Synthetic promoter for xylose-regulated gene expression
Inventors
Assignees
US Department of Agriculture USDA
Publication Number
US-9506097-B1
Publication Date
2016-11-29
Expiration Date
2035-07-10
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Abstract
Disclosed are isolated nucleic acid molecules that have promoter activity specific to xylose. The synthetic promoters, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, promote the expression of a coding region of interest in transformed yeast cells.
Core Innovation
The invention provides isolated nucleic acid molecules with xylose-specific promoter activity, comprising DNA sequences selected from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. These synthetic promoters promote expression of coding regions of interest in transformed yeast cells, enabling gene expression control responsive to xylose availability.
The problem addressed is that naturally occurring yeast strains, such as Saccharomyces cerevisiae, cannot metabolize xylose effectively, and uncontrolled high-level gene expression for xylose fermentation can be detrimental to yeast growth and fermentation efficiency. Existing yeast promoters are mostly constitutive or inducible without xylose specificity, creating a need for a promoter that regulates gene expression in response to xylose to balance metabolic efficiency and cell growth.
Claims Coverage
The patent includes five main inventive features related to isolated nucleic acid molecules, vectors, cells, methods of expression, and transformed yeast with xylose-inducible promoters.
Isolated nucleic acid molecule with xylose-specific promoter activity
An isolated nucleic acid molecule has promoter activity specific to xylose and comprises a DNA sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, operatively linked to at least one heterologous nucleic acid sequence of interest.
Vector comprising the xylose-specific promoter sequence
A vector includes a polynucleotide consisting of one of the sequences SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 that functions as a xylose-specific promoter.
Cell comprising the vector with xylose-specific promoter
A transformed cell contains the vector that includes the xylose-specific promoter sequence from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and the vector can be stably integrated into the cell's genome.
Method for expressing a coding region in transformed yeast under control of xylose-specific promoter
A method comprises providing a transformed yeast cell having a recombinant construct with a promoter region containing SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 operably linked to a coding region of interest and growing the yeast under conditions where the construct is expressed.
Transformed yeast comprising xylose-inducible promoter and heterologous gene
A transformed yeast contains a promoter comprising SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 operably linked to a heterologous gene, where the promoter activates transcription of the gene when xylose is present, with yeast genera including Saccharomyces, Kluyveromyces, Candida, and others.
The claims collectively cover the synthetic xylose-specific promoters, their incorporation into vectors and yeast cells, methods for expression regulation via these promoters, and transformed yeast strains utilizing these promoters for controlled gene expression in response to xylose availability.
Stated Advantages
The xylose-inducible promoter allows controlled gene expression in response to xylose availability, preventing detrimental effects of uncontrolled, high-level gene expression on yeast growth and fermentation.
Use of the synthetic promoter facilitates efficient metabolism energy management in yeast by inducing gene expression only when xylose is present.
The addition of nuclear localization signals and chromatin-modifying proteins to the repressor protein enhances transcriptional repression when xylose is absent, improving regulation fidelity.
Documented Applications
Expressing heterologous genes encoding enzymes and transporters involved in xylose metabolism and related metabolic pathways such as xylanases, xylose reductases, xylose transporters, glucose transporters, and others in transformed yeast.
Regulated recombinant protein production in yeast where gene expression is controlled by xylose presence, improving industrial applications involving biomass conversion and fermentation processes.
Engineering yeast strains of various genera (including Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Spathaspora, Yarrowia, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces, Debaryomyces, Schwanniomyces, Pachysolen, Torulaspora, Hansenula, and Pichia) for xylose-responsive gene expression systems.
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