Compositions and methods for manipulating carbon flux in cells Number:7,435,168 from the United States Patent and Trademark Office (PTO) owispatent Nucleotide sequences and genetic constructs that can be used to regulate genes encoding enzymes that change carbon flux through metabolic pathways that lead to lactic acid or fumarate production in a host cell, such as a R. oryzae cell, are provided. Methods of manipulating carbon flux in a cell also are provided.<H manipulating, Compositions, Compositions, an, 7435168.html, Patent, pto, 7435168

Title: Compositions and methods for manipulating carbon flux in cells

Abstract: Nucleotide sequences and genetic constructs that can be used to regulate genes encoding enzymes that change carbon flux through metabolic pathways that lead to lactic acid or fumarate production in a host cell, such as a R. oryzae cell, are provided. Methods of manipulating carbon flux in a cell also are provided.

Patent Number: 7,435,168 Issued on 10/14/2008 to Fatland-Bloom, et al.

Inventors: Fatland-Bloom; Beth (Decatur, IL), Rai; Gyan (Newburgh, IN), Rayapati; P. John (Monticello, IL), Tonukari; Nyerhovwo John (Delta State, NG)
Assignee: Archer-Daniels-Midland Company (Decatur, IL)

Appl. No.: 11/334,713

Filed: January 17, 2006

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60643982	Jan., 2005

Current U.S. Class: 435/252.3 ; 435/139; 435/145; 435/252.32; 435/252.33; 435/252.34; 435/252.35; 435/254.2; 435/254.21; 435/254.23; 435/254.9; 435/471; 435/483; 435/69.1; 435/91.1; 536/23.2; 536/24.1

Current International Class: C12P 21/06 (20060101); C12N 1/00 (20060101); C12P 7/56 (20060101); C12P 7/46 (20060101); C12N 1/20 (20060101); C12N 15/74 (20060101); C12P 19/34 (20060101)

References Cited [Referenced By]

Other References

Caplen et al., Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. PNAS., 2001, vol. 98 (17): 9742-9747. cited by examiner .
Persengiev et al., Nonspecific, concentration-dependent stimulation and represion of mammalian gene expression by small interfering RNAs (siRNAs). RNA, 2004, vol. 10: 12-18. cited by examiner .
Brummelkamp et al., A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, vol. 296: 550-553. cited by examiner.

Primary Examiner: Prouty; Rebecca E.
Assistant Examiner: Raghu; Ganapathirama
Attorney, Agent or Firm: K&L Gates LLP

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 60/643,982, filed Jan. 14, 2005, which is incorporated by reference herein in its entirety.

Claims

What is claimed is:

1. An isolated or recombinant nucleic acid molecule comprising an expressed sequence operably linked to SEQ ID NO:2.

2. The isolated or recombinant nucleic acid molecule of claim 1, wherein the expressed sequence encodes an mRNA.

3. The isolated or recombinant nucleic acid molecule of claim 2, wherein the mRNA encodes a protein selected from the group consisting of lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase.

4. The isolated or recombinant nucleic acid molecule of claim 1, wherein the expressed sequence encodes an siRNA or an antisense RNA.

5. The isolated or recombinant nucleic acid molecule of claim 2, wherein the isolated or recombinant nucleic acid molecule is fused to a gene that encodes an enzyme that increases carbon flux to fumarate.

6. The isolated or recombinant nucleic acid molecule of claim 2, wherein the isolated or recombinant nucleic acid molecule is fused to a gene that encodes an enzyme that increases carbon flux to lactic acid.

7. A vector comprising: SEQ ID NO:2; and a coding region operably linked to SEQ ID NO:2.

8. The vector of claim 7, wherein the coding region is selected from the group consisting of an open reading frame, a sequence encoding an antisense RNA, and a sequence encoding an interfering RNA.

9. The vector of claim 7, wherein the coding region comprises a nucleic acid molecule encoding a protein selected from the group consisting of lactate dehydrogenase, pyruvate carboxylase and phosphoenolpyruvate carboxylase.

10. An isolated recombinant host cell comprising the vector of claim 7.

11. The isolated recombinant host cell of claim 10, wherein the isolated recombinant host cell is of a genus selected from the group consisting of Rhizopus, Saccharomyces, Streptomyces, Pichia, Aspergillus, Lactobacillus, Escherichia coli, Corynebacterium, Brevibacterium, Pseudomonas, Proteus, Enterobacter, Citrobacter, Erwinia, Xanthomonas, Flavobacterium, Streptococcus, Lactococcus, Leuconostoc, and Enterococcus.

Description

FIELD OF THE INVENTION

The present invention relates to novel nucleic acids and related methods that can be used to regulate genes encoding enzymes that manipulate carbon flux through metabolic pathways.

BACKGROUND

Metabolic engineering of microorganisms is an effective means to produce commercially a number of chemicals useful for a variety of applications, including production of polymer monomers and food additives (see, e.g., Lee, S. Y., et al. Macromol. Biosci. 4:157-164 (2004)).

As an example, fumaric acid is an organic acid widely found in nature. In humans and other mammals, fumaric acid is a key intermediate in the tricarboxylic acid cycle for organic acid biosynthesis (also known as the Krebs cycle or the citric acid cycle). Fumaric acid is also an essential ingredient in plant life. Fumaric acid is the strongest organic food acid in titratable acidity and in sourness. In one example, commercial fumaric acid is made from N-butane that is oxidized to maleic acid that is then isomerized to fumaric acid. Production of fumaric acid by bioprocess methods has potential to avoid synthetic production processes that often are more costly than bioprocess methods.

As an additional example, lactic acid (lactate) is used in the food industry as an additive for preservation, flavor, and acidity. It is also used for the manufacture of poly-lactic acid, a biodegradable plastic, and ethyl lactate, an environmentally friendly nonchlorinated solvent. Worldwide, in excess of 100,000 tons of lactic acid is produced annually, with predictions of an increasing demand. The growth in demand is attributable to the poly-lactic acid and ethyl acetate products.

In a number of microorganisms, lactic acid is normally produced from pyruvic acid (pyruvate). The reaction also occurs in the cells of higher organisms when oxygen is limited. Glycolysis is the sequence of reactions that converts glucose into pyruvic acid (pyruvate). Glycolysis can be carried out anaerobically. Pyruvic acid has a number of fates depending on where the chemical reaction takes place and whether the reaction takes place in the presence or absence of oxygen.

As shown in FIG. 1, under aerobic conditions, pyruvic acid can be converted to acetyl-CoA by pyruvate dehydrogenase. Under anaerobic conditions, pyruvic acid can be converted to ethanol (alcoholic fermentation) or lactic acid (e.g., in contracting muscle). The conversion of pyruvic acid to lactic acid is catalyzed by lactate dehydrogenase (LDH). The efficiency of lactic acid fermentation can be quantified as the percent yield of lactate from glucose or as a decrease in the levels of co-products (e.g., glycerol, ethanol, and fumarate) found in the fermentation broth.

Lactic acid is often manufactured using Lactobacilli, which typically has specialized growth requirements and is unable to produce significant amounts of lactic acid below pH 4. (Skory, C. D. J. Ind. Microbiol. Biotechnol. 30:22-27 (2003)). Alternatively, maintenance of neutral pH results in decreased product solubility in the form of salts and requires further processing to regenerate the acid from the resulting lactate salt.

Saccharomyces cerevisiae is a hearty, acid-tolerant microorganism that is amenable to industrial processes. In these microorganisms, however, the major product of pyruvate metabolism is ethanol, by way of pyruvate decarboxylase. Skory reported the production of lactic acid in a yeast, S. cerevisiae, expressing an ldh gene derived from Rhizopus oryzae. (J. Ind. Microbiol. Biotechnol. 30:22-27, (2003)). Skory demonstrated an increase in lactic acid production in the recombinant yeast. Nevertheless, despite the increase in lactic acid production, the majority of carbon was diverted into ethanol. In the same report, when lactic acid production was studied in a S. cerevisiae mutant strain deficient in ethanol production, diminished ethanol production was observed, but the efficiency of lactic acid production also decreased.

Anderson et al. demonstrated that ldh activity had little or no effect on the flux of carbon to lactic acid in Lactococcus lactis. Eur. J. Biochem., 268:6379-6389 (2001). Despite increasing the expression and activity of ldh to beyond that found in wild-type L. lactis, researchers observed no change in the flux of carbon to lactic acid.

Lactic acid can be synthesized chemically, but such synthesis results in a mixture of D and L isomers. The products of microbiological fermentation depend on the organism used and also may include a mixture of the two isomers or individual isomers in a stereospecific form. The desired stereospecificity of the product depends on the intended use; however, L-(+)-lactic acid is the form desired for most applications (Skory, C. D. Appl. Environ. Microbiol. 66:2343-2348 (2000)).

U.S. Pat. No. 6,528,636 describes R. oryzae (ATCC 9363) as a lactic acid producer found in the Rhizopus genus. Rhizopus is a filamentous fungus that is commercially versatile and used in the production of fermented foods, industrial enzymes such as glucoamylase and lipase, corticosteroids, chemicals such as glycerol and ethanol, as well as organic acids such as lactic acid and fumaric acid.

Production levels of different metabolites vary tremendously among the Rhizopus species, with some species producing predominantly lactic acid and others producing primarily fumaric acid. An ideal lactic acid-producing Rhizopus strain would produce little or none of these metabolites, since their production depletes sugars that could be used for conversion to lactic acid.

Ethanol is believed to be produced by most Rhizopus species primarily in low oxygen conditions. While Rhizopus is not typically considered an organism that grows under anaerobic conditions, it does possess ethanol fermentative enzymes that allow the fungus to grow for short periods in the absence of oxygen.

U.S. Pat. No. 4,877,731 discusses that fumaric acid production has been well studied in Rhizopus and that the fumarase gene also has been isolated. Synthesis of fumarate is believed to occur primarily through the conversion of pyruvate to oxaloacetate by pyruvate carboxylase. Conditions leading to increased fumaric acid usually are associated with aerobic growth in high glucose levels and low available nitrogen. Accumulation of fumarate often is a problem with lactic acid production, because its low solubility can lead to detrimental precipitations that compromise fermentative efficiency.

Glycerol is also a by-product that often is produced by Rhizopus grown in high glucose-containing medium. Glycerol is thought to accumulate in Rhizopus in a manner similar to that found in Saccharomyces (U.S. Pat. No. 6,268,189).

Oxaloacetate is also produced by Rhizopus. Pyruvate carboxylase [EC 6.4.1.1] is a member of the family of biotin-dependent carboxylases which catalyzes the carboxylation of pyruvate to form oxaloacetate with concomitant ATP cleavage. The resulting oxaloacetate can be used for the synthesis of glucose, fat, and some amino acids or other derivatives. The enzyme is highly conserved and is found in a wide variety of prokaryotes and eukaryotes. During fermentation by Rhizopus oryzae, pyruvate is primarily converted to lactic acid, but other by-products such as fumaric acid, ethanol and glycerol are also produced. In this fungus, there is evidence that fumaric acid production is predominantly from cytosolic oxaloacetate that is converted from pyruvate by pyruvate carboxylase (Osmani, S. A., et al., Eur. J. Biochem. 147:119-128 (1985)).

Active pyruvate carboxylase consists of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain (Jitrapakdee, S., et al., Biochem. J. 340:1-16 (1999)). Pyruvate carboxylases contain the prosthetic group biotin, which is covalently bound to the amino group of a specific lysine residue. The overall reaction catalyzed by pyruvate carboxylase involves two partial reactions that occur at spatially separate subsites within the active site, with the covalently bound biotin acting as a mobile carboxyl group carrier. In the first partial reaction, biotin is carboxylated using ATP and HCO.sub.3.sup.- as substrates, while in the second partial reaction, the carboxyl group from carboxybiotin is transferred to pyruvate (Attwood, P. V., Int. J. Biochem. Cell Biol. 27:231-249 (1995)).

##STR00001##

Pyruvate carboxylase was first described by (Utter, M. F., et al., J. Biol. Chem. 235:17-18 (1960)) in the course of defining the gluconeogenic pathway in chicken liver. Native pyruvate carboxylase from a number of sources, including bacteria, yeast, insects and mammals, consists of four identical subunits of approximately 120-130 kDa. Pyruvate carboxylases from many sources possess a reactive lysine residue that is essential for full enzymatic activity. Sequencing of cDNA encoding pyruvate carboxylase, as well as limited proteolysis and primary structure comparisons, have shown that pyruvate carboxylases from different species contain ATP, pyruvate, and biotin binding domains (Jitrapakdee and Wallace (1999); Koffas, M. A., et al., Appl. Microbiol. Biotechnol. 50:346-352 (1998)). In S. cerevisiae there are two pyruvate carboxylase isoenzymes (PYC1 and PYC2) encoded by separate genes (Stucka, R., et al., Mol. Gen. Genet. 229:307-315 (1991); Walker, M. E., et al., Biochem. Biophys. Res. Commun. 176:1210-1217 (1991)) while in mammals, no tissue-specific isoenzymes have been reported. Pyruvate carboxylase is most effectively activated by long-chain acyl-CoA derivatives, such as palmitoyl-CoA, and is inhibited by aspartate and 2-oxoglutarate (Osmani, S. A., et al., Ann. N.Y. Acad. Sci. 447:56-71 (1985)).

Fermentations with the fungus Rhizopus are often advantageous because the organism is able to produce optically metabolites, such as pure L-(+)-lactic acid. Therefore, the quality of the final product is considered to be superior to that obtained by bacterial fermentations. Furthermore, L-(+)-lactic acid is more desirable for making poly-lactic acid. (U.S. Pat. No. 6,268,189). Additionally, Rhizopus can grow in chemically simple medium without the need for complex components such as yeast extracts (Skory, C. D. Curr. Microbiol. 47:59-64 (2003)). Nevertheless, the efficiency of lactic acid and fumaric acid production (the amount of available carbon diverted to lactate or fumarate production) in Rhizopus generally is low as compared to bacterial fermentations. There also is little known in the art about gene regulatory elements for Rhizopus. There is a need for a method of increasing the efficiency and amount of lactate and fumarate production in Rhizopus.

SUMMARY

Provided herein are genes and genetic elements useful in modifying host cells, such as, without limitation, microorganisms. Further, the methods and compositions of the invention are useful for overexpressing, for example, and without limitation, specific metabolites in the microorganism, such as, without limitation, fumaric acid, lactic acid, and glycerol. Methods of manipulating carbon flux in a microorganism such as R. oryzae also are provided.

In one embodiment, an isolated polynucleotide is provided comprising a promoter such as a Rhizopus transcription elongation factor (tef) gene promoter or, in another embodiment, Rhizopus ribosomal RNA cluster (rRNA cluster) gene promoter. In one embodiment, the isolated polynucleotide comprises a promoter such as a Rhizopus oryzae transcription elongation factor (tef) gene promoter contained within a sequence shown in one of FIGS. 2, 3 and SEQ ID NO:1 and SEQ ID NO:2 as well as a Rhizopus oryzae ribosomal RNA cluster (rRNA cluster) gene promoter contained within a sequence shown in FIG. 10, SEQ ID NO:10 and SEQ ID NO:11. The isolated polynucleotide can comprise an expressed sequence, such as an open reading frame or a sequence encoding an antisense RNA or an interfering RNA operably linked to the promoter. In other embodiments, the expressed sequence encodes one of an siRNA and an antisense RNA directed to one of pyruvate dehydrogenase and pyruvate decarboxylase. In certain embodiments, the open reading frame encodes, for example, lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase. The polynucleotide may be contained within a vector and/or a host cell.

Also provided is the sequence of a novel pyruvate carboxylase gene (SEQ. ID NO:6) and a protein product encodedthereof (SEQ ID NO:8) obtained from R. oryzae.

In another embodiment, a method is provided for manipulating carbon flux in a microorganism comprising: culturing a cell containing a polynucleotide capable of expressing a sequence for manipulating carbon flux in a cell (for example, a sequence as described supra) and recovering one of lactic acid, glycerol and fumaric acid from the culture medium.

In another embodiment, a selectable marker for more efficient metabolic engineering of Rhizopus is provided.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as described and claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of common metabolic pathways in R. oryzae, with a PEP carboxylase pathway introduced by expression of phosphoenolpyruvate carboxylase gene (pepc) shown by the dotted line.

FIG. 2 shows the full length sequence of the tef gene promoter isolated from R. oryzae. (SEQ ID NO:1). The TATA box and ATG start codon are shown underlined.

FIG. 3 shows a truncated sequence of the tef gene promoter isolated from R. oryzae. (SEQ ID NO:2). The TATA box and ATG start codon are shown underlined.

FIG. 4 shows a portion of the external transcribed spacer (ETS) region of the 18s subunit of the ribosome isolated from R. oryzae. (SEQ ID NO:3).

FIG. 5 shows a comparison of nucleotide sequences pyruvate dehydrogenase from the genomic sequence "g" and the expressed sequence "c" of R. oryzae (SEQ ID NO:4 and SEQ ID NO:5, respectively).

FIG. 6 shows a cDNA sequence (SEQ ID NO:6), genomic DNA sequence (SEQ ID NO:7), and a protein sequence (SEQ ID NO:8) of R. oryzae pyruvate carboxylase. The open reading frame encodes a protein of 1178 amino acids. The intron, 61 bp, is typed in italic lowercase.

FIG. 7 shows the cDNA and protein sequence of Medicago sativa phosphoenolpyruvate carboxylase (SEQ ID NO:9).

FIG. 8 shows conserved domains among R. oryzae, S. cerevisiae, A. niger, A. terreus, P. pastoris, and S. pombe pyruvate carboxylase proteins. The two ATP binding domains (amino acids 187-193 and 311-318 of the protein sequence provided in FIG. 6, underlined) and the biotin binding domain (amino acids 1138-1141 of the protein sequence provided in FIG. 6, underlined) are 100% conserved, while the pyruvate binding domain (amino acids 603-625 of the protein sequence provided in FIG. 6, underlined, with W.sub.622 being the putative pyruvate binding site) is 89% conserved among these fungal proteins.

FIG. 9 is a Southern blot of total genomic DNA from R. oryzae digested with restriction enzymes PstI, BamHI, or EcoRI showing relative copy numbers of the pyruvate carboxylase (pyrC) containing plasmid.

FIG. 10 shows a full length sequence of nucleotides 1-1043 of the rRNA cluster gene promoter region isolated from R. oryzae (SEQ ID NO:10). The rRNA cluster core promoter is shown in italics (SEQ ID NO:11).

DETAILED DESCRIPTION

Provided herein are methods and compositions of matter useful in the manipulation of carbon flux in microorganisms, typically in members of the Rhizopus genus, and most typically in R. oryzae. As a non-limiting example, the manipulation of R. oryzae metabolic pathways depicted in FIG. 1 is facilitated by the methods and compositions of matter described herein. Tools for manipulating carbon flux described herein include novel promoters and/or gene sequences, as well as portions thereof and sequences complementary thereto which can be used in antisense and siRNA methods.

It is to be understood that certain descriptions of the present invention have been simplified to illustrate only those elements and limitations that are relevant to a clear understanding of the present invention, while eliminating, for purposes of clarity, other elements. Those of ordinary skill in the art, upon considering the present description of the invention, will recognize that other elements and/or limitations may be desirable in order to implement the present invention. However, because such other elements and/or limitations may be readily ascertained by one of ordinary skill upon considering the present description of the invention, and are not necessary for a complete understanding of the present invention, a discussion of such elements and limitations is not provided herein. As such, it is to be understood that the description set forth herein is merely exemplary to the present invention and is not intended to limit the scope of the claims.

Other than in the examples herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and temperatures of reaction, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word "about", even though the term "about" may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the standard deviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (end points may be used). When percentages by weight are used herein, the numerical values reported are relative to the total mass weight. Those of skill in the art recognize that percent mass weight and actual mass weight are interconvertable.

All referenced patents, patent applications, publications, sequence listings, electronic copies of sequence listings, or other disclosure material are incorporated by reference in whole but only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material. The articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more elements, and thus, possibly, more than one element is contemplated, and may be employed or used.

As used herein, the term "auxotroph" refers to an organism that requires a specific growth factor (for example, an amino acid or sugar) for its growth. A "bradytroph" refers to an organism that does not necessarily require a specific growth factor for its growth, but which produces a certain growth factor in lower amounts than a wild-type (w.t.) organism.

As used herein, the term "fumaric acid" refers to trans 1,2-ethylenedicarboxylic acid in either the free acid or salt form. The salt form of fumaric acid is referred to as "fumarate" regardless of the anion, for example and without limitation, carbonate (e.g., neutralizing via calcium carbonate) or hydroxide (e.g., neutralizing via ammonium hydroxide).

By the term "lactic acid" is meant 2-hydroxypropionic acid in either the free acid or salt form. The salt form of lactic acid is referred to as "lactate" regardless of the anion, for example and without limitation, carbonate (e.g., neutralizing via calcium carbonate) or hydroxide (e.g., neutralizing via ammonium hydroxide).

By the term "gene" is meant a segment of nucleic acid, DNA or RNA, which encodes and is capable of expressing a specific gene product. A gene often produces a protein or polypeptide as its gene product, but in its broader sense, a gene can produce any desired product, whether the product is a protein, polypeptide or nucleic acid. Functional or structural nucleic acid, such as, without limitation, rRNA, ribozymes, antisense RNA or interfering RNA (e.g., siRNA) also may be considered "gene products."

A "gene" contains an "expressed sequence" that can encode not only a protein or polypeptide, but a structural or functional nucleic acid, such as an antisense or siRNA. A gene may also contain sequences containing regulatory elements, such as, without limitation, promoters, enhancers and terminators; such regulatory elements may be "operably linked," most typically in an appropriate proximity to each other. Such promoters operate in cis (attached to each other on the same nucleic acid molecule) to cause expression of "a gene product." The choice of gene constituents, such as the particular combination of regulatory elements and expressed sequence, will dictate the conditions of expression. For example, a constitutive promoter, such as the CMV (cytomegalovirus) promoter, coupled to an expressed sequence will cause constitutive expression of the expressed sequence when transferred into a suitable host cell. A promoter is considered constitutive if it functions to promote transcription of a gene under normal growth conditions. A constitutive promoter is not tissue specific or developmentally specific, has broad cross-species tropism, and typically does not vary substantially in its expression under normal growth conditions.

A "gene" can include introns or other DNA sequences that can be spliced from the final RNA transcript. An expressed DNA sequence that encodes a protein or peptide ("protein encoding sequence") includes an open reading frame (ORF). The protein encoding sequence may comprise intervening introns. Further, the term "gene" includes expressed sequences as well as non-expressed sequences. All DNA sequences provided herein are understood to include complementary strands unless otherwise noted. Furthermore, RNA sequences can be prepared from DNA sequences by substituting uracil for thymine, and are included in the scope of this definition and the invention, along with RNA copies of the DNA sequences of the invention isolated from cells.

By the term "oligonucleotide" is meant a nucleic acid of from about 7 to about 50 bases though they are more typically from about 15 to about 35 bases. Oligonucleotides are useful as probes or primers for use in hybridization or amplification assays such as Southern or Northern blots; molecular beacon; polymerase chain reaction (PCR); reverse transcriptive PCR (RT-PCR); quantitative RT-PCR (QRT-PCT), e.g., TAQMAN; isothermal amplification methods, such as NASBA (nucleic acid sequence-based amplification); and rolling circle amplification, including use of padlock probes. The oligonucleotides of the invention can be modified by the addition of peptides, labels (including fluorescent, quantum dot, or enzyme tags), and other chemical moieties and are understood to be included in the scope of this definition and the invention.

As used herein, in the context of the novel nucleotide sequences described herein, a nucleic acid is "specific to" a given sequence, such as the pyruvate carboxylase cDNA and genomic sequences provided, if it can hybridize specifically to a given sequence under stringent conditions, such as, without limitation, 0.2.times.SSC at 65.degree. C. or in a PCR reaction under typical reaction (annealing) temperatures. Typically, one sequence is "specific" to a reference sequence if the nucleic acid has 90 to 100% homology (sequence identity) to the reference sequence.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity". As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the National Center for Biotechnology Information website on the world wide web at ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix; or any equivalent program thereof. By "equivalent program" is intended to mean any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.

In the context of the sequences provided herein, a sequence is specific to that reference sequence if, under any given reaction condition that can be used to distinguish one sequence from another, such as, without limitation, PCR, Southern blot or Northern blot, the nucleic acid can hybridize specifically to a nucleic sequence provided herein, but not to other sequences, such as sequences from other species including without limitation those of S. cerevisiae, A. niger, A. terreus, P. pastoris, and S. pombe. Thus, in a nucleic acid detection assay, a probe/primer is "specific to" a sequence if it can bind to a specific transcript or desired family of transcripts extracted from a specimen, to the practical exclusion (i.e., does not interfere substantially with the detection assay) of other sequences. In a PCR assay, primers are specific to a reference sequence if they specifically amplify a portion of that sequence, to the practical exclusion of other sequences in a sample.

As used herein, a "primer" or "probe" for detecting a specific nucleic acid species is any primer, primer set, and/or probe that can be utilized to detect and/or quantify the specific nucleic acid species. A "nucleic acid species" can be a single nucleic acid species, corresponding to a single gene, or can be nucleic acids that are detected by a single common primer and/or probe combination.

By the term "host cell" is meant any prokaryotic or eukaryotic cell where a desired nucleic acid sequence has been introduced into the cell. The metabolic processes and pathways of such a host cell are capable of maintaining, replicating, and/or expressing a vector containing a foreign gene or DNA molecule. There are a variety of suitable host cells, including but not limited to bacterial, fungal, insect, mammalian, and plant cells, that can be utilized in various ways (for example, as a carrier to maintain a plasmid comprising a desired sequence). Representative microbial host cells include, but are not limited to, fungal cells such as Rhizopus ssp., Saccharomyces ssp., Streptomyces ssp., Pichia ssp., Aspergillus ssp., and bacterial cells such as Lactobacillus ssp., Escherichia ssp., Corynebacterium ssp., Brevibacterium ssp., Pseudomonas ssp., Proteus ssp., Enterobacter ssp., Citrobacter ssp., Erwinia ssp., Xanthomonas ssp., Flavobacterium ssp., Streptococcus ssp., Lactococcus ssp., Leuconostoc ssp., and Enterococcus ssp. In one embodiment, the host cell is Rhizopus oryzae. In another embodiment, the host cell is Escherichia coli.

By the term "polynucleotide" is meant any single-stranded sequence of nucleotide, connected by phosphodiester linkages, or any double-stranded sequences comprising two such complementary single-stranded sequences held together by hydrogen bonds. Unless otherwise indicated, each polynucleotide sequence set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T). The term "polynucleotide" encompasses DNA molecules or polynucleotide, sequences of deoxyribonucleotides, and RNA molecules or polyribonucleotides and combinations thereof

By the term "promoter" is meant a DNA sequence within a larger DNA sequence that provides or defines a site to which RNA polymerase can bind and initiate transcription. The promoters described herein can be used to over-express or up-regulate, for example, and without limitation, genes encoding enzymes that increase carbon flux to lactic acid, fumarate, and other desired metabolites during changes in fermentation conditions.

By the term "carbon flux" is meant the biochemical pathway by which carbon is metabolized in an organism. A change in carbon flux, therefore, is a change in the metabolic processing of carbon in response to a change in the organism or its environment. Carbon flux may be changed in any manner, including but not limited to changing the environment of the organism, such as limiting oxygen and/or changing the expression of genes and gene products in the organism (e.g. introducing heterdogous gene sequences).

An "equivalent" of a given reference nucleotide sequence or element contained therein is a nucleotide sequence containing, as compared to the reference nucleotide sequence, all elements of that reference nucleotide sequence, such that the characteristic function of that reference nucleic acid or peptide is retained. Those of skill in the art understand that a functional protein may be encoded by equivalent DNA sequences due to degeneracy in the genetic code. For example, one codon may be substituted for another, yet encode the same amino acid, such as, for example and without limitation, in reference to the Ala codon, the substitution of GCC or GCG for GCA. In the case of proteins, a sequence can contain amino acids that represent conservative amino acid substitutions, including but not limited to, the conservative substitution groups: Ser and Thr; Leu, Ile and Val; Glu and Asp; and Gln and Asn. A sequence as claimed herein thus includes the referenced sequence as well as its equivalents due to degeneracy in the genetic code. Conservative substitutions also can be determined by other methods, such as, without limitation, those used by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM Substitution Scoring Matrix, and the BLOSUM 62 matrix (see also, for example, Altschul et al., Methods in Enzymology 266:460-479 (1996)). Importantly, "equivalents" and "conserved equivalents" of a reference nucleic acid or peptide/protein substantially retain or enhance the function of the reference nucleic acid or peptide/protein.

As used herein, a "tef promoter" or "tef Pol II promoter" is the promoter for transcription of translation elongation factor. See, for example, FIGS. 2 and 3; and SEQ ID NO:1 and SEQ ID NO:2. Likewise, an "rRNA cluster promoter " is the promoter for transcription of ribosomal RNA such as the 5s (comprising the NTS1 promoter region) and 18s (comprising the NTS2 region) ribosomal RNA. Those of skill in the art recognize that ribosomal DNA (rDNA) in eukaryotes is arranged in tandemly repeated units containing the coding regions for 18S, 5.8S, and 28S ribosomal RNA separated by spacers. A large intergenic spacer (IGS) separates the 28S and 18S coding regions, and contains signals for transcription initiation and termination. The structure of the 35S pre-mRNA cluster is: NTS1::5S::NTS2::5'ETS::18S::ITS1::5.8S::ITS2::28S::3'ETS. The internal transcribed spacers (ITS), which separate the 5.8S gene from the 18S and 28S genes on either side of it, contain motifs responsible for the correct splicing of the mature 18S, 28S and 5.8S rRNA molecules from the primary rRNA transcript wherein the promoter regions drive expression of such rRNA. Examples of an rRNA cluster promoter sequence include that shown in FIG. 10 and the sequence listed in SEQ ID NO:10 and SEQ ID NO:11.

In the context of the promoters described herein, equivalents of those promoters substantially retain the promoter activity, host cell tropism and strength of the promoter. Methods of making "equivalent" promoters include any of the large variety of genetic engineering and/or mutational methods known to those of skill in the art. These methods can be used to create nucleic acid substitutions, deletions or insertions that do not substantially affect the promoter function. For example, and without limitation, in the case of the tef promoter (see, for example, FIGS. 2 and 3; and SEQ ID NO:1 and SEQ ID NO:2), in the region located between the TATA box and the downstream transcription start site (AUG), one or more nucleotides may be inserted, deleted or substituted without substantially decreasing promoter function. Similarly, other cis-acting elements present in the tef promoter, such as those found 5' to the TATA box (bases 735 to 739 of SEQ ID NO:1, with the ATG start codon at bases 777 to 779; bases 208 to 213 of SEQ ID NO:2, with the ATG start codon at bases 251 to 253), may be retained, yet one or more nucleotides between those cis-acting elements may be inserted, deleted or modified without substantially decreasing promoter function. Even small 1 or 2 nucleotide substitutions, insertions and deletions within promoter elements may be tolerated without substantial loss of promoter function. As such, "equivalents" of the tef promoter contain sequences having at least about 90%, preferably at least about 95% and most preferably at least about 97.5% sequence identity with the sequences of the invention. Both sequences presented in SEQ ID NO:1 and 2 retain the essential promoter characteristics of the tef promoter.

As with the tef promoter, certain portions of the rRNA cluster promoter are necessarily substantially conserved in "equivalents," while others are not. As discussed herein, and as is well-known in the art, Pol I promoters such as the rRNA cluster promoters contain a core element and an upstream control element ("UCE"). As such, nucleotide sequences between those elements need not be conserved, only their general spacing. Thus, outside the core and UCE sequences, any nucleotide can be deleted, inserted or substituted, so long as the ability of the promoter to cause expression of an operably linked expressed sequence is not substantially affected. Thus, for the tef promoter and the rRNA cluster promoter, an "equivalent" thereof retains, substantially, the ability of the promoters contained within the sequences to cause expression of gene product in a host cell. As discussed above, methods for producing such equivalents, for example, by PCR-based or oligonucleotide-based mutational methods or other methods well known in the art. A person of ordinary skill in the art would be able to produce such equivalents with little difficulty. Testing for efficacy of the equivalent promoters can be performed in many ways known to those of average skill in the art. For the tef promoter, promoter function can be determined in E. coli, yeast and Rhizopus species, or another suitable host cell. Similarly, the rRNA cluster promoter can be tested in E. coli, yeast, and Rhizopus cells, or in any other suitable host cell. Expression levels can be determined by, for example and without limitation, Northern blot, by quantitative RT-PCR (e.g., TAQMAN) or by expression of an indicator gene product.

By the term "vector" is meant a means for introducing a foreign nucleotide sequence into a cell, including without limitation, a plasmid or virus. Such vectors can operate under the control of a host cell's gene expression machinery. A vector contains sequences that facilitate replication and/or maintenance of a segment of foreign nucleic acid in the host cell. Generally, the vector is introduced into a host cell for replication and/or expression of the segment of foreign DNA or for delivery of the foreign DNA into the host genome. A typical plasmid vector contains: (i) an origin of replication, so that the vector can be maintained and/or replicated in a host cell; (ii) a selectable marker, such as an antibiotic resistance gene to facilitate propagation of the plasmid; and (iii) a polylinker site containing several different restriction endonuclease recognition and cut sites to facilitate cloning of a foreign DNA sequence. Yep353, discussed below in the Examples, is one such plasmid vector.

RNA interference (RNAi) is a powerful and robust method for disrupting gene expression. It is based on a highly conserved gene silencing method that uses double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA, see, e.g., Martinez J, et al., Cell 110(5):563-74 (2002)) as a signal to trigger the degradation of homologous cellular RNA. The mediators of the sequence-specific degradation are 21- to 23-nucleotide (nt) dsRNA small interfering RNAs (siRNA). Selection of appropriate siRNA sequences and preparation of the siRNA are discussed in detail in Elbashir, S. M. et al., Methods 26: 199-213 (2002) and in U.S. Patent Application Nos. 2002/0173478, 2002/0182223, 2002/0183276, 2002/0160393 and 2002/0162126.

Xia et al. describes construction of suitable plasmid containing a gene for expression of an siRNA. That reference also describes recombinant viral vectors and delivery systems The reference describes appropriate expression of an siRNA hairpin which down-regulation of the expression of a target .beta.-glucuronidase gene in mouse brain and liver, thereby providing proof of concept of the usefulness of siRNA technology as a gene therapy for human diseases (Xia et al., Nature Biotechnology, 20:1006-1010 (2002)). See also, for example, U.S. Patent Application Nos. 2004/0241854 and 2004/0053876. Vectors for siRNA production are widely available from commercial sources, such as, without limitation, Ambion, Inc. of Austin Tex., Invivogen of San Diego, Calif., and GenScript Corporation of Piscataway, N.J. Vectors containing appropriate promoters, such as Pol III promoters, include for example and without limitation, H1 and U6 promoters and have proven especially useful in producing sufficient quantities of siRNA. A typical siRNA "gene" would therefore comprise an appropriate promoter operably linked to a sequence encoding an siRNA. Ambion's Technical Bulletin #506 ("siRNA Design Guidelines") provides non-limiting examples of siRNA design considerations. Computer software for generating suitable siRNA sequences from, for example and without limitation, a cDNA or ORF sequence also is commercially available.

Using well-established methods for determining effective siRNA sequences, siRNA sequences can be made to silence R. oryzae pyruvate dehydrogenase and pyruvate decarboxylase. One non-limiting example of an siRNA sequence designed to silence the pyruvate dehydrogenase sequence from R. oryzae (FIG. 5) is:

TABLE-US-00001 Sense 5'-CAGACGAUGACCUUCCUUA (SEQ ID NO:12) Antisense 5'-UAAGGAAGGUCAUCGUCUG (SEQ ID NO:13)

One non-limiting example of an siRNA sequence designed to silence pyruvate decarboxylase from Rhizopus oryzae (GenBank Accession Nos. AF282846 and AF282847) is:

TABLE-US-00002 Sense 5'-CUUUGAUGUGUUCUUCAAC (SEQ ID NO:14) Antisense 5'-GUUGAAGAACACAUCAAAG (SEQ ID NO:15)

In one example, the sense/antisense pairs provided above may be expressed under the control of the P.sub.TEF promoter or rRNA cluster promoter in a vector construct, such as for example and without limitation in pPYR225b containing the pyrG gene for selection.

Along with RNAi, antisense RNA is another method of interference with gene function. In antisense technology, RNA complementary to portions of mRNA are introduced into a cell, thereby down-regulating production of the protein product of the mRNA. Unlike RNAi technology, antisensense does not completely silence the target gene in most cases. Production of useful antisense constructs and reagents are well within the abilities of those of ordinary skill in the art. At least 450 U.S. patents directed to antisense technologies and applications thereof have been issued to date.

In one example, U.S. Pat. No. 6,838,283 describes antisense modulation of survivin, which is accomplished by providing antisense compounds which specifically hybridize with survivin mRNA. As described in that patent, the specific hybridization of an antisense sequence with its target nucleic acid ("target nucleic acid" encompasses DNA encoding the gene to be modulated), as well as RNA (including pre-mRNA and mRNA) interferes with the normal function of the nucleic acid. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of the gene to be modulated. "Modulation" therefore means either an increase or a decrease in the expression of a gene or its product.

In some embodiments, the target is a nucleic acid molecule encodes, for example, pyruvate dehydrogenase, wherein expression of the molecule shunts pyruvate towards the production of lactate, ethanol and/or fumarate and away from the mitochondrial Krebs cycle. In yet other embodiments, the nucleic acid molecule encodes pyruvate decarboxylase, thereby shunting pyruvate away from ethanol production. Down-regulation of both pyruvate dehydrogenase and pyruvate decarboxylase favors production of fumarate. It is necessary to determine a site or sites within a gene for the antisense interaction to occur such that the desired inhibition of gene expression will result. Within the context of the present invention, an intragenic target for the antisense compound can be the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the target gene. The ORF can be pyruvate dehydrogenase or pyruvate decarboxylas,e though the ORF of any given gene may be used. The translation initiation codon or "start codon" can be 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule) or any equivalent, for example, genes having a start codon RNA sequence of 5'-GUG, 5'-UUG, 5'-CUG, 5'-AUA, and 5' ACG. Some genes have two or more alternate start codons, which may also be used to initiate translation. As used herein, "start codon" and "translation initiation codon" refer to the codon or codons that are used to initiate translation of an mRNA molecule transcribed from a target gene, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three (RNA) sequences: 5'-UAA, 5'-UAG, and 5'-UGA (i.e., the corresponding DNA sequences are 5'-TAA, 5'-TAG, and 5'-TGA, respectively).

The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively by antisense. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene. Similarly, the 3' untranslated region (3'UTR) may be targeted, e.g., the portion of an mRNA in the 3' direction from the translation termination codon, including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of a eukaryotic mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'--5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself, as well as the first 50 nucleotides adjacent to the cap. The 5' cap region may also be a preferred target region.

Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target; that is, they hybridize sufficiently well and with sufficient specificity, to give the desired effect. As used herein, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementarity," as used herein, refers to pairing between two nucleotides according to the rules of nucleotide base-pairing (i.e., A:T/U; C:G). For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA may hybridize to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementarity" are terms which are used to indicate a sufficient degree of precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a down-regulation of the expression of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, that is, under conditions in which the host cell is grown.

A typical antisense construct contains a transcribed portion of the gene to be modulated in antisense orientation. Thus, a typical antisense construct contains a promoter operably linked to a transcribed sequence or a portion thereof as the expressed sequence and a transcription terminator (polyadenylation signal, for example), where the transcribed sequence is oriented in the 3' to 5' direction as compared to the wild-type transcribed sequence.

Eukaryotic cells regulate the expression of genes in many ways. The expression of many eukaryotic genes, however, is controlled primarily at the level of transcription. Promoters can specify the time and manner in which transcription can occur from a particular gene. Therefore, genes can be effectively regulated by strong promoters. Promoters that drive such expression of genes in Rhizopus were heretofore not known.

Two Rhizopus genes described in public databases include the open reading frames of the translation elongation factor (tef) gene (GenBank Accession No. AF157289) and the ribosomal RNA cluster (rRNA cluster) gene (GenBank Accession No. AB109757). These two genes are expressed at high levels in all eukaryotic cells regardless of growth state or most environmental changes.

The rRNA cluster is a tandem repeat of identical copies of a single gene. These genes, which encode the precursor of the 18S, 5.8S and 28S ribosomal RNAs, are transcribed in the nucleolus by RNA Polymerase I ("Pol I"). Pol I produces a single primary transcript that is processed post-transcriptionally to generate all three RNAs. The promoter