US20050289666A1

US20050289666A1 - Starch-inducible promoters, recombinant gene constructs, and methods of regulating gene expression

Info

Publication number: US20050289666A1
Application number: US11/122,494
Authority: US
Inventors: Brian Hooker; Keith Miller; Johnway Gao; Rodney Skeen; Daniel Anderson
Original assignee: Individual
Current assignee: Battelle Memorial Institute Inc
Priority date: 2000-08-04
Filing date: 2005-05-04
Publication date: 2005-12-29
Also published as: CA2417868A1; WO2002012516A3; US6900305B2; US20020155549A1; EP1311671A2; AU2001278164A1; WO2002012516A2

Abstract

The invention includes an isolated promoter containing nucleotides 1827 through 2147 of SEQ ID NO.: 1. The invention also includes methods of regulating expression of a gene by fusing the isolated promoter to a coding sequence to form a fused construct, and introducing the fused construct into a host such that the promoter regulates the expression of the gene product within the host. The invention additionally includes a recombinant gene comprising an isolated promoter containing at least nucleotides 1827 through 2147 of SEQ ID NO.: 1, operably linked to a coding region encoding a gene product of interest. The invention includes a host cell containing the recombinant gene.

Description

RELATED PATENT DATA

This patent resulted from a Continuation-In-Part of U.S. patent application Ser. No. 09/921,944, which was filed Aug. 2, 2001; which is a Continuation-In-Part of U.S. patent application Ser. No. 09/632,314, filed Aug. 4, 2000, now abandoned. The entirety of each of these previous applications is hereby incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under Contract DE-AC06 76RLO 1830 awarded by the United States Department of Energy. The Government has certain rights in the invention.

TECHNICAL FIELD

The invention pertains to isolated promoters, recombinant polynucleotide constructs, methods of regulating expression of a gene in recombinant host cells, and methods of expressing a gene product.

BACKGROUND OF THE INVENTION

Yeast are becoming increasingly utilized as expression systems for production of proteins and other commercially useful products. Since yeast are eukaryotic organisms, they can advantageously be utilized to produce eukaryotic derived proteins as well as prokaryotic proteins, and can often produce secreted proteins and/or post-translationally modified proteins which are difficult or impossible to produce in non-eukaryotic expression systems.
Yeast cells can allow relatively inexpensive production of large amounts of protein as compared to other eukaryotic expression systems such as mammalian or insect cell systems. As compared to alternative eukaryotic expression systems, yeast systems can often allow easy culturing and handling, and can also allow the protein(s) of interest to be easily purified.
A helpful tool in production of heterologous proteins in most expression systems is a strong gene promoter which can allow high levels of gene expression, resulting in high levels of production of the protein encoded by the gene. In many instances, it can be desirable to express high levels of a heterologous protein at a particular time or over a particular time range, while a basal level of expression (low or no expression of the heterologous gene) occurs at other times. Accordingly, an inducible promoter can be desirable such that expression of the heterologous protein can be enhanced or triggered by one or more inducing agents at a chosen time or growth stage of the host.
In addition to the advantageous of yeast expression systems set forth above, some yeast promoters can also function to regulate gene expression in one or more of bacteria, mold, plants and/or plant cells. However, the number of available promoters having an ability to function in yeast, or in yeast as well as in prokaryotic cells, molds and/or plants is limited at the present time. Further limited is the availability of promoters having the ability to allow strong and/or inducible expression in yeast, or in both yeast and alternative systems. Accordingly, it would be desirable to develop additional promoters for utilization in yeast an alternative systems, and to develop additional expression systems.

SUMMARY OF THE INVENTION

In one aspect the invention encompasses a method of regulating expression of a gene product. The method includes providing a coding region which includes a nucleic acid sequence that encodes a gene product. The coding region is fused with an isolated promoter to form a fused construct; the isolated promoter includes nucleotides 1827 through 2147 of SEQ ID. NO.: 1. The method includes introducing the fused construct into a host such that the promoter regulates the expression of the gene product within the host.
In one aspect the invention encompasses an isolated gene promoter comprising at least nucleotides 1827 through 2147 of SEQ ID NO.: 1.
In one aspect the invention encompasses a recombinant gene comprising an isolated promoter containing at least nucleotides 1827 through 2147 of SEQ ID NO.: 1, and a coding region comprising a nucleic acid sequence encoding a gene product other that the Schwanniomyces occidentalis ATCC 26077 glucoamylase gene product, the isolated promoter being operably linked to the coding region.
In one aspect the invention encompasses a method of expressing a gene product by providing a starch inducible promoter including at least nucleotides 1827 through 2147 of SEQ ID NO.: 1 and operably linking the inducible promoter to a coding DNA sequence to form a recombinant gene. The coding DNA sequence encodes a protein of interest. The recombinant gene is introduced into a host cell and expression of the recombinant gene is induced.
In one aspect the invention encompasses a host cell containing a promoter operably linked to a coding sequence which encodes a gene product other than the Schwanniomyces occidentalis ATCC 26077 glucoamylase gene product. The promoter comprises at least nucleotides 1827 through 2147 of SEQ ID NO.: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
Preferred embodiments of the invention are described below with reference to the following accompanying drawings.
FIG. 1 is a flowchart diagram illustrating a particular aspect of the present invention.
FIG. 2 shows a schematic illustration of an inverse PCR method for obtaining an isolated promoter.
FIG. 3 is a photograph image of a gel separation of PCR clones of the Schwanniomyces occidentalis glucoamylase promoter.
FIG. 4 is a schematic illustration of construction of a plasmid vector pGA2066.
FIG. 5 is a schematic illustration of construction of a plasmid vector pGA2100.
FIG. 6 is a schematic illustration of construction of a plasmid vector pGA2101.
FIG. 7 is a schematic linear representation of a recombinant DNA construct containing a 1.5 kbp glucoamylase promoter sequence operably linked to the beta-glucuronidase reporter gene.
FIG. 8 is a schematic linear representation of a recombinant DNA construct containing a 1.0 kbp glucoamylase promoter sequence operably linked to the beta-glucuronidase reporter gene.
FIG. 9 is a schematic linear representation of a recombinant DNA construct containing a 0.3 kbp glucoamylase promoter sequence operably linked to the beta-glucuronidase reporter gene.
FIG. 10 is a schematic linear representation of a recombinant DNA construct containing a 1.2 kbp glucoamylase promoter sequence which lacks the 0.3 kb minimal promoter sequence, operably linked to the beta-glucuronidase reporter gene.
FIG. 11 is a schematic linear representation of a recombinant DNA construct containing a 0.7 kbp glucoamylase promoter sequence (which lacks the 1.0 kb promoter fragment sequence) operably linked to the beta-glucuronidase reporter gene.
FIG. 12 is a scatter plot of forward scatter height (FSH) relative to side scatter height (SSH) for light scatter analysis of cell viability for a control non-transformed cell line and five transformed cell lines. The five transformants include: transformant 470, which contains a 1.0 kb GAM promoter fragment; transformant 478 which contains a 0.3 kb minimal GAM promoter fragment; transformant 481, which contains a 1.2 kb GAM promoter fragment; transformant 484 which contains a 0.7 kb fragment of the GAM promoter; and transformant 469, which contains 1.5 kb of the GAM promoter.
FIG. 13 is a histogram depicting fluorescence intensity relative to the number of cells counted (counts) for five transformed cell lines shown in FIG. 12, relative to the non-transformed control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This disclosure of the invention is submitted in furtherance of the constitutional purposes of the U.S. Patent Laws “to promote the progress of science and useful arts” (Article 1, Section 8).
The invention encompasses recombinant gene expression involving isolated polynucleotide promoter sequences. For purposes of the present description the term “expression” refers to transcription of a gene to produce corresponding RNA and translation of the mRNA to produce the gene product (i.e. peptide, polypeptide or protein), or a portion of the transcription and/or translation process. The term “isolated” can refer to a naturally occurring molecule such as, for example, a polynucleotide or a polypeptide that has been recovered from an organism which produced it, or alternatively can refer to a synthetic molecule. The invention also encompasses formation of polynucleotide constructs containing isolated polynucleotide promoter sequences, polynucleotide constructs, host cells containing the polynucleotide constructs, and expression systems which utilize the polynucleotide constructs.
Isolated promoters in accordance with the invention comprise all or a functional portion of the nucleotide sequence set forth in SEQ ID NO.: 1. SEQ ID NO.: 1 corresponds to a 2182 nucleotide sequence including the ATG start codon (nucleotides 2148-2150) and an additional 32 nucleotides (2151-2182) from the coding region of the Schwanniomyces occidentalis (formerly Schwanniomyces castellii), ATCC 26077 glucoamylase gene. Nucleotides 1-2147 of SEQ ID NO.: 1 can be referred to as being “upstream” or 5′ relative to the coding region of the glucoamylase (GAM) coding region. A 1662 base sequence located immediately 5′ to the start codon (corresponding to nucleotides 486-2147 of SEQ ID NO.: 1), is identified being the GAM promoter region based on open reading frame analysis. For purposes of the description, the term “promoter” refers to a nucleic acid sequence located upstream or 5′ to a translational start codon (ATG) of an open reading frame or coding region of a gene where the promoter region is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A constitutive promoter is one which is functional in a native organism or host in an absence of specific inducing agents. An inducible promoter is a promoter having promoter activity that is initiated or enhanced by an inducing agent. For purposes of the description the term “minimal promoter activity” refers to a detectible ability to initiate, direct or promote gene transcription. Additionally, the term “minimal promoter sequence” refers to a minimal nucleic acid sequence sufficient to direct transcription of a nucleic acid sequence.
The term “fragment” as used herein can describe a nucleic acid or a portion of a nucleic acid sequence that is less than a full length. For example, a promoter fragment can refer to a portion of a promoter sequence that has promoter activity.
The Schwanniomyces occidentalis nucleic acid sequences described herein can be obtained from the American Type Culture Collection (ATCC), Manassas, Va., under ATCC No. 26077. This yeast strain has been shown to completely hydrolyze and utilize starch as a nutrient source. The glucoamylase gene from which the isolated promoter sequences of the invention are derived is a starch inducible promoter. The promoters of the invention utilized in conjunction with the methodology described below can be used to produce expression systems for expression of recombinant genes to produce proteins of interest. In particular expression systems of the invention, the expression of a protein of interest can be induced by providing starch to the expression host.
A process encompassed by the present invention is described generally with reference to the block diagram of FIG. 1. At an initial step (A) an isolated polynucleotide comprising a promoter sequence is provided. The isolated promoter sequence can comprise all or a functional portion of the promoter region of SEQ ID NO.: 1. The terms ‘functional portion’ or “functional fragment” as used in the present description refers to any portion of a promoter sequence having at least minimal promoter activity. When utilized in conjunction with a particular host, a functional promoter is one that can produce at least minimal transcription within the particular host.
In particular embodiments of the present invention, the isolated promoter utilized in step A of FIG. 1 will contain all or a portion of nucleotide sequence 1-2147 of SEQ ID NO.: 1. As indicated above, a 1662 nucleotide sequence containing nucleotides 486-2147 is determined to be the starch inducible promoter region of the GAM gene of strain 26077. Accordingly, the isolated promoter is step (A) can comprise an entirety or a functional portion of the nucleotide sequence from nucleotide 486-2147 of SEQ ID NO.: 1. Alternatively, an isolated promoter can be provided in step A which comprises a fragment of SEQ ID NO.: 1 which is unique relative to other known promoter sequences. The unique fragment can comprise for example, particular functional portions of SEQ ID NO.: 1 which are described in more detail below.
As will be understood by those of ordinary skill in the art, the promoter sequence set forth in SEQ ID NO.: 1 can be manipulated by deleting, inserting or modifying one or more nucleic acids within the sequence utilizing conventional methods to result in a sequence that retains promoter functionality. The retained functionality can in certain instances, even be enhanced by such modification. Accordingly, the invention encompasses utilizing such modified sequences for the isolated promoter in step (A). Preferably where a modified isolated promoter utilized in step (A) the promoter will have at least 70% identity to the corresponding fragment set forth in SEQ ID NO.: 1. Typically, the promoters of the invention will include a sequence having at least 80% identity to a functional fragment of SEQ ID NO.: 1, and can in particular instances comprises a sequence having at least 90% identity to a functional fragment of SEQ ID NO.: 1.

Referring to FIG. 2, an exemplary method for isolation of a promoter sequence for use in step (A) of FIG. 1 is shown. Genomic DNA can be isolated from, for example, cultured Schwanniomyces occidentalis strain ATCC 26077. The isolated genomic DNA can be purified and used for subsequent isolation of the GAM promoter. For instance, the genomic DNA (FIG. 2 top) can be digested with restriction enzymes to produce fragments of the genomic DNA. Such fragments can be self ligated to form the circular DNA shown in FIG. 2 (center). Inverse polymerase chain reaction (PCR) can then be performed to introduce desired restriction enzyme sites into the fragments. Exemplary reverse primers appropriate for use during such PCR process are set forth in SEQ ID NOS.: 4 and 5. Exemplary forward primers for utilization during such inverse PCR process are set forth in SEQ ID NOS.: 6 and 7.

TABLE 1


Primer Pairing and Restriction Enzymes for Inverse PCR
Reactions

PCR Sample	Restriction Enzyme	Primer Pairing

1	Bcl I	SEQ ID No.: 4/SEQ ID No.: 7
2	Bcl I	SEQ ID No.: 5/SEQ ID No.: 7
3	BstB I	SEQ ID No.: 4/SEQ ID No.: 6
4	BstB I	SEQ ID No.: 5/SEQ ID No.: 7
5	Hinc II	SEQ ID No.: 4/SEQ ID No.: 7
6	Hinc II	SEQ ID No.: 5/SEQ ID No.: 7
7	Hpa I	SEQ ID No.: 4/SEQ ID No.: 7
8	Hpa I	SEQ ID No.: 5/SEQ ID No.: 7
9	Sac I	SEQ ID No.: 4/SEQ ID No.: 7
10	Xmn I	SEQ ID No.: 4/SEQ ID No.: 7
11	Xmn I	SEQ ID No.: 5/SEQ ID No.: 7

Table 1 shows exemplary restriction enzymes (column 2) that can be utilized during digestion of genomic DNA, and shows pairing of the exemplary primers for utilization during inverse PCR processing. It is to be understood that the invention encompasses use of alternative or additional restriction enzymes during the digestion and also encompasses use of alternative primers and primer pairing.
As shown in FIG. 2, the restriction enzyme digestion, self ligation and subsequent inverse PCR can be used to produce the isolated promoter for utilization in step (A) of FIG. 1. Alternatively, the isolated promoter obtained can undergo further processing to produce fragments thereof and/or modify such sequence for production of alternative isolated promoters which can be utilized in step (A).
Referring again to FIG. 1, a polynucleotide sequence that encodes a protein of interest can be provided in a step (B) and can be utilized in conjunction with the isolated promoter provided in step (A) for polynucleotide construct formation step (C). The encoding polynucleotide provided in step (B) is not limited to any specific polynucleotide sequence. The coding sequence can comprise sequence encoding any protein, polypeptide, or peptide of interest, or can encode two or more proteins/peptides of interest. In accordance with the invention, a protein of interest can be an enzyme, a structural protein, an enzyme cofactor, a receptor, a ligand, a regulatory protein, a peptide, a portion of a protein, etc. In particular instances, the methodology and expression systems in accordance with the invention can be useful for producing therapeutically beneficial proteins/peptides.
The phylogenetic origin of the coding sequence can be any desired species, either prokaryotic or eukaryotic. In particular embodiments, the native origin of the coding sequence can be a different species than the originating species of the isolated promoter, can be different species than the originating species of the eventual host (discussed below), or both.
Polynucleotide construct formation step (C) can include forming a recombinant molecule by fusing the isolated promoter provided in step (A) with the coding sequence provided in step (B). Preferably, the isolated promoter is operably linked to the provided coding sequence. For purposes of the description the term “operably linked” refers to a nucleic acid sequence which is arranged or joined with a second sequence such that the first nucleic acid sequence affects the function or processing of the second nucleic acid sequence. A promoter sequence is operably linked to a coding sequence if the promoter regulates or mediates transcription of the coding sequence. Operable linkage of a polynucleotide to a promoter to form a recombinant polynucleotide construct in step (C) can allow expression of the polynucleotide and production of the encoded polypeptide to be controlled by the promoter. The term “recombinant” as used in the present description can refer to a nucleic acid molecule which is made at least in part by artificial combination of two or more segments. The term “gene” refers to a DNA sequence or molecule which includes an encoding region and one or more regions involved in regulation of expression of the coding sequence. The term gene can refer to a native gene (where native refers to naturally occurring nucleic acid) or can refer to a DNA molecule having at least some synthetic or recombinant portion.
Recombinant DNA construction formation step (C) can additionally comprise formation of a vector. For purposes of the present invention, a vector can comprise a plasmid, cosmid, phage or yeast artificial chromosome (YAC). A particular vector to be formed in step (C) can be determined based on the intended use of the vector. For example, vector formation in step (C) can be determined by appropriateness of the vector for introduction into a host in step (D) (discussed below).
Once the desired polynucleotide construct is formed in step (C), the construct can be utilized for introduction into a host cell in step (D). Introduction into a host cell is not limited to a specific method of introduction. In particular embodiments, introducing a construct into a host cell can comprise transformation of a host cell. The terms “transgenic” and “transformed” can refer to any cell tissue, organism or seed into which foreign or recombinant DNA has been introduced. Transformation can be achieved utilizing one or more of electroporation, sonication, T-DNA mediated transformation, particle-bombardment mediated transformation, microinjection, virus-mediated transformation, whiskers mediated transformation, liposome mediated transformation, chemical mediated transformation and plasma transformation. When referring to plant cells, plant tissue, whole plants or other plant parts the designated T0 can refer to the primary transformant and the designation T1 can refer to the first generation produced from the primary transformant T0.
The host cell into which the polynucleotide construct is introduced in step (D) is not limited to a particular type of cell. Such host cell can be prokaryotic or eukaryotic. Additionally, the host cell can be from a different species than the species from which the isolated promoter has been isolated, can be different from the species from which the coding polynucleotide is isolated or can be different from both the originating species of the promoter and the originating species of the coding polynucleotide.
In particular embodiments, the host cell can be a species belonging to a phylogenetic kingdom that is different than the phylogenetic kingdom from which at least one of the isolated promoter and the isolated coding polynucleotide originate. For example, when the isolated promoter comprises all or a portion of the promoter region of SEQ ID NO.: 1 isolated from the yeast (belonging to the fungi kingdom) Schwanniomyces occidentalis 26077, the host cell can comprise, for example, a cell belonging to a species within the kingdom Plantae.
Alternatively, when an isolated promoter is isolated from Schwanniomyces occidentalis, the host cell can belong to a species within the same phylogenetic kingdom. When belonging the same kingdom (Fungi) the host cell species can be the same or can be different from the species which the promoter is isolated. For example, when the isolated promoter provided in step (A) is the Schwanniomyces occidentalis GAM promoter or a portion thereof, the host cell in step (D) can also be Schwanniomyces occidentalis (either strain 26077 or an alternative strain), can be a second species within the Schwanniomyces genus, or can belong to an independent genus or even an independent phylum within the Fungi kingdom.
The invention also encompasses introducing the constructs formed in step (C) into a prokaryotic host such as bacteria. The introduction of the construct of the present invention into bacterial can be useful for manipulation and/or amplification of the construct. For purposes of the present invention the term “amplification” of a nucleic acid or nucleic acid sequences refers to the production of additional copies of the nucleic acid or sequence. Amplification can utilize host mediated amplification and/or polymerase chain reaction technology.
As shown in FIG. 1, once the recombinant polynucleotide construct has been introduced into the host cell the polypeptide encoded by the polynucleotide can be expressed by the host cell in an expression step (E). Alternatively, the host cell can be utilized to assist transformation of a subsequent host cell or to amplify or manipulate the polynucleotide construct (not shown). Expression within a host cell in accordance with the invention can be constitutive expression or induced expression. In particular instances, expression can be constitutive at a low level and can be inducible such that the level of expression increases upon inducing with an appropriate inducing agent.
In embodiments of the present invention where the encoded protein is expressed in the host cell, the expressed protein is not limited to any particular type, and can be for example, an intracellular peptide, an excreted or extracellular peptide or a transmembrane peptide. Alternatively, the expressed peptide can confer resistance to the host cell. Such conferred resistance can be resistance to one or more of an antibiotic, a herbicide, a toxin, a parasite and a pathogen. The invention also encompasses introducing constructs where the coding sequence codes for a protein that can regulate or control expression of native genes within the host cell, or can regulate or control expression of the introduced (heterologous) genes.
The invention encompasses recombinant constructs and hosts containing the constructs where the protein encoded by the constructs confers a new (non-native) phenotype to the host cell. Additionally encompassed are constructs and hosts where the expressed protein has a sequence, a function, or an effect that is similar to or identical to one or more native polypeptides produced by the host.
In one embodiment of the invention, the isolated promoters of the invention can be utilized to form recombinant genes which can be introduced and expressed in Schwanniomyces occidentalis. As indicated above, the native GAM promoter is an inducible promoter. The native GAM promoter can be induced to initiate or enhance transcription by agents such as maltose and starch. In native Schwanniomyces occidentalis the expression level of the glucoamylase gene can be increased by 100 fold when cells are shifted from a glucose culture medium to a maltose culture medium. Accordingly, the use of the isolated promoters of the invention in recombinant genes can allow host Schwanniomyces occidentalis cells to be induced to produce a protein of interest. As further discussed below, the promoter region of SEQ ID NO.: 1 and various functional fragments thereof can function as constitutive or inducible promoters in Schwanniomyces occidentalis or in alternative yeast strains.
In addition to being useful in yeast expression systems, the isolated promoters of the invention can also be utilized to form recombinant molecules for introduction and expression in non-yeast and even non-fungi systems. In particular, the isolated promoters of the invention can be utilized for heterologous expression in plants.

EXAMPLES

Example 1

Expression utilizing an isolated promoter in a yeast host cell.
The glucoamylase gene promoter was isolated from Schwanniomyces occidentalis strain ATCC 26077 utilizing the exemplary method discussed above with reference to FIG. 2. The method included growing Schwanniomyces occidentalis cells overnight in a culture medium, harvesting the cells and isolating and purifying genomic DNA utilizing spheroplasting technology. Inverse PCR methodology was utilized to obtain the sequence shown in SEQ ID NO.: 1 which is inclusive of the promoter region of the GAM gene. The PCR methodology utilized the reverse primer set forth in SEQ ID NOS.: 4 and 5 and the PCR forwarding primers set forth in SEQ ID NOS.: 6 and 7.
The isolation included digestion of genomic DNA with restriction enzymes including Bcl I, Bst B I, Hinc II, Hpa I, Sac I and Xmn I having restriction sites upstream of the glucoamylase gene. After digestion, the DNA samples were purified and self ligated using T4 DNA ligase followed by inverse PCR. Primer pairing and restriction enzyme for eleven independent samples is indicated in Table 1.
Following PCR, the DNA products were separated in an agarose gel by electrophoresis, the results of which are shown in FIG. 3. The lane numbers shown in FIG. 3 correspond to the inverse PCR reactions set forth in Table 1, with lane S being a DNA size marker. The isolated GAM promoter clones are apparent as dark bands in the photograph. Lanes 1, 2, 5, 6, 7 and 8 show strong bands corresponding to ligated DNA samples previously cleaved by Bcl I, Hinc II, or Hpa I. The sizes of the resulting clones range from about 0.4 kb to about 4.4 kb with the strongest bands being from about 1.7 kb to about 2.3 kb.
For sequencing and analysis, the PCR product from reaction 2 shown in FIG. 3 was initially inserted into transient vector pGEM-T (Promega, Madison, Wis.) to form pGA2066 as shown in FIG. 4. This vector was utilized for initial amplification, cloning and sequencing of the promoter. The determined sequence of the isolated DNA fragment containing the GAM promoter is set forth in SEQ ID NO.: 1. The clone has a length of 2182 base pairs which includes the GAM transcription initiation codon (corresponding to nucleotides 2148-2150 of SEQ ID No.:1) and an additional 32 bases within the coding region of the GAM gene nucleotides 2151-2182 of SEQ ID No.:1). Of the remaining sequence set forth in SEQ ID NO.: 1 (nucleotides 1-2147) a 1662 nucleotide sequence containing nucleotides 486-2147 was determined to be the GAM promoter and is referred to as the GAM promoter region.
Analysis of the 1662 base pair GAM promoter region reveals seven CAT boxes (nucleotides 1571-1574; nucleotides 1709-1712; nucleotides 1806-1809; nucleotides 1816-1819; nucleotides 1776-1779; nucleotides 1963-1966; and nucleotides 2015-2018) and nine TATA boxes (nucleotides 1561-1565; nucleotides 1626-1643; nucleotides 1730-1734; nucleotides 1864-1868; nucleotides 1884-1887; nucleotides 1937-1943; nucleotides 2034-2043; nucleotides 2081-2090; and nucleotides 2135-2139). These identified CAT boxes and TATA boxes are within a 600 base pair fragment within SEQ ID NO.: 1 relative to the initiation codon (within the sequence from nucleotide 1548-2147).
A sequence alignment and comparison was performed to determine differences between the isolated GAM ATTC 26077 promoter, and a known sequence corresponding to a fragment of a GAM promoter from Schwanniomyces occidentalis stain ATCC 26076. The strain 26076 promoter has been shown to lack ability to direct transcription in Saccharomyces cerevisiae. The comparison utilized a fragment of the isolated promoter from strain ATCC 26077 set forth in SEQ ID NO.: 2. This fragment corresponds to nucleotides 1823-2105 of SEQ ID NO.: 1. SEQ ID NO.:2 was aligned with and compared to a 325 nucleotide sequence (SEQ ID NO.:3) from the GAM promoter of strain ATCC 26076. Comparison revealed difference between the two sequences at positions corresponding to nucleotides 1984-1986, 1992-1993 and 2113 or SEQ ID NO. 1. Since the isolated GAM promoter from ATCC 26077 is able to direct transcription in Saccharomyces (see below), the positions of sequence difference indicates that these positions are important for providing promoter function, especially in hosts other than Schwanniomyces occidentalis.
The promoter activity of various fragments of SEQ ID NO.: 1 was analyzed by fusing various promoter fragments to a bacterial glucuronidase gene. Initially two fragments were utilized. A first fragment contains a 1.5 kb nucleotide sequence which includes a portion of the GAM promoter corresponding to nucleotides 647-2147 of SEQ ID NO.: 1, and a second 1.0 kb fragment including nucleotides 1297-2147 of SEQ ID. NO.: 1. These two clones (referred to as GAM15 and GAM10 respectively) were cloned from pGA2066 utilizing forward primers having the sequence set forth in SEQ ID NOS.: 8 and 9, with the primer sequence set forth in SEQ ID NO.: 8 being utilized for the 1.5 kb GAM promoter and the sequence set forth in SEQ ID NO.: 9 being utilized as a forward primer to obtain the 1.0 kb GAM promoter. The cloning additionally utilized a reverse primer having the sequence set forth in SEQ ID NO.: 10 which was utilized for each of the 1.5 kb and 1.0 kb promoters. The specified forward and reverse primers introduced a Spe I restriction enzyme site at the 5′ end of the promoter sequence and introduced a Hind III restriction enzyme site at the 3′ end of the promoter sequences.
The forward and reverse primers above were used in conjunction with PCR to produce polynucleotides corresponding to the 1.5 kb and 1.0 kb promoters having appropriate ends to allow insertion into vector pGA2028D to form plasmid vectors pGA2100 containing the 1.5 kb GAM promoter as illustrated in FIG. 5, and the vector pGA2101 containing the 1.0 kb GAM promoter as illustrated in FIG. 6. Each of the resulting vectors has a DNA replicon designated ‘2 micron’ for plasmid replication in Saccharomyces strains. The vector region designated ‘ColE1’ is the origin for plasmid replication during gene manipulation in E. coli strains, ‘f1 ori’ is the phage origin and ‘gus’ is the bacterial glucuronidase gene. Tcyc1 is the transcription terminator and Zeocin is the Zeocin resistance gene. Formation of each of the two plasmids including operably linking the isolated promoter fragment to the coding portion of the E. coli beta-glucuronidase (GUS) gene.
Plasmid vectors pGA2100 and pGA2101 were independently utilized to transform a Saccharomyces host. Each plasmid was introduced into a Saccharomyces hybrid yeast strain (obtained from James R. Mattoon of University of Colorado) utilizing plasma transformation of the host. After transformation, the Saccharomyces cells were plated onto appropriate selective medium containing glucose, and incubated at 30° C. for 4 days.
Transformed colonies were chosen for determining activity of the introduced GAM promoter sequences utilizing glucuronidase (GUS) activity analysis. For this analysis, protein samples were collected from isolated colonies by suspending a single transformed colony and subsequently disrupting the cells. After disruption, the sample was centrifuged and the supernatant containing protein was analyzed for protein content and GUS activity.
The determined GUS activity for individual colonies containing either the GAM15 or GAM10 promoters are set forth in Table 2. The GUS specific activity (units of glucuronidase activity per milligram of total protein, where 1 unit of glucuronidase activity is the amount of glucuronidase that converts 1 pmole of 4-methylumbelleiferul-beta-D-glucuronide (MUG) to 4-methylumbelliferone) is reported for two isolated colonies containing the 1.5 kb fragment and four isolated colonies containing the 1.0 kb fragment. The measured GUS activity was compared to a control assay which utilized non-transformed cells (C*).
The GUS analysis indicate that the 1.5 kb fragment and the 1.0 kb fragment function as promoters to direct transcription of the recombinant GUS gene. These results additionally indicate that the GAM promoter fragments isolated from Schwanniomyces occidentalis are able to function as promoters in other fungal species.

TABLE 2

GUS-activity analysis of transformants grown in glucose medium

Isolated GUS specific activity Average activity

Transformant Promoter (unit/mg) (units/mg)

C* N/A 6 6

1 GAM15 54 60

2 GAM15 66

3 GAM10 100 97 ± 15

4 GAM10 99

5 GAM10 111

6 GAM10 76

C* Non-transformed control

Example 2

Inducible expression from isolated promoters in yeast host cells.
Transformed Saccharomyces colonies containing either the 1.5 kb promoter fragment or the 1.0 kb promoter fragment were chosen for determining activity and inducibility of the introduced promoter utilizing the glucuronidase activity analysis described above. Transformed colonies were first grown in a medium containing glucose. Cells were washed and subsequently transferred into culture medium containing 2% potato starch for GUS gene expression analysis. The results of the GUS activity assays are summarized in Table 3.

The GUS specific activity is reported for six isolated colonies transformed with the 1.5 kb fragment and six isolated transformed colonies containing the 1.0 kb GAM promoter fragment. The specific activities are reported relative to a control host cell which has not been transformed with the GUS expression vector.

TABLE 3


GUS-activity analysis of transformants grown in starch medium

	Isolated	GUS specific activity	Average activity
Transformant	Promoter	(unit/mg)	(units/mg)

C*	N/A	0.0	0.0
1	GAM15	1394	890 ± 350
2	GAM15	1405
3	GAM15	582
4	GAM15	854
5	GAM15	685
6	GAM15	963
7	GAM10	1398	1521 ± 327
8	GAM10	1645
9	GAM10	2123
10	GAM10	1250
11	GAM10	1432
12	GAM10	1277

C* Non-transformed control

Comparing the results presented in Table 3 obtained for colonies grown in starch medium with the results presented in Table 2 for colonies grown in glucose medium, the results indicate that the two promoter fragments are each highly induced by starch and maintain their starch inducibility even in a non-Schwanniomyces host. Accordingly, the promoters of the invention can advantageously allow use of starch as an inducing agent in alternative expression systems to allow controlled and highly inducible expression utilizing a relatively inexpensive inducing agent.

Example 3

Promoter Deletion Analysis
The glucoamylase promoter-gus reporter gene construct containing the 1.5 kb promoter fragment described above was utilized for promoter deletion analysis. Restriction endonuclease digestion of the GAM15 construct plasmid was utilized to produce four deletion constructs to determine the functionality of shorter fragments and particular regions within the 1.5 kb promoter fragment.
As indicated above, the 1.5 kb promoter fragment contains nucleotide sequence corresponding to nucleotides 647-2147 of SEQ ID NO.: 1. A portion of the plasmid containing the 1.5 kb glucoamylase promoter fragment with the plasmid being designated as the 469 plasmid is shown in FIG. 7. Four additional constructs were derived from this plasmid by restriction endonuclease digestion. A 1.0 kb glucoamylase promoter fragment operably linked to the GUS reported gene was formed by digestion with Nco I/Nae I to produce plasmid 470 depicted in FIG. 8. The 1.0 kb construct includes GAM promoter sequence corresponding to nucleotides 1297-2147 of SEQ ID NO.:1 Digestion of plasmid 469 with Nae I/Bgl II was utilized to form a 0.3 kb ‘minimal glucoamylase promoter’ operably linked to the GUS reporter gene in a plasmid construct designated 478 shown in FIG. 9. The 0.3 kb minimal promoter corresponds to nucleotides 1826-2147 of SEQ ID NO.: 1. Bgl Il/Hind III digestion of the 469 plasmid was utilized to form a 1.2 kb promoter fragment which lacks the 300 bp minimal promoter sequence. The 1.2 kb fragment corresponds to nucleotides 647-1826 of SEQ ID NO.:1, and is operably linked to the GUS reporter gene in a plasmid construct 481 depicted in FIG. 10. A 0.7 kb promoter fragment was prepared by digestion of plasmid 469 with Nco I/Hind III to produce the plasmid construct 484 shown in FIG. 11. The 0.7 kb promoter sequence contains nucleotides 647-1297 of SEQ ID NO.:1 (and lacks the 1.0 kb promoter fragment sequence).
The newly constructed deletion plasmids were isolated and purified and were used to transform Saccharomyces as described above with respect to the 1.5 and previous 1.0 kb promoter fragment transformants. Transformants were selected by growth on Zeocin plates. Individual colonies were then chosen, each containing one of the five plasmids and were grown in liquid culture overnight at 30° C. Non-transformed yeast was utilized as a control.
After growth overnight, the yeast were pelleted and re-suspended in medium containing 0.2% soluble starch for four hours at 25° C. to induce GUS expression. In order to analyze GUS activity of the transformed cell line, the yeast were pelleted and resuspended in a staining buffer containing 1% fluorescein diglucuronide (FDGIcU, available from Molecular Probes, Eugene Oreg.) and incubated at room temperature for 15 minutes.
After incubation, the cells were again pelleted by centrifugation and were subsequently resuspended in a non-FDGIcU buffer. The cells in the resulting suspension were analyzed by flow cytometry on the FL1 channel (indicated as myc/GaM488 in FIG. 13.). The intact FDGIcU molecule does not fluoresce. However, upon being cleaved by the enzyme β-glucuronidase, fluorescein moiety is fluorescent. Accordingly, fluorescence intensity of the transformed yeast is proportional to the amount of glucuronidase enzyme present within the yeast cells.

Referring to FIG. 12, yeast cells were sorted in the R1 sort-gate using light scatter analysis to assess cell viability. Populations of intact viable cells were obtained by sorting and were analyzed using cytometry. The fluorescence intensity relative to the number of cells (counts) is presented in FIG. 13 for each of the five transformants relative to a control (non-transformed) cell line. The results of the GUS analysis are summarized in Table 4.

TABLE 4


GUS-activity analysis of GAM promoter fragment transformants

		Geometric Mean
Yeast line	GAM Promoter fragment	Fluorescence Intensity

Control	N/A	4.82
478	0.3 kb (nucleotides 1827-2147	13.91
	of SEQ ID NO.: 1)
469	1.5 kb (nucleotides 647-2147	16.1
	of SEQ ID NO.: 1)
481	1.2 kb (nucleotides 647-1826	13.82
	of SEQ ID NO.: 1)
470	1.0 kb (nucleotides 1297-2147	16.55
	of SEQ ID NO.: 1)
484	0.7 kb (nucleotides 647-1297	15.09
	of SEQ ID NO.: 1)

The results above indicate that the promoters corresponding to the 1.5 kb GAM promoter fragment and the 1.0 kb GAM promoter fragment have the highest promoter activity. The 300 base pair fragment shows measurable activity above the control indicating that such fragment can serve as a functional promoter. The 0.7 kb fragment which lacks the sequence of the 1.0 kb promoter fragment, is additionally functional as a promoter having activity between that of the 300 kb minimal promoter region and the 1.0 and 1.5 kb fragments. The lowest activity is observed for the 1.2 kb promoter fragment which lacks the 300 kb minimal promoter region. Accordingly, promoters of the invention and various constructs and expression systems in accordance with the invention can preferably comprise at least the 300 kb fragment, corresponding to nucleotides 1827-2147 of SEQ ID NO.: 1. Alternatively, or in addition to nucleotides 1827-2147 of SEQ ID NO.: 1, isolated promoters of the invention can comprise a promoter fragment corresponding to nucleotides 647-1297 of SEQ ID NO.: 1. In some applications, an isolated promoter of the invention can comprise at least nucleotides 1297-2147 of SEQ ID NO.: 1.

Example 4

Expression in a plant cell protoplast host utilizing an isolated yeast promoter.
Protoplasts were prepared from Nicotiana tabacum cell suspension cultures. Super-coiled plasmid DNA, either pGA2100 or pGA2101 described above, was combined with salmon sperm DNA as a carrier, and was utilized for electroporation transformation of the protoplasts. The protoplasts were subsequently cultured for 48 hours at 28° C. in modified Murashige and Skoog (MS) medium in the presence of sucrose. The protoplasts were then tested for GUS activity.

The GUS activity of the protoplasts was analyzed by extracting protein samples from the protoplasts using sonication of suspended protoplasts, centrifugation, and collection of the protein in the resulting supernatant. The supernatant was analyzed for GUS activity as described above in previous examples. Four transformant protoplast samples were compared to a control, non-transformed protoplasts samples. The results of the GUS activity analysis on the transformed protoplasts is presented in Table 5.

Samples

1 and 2 correspond to transformants containing the 1.5 kb GAM fragment and

samples

3 and 4 correspond to transformant protoplasts containing the 1.0 kb promoter fragment.

TABLE 5


GUS-activity analysis of transformants grown in glucose medium

	Isolated		GUS specific
Transformant	Promoter	Culture medium	activity (unit/mg)

C*	N/A	sucrose	10.2
1	GAM15	sucrose	49.2
2	GAM15	sucrose	36.0
3	GAM10	sucrose	60.0
4	GAM10	sucrose	51.2

C* Non-transformed control protoplasts

As indicated by the results shown in Table 5, the isolated Schwanniomyces occidentalis promoter and fragments thereof are able to function as a promoter in plant protoplast cultures.
The combined results of the examples set forth above indicate that the GAM promoter isolated from Schwanniomyces occidentalis and fragments thereof as small as 300 kb (nucleotides 1827-2147 of SEQ ID NO.: 1) can be utilized as a functional promoter in other fungal species and also in species belonging to other phylogenetic kingdoms including plants.
In compliance with the statute, the invention has been described in language more or less specific as to structural and methodical features. It is to be understood, however, that the invention is not limited to the specific features shown and described, since the means herein disclosed comprise preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted in accordance with the doctrine of equivalents.

Claims

1. A method of regulating expression of a gene product comprising:

providing a coding region comprising a nucleic acid sequence that encodes a gene product;

fusing the coding region with an isolated promoter to form a fused construct, the isolated promoter comprising nucleotides 1827 through 2147 of SEQ ID NO.: 1; and

introducing the fused construct into a host such that the promoter regulates the expression of the gene product within the host.

2. The method of claim 1 wherein the host is a yeast cell, and wherein the introducing comprises integrating the fused construct into genomic DNA.

3. The method of claim 2 wherein the yeast is a species other than Schwanniomyces occidentalis.

4. The method of claim 1 wherein the isolated promoter further comprises nucleotides 1297 through 1826 of SEQ ID NO.: 1.

5. The method of claim 1 further comprising inducing expression of the gene providing starch to the host.

6. An isolated gene promoter comprising at least nucleotides 1827 through 2147 of SEQ ID NO.:1.

7. A vector comprising the isolated promoter of claim 6.

8. The vector of claim 7 wherein the vector is a plasmid vector.

9. A recombinant gene comprising:

the isolated promoter of claim 6; and

a coding region comprising a nucleic acid sequence encoding a gene product other than the Schwanniomyces occidentals ATCC 26077 glucoamylase gene product, the isolated promoter being operably linked to the coding region.

10. A host cell comprising the recombinant gene of claim 9.

11. A method of regulating expression of a gene product comprising:

fusing the coding region with an isolated promoter to form a fused construct, the isolated promoter comprising at least nucleotides 647-1297 of SEQ ID NO.: 1; and

12. A method of expressing a gene product comprising:

providing a starch inducible promoter comprising at least nucleotides 1827 through 2147 of SEQ ID NO.: 1;

operably linking the starch inducible promoter to a coding DNA sequence to form a recombinant gene, the coding DNA sequence encoding a product of interest;

introducing the recombinant gene into a host cell; and

inducing expression of the recombinant gene.

13. The method of claim 12 wherein the inducing comprises providing starch into a growth medium.

14. The method of claim 13 wherein the starch is the primary carbon source within the growth medium.

15. The method of claim 12 wherein the host cell is a yeast.

16. The method of claim 12 wherein the host is a plant cell.

17. The method of claim 12 wherein the host is a plant protoplast.

18. The method of claim 12 wherein the host is a Nicotiana tabacum cell.

19. The method of claim 12 wherein the gene product is selected from the group consisting of an enzyme, an enzyme cofactor, a ligand, and a receptor.

20. The method of claim 12 wherein the gene product is a structural protein.

21. A host cell comprising a promoter operably linked to a coding sequence which encodes a gene product other than Schwanniomyces occidentalis ATCC 26077 glucoamylase gene product, the promoter comprising at least nucleotides 1827 through 2147 of SEQ ID NO.: 1.

22. The host cell of claim 21 wherein the host cell is a plant cell.

23. A method of regulating expression of a gene product comprising:

fusing the coding region with an isolated promoter to form a fused construct, the isolated promoter comprising nucleotides 486 through 2147 of SEQ ID NO.: 1; and

24. The method of claim 23 wherein the host is a yeast.