamomyces ciferrii mutant strain having an improved ability to produce tetraacetylphytosphingosine (TAPS), and a method for producing TAPS using the same. The Wickerhamomyces ciferrii mutant strain provided by the present invention exhibits excellent TAPS productivity, and thus may be widely used in development of various products that use TAPS.