Ureohydrolase Selectable Markers for Yeast Genetic Engineering
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Solution Overview
Problem
Current genetic engineering techniques for yeast strains face limitations due to a lack of suitable selectable markers, particularly for wild-type, allopolyploid, and aneuploid prototrophic strains, as traditional markers like auxotrophic and antibiotic resistance markers are often undesirable, leaving a limited number of options for introducing genomic alterations.
Innovation Solution
The development of a nucleic acid molecule encoding guanidinobutyrase or guanidino-amide hydrolase as dominant selectable markers, which are operably linked to heterologous promoter and terminator sequences, allowing for the use of guanidinobutyrate or agmatine as sole nitrogen sources for selection, providing a convenient and efficient method for introducing genomic alterations in yeast strains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional selectable markers (auxotrophic and antibiotic resistance markers) are used for genetic engineering in yeast, then the selection process is well-established and reliable, but the number of suitable markers is limited and they are often undesirable for wild-type, allopolyploid, and aneuploid prototrophic strains
Solution Approach 1:
The patent changes the biochemical parameter by introducing a novel metabolic pathway capability - the ability to utilize guanidinobutyrate or agmatine as sole nitrogen sources. This is achieved by expressing heterologous ureohydrolase enzymes (guanidinobutyrase or agmatinase) that catalyze the hydrolysis of these compounds, thereby creating a new selectable marker system that expands the range of available markers beyond traditional auxotrophic and antibiotic resistance markers.
2Adaptability or versatility
If new selectable markers are developed to expand marker options, then the versatility for genetic manipulation increases, but the complexity of the marker system and its implementation increases
Solution Approach 1:
The patent segments the selection process into two distinct biochemical pathways: one based on guanidinobutyrase activity for guanidinobutyrate utilization, and another based on agmatinase activity for agmatine utilization. Each pathway can be independently implemented using separate nucleic acid molecules encoding the respective ureohydrolase enzymes, allowing flexible combination and selection based on specific experimental needs.
Solution Approach 2:
The patent uses heterologous promoter and terminator sequences as intermediaries to regulate the expression of the ureohydrolase encoding sequences. These regulatory elements mediate between the structural genes and the host cell's transcriptional machinery, enabling controlled expression of the foreign enzymes in yeast cells without requiring modification of the yeast's native regulatory systems.
3Reliability
If ureohydrolase enzymes are used as selectable markers, then the number of available markers increases and traditional marker limitations are overcome, but the biochemical complexity and selection methodology becomes more complex
Solution Approach 1:
The patent implements a self-service selection system where the yeast cells themselves perform the detection function. Cells expressing the ureohydrolase enzymes automatically demonstrate their selected status by their ability to grow on guanidinobutyrate or agmatine as sole nitrogen sources. The enzymatic activity is directly coupled to the essential function of nitrogen assimilation, so only cells with functional enzyme expression can survive and proliferate under selective conditions.
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 enables the use of guanidinobutyrase and agmatinase as dominant selectable markers, expanding the range of suitable markers for genetic manipulation in yeast, particularly for strains where traditional markers are not viable, facilitating more efficient genetic engineering and altering of yeast genomes.
Implementation Method 1
The invention relates to the fields of molecular biology and genetic engineering of microorganisms, especially of yeast... arginase (EC 3.5.3.1), an ureohydrolase that converts arginine to ornithine and urea... agmatinase (EC 3.5.3.11), which participates in an alternative pathway for arginine catabolism
Data Source
AI summary
The invention relates to a nucleic acid molecule encoding a novel selection marker. Said marker is a guanidinobutyrase from Kluyveromyces lactis, which, when expressed in Saccharomyces, allows the growth of the yeast in the presence of guanidinobutyrate as the sole nitrogen source. Said marker can be used in a method for producing a microorganism having an altered genome. The invention further relates to a set of constructs, comprising a first construct comprising a recognition site for an endonuclease, a first region of homology with a target gene of a microorganism, and a first part of a nucleotide sequence encoding the selection marker, and a second construct comprising a second part of the nucleotide sequence encoding the selection marker, a second region of homology with the target gene of the microorganism, and a copy of the endonuclease recognition site. The invention further relates to methods for altering a target gene in a microorganism, to methods for producing a microorganism, and to microorganisms that are produced by the methods of the invention.