# Patent

## 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.

## Background
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.


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 HCO3− 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)).




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 W622 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×SSC at 65° 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 β-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:










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:










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 PTEF 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 region of the rRNA cluster genes spans about 150 base pairs just upstream of (5′ to) the transcription initiation start site. These promoters are recognized by two transcription factors, upstream binding factor (“UBF”) and promoter selectivity factor-1 (“SL-1”), which bind cooperatively to recruit Pol I to form a transcription initiation complex.
In a particular embodiment, Pol I, along with transcription factors and enhancer elements, binds to the novel promoters of the rRNA cluster genes to regulate expression of the genes. Pol I transcription is localized to the nucleolus and is not inhibited by α-amanitin, a toxic peptide found in certain types of mushrooms. Pol I, alone, cannot initiate or terminate transcription. UBF and SL-1 are necessary and sufficient for full transcription by Pol I. Pol I promoters contain an essential core element immediately surrounding the transcription start site and an upstream control element (UCE) beginning about 100 bases upstream of the start site. UBF binds both the UCE and an upstream portion of the core elements.
Pol I termination of transcription occurs at well-defined sites. The termination sites, called Sal boxes, specifically terminate transcription and comprise an 18 base pair consensus sequence. The Sal box serves as the binding site for transcription termination factor I (TTFI). A single Sal box, which is in the proper orientation and to which TTFI is bound, is sufficient for termination of transcription.
Transcription Elongation Factors (TEFs) are universally conserved proteins that promote the GTP-dependent binding of an aminoacyl-tRNA to ribosomal A-site in protein synthesis. Especially conserved is the N-terminus of the protein containing the GTP binding domain. TEFs are very abundant in cells, comprising about 4-6% of total soluble proteins. Tef genes have been isolated from several organisms. In some organisms, they constitute a multigene family. A number of tef pseudogenes also have been isolated from some organisms. Tef is constitutively expressed, except in aging and quiescent cells. Tef is not known to be regulated by the growth substrates of the host.
Tef promoters are RNA Polymerase II (Pol II) promoters. That is, Pol II is responsible for transcription of the tef gene. Pol II is responsible for synthesizing the precursors to messenger RNA (mRNA) and several small nuclear RNA molecules localizes to the nucleoplasm. Like Pol I, Pol II requires a number of transcription factors to assemble on the promoter to initiate transcription. One of the best characterized Pol II promoter elements is the TATA box. The TATA box consists of a specific sequence of nucleotides (TATAAA) located approximately 25 base pairs upstream of the transcription initiation site. It is present in most eukaryotic genes that encode mRNA.
The mRNAs transcribed by Pol II are polyadenylated. Polyadenylation is signaled by a poly (A) addition (AAUAAA) (also known as a poly (A) site) at the 3′ end of the processed mRNA. The poly (A) site not only contributes to the addition of the poly (A) tail, but also to transcription termination. Transcription is terminated 200 to 2,000 bases downstream of the poly (A) site.
In particular embodiments, the present invention is directed to isolated polynucleotides that include a promoter from the Rhizopus transcription elongation factor (tef) gene and/or from the Rhizopus ribosomal RNA cluster (rRNA cluster) gene. Such sequences may be isolated from any species such as Rhizopus delemar, Rhizopus niveus or Rhizopus oryzae. 
In certain embodiments, an isolated polynucleotide may comprise an expressed sequence, such as an ORF, operably linked to the promoter. In particular embodiments, the promoter is operably linked to a protein coding sequence that encodes an enzyme that increases carbon flux to lactic acid or fumarate production. The increase in carbon flux is a result of an increase in the transcription of the gene encoding that enzyme. The protein coding sequence may encode, for example and without limitation, pyruvate carboxylase (e.g., SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8), phosphoenolpyruvate carboxylase (e.g., SEQ ID NO:9), pyruvate dehydrogenase (e.g., SEQ ID NO:4 and SEQ ID NO:5), glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and/or pyruvate kinase. The gene also may encode enzymes that catalyze reactions that regenerate nicotinamide adenine dinucleotide (NAD), for example, lactate dehydrogenase (ldh).
In other particular embodiments; an isolated polynucleotide comprises the nucleotide sequence of base pairs 1-877 or 1-351 of the tef gene promoter of Rhizopus oryzae (FIGS. 2 and 3; SEQ ID NO: 1 and SEQ ID NO: 2, respectively). The isolated polynucleotide can also be the nucleotide sequence of base pairs 1-1043 of the rRNA cluster gene promoter of Rhizopus oryzae (FIG. 10; SEQ ID NO: 10; SEQ ID NO: 11) or the core rRNA promoter found therein (SEQ ID NO:11). Alternatively, the isolated polynucleotide can also be fused to a reporter gene, for example, but not limited to, the β-galactosidase (lac-Z) reporter gene from Escherichia coli. 
The invention also includes vectors comprising an isolated polynucleotide, wherein the polynucleotide comprises a promoter derived from a Rhizopus tef gene or a Rhizopus rRNA cluster gene. In certain embodiments, the vectors include a multiple cloning site (MCS) 3′ to the promoter, permitting insertion of an expressed sequence into the vector to produce the expression product of the expressed sequence, such as a protein or functional nucleic acid. In yet another embodiment, the vector contains both a promoter derived from a Rhizopus tef gene and from a Rhizopus rRNA cluster gene, optionally including an MCS 3′ to both promoters, permitting insertion of two different expressed sequences in the same vector.
In other embodiments, the vector includes an ORF or coding sequence, with or without introns, for expressing an enzyme that affects carbon flux in a host cell containing the vector. In one embodiment, the expressed sequence encodes an enzyme, such as ldh, that increases carbon flux to lactic acid production during changes in fermentation conditions. The increase in carbon flux to lactic acid would result from an increase in the transcription of the gene. Any ldh gene can be used so long as that ldh accepts pyruvic acid as a substrate. For example, any of the genes encoding bacterial ldh described herein can be used. In one embodiment, the ldh is derived from microorganisms, including but not limited to, Rhizopus oryzae (GenBank Accession Nos. AF226154 (ldhA) and AF226155 (ldhB)) or Lactobacillus casei (GenBank Accession No. M76708). In yet another embodiment, a host cell comprises a gene in which the tef or rRNA cluster promoter is operably linked to a gene that encodes an enzyme that modifies carbon flux in the host cell, for example and without limitation, the ldh enzyme or any other suitable gene described above and known in the art.
Likewise, a coding sequence for pyruvate carboxylase (e.g., SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8), phosphoenolpyruvate carboxylase (e.g., SEQ ID NO:9) would increase flux of carbon from pyruvate or phosphoenolpyruvate, respectively, to oxaloacetate, resulting in increased production of fumarate through action of malate dehydrogenase and fumarase (see FIG. 1). Further, carbon flux can be changed by changing expression of pyruvate dehydrogenase (e.g., SEQ ID NO:4 and SEQ ID NO:5) in the TCA cycle (see FIG. 1). In yet other embodiments, fumarase (e.g., R. oryzae fumR; GenBank Accession No. X78576) may be overexpressed to further increase carbon flux to fumarate. In some other embodiments, the tef and rRNA cluster gene promoters can be fused to a β-galactosidase lac-Z reporter gene from, for example, Escherichia coli. 
Alternatively, carbon flux can be increased by expressing a gene product that interferes with shunting of pyruvate and its precursors into undesirable metabolic pathways, for example by interfering with the enzymes involved in the conversion of pyruvate into ethanol, or enzymes involved in conversion of 3-phosphoglycerate to glycerol. Genes of interest also include pyruvate decarboxylase genes, such as those of R. oryzae (GenBank Accession Nos. AF282846 (pdcA) and AF282847(pdcB)).
In another embodiment, the present invention is directed to a host cell comprising an isolated polynucleotide, wherein the polynucleotide comprises a promoter derived from a Rhizopus tef gene promoter or a Rhizopus rRNA cluster gene promoter. Microorganisms capable of acting as a host cell include, but are not limited to, 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.
Nucleic acids can be introduced into cells according to standard methodologies including electroporation, or any other transformation or nucleic acid transfer method known in the art. For example, R. oryzae can be transfected by electroporation. R. oryzae cells can be permanently transformed by insertion of a gene of interest into the cell by electroporation, so long as the introduced DNA integrates into the host cell genome. This is accomplished, without any intention to be bound by this theory, by homologous recombination of the introduced DNA with the genomic DNA via single or double crossover, or is randomly integrated. The efficiency of transformation is increased when the introduced DNA is linearized and contains non-complementary ends, as is the case when a DNA fragment containing a gene is excised from a plasmid using two different restriction endonucleases which yield non-complementary ends. In such instances, the sequence can be purified from the plasmid backbone prior to transfection. Circularized DNA tends to concatamerize in R. oryzae, yielding large, circular extrachromosomal elements, which are eventually lost from the host cell during successive passage of the transfected cell line. Linearized DNA having complementary ends can also re-circularize and concatamerize (not necessarily in that order) and be lost in the same manner as an extrachromosomal element during successive passage of the transfected host cell line.
Host cells may be cultured under any conditions, such as those known in the art. As stated previously, fermentation conditions can affect the flux of carbon in an organism. For example, strong aeration shifts the flux of carbon to production of acetic acid and acetoin, and away from lactic acid production in lactic acid-producing bacteria. Fermentation conditions include, without limitation: the level of aeration, pH, and oxygen saturation level of the medium, as well as the amount of carbon and other growth factors available in the medium. The carbon source can be, for example and without limitation, various sugar alcohols, polyols, aldol sugars or keto sugars, including but not limited to arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinate or methylamine or other substrates which may be determined by one skilled in the art. As described herein, many organisms will thrive on common growth media. For example and without limitation, R. oryzae can be grown in LB (Luria-Bertani) Broth.
Host cells may also be engineered to change carbon flux. Provided in one embodiment is a method of increasing carbon flux to lactic acid comprising culturing in a culture medium a host cell comprising a Rhizopus tef gene promoter, a Rhizopus rRNA cluster gene promoter, or another promoter operably linked to an ldh coding sequence and recovering lactic acid from the culture medium. Likewise, a method of increasing carbon flux to fumarate is provided comprising culturing in a culture medium a host cell comprising a Rhizopus tef gene promoter, a Rhizopus rRNA cluster gene promoter, or another promoter operably linked to a pyruvate carboxylase or phosphoenolpyruvate carboxylase coding sequence and recovering fumarate from the culture medium.
In another embodiment, regulation of the expression of a gene product includes providing a coding region that encodes a gene product; operably linking the coding region to an isolated tef gene promoter or an rRNA cluster gene promoter to form a promoter-coding region within genomic DNA in cells wherein the promoter regulates the expression of the gene product in the cells. In some embodiments, the promoter-coding region can be integrated into a genomic DNA in cells wherein the promoter regulates the expression of the gene product in the cells.
In yet another embodiment, the methods of manipulating carbon flux in a cell, such as, without limitation a R. oryzae cell, are provided. Referring to FIG. 1, expression of a number of genes may be utilized to engineer a cell with altered metabolic pathways. As discussed in detail above, the expression of certain genes native to the host cell, for example and without limitation, R. oryzae, such as ldh (to produce lactate), pyruvate carboxylase (to produce oxaloacetate), fumarase (for example and without limitation, R. oryzae fumR, GenBank Accession No. X78576; and Freidberg, et al., Gene. 163(1):139-44(1995)) (to produce fumarate), or, glycerol-3-phosphate dehydrogenase (to produce glycerol) can be increased by the methods described herein. In a similar manner, genes not native to the host cell may be introduced into the host cell under constitutive or inducible control of a promoter with the goal of increasing carbon flux to a desired end-product metabolite, such as fumarate. In one example for production of fumarate, alfalfa (Medicago sativa) phosphoenolpyruvate (PEP) carboxylase is introduced into an R. oryzae cell to shunt carbon from PEP directly to oxaloacetate, preventing diversion of pyruvate to the Krebs cycle and to ethanol and lactate production (see FIG. 1, dotted line).
In a further example, antisense or RNAi technologies may be used alone, or in combination with increased gene expression of lactate dehydrogenase, PEP carboxylase or pyruvate carboxylase to further divert carbon from one metabolic pathway to another. It is noted that under some conditions, complete gene silencing may prevent sufficient cell culture growth unless a specific metabolite is provided in the culture medium (auxotroph). Thus, production of a bradytroph may be optimized in many instances with antisense technology or RNAi technology. One particular candidate enzyme for antisense or RNAi targeting is pyruvate dehydrogenase, which converts pyruvate to acetyl coenzyme A (acetyl-coA), which donates its acetyl group to oxaloacetate to form citrate in the citric acid cycle, resulting in overproduction of fumarate. A cell co-transfected with genes for overexpressing pyruvate carboxylase and for down-regulating expression of pyruvate dehydrogenase is expected to shunt carbon to fumarate. Use of an inducible promoter, such as the TET-ON or TET-OFF promoter (BD Biosciences Clontech) can avoid the growth inhibition connected with the silencing of pyruvate dehydrogenase. In such a case, the cells can be grown to a desired density in culture before pyruvate dehydrogenase (and the Krebs cycle) is silenced.
Another embodiment includes a method to construct selectable markers for more efficient metabolic engineering of a microorganism, comprising introducing into a lactose auxotroph host cell a nucleic acid comprising lac-Z (encoding β-galactosidase) operably linked to a promoter derived from a Rhizopus tef gene or a Rhizopus rRNA cluster gene. The nucleic acid can be a vector containing a second gene for expression in the host cell. The ability of a transfected host cell to grow on lactose would facilitate selection of transfected host cells.
In yet an additional embodiment, the compositions of the invention may be produced at a first geographic location and transported or shipped to a second geographic location. For instance, a facility at the first geographic location may be able to produce a product more economically than a facility at the second location due to various factors. The factors may include, inter alia, lower costs of materials (i.e., the mannitol), lower costs of energy (i.e., electricity or gas), lower costs of labor (i.e., wages paid to employees), lower costs of environmental controls or effects, or any other requirement for production of the compositions of the invention. Further, a certain product may be well suited for production in the first geographic location and desired, but not produced well, in the second geographic location. As a non-limiting example, residents of Alaska may desire bananas produced in Central America. Thus, the costs of producing the products in a first geographic location may be less than the costs of producing the products in a second geographic location, resulting in the production costs of the product being less in the first geographic location.
In such an instance, the compositions of the invention may be produced at the first geographic location and shipped to the second geographic location, such as by transport over water with ships or barges, trucking, flying, or other means of transportation. The geographic location may be a county, a state, a country, a continent and/or combinations of any thereof. In this manner, the product may be produced in a first country and transported and/or sold in a second country.
The following are examples of methods and compositions of the invention. The examples are not meant to limit the scope of the invention, as defined by the claims.
EXAMPLE 1
Isolation of the tef Gene Promoters and Rhizopus ETS Region
Promoter regions of the tef and regions of the External Transcribed Sequences (ETS) were cloned by cutting total genomic DNA of R. oryzae with restriction endonuclease. The DNA was ligated to adapters (LA PCR in vitro Cloning Kit, Takara Mirus Bio, Inc. of Madison, Wis., see also U.S. Pat. No. 5,436,149) and the promoter regions were amplified with the polymerase chain reaction using one primer complementary to known gene sequences and one primer complementary to the adapter, as follows:








(SEQ ED NO:16)











C2 cassette primer




5′-CGTTAGAACGCGTAATACGACTCACTATAGGGAG;



(Takara)



 








(SEQ ID NO:17)











TEF reverse primer




5′-GTAATCATGTTCTTGATGAAATCACGG;



 








(SEQ ID NO:18)











ETS reverse primer




5′-GATTCACTGAATATGCAATTCACACTAG.






Three products were amplified using the respective primers. The resulting products were a 351 base pair tef polynucleotide (FIG. 2), a 877 base pair tef polynucleotide (FIG. 3) and an ETS polynucleotide (FIG. 4). The 351 base pair tef polynucleotide (FIG. 2) was then inserted into the multiple cloning site to the E. coli β-galactosidase lac-Z reporter gene of YEP353 plasmid (GenBank Accession No. U03500).
Yep353 (GenBank Accession No. U03500) is a shuttle vector that has origins of replication for bacteria and yeast. It has a multiple cloning site situated in front of the reporter gene lacZ. When a piece of DNA that responds to transcriptional machinery is cloned into it in the proper direction, the lacZ gene is expressed and β-galactosidase activity is quantifiable. If nothing is cloned in the MCS, or if the insert DNA in the MCS does not behave as a promoter in these organisms, then no activity is expressed.
The 351 base pair tef polynucleotide showed strong expression in E. coli and yeast. E. coli cultures containing the YEP353 PTEF:lacZ plasmid were grown on LB plates containing X-gal. The strong promoter capability of tef in E. coli and yeast indicates that it has a broad host cell tropism, making the promoter useful in a large variety of organisms.
EXAMPLE 2
Effect of Promoter Constructs on Lactic Acid Production
In another construct, the 351 base pair tef polynucleotide is fused to ldh genes from Rhizopus oryzae and Lactobacillus casei. The effect of over-expression of these genes on lactic acid production can be evaluated.
EXAMPLE 3
Reporter-Promoter Constructs
In another construct, the 351 base pair tef polynucleotide was fused to the β-galactosidase lac-Z reporter gene from Escherichia coli. This construct can be used to select for transformants that show a gain of ability to use lactose as a carbon source and can facilitate more effective metabolic engineering. With this construct, an auxotroph will not need to be created before genetic engineering begins.
EXAMPLE 4
Construction of PTEF:pyrC:TPGK Plasmid—Using Ligation Independent Cloning
TEF promoter (PTEF) is amplified by PCR using primers that will create a smaI site on the 5′ end and add the ATG codon and 10 bases of the 5′ end of pyruvate carboxylase to the 3′ end of the amplicon (Product 1=SmaI-PTEF-ATG-10 bp of pyrC). Pyruvate carboxylase (pyrC) from cDNA clone is amplified by PCR using primers that will add 10 base pairs of the 3′ end of PTEF and the ATG codon to the 5′ end of the amplicon and an speI restriction site to the 3′ end of the amplicon (Product 2=10 bp of PTEF-ATG-pyrC-SpeI). PCR products 1 and 2 are mixed with PTEF smaI forward primer and pyrC spel reverse primer. PTEF:pyrC product is amplified by PCR. The smaI-PTEF:pyrC-speI, PCR product 3, and pyr225b vector are cut with small and speI and ligated. S. cerevisiae PGK terminator (TPGK, terminator on vector YIpDCE1 GenBank-AF039102) is amplified by using primers that will introduce speI and sacI restriction sites. Pyr225b containing PTEF:pyrC and the amplified Tpgk terminator are cut with speI and sacI and ligated. pPYR225B vector (pBluescript KS-containing a 2.25 Eco RI genomic Rhizopus fragment (GenBank AF497632) contains the pyrG gene.
The resulting plasmid is linearized within the pyrG gene to facilitate Type I or single crossover into genomic DNA, and used to transform a pyrG deletion mutant generated from Rhizopus oryzae wild-type strain ATCC 10260 (NRRL 1526).
EXAMPLE 6
Construction of PTEF:pepc:TPGK Plasmid—Using Ligation Independent Cloning
The plasmid construction outlined in Example 5 above can be used to build a similar construct containing the alfalfa PEP carboxylase coding sequence (see U.S. Pat. No. 6,599,732, SEQ ID NO: 1). The pyrC fragment of PTEF:pyrC:TPGK can be excised with restriction endonucleases sbfI and apaI and replaced with a PEP carboxylase ORF that has been PCR-modified to contain sbfI and apaI restriction sites.
EXAMPLE 7
Rhizopus oryzae Pyruvate Carboxylase
The isolation and characterization of Rhizopus oryzae genomic and cDNA is described herein. Both the nucleic acid molecule and the encoded pyruvate carboxylase protein are provided. The properties of this enzyme and potential application for fumaric acid and during lactic acid production are discussed.
As part of an effort to characterize the genes encoding the enzymes in the pathway leading to the synthesis of lactic acid, fumaric acid, ethanol and glycerol during fermentation, a pyruvate carboxylase gene was isolated from R. oryzae and the relatedness of its deduced protein to other known orthologs was studied. Two degenerate oligonucleotide primers were synthesized based on conserved regions pyruvate carboxylase-related amino acid sequences of A. bisporus (GenBank Accession No.: AJ276430), A. terreus (GenBank Accession No.: AF097728), P. pastoris (GenBank Accession No.: Y11106), and S. pombe (GenBank Accession No.: D78170,). Amplification by polymerase chain reaction (PCR) with R. oryzae genomic DNA as template yielded a product of the predicted size (648 bp). Additional PCR reactions using gene-specific and degenerate primers were used to isolate the pyruvate carboxylase gene and cDNA fragments from R. oryzae. The cDNA, genomic DNA, and encoded amino acid sequence of the protein, were described (SEQ ID NOS:6, 7, and 8, respectively). The R. oryzae gene (designated PYC) has an intron of 61 bp. The 3′-untranslated region is 91 bp and the 5′-untranslated region is 168 bp in length (FIG. 6).
Rhizopus oryzae strain 28.51 was maintained on YM agar plates (per liter:3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g dextrose, and 20 g agar). The fungus was grown in YML liquid media (per liter: 3 g yeast extract, 3 g malt extract, 5 g peptone, and 10 g dextrose) at room temperature with shaking (100 to 150 rpm) or YM agar plates at 30° C.
DNA and total RNA were extracted from frozen spores (−80° C.) of R. oryzae. Genomic DNA was isolated using the OmniprepTM purification system (Geno Technology, Inc., St. Louis, Mo.) or by a CTAB buffer (100 mM Tris-HCl, pH 7.5, 1% mixed alkyltri-methyl ammonium bromide (Sigma, St. Louis, Mo.), 0.7M NaCl, 10 mM EDTA 1% β-mercaptoethanol (v/v)) plus 0.03% proteinase K. The frozen spores were ground by mortar and pestle and extracted in the CTAB buffer followed by incubation at 65° C. for 30 min. One volume of chloroform/isoamyl alcohol (24:1) was added, gently mixed for 5 min., and centrifuged at 3,000 rpm for 20 min. The supematant was removed and a 2⁄3 volume of 2-propanol was added and recentrifuged as above. The precipitated DNA was rinsed with 75% ethanol and suspended in 0.5 ml sterile water. Contaminating RNA was removed by addition of 5 μl of 10 mg/ml RNAse A and incubation at 37° C. for about 30 min.
Total RNA was isolated using RNAqueousTM Kit (Ambion, Inc., Austin, Tex.) and mRNA was purified from the total RNA using the PolyATtractTM mRNA Isolation Systems (Promega Corporation, Madison, Wis.). The methods used for DNA and RNA electrophoresis have been described elsewhere (Sambrook, J., Fritsch, E. F., and Maniatis, T., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989)).
PCR was performed in a GeneAmp PCR System 9700TM (Applied Biosystems, Foster City, Calif.) using Taq DNA polymerase (Life Technologies, Gaithersburg, Md.) and two degenerate primers based on conserved amino acid sequences of pyruvate carboxylase homologs from Aspergillus agricarus, A. terreus, Pichia pastoris, and Schizosaccharomyces pombe. Forward primer 5′CARAGRAGRCAYCARAARGT 3′ (SEQ ID NO:19) is based on the amino acid sequence “QRRHQKV,” and reverse primer 5′ TCRTCDATRAANGTNGTCCA 3′ (SEQ ID NO:20) is based on the amino acid sequence “WTTFIDD” (where Y=T or C; R=G or A; D=A, G or T; N=A, T, G, or C) (SEQ ID NO:21). The degenerate primers were used in Touchdown PCR (Don, R. H., et al., Nucleic Acids Res. 19:4008 (1991)) to amplify a 648-bp fragment from R. oryzae genomic DNA. Touchdown PCR was performed under the following conditions: initial denaturation at 94° C. for 3 min; 38 cycles of denaturation, 94° C. for 30 sec; annealing for 30 sec; and polymerization at 72° C. for 2 min. The annealing temperature ranged from 55° C. to 45° C. with a decrease of 1° C. every three cycles. This was followed by 14 cycles of denaturation at 94° C. for 1 min; annealing at 45° C. for 30 sec.; and polymerization at 72° C. for 2 min. The PCR product was cloned into pGEM T-easyTM vector (Promega, Madison, Wis.). Additional PCR products were isolated using pyruvate carboxylase (PYC) gene-specific primers, genomic DNA or cDNA and other degenerate primers.
The 5′ end of the pyruvate carboxylase (PYC) cDNA was determined using the GeneRacerTM kit, following the instructions of the manufacturer (Invitrogen Corporation, Carlsbad, Calif.). A PYC-specific oligonucleotide of sequence 5′-CCAATACGACCGAGTTGATAGGATTCAT-3′ (SEQ ID NO:22) was used to prime first-strand cDNA synthesis, which was then amplified by PCR using a nested primer of the sequence 5′-GCATAGATAATGTATCTTCATGA-3′ (SEQ ID NO:23).
Automated fluorescence DNA sequencing was done at the W. M. Keck Center for Comparative and Functional Genomics Facility, University of Illinois at Urbana-Champaign. Sequence data were analyzed with DNASTARTM software (DNASTAR, Inc., Madison, Wis.).
The open reading frame of the product of PYC, PYCp, is 1178 amino acids and has a molecular mass of 130 kD. PYCp has  ̃61 to 67% overall identity with S. cerevisiae (Morris, C. P., et al., Biochem. Biophys. Res. Commun. 145:390-396 (1987)); Aspergillus niger (Panneman, H., Ruijter, G. J. G., Van den Broeck, H. C. and Visser, J., unpublished); A. terreus (Li, Y. F., Chen, M. C., Lin, Y. H., Hsu, C. C. and Tsai, Y. C., unpublished); P. pastoris (Menendez, J., et al., Yeast 14:647-654 (1998)); and S. pombe (Saito, A., et al., unpublished) pyruvate carboxylase proteins. The similarity is very strong throughout the protein sequence (FIG. 8). The two ATP and biotin binding domains are 100% conserved, while the pyruvate binding domain is 89% conserved among these fungal proteins (FIG. 8), like its yeast homolog (Lim, F., et al., Arch. Biochem. Biophys. 258:259-264 (1987)). The PSORT program (Nakai, K., et al., Genomics 14:897-911 (1992)) strongly predicts the subcellular localization of R. oryzae pyruvate carboxylase to the cytoplasm. The computed probability of PYCp having a cytoplasmic localization is 78%. Hybridization of a PYC probe to a blot of R. oryzae genomic DNA digested with different restriction enzymes (PstI, BamHI, or EcoRI) resulted in a single band in one case and multiple bands in the others. Preliminary data indicates that there may be a single copy of this pyruvate carboxylase gene in R. oryzae (FIG. 9).
The production of fumaric acid by R. oryzae has been shown to result from a cytosolic pathway during which pyruvate is converted to oxaloacetate by pyruvate carboxylase (Osmani and Scrutton, Ann NY Acad Sci 447: 56-71 (1985)). Therefore, this gene expression can be enhanced by introducing multiple copies or expressing it from a strong promoter to increase fumaric acid production. Moreover, the disruption of this gene can also lead to the reduction of fumaric acid produced during lactic acid production by R. oryzae. 
EXAMPLE 8
Cloning and Construction of PrRNA cluster Plasmid
A search of GenBank for the 28S-IGS-18S region for yielded a Rhizopus oligosporus sequence (GenBank Accession No. AY847625). A cloned a portion of the 18S gene upstream from the GenBank sequence ab109757 was used for the search. Two putative rRNA clusters were found (AY847625 and and other fungal 5S sequences). These were aligned with the two putative rRNA clusters to identify the promoter region of interest. Thus, by using GenBank sequence AY847625 from R. oligosporus to blast the public but un-annotated genome sequence for R. oryzae (Broad Institute), the putative desirable 18S promoter sequence of NTS2 was identified. Primers were designed against this sequence and a fragment isolated. The forward primer was (EcoRi restriction site in italics):










TCGAATTCGGGGGACCACATGGGAATAC (SEQ ID NO:24)







The reverse primer was (PstI restriction site in italics):










TGGCTGCAGGTCATGTTGGCAGGATC (SEQ ID NO:25)






Using the methods described in Example 1, the isolated fragment (SEQ ID NO:10) was operably linked in frame to the lacZ expression marker and cloned into a Yep353 plasmid. Expression of lacZ was detected in E. coli, but not yeast. This is as predicted since in eukaryotes the rRNA core promoter (e.g., SEQ ID NO:11) recruits polymerase I (Pol I) and Pol I transcripts do not have a 7-methylguanylate cap nor are they recruited to ribosomes for translation. Conversely, in prokaryotes, transcription and translation are simultaneous and capping is not present so translation of the protein occurs. Thus, the rRNA promoter can be used as a promoter in eukaryotes for transcription of antisense or RNAi constructs.

Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. It will be appreciated by those skilled in the art that changes could be made to the embodiments described herein without departing from the broad concept of the invention. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover modifications that are within the spirit and scope of the invention as defined by the claims.

## Claims

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.