Promoter useful for high expression of a heterologous gene of interest in Aspergillus niger
Inventors
Gladden, John M. • Campen, Saori Amaike • Zhang, Jinxiang • Magnuson, Jon K. • Baker, Scott E. • Simmons, Blake A.
Assignees
Pacific Northwest National Laboratory • Battelle Memorial Institute Inc • National Technology and Engineering Solutions of Sandia LLC • University of California San Diego UCSD
Publication Number
US-11414653-B2
Publication Date
2022-08-16
Expiration Date
2038-09-06
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Abstract
The present invention provides for an Aspergillus niger host cell comprising a gene of interest operatively linked to an ecm33 promoter of an ascomycete fungi, wherein the gene of interest is heterologous to the ecm33 promoter and/or to Aspergillus niger. In some embodiments, the gene of interest is a glycoside hydrolase enzyme. In some embodiments, the glycoside hydrolase enzyme is a glucosidase.
Core Innovation
The invention provides an Aspergillus niger host cell comprising a gene of interest operatively linked to an ecm33 promoter of an ascomycete fungus, where the gene of interest is heterologous to the ecm33 promoter and/or to Aspergillus niger. The ecm33 promoter, identified through secretome analysis and comprising the nucleotide sequence of SEQ ID NO:1, is shown to drive high expression of heterologous genes including glycoside hydrolase enzymes such as glucosidases. The invention also includes nucleic acids, vectors, and methods for expressing heterologous genes using this promoter in Aspergillus niger host cells.
The problem addressed by the invention is the need for efficient and economical enzyme production for lignocellulosic biomass deconstruction in biofuel applications. Existing promoters like glucoamylase (glaA) promoter are inducible and limited to specific growth conditions such as presence of starch or maltose, restricting enzyme production under diverse media conditions, especially those relevant to biomass hydrolysis. There is a need for strong, constitutive promoters that enable high heterologous enzyme expression in Aspergillus niger under various carbon sources including maltose, glucose, and xylose to reduce enzyme production costs and support industrial lignocellulolytic biofuel production.
The invention solves this by identifying and characterizing the ecm33 promoter as a strong, constitutive promoter that is not sugar inducible but drives equal or greater expression of heterologous enzymes compared to widely used promoters such as glaA and gpdA. The promoter was identified by proteomic analysis of the Aspergillus niger secretome, and promoter mutants with heterologous beta-glucosidase gene A5IL97 showed that the ecm33 promoter achieved higher mRNA and protein expression under multiple growth conditions. Furthermore, the promoter is regulated via binding of the transcription factor AtfA involved in MAPK signaling, providing insight into its regulatory control and potential for enhanced heterologous protein production.
Claims Coverage
The patent contains two independent claims covering both the Aspergillus niger host cell and a method for expressing a heterologous gene using the ecm33 promoter.
Aspergillus niger host cell comprising a heterologous gene under control of the ecm33 promoter
An Aspergillus niger host cell comprising a nucleic acid encoding a gene of interest operatively linked to an ecm33 promoter of an ascomycete fungus, wherein the gene of interest is heterologous to the ecm33 promoter and/or to the Aspergillus niger host cell.
Method of expressing a heterologous gene using the ecm33 promoter in Aspergillus niger
A method of expressing a heterologous gene of interest in an Aspergillus niger host cell by introducing a nucleic acid encoding the gene of interest operatively linked to an ecm33 promoter of an ascomycete fungus, with the gene being heterologous to the promoter and/or host cell, followed by culturing the host cell in a medium suitable for gene expression, optionally followed by separation or purification of the gene product.
The claims cover the use of the ecm33 promoter from ascomycete fungi to drive expression of heterologous genes in Aspergillus niger host cells and the associated methods of gene expression, emphasizing the promoter’s function in high-level, constitutive expression of heterologous enzymes.
Stated Advantages
The ecm33 promoter drives higher expression and secretion of heterologous enzymes compared to the commonly used glucoamylase (glaA) promoter under diverse media conditions including maltose, glucose, and xylose.
It is a strong constitutive promoter that allows enzyme expression without the need for inducers such as starch or maltose, thereby expanding the range of usable growth media for heterologous protein production.
Binding of the transcription factor AtfA to the ecm33 promoter links expression to the MAPK signaling pathway, which may support regulation and enhancement of enzyme production.
The invention enables development of improved heterologous enzyme production systems in Aspergillus niger for industrial applications, improving cost-effectiveness and feasibility of producing enzymes for lignocellulosic biofuel production.
Documented Applications
Use in biorefinery processes utilizing lignocellulose as feedstock to produce fuels and chemicals through efficient enzyme production.
High titer heterologous enzyme production, particularly glycoside hydrolases like glucosidases, in Aspergillus niger for biotechnological and industrial enzyme applications.
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