WO2001027258A2 - Systeme et procedes d'expression de proteines a haut rendement - Google Patents

Systeme et procedes d'expression de proteines a haut rendement Download PDF

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Publication number
WO2001027258A2
WO2001027258A2 PCT/US2000/028578 US0028578W WO0127258A2 WO 2001027258 A2 WO2001027258 A2 WO 2001027258A2 US 0028578 W US0028578 W US 0028578W WO 0127258 A2 WO0127258 A2 WO 0127258A2
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Prior art keywords
cell
carboxylase
protein
metabolically engineered
peptide
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PCT/US2000/028578
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English (en)
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WO2001027258A3 (fr
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Mark A. Eiteman
Elliot Altman
Ravi R. Gokarn
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The University Of Georgia Research Foundation, Inc.
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Priority to MXPA02003634A priority Critical patent/MXPA02003634A/es
Priority to JP2001530463A priority patent/JP2003511067A/ja
Priority to CA002387605A priority patent/CA2387605A1/fr
Priority to AU12068/01A priority patent/AU1206801A/en
Priority to BR0014758-3A priority patent/BR0014758A/pt
Priority to EP00973568A priority patent/EP1235903A2/fr
Publication of WO2001027258A2 publication Critical patent/WO2001027258A2/fr
Publication of WO2001027258A3 publication Critical patent/WO2001027258A3/fr
Priority to HK03101556.1A priority patent/HK1052198A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • Enzymes important in the dairy industry include rennet, lactase, papain and pectinesterases; in starch processing, ⁇ -amylase, glucoamylase and glucose isomerase; in the detergent industry, protease, lipase and amylase; in textiles, amylase; and in pulp and paper, cellulases.
  • Major peptide drugs include erythropoietin, insulin, granulocyte colony stimulating factor, human growth hormone and interferon.
  • Escherichia coli is the most widely used organism for recombinant protein production. It is well-characterized, fast and inexpensive to grow, and relatively easy to alter genetically. Some strains can produce as much as 30 percent of their total protein as the expressed gene product. Proteins produced from recombinant E. coli include insulin, interferons, growth hormones, interleukins, hydrolases, reductase, and transferases.
  • acetyl-CoA In the presence of oxygen, as is typically the case for recombinant protein production, acetyl-Co A and the 4-carbon metabolite oxaloacetete combine to yield the 6-carbon metabolite citrate, thereby fueling the tricarboxylic acid (TCA) cycle and providing energy and key metabolites for the cell (see Fig. 1).
  • acetyl CoA can undergo a two-step process to form acetate, while oxaloacetate can be used to generate several arnino acids.
  • Pyruvate carboxylase is a biotin-dependent enzyme which can accomplish anaplerotic reactions by converting pyruvate and carbon dioxide into oxaloacetate. This enzyme has been found in many different cell types ranging from bacteria to human. It has been surprisingly found that increasing pyruvate carboxylase activity within a host cell results in an enhancement of polypeptide production. Thus, the invention involves increasing pyruvate carboxylase activity within a host cell that produces a protein or peptide. The enhancement in polypeptide production by the invention is fully expected to be independent of the type of host cell, the source of the pyruvate carboxylase gene, and the nature of the protein or peptide expressed.
  • the present invention thus represents an unprecedented advance in recombinant protein engineering; virtually any cell that is used to produce proteins or peptides can be genetically engineered in accordance with the invention to improve protein or peptide yield.
  • the present invention thus includes a method for enhancing protein or peptide production in a host cell used for protein production.
  • the method involves metabolically engineering the host cell by introducing into the cell a native (i.e., endogenous) and/or foreign (i.e., heterologous) nucleic acid fragment that functionally encodes a pyruvate carboxylase so as to overproduce pyruvate carboxylase in the host cell, relative to a cell that has not been so engineered.
  • engineering the host cell to overproduce pyruvate carboxylase enhances production of the protein or peptide of interest.
  • the DNA of a cell that endogenously expresses a pyruvate carboxylase can be mutated to increase expression of the native pyruvate carboxylase gene and hence the pyruvate carboxylase enzyme so as to cause the cell to exhibit enhanced protein or peptide production.
  • Overexpression of pyruvate carboxylase is preferably effected by fransforming a host cell with a DNA fragment encoding a pyruvate carboxylase that is derived from an organism that endogenously expresses pyruvate carboxylase, such as Rhizobium etli, Corynebacterium glutamicum, Methanobacterium thermoautotrophicum, or Pseudomonas fluorescens.
  • Pyruvate carboxylase can be expressed within the metabolically engineered cell from an expression vector, or alternatively from a DNA fragment that has been chromosomally integrated into the host cell's genome.
  • the host cell is not limited in any way and can be any type of cell that is used for the production of proteins or peptides.
  • the metabolically engineered cell is a bacterial cell such as an E. coli or Bacillus subtilis cell, a yeast cell, a plant cell, an insect cell, or a mammalian cell such as a mouse or human cell.
  • the present invention further includes a method for making a protein or peptide.
  • the method for making the protein or peptide includes providing a metaboUcally engineered cell that overexpresses pyruvate carboxylase followed by culturing the metabolically engineered cell to cause increased expression of the protein or peptide.
  • the method includes fransforming the host cell with a nucleic acid fragment containing a nucleotide sequence encoding a pyruvate carboxylase enzyme to yield the metabolically engineered cell.
  • the protein or peptide can be isolated from the ceU.
  • the DNA of a host cell that endogenously expresses a pyruvate carboxylase can be mutated to alter transcription of the native pyruvate carboxylase gene so as to cause overproduction of the native enzyme.
  • a novel protein expression system is also provided by the invention.
  • the protein expression system includes any cell or cell culture that is useful for the expression of proteins or peptides, wherein the ceU or cells have been modified to overexpress pyruvate carboxylase in accordance with the invention, thereby enhancing protein or peptide production.
  • the protein expression system of the invention is capable of producing higher yields of proteins or peptides than the expression system utilizing the analogous cell or cell culture that does not overexpress pyruvate carboxylase.
  • the invention further provides a metaboUcally engineered cell for use in the production of a protein or peptide, wherein the cell has been metabolically engineered to overexpress PEP carboxylase so as to cause enhanced production of the protein or peptide compared to a comparable cell that does not overexpress PEP carboxylase.
  • the cell is one that does not use PEP to effect transport of glucose into the cell.
  • a method for making a protein or peptide that includes culturing a metabolically engineered cell that overexpresses PEP carboxylase for a time and under conditions to produce the protein or peptide.
  • Figure 1 shows an aerobic metabolic pathway in E. coli depicting glycolysis, the TCA cycle, and biosynthesis of oxaloacetate-derived biochemicals; dashed lines signify that multiple steps are required to biosynthesize the compound while solid lines signify a one-step conversion; the participation of PEP in glucose uptake is shown by a light line; the pathway as shown is not stoichiometric, nor does it include cofactors.
  • Figure 2 is a graph showing kinetic analysis of pyruvate carboxylase activities for MG1655 pUC18 (O) and MG1655 pUC18-pyc (•) with respect to pyruvate.
  • Figure 3 is a graph showing the effects of increasing aspartate concentrations on the activity of pyruvate carboxylase.
  • Figure 4 is a graph showing kinetic analysis of pyruvate carboxylase with respect to ATP and ADP; pyruvate carboxylase activity was determined in the absence of ADP (•) and in the presence of 1.5 mM ADP (O).
  • Figure 5 is a petri plate showing growth of appc null E. coli strain which contains either pUC 18 or the pUC 1 S-pyc construct on minimal media that utilizes glucose as a sole carbon source.
  • Figure 6 shows an anaerobic pathway in E. coli depicting glycolysis and biosynthesis of selected oxaloacetate-derived biochemicals; the participation of PEP in glucose uptake is shown by the dashed line; the pathway as shown is not stoichiometric, nor does it include all cofactors.
  • Figure 7 is a graph showing the effect of nicotinamide nucleotides on pyruvate carboxylase activity: NADH (O), NAD + ( D), NADPH ( ⁇ ) and NADP+ (o) .
  • Figure 8 is a graph showing the growth pattern and selected fermentation products of wild-type strain (MG1655) under strict anaerobic conditions in a glucose-limited (10 g/L) medium; concentrations of glucose (•), succinate ( ⁇ ), lactate (O), formate (D) and dry cell mass ( ⁇ ) were measured.
  • Figure 9 is a graph showing growth pattern and selected fermentation products of wild-type strain with pUC18 cloning/expression vector (MG1655/pUC18) under strict anaerobic conditions in a glucose-limited (10 g/L) medium; concentrations of glucose (•), succinate ( ⁇ ), lactate (O), formate (D) and dry cell mass ( ⁇ ) were measured.
  • Figure 10 is a graph showing growth pattern and selected fermentation products of wild-type strain with pyc gene (MG1655/pUC18- ⁇ yc) under strict anaerobic conditions in a glucose-limited (10 g/L) medium; concentrations of glucose (•), succinate ( ⁇ ), lactate (O), formate (D) and dry ceU mass ( ⁇ ) were measured.
  • Figure 11 is a graph showing growth pattern and threonine production in the threonine producing strain ⁇ IM-4 (ATCC 21277) containing either pTrc99A or pTrc99A- ⁇ yc under strict aerobic conditions in a glucose-limited (30 g/L) medium; optical density in the pTrc99A containing strain (O), optical density in the pTrc99A-pyc containing strain (D), threonine concentrations in the pTrc99A containing strain (•), and threonine concentrations in the pTrc99A-pyc containing strain ( ⁇ ) were measured.
  • Figure 12 is a graph depicting production of ⁇ -galactosidase from E. coli
  • Figure 13 is a graph depicting production of catechol 2,3-dioxygenase from E. coli MG1655 pTrc99A-£yc pACYC- y/E (•) and E. coli MG1655 pTrc99A pAC YC-xylE (O) grown in 100 mL shake flasks.
  • Figure 14 is a graph depicting cell mass concentration from E. coli MG1655 pTrc99A-/?yc pACYC-/ ⁇ cZ (•) and E. coli MG1655 pTrc99A pACYC-/ ⁇ cZ (O), together with production of ⁇ -galactosidase for E. coli MG1655 pTrc99A-/ ⁇ yc pACYC-/ ⁇ cZ (*) and E. coli MG1655 pTrc99A pACYC- lacZ ( ⁇ ), grown in 2 L fermenters using sodium hydroxide for pH control.
  • Figure 15 is a graph depicting glucose concentration from E. coli MG1655 pTrc99A-/ ⁇ c pACYC-/ cZ (•) and E. coli MG1655 pTrc99A pACYC-/ ⁇ cZ (O), together with acetate concentration for E. coli MG1655 pTrc99A-£yc pACYC-/ ⁇ cZ(*) and E. coli MG1655 pTrc99A pACYC-/ ⁇ cZ ( ⁇ ), grown in 2 L fermenters using sodium hydroxide for pH control.
  • Figure 16 is a graph depicting cell mass concentration from E. coli MG1655 pTrc99A- ⁇ yc pACYC-/ ⁇ cZ (•) and E. coli MG1655 pTrc99A pACYC-/ ⁇ cZ (O), together with production of ⁇ -galactosidase for E. coli MG1655 pTrc99A-pvc pACYC-/ ⁇ cZ (A) and E. coli MG1655 pTrc99A pACYC- lacZ ( ⁇ ) grown in 2 L fermenters using sodium carbonate for pH control.
  • Figure 17 is a graph depicting glucose concentration from E. coli MG1655 pTrc99A-/> >c pACYC-/ cZ (•) and E. coli MG1655 pTrc99A pACYC-f ⁇ cZ (O), together with acetate concentration for E. coli MG1655 pTrc99A-£yc pAC YC-lacZ (*) and E. coli MG1655 pTrc99A pACYC-lacZ ( ⁇ ), grown in 2 L fermenters using sodium carbonate for pH control.
  • Figure 18 is a graph depicting fermentation of E.
  • Figure 19 is a graph depicting fermentation of E. coli MG1655 pTrc99A- pyc pACYC-lacZ on defined glucose media (Media C2, Example LX). Symbols: (•) glucose, ( ⁇ ) ⁇ -galactosidase, (O) optical density, ( ⁇ ) acetate.
  • pyruvate carboxylase and "pyruvate carboxylase enzyme” mean an enzyme that has pyruvate carboxylase activity, i.e., that is able to catalyze carboxylation of pyruvate to yield oxaloacetate.
  • pyruvate carboxylase thus includes naturally occurring pyruvate carboxylase enzymes, along with fragments, derivatives, or other chemical, enzymatic or structural modifications thereof, including enzymes encoded by insertion, deletion or site mutants of naturally occurring pyruvate carboxylase genes, as long as pyruvate carboxylase activity is retained.
  • Pyruvate carboxylase activity is conveniently measured by the coupled method of J. Payne et al. (J. Gen. Microbiol. 59:97-101
  • pyruvate carboxylase gene means a gene that functionally encodes a pyruvate carboxylase enzyme.
  • a protein or peptide that is "functionally encoded" by a gene or other nucleic acid is one which, when introduced into a host cell, is capable of being expressed by the host cell.
  • the nucleic acid encoding the protein or peptide can include or encode transcriptional and translational regulatory elements such as promoters, operators, enhancers, termination signals, transcription start and stop codons, and the like, that permit constitutive or inducible transcription and translation of the encoded protein.
  • An enzyme is "overexpressed” or “overproduced” in a host cell of the invention when the enzyme is expressed in the host cell at a level higher than the level at which it is expressed in a comparable wild-type cell. In cells that do not endogenously express a particular enzyme, any level of expression of that enzyme in the cell is deemed an "overexpression” or “overproduction” of that enzyme for purposes of the present invention.
  • Genetically engineered cells are referred to herein as "metaboUcally engineered” cells when the genetic engineering is directed to disruption or alteration of a metabolic pathway so as to cause a change in the metabolism of carbon.
  • the pyruvate carboxylase that is overexpressed by a metabolically engineered cell in accordance with the invention can be either endogenous or heterologous.
  • a "heterologous" enzyme is one that is encoded by a nucleotide sequence that is not normally present in the cell. For example, a bacterial cell that has been transformed with and expresses a gene from a different species or genus that encodes a pyruvate carboxylase contains a heterologous pyruvate carboxylase.
  • the heterologous nucleic acid fragment may or may not be integrated into the host genome.
  • Pyruvate carboxylase enzymes and, in some cases, genes that have been characterized include human pyruvate carboxylase (GenBank K02282; S. Freytag et al.. J. Biol. Chem.. 259. 12831-12837 (1984)); pyruvate carboxylase from Saccharomyces cerevisiae (GenBank X59890, J03889, and M16595; R. Stucka et al., Mol. Gen. Genet..229. 305-315 (1991); F. Lim et al.. J. Biol. Chem..263. 11493-11497 (1988); D.
  • the pyruvate carboxylase that is overexpressed by the metabolically engineered cell in accordance with the invention is derived from either R. etli or P.
  • the pyruvate carboxylase in R. etli is encoded by Ob ⁇ pyc gene (M. Dunn et al., J. Bacteriol.. 178. 5960-5970 (1996)).
  • the R. etli enzyme is classified as an ⁇ 4 pyruvate carboxylase, which is inhibited by aspartate and requires acetyl CoA for activation.
  • the metabolically engineered cell expresses an ⁇ 4 ⁇ 4 pyruvate carboxylase.
  • pyruvate carboxylases do not require acetyl CoA for activation, nor are they inhibited by aspartate, rendering them particularly well-suited for use in the present invention.
  • fluorescens is one organism known to expresses an ⁇ 4 ⁇ 4 pyruvate carboxylase.
  • the metabolically engineered cell of the invention therefore is preferably one that has been transformed with a nucleic acid fragment isolated from P. fluorescens which contains a nucleotide sequence encoding a pyruvate carboxylase expressed therein, more preferably the pyruvate carboxylase isolated and described in R. Silvia et al., J. Gen. Microbiol.. 93, 75-81 (1976).
  • the metabolically engineered cell of the invention overexpresses pyruvate carboxylase.
  • the metabolically engineered cell preferably expresses pyruvate carboxylase at a level higher than the level of pyruvate carboxylase expressed in a comparable wild-type cell.
  • This comparison can be made in any number of ways by one of skUl in the art and is done under comparable growth conditions.
  • pyruvate carboxylase activity can be quantified and compared using the method of Payne and Morris (3. Gen. Microbiol.. 59, 97-101 (1969)).
  • the metabolically engineered cell that overexpresses pyruvate carboxylase will yield a greater activity than a wild-type ceU in this assay.
  • the amount of pyruvate carboxylase can be quantified and compared by preparing protein extracts from the cells, subjecting them to SDS-PAGE, transferring them to a Western blot, then detecting the biotinylated pyruvate carboxylase protein using detection kits which are commercial available from, for example, Pierce Chemical Company (Rockford, EL), Sigma Chemical Company (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN) for visualizing biotinylated proteins on Western blots.
  • detection kits which are commercial available from, for example, Pierce Chemical Company (Rockford, EL), Sigma Chemical Company (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN) for visualizing biotinylated proteins on Western blots.
  • pyruvate carboxylase expression in the non- engineered, wild-type cell may be below detectable levels.
  • the metabolically engineered cell utilized in the invention is not limited in any way to any particular type or class of cell. It can be a eukaryotic cell or a prokaryotic cell, without limitation; it can thus include a cell of a bacterium, a plant, a protist (such as a protozoan or an algae), a fungus (such as yeast), or an animal.
  • An animal cell includes, for example, the cell of a vertebrate or an invertebrate, such as an insect cell or a mammalian cell, preferably a mouse or a human cell.
  • a bacterial cell includes, for example, the cell of a bacterium or an archaebacterium.
  • the cell is a bacterial cell, a yeast cell, a plant cell, an insect cell, or a mammalian cell.
  • Particularly preferred bacterial cells are E. coli cells and B. subtilis cells.
  • Preferred mammalian cells are human cells and mouse cells.
  • Many organisms can synthesize oxaloacetate from either PEP via the enzyme PEP carboxylase, or from pyruvate via the enzyme pyruvate carboxylase.
  • Representatives of this class of organisms include C. glutamicum, R. etli, P. fluorescens, Pseudomonas citronellolis, Azotobacter vinelandii,
  • Aspergillus nidulans, and rat liver cells cannot synthesize oxaloacetate directly from pyruvate because they lack the enzyme pyruvate carboxylase.
  • E. coli, Salmonella typhimurium, Fibrobacter succinogenes, and Ruminococcus flavefaciens are representatives of this class of organisms.
  • the metabolic engineering approach of the present invention can be used to redirect carbon to oxaloacetate and, as a result, enhance protein and peptide yields.
  • Another alternative involves interfering with the metabolic pathway used to produce acetate from acetyl CoA.
  • acetyl CoA Disrupting this pathway should result in higher levels of acetyl CoA, which may then indirectly result in increased amounts of oxaloacetate.
  • the pyruvate carboxylase enzyme that is expressed in the host cell is one that is activated by acetyl CoA
  • higher levels of acetyl CoA in these mutants leads to increased activity of the enzyme, causing additional carbon to flow from pyruvate to oxaloacetate.
  • acetate" mutants are preferred host cells.
  • the metabolically engineered cell used in the invention is made by fransforming a host cell with a nucleic acid fragment comprising a nucleotide sequence encoding a pyruvate carboxylase enzyme.
  • Methods of transformation for bacteria, plant, and animal cells are well known in the art. Common bacterial transformation methods include electroporation and chemical modification. Transformation yields a metabolically engineered cell that overexpresses pyruvate carboxylase.
  • the cells are further transformed with a nucleic acid fragment comprising a nucleotide sequence encoding an enzyme having PEP carboxylase activity.
  • the nucleic acid fragment is introduced into the cell using a vector, although "naked DNA” can also be used.
  • the nucleic acid fragment can be circular or linear, single-stranded or double stranded, and can be DNA, RNA, or any modification or combination thereof.
  • the vector can be a plasmid, a viral vector or a cosmid. Selection of a vector or plasmid backbone depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, plasmid reproduction rate, and the like. Suitable plasmids for expression in E.
  • coli for example, include pUC(X), pKK223-3, pKK233-2, pTrc99A, and pET-(X) wherein (X) denotes a vector family in which numerous constructs are available.
  • pUC(X) vectors can be obtained from Pharmacia Biotech (Piscataway, NH) or Sigma Chemical Co. (St. Louis, MO).
  • pKK223-3, pKK233-2 and pTrc99A can be obtained from Pharmacia Biotech.
  • pET-(X) vectors can be obtained from Promega (Madison, WI) Stratagene (La Jolla, CA) and Novagen (Madison, WI).
  • the vector preferably includes an origin of replication (known as an "ori") or repUcon.
  • an origin of replication known as an "ori"
  • repUcon an origin of replication
  • ColEl and PI 5 A replicons are commonly used in plasmids that are to be propagated in E. coli.
  • the nucleic acid fragment used to transform the cell according to the invention can optionally include a promoter sequence operably linked to the nucleotide sequence encoding the enzyme to be expressed in the host ceU.
  • a promoter is a DNA fragment which causes transcription of genetic material. Transcription is the formation of an RNA chain in accordance with the genetic information contained in the DNA. The invention is not limited by the use of any particular promoter, and a wide variety are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding sequence.
  • a promoter is "operably linked" to a nucleic acid sequence if it is does, or can be used to, control or regulate transcription of that nucleic acid sequence.
  • the promoter used in the invention can be a constitutive or an inducible promoter. It can be, but need not be, heterologous with respect to the host cell.
  • Preferred promoters for bacterial transformation include lac, lac ⁇ JV5, tac, trc, T7, SP6 and ara.
  • the nucleic acid fragment used to transform the host cell can, optionally, include a Shine Dalgarno site (e.g., a ribosome binding site) and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the enzyme. It can, also optionally, include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding arninoacetyl-tRNA, thus ending polypeptide synthesis.
  • the nucleic acid fragment used to transform the host cell can optionally further include a transcription termination sequence.
  • the rrnB terminators which is a stretch of DNA that contains two terminators, Tl and T2, is the most commonly used terminator that is incorporated into bacterial expression systems (J. Brosius et al., J. Mol. Biol.. 148. 107-127 (1981)).
  • the nucleic acid fragment used to transform the host cell optionally includes one or more marker sequences, which typically encode a gene product, usually an enzyme, that inactivates or otherwise detects or is detected by a compound in the growth medium.
  • a marker sequence can render the transformed cell resistant to an antibiotic, or it can confer compound-specific metabolism on the transformed cell.
  • Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicUUn, chloramphenicol and tetracycline.
  • Pyruvate carboxylase can be expressed in the host cell from an expression vector containing a nucleic acid fragment comprising the nucleotide sequence encoding the pyruvate carboxylase.
  • the nucleic acid fragment comprising the nucleotide sequence encoding pyruvate carboxylase can be integrated into the host's chromosome.
  • Nucleic acid sequences, whether heterologous or endogenous with respect to the host cell can be introduced into the cell's chromosome using, for example, homologous recombination.
  • the gene of interest and a gene encoding a drug resistance marker are inserted into a piece of DNA on a plasmid that is homologous to the region of the chromosome within which the gene of interest is to be inserted.
  • this recombinagenic DNA is introduced into the bacteria, and clones are selected in which the DNA fragment containing the gene of interest and drug resistant marker has recombined into the chromosome at the desired location.
  • the gene and drug resistant marker can be introduced into the bacteria via transformation either as a linearized piece of DNA that has been prepared from any cloning vector, or as part of a specialized recombinant suicide vector that cannot replicate in the bacterial host, preferably a recD ' bacterial host.
  • Clones are then verified using PCR and primers that amplify DNA across the region of insertion. PCR products from non-recombinant clones will be smaller in size and only contain the region of the chromosome where the insertion event was to take place, while PCR products from the recombinant clones will be larger in size and contain the region of the chromosome plus the inserted gene and drug resistance.
  • the metabolically engineered cell used in the invention is made by mutating the DNA of a cell that endogenously expresses a pyruvate carboxylase to alter transcription of the native pyruvate carboxylase gene so as to cause overproduction of the native enzyme.
  • a mutated chromosome can be obtained by employing either chemical or transposon mutagenesis and then screening for mutants with enhanced pyruvate carboxylase activity using methods that are well-known in the art.
  • the metabolically engineered cell used in the invention also overexpresses PEP carboxylase.
  • the metabolically engineered cell optionally expresses PEP carboxylase at a level higher than the level of PEP carboxylase expressed in a comparable wild-type cell.
  • overproduction of PEP carboxylase alone has been shown to hamper glucose uptake in E. coli.
  • overproduction of PEP carboxylase alone or in combination with overproduction of pyruvate carboxylase may nonetheless improve protein or peptide production in other cells, such as those that do not rely on PEP for glucose uptake.
  • overproduction of PEP carboxylase is not expected to negatively impact uptake of the alternative carbon source.
  • overproduction of PEP carboxylase alone may adversely affect glucose uptake, it is possible that simultaneous overproduction of both pyruvate carboxylase and PEP carboxylase may not.
  • PEP carboxylase activity can be assayed, quantified and compared.
  • PEP carboxylase activity is measured in the absence of ATP using PEP instead of pyruvate as the substrate, by monitoring the appearance of CoA-dependent thionitrobenzoate formation at 412 nm (see Example II).
  • the metabolically engineered cell that overexpresses PEP carboxylase will yield a greater PEP carboxylase activity than a wild-type cell.
  • the amount of PEP carboxylase can be quantified and compared by preparing protein extracts from the cells, subjecting them to SDS-PAGE, transferring them to a Western blot, then detecting the PEP carboxylase protein using PEP antibodies in conjunction with detection kits available from Pierce Chemical Company (Rockford EL), Sigma Chemical Company (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN) for visualizing antigen-antibody complexes on Western blots.
  • the metabolically engineered ceU expresses PEP carboxylase derived from a cyanobacterium, more preferably Anacystis nidulans.
  • the invention includes a method for making a protein or peptide by enhancing or augmenting production of the protein or peptide in a cell that is, prior to transformation as described herein, capable of biosynthesizing the protein or peptide.
  • a metaboUcaUy engineered ceU that overexpresses pyruvate carboxylase in accordance with the invention and which also produces a protein or peptide of interest is cultured to cause expression of the protein or peptide.
  • Any type of cell culture or fermentation can be used, including but not limited to batch fermentations, fed-batch fermentations, continuous cultures, and perfusion cultures.
  • the method of the invention allows enhanced production of the protein or peptide to be achieved, for example, by causing an increase in the protein activity or amount per cell, by causing an increase in the protein activity or amount per milUter of medium, by allowing cultures or fermentations to continue efficiently for longer periods of time, or through a combination of these effects.
  • Protein or peptide production is enhanced compared to the level of production that can be achieved a comparable cell that does not overexpress pyruvate carboxylase.
  • a higher yield of the protein or peptide is obtained in the metabolically engineered cell relative to similarly culturing a comparable host cell that does not overexpress pyruvate carboxylase.
  • yield as used in connection with protein or peptide production in fermentations is usually expressed as a quotient of volumetric units, e.g., as units of activity per unit volume (typically milliUter) of media, divided by the change in substrate concentration during the fermentation, typically expressed in units of grams of substrate per liter of media.
  • volumetric units e.g., as units of activity per unit volume (typically milliUter) of media, divided by the change in substrate concentration during the fermentation, typically expressed in units of grams of substrate per liter of media.
  • a fermentation of E. coli producing ⁇ -galactosidase could yield 2667 enzyme units
  • the method further includes metaboUcaUy engineering the host ceU to overexpress pyruvate carboxylase, as described above, prior to culturing. Also optionaUy, the method further includes isolating the protein or peptide from the cultured ceUs. Proteins and peptides can be isolated from the cells using protocols, methods and techniques that are well-known in the art.
  • protein isolation techniques include precipitations such as ammonium sulfate precipitation, filtration techniques, dialysis, phase extractions, chromatographic techniques including anion or cation exchange, hydroxyapatite, gel filtration, and affinity chromatography.
  • the invention further includes a method for increasing protein or peptide production in a host cell that, prior to applying the method of the invention, produces a given yield of protein or peptide of interest.
  • the protein or peptide of interest can be a native or a recombinant protein, and the host cell can be a wild-type cell or a cell that has been genetically engineered.
  • the protein- or peptide-producing host cell is transformed with a nucleic acid fragment comprising a nucleotide sequence functionally encoding a pyruvate carboxylase enzyme to yield a metabolically engineered cell that produces a higher yield of the protein or peptide, compared with the host cell prior to transformation.
  • the method involves mutating a pyruvate carboxylase gene of the host cell such that the host cell overexpresses pyruvate carboxylase to yield a metabolically engineered cell that produces a higher yield of the protein or peptide compared with the host cell prior to transformation.
  • Protein- or peptide-producing host cells whose protein or peptide production can be enhanced in accordance with the method of the invention are as described in detail hereinabove and are not limited in any way to a particular type or class of cell.
  • proteins and peptides that are produced or overproduced in, and isolated from, the metabolically engineered cells according to the method of the invention are not limited in any way and include native, mutant and recombinant polypeptides, including fusion proteins. They can be labeled with a detectable label, such as a radiolabel or fluorescent label, and can include known or unknown amino acid sequences and activities, and predete ⁇ nined or randomized amino acid sequences. While any desired polypeptide can be made according to the present invention, the invention is particularly well-suited to production of industrial enzymes, research and diagnostic enzymes, and therapeutic proteins and peptides.
  • Industrial enzymes are those used in industrial processes or settings or in the production of consumer goods and services, such as amylase, glucoamylase, glucose isomerase, protease, lipase and ceUulase.
  • Research and diagnostic enzymes include those enzymes used in scientific or research settings such as restriction enzymes, for example Hind EH, EcoR I and BamHl and DNA RNA modifying enzymes, for example DNA or RNA polymerases, methylases, ligases, exonucleases, and kinases.
  • Other proteins used in research include myoglobin, amidase, streptavidin ras protein, cholinesterase, and various human growth factors.
  • Therapeutic proteins or protein drugs are those used for medical, nutritional or veterinary purposes and include, for example, erythropoietin, insulin, granulocyte colony stimulating factor, human growth hormone and interferon.
  • Recombinant proteinaceous drugs that are currently produced in E. coli include aldesleukin (interleukin-2; EL-2), asparaginase, denileukin diftitox, filgrastim, growth hormone, insulin, interferon alfa-2a, interferon alfa-2b, interferon beta, interferon gamma- lb, oprelvekin (interleukin 11), and reteplase.
  • recombinant proteinaceous drugs include alteplase, ancestim, basiliximab, becaplermin, coagulation factor VEIa, daclizumab, dornase alfa (recombinant human deoxyribonuclease; DNAse), epoetin alfa (erythropoietin; EPO), etanercept - a TNF ⁇ inhibitor, follitropins hepatitis B vaccine, imiglucerase, infliximab, lepirudin, lyme disease vaccine (recombinant OspA), palivizumab, rituximab, sargramostim (granulocyte macrophage colony stimulating factor; GM-CSF) and trastuzumab.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the invention further provides a novel protein expression system characterized by a protein-expressing or peptide-expressing cell that overexpresses pyruvate carboxylase.
  • a protein-expressing or peptide-expressing cell that overexpresses pyruvate carboxylase.
  • Any cellular protein expression system capable of expressing a protein or peptide of interest can be modified in accordance with the invention by altering the protein- or peptide-producing cell to overexpress pyruvate carboxylase as described herein, to yield a protein expression system with enhanced protein or peptide yields.
  • Example I Expression of the R. etli Pyruvate Carboxylase Enzyme Enables E. coli to Convert Pyruvate to Oxaloacetate
  • the pyc gene from R. etli was cloned into an E. coli expression vector and several experiments were conducted to determine whether active pyruvate carboxylase enzyme can be expressed in E. coli.
  • the R. etlipyc gene which encodes pyruvate carboxylase, was amplified using the polymerase chain reaction (PCR).
  • Pfu polymerase (Stratagene, La Jolla, CA) was used instead of Taq polymerase and the pPCl plasmid served as the DNA template.
  • Primers were designed based on the published pyc gene sequence (M. Dunn et al., J. BacterioL. 178. 5960-5970 (1996)) to convert the pyc translational start signals to match those of the lacZ gene.
  • primers also introduced a Kpnl (GGTACC) restriction site at the beginning of the amplified fragment and a BglU (AGATCT) restriction site at the end of the amplified fragment; forward primer 5' TAC TAT GGT ACC TTA GGA AAC AGC TAT GCC CAT ATC CAA GAT ACT CGT T 3' (SEQ ID NO: 1), reverse primer 5' ATT CGT ACT CAG GAT CTG AAA GAT CIA ACA GCC TGA CTT TAC ACA ATC G 3' (SEQ ED NO:2) (the Kpnl, Shine Dalgamo, ATG start, and BglU sites are underlined).
  • the resulting 3.5 kb fragment was gel isolated, restricted with Kpnl and BglU and then Ugated into gel isolated pUCl 8 DNA which had been restricted with Kpnl and BamUl to form the pUC 1 -pyc construct.
  • This construct identified as "Plasmid in E. coli ALS225 pUC18-pyc", was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA, 20110-2209, USA, and assigned ATCC number 207111. The deposit was received by the ATCC on February 16, 1999. Protein gels and Western blotting. Heat-denatured cell extracts were separated on 10% SDS-PAGE gels as per Altman et. al. (J.
  • ALS225 E. coli cells containing either pUCl 8 or pUCl S-pyc were grown to mid-log in rich media at 37°C both in the presence and absence of EPTG Because ALS225 contains lacM ⁇ on the F, significant induction of the pUC18-pvc construct should not occur unless EPTG is added.
  • Protein extracts were prepared, subjected to SDS PAGE, and Western blotted. Proteins which had been biotinylated in vivo were then detected using the Sigma-Blot protein detection kit (Sigma Chemical Corp., St. Louis, MO). The instructions of the manufacturer were followed except that during the development of the western blots the protein biotinylation step was omitted, thus allowing for the detection of only those proteins which had been biotinylated in vivo.
  • PC Pyruvate carboxylase enzyme assay.
  • 100 mL of mid-log phase culture was harvested by centrifugation at 7,000 x g for 15 minutes at 4°C and washed with 10 mL of 100 mM Tris-Cl (pH 8.0).
  • the cells were then resuspended in 4 mL of 100 mM Tris-Cl (pH 8.0) and subsequently subjected to cell disruption by sonication.
  • the cell debris was removed by centrifugation at 20,000 x g for 15 minutes at 4°C.
  • the pyruvate carboxylase activity was measured by the method of Payne and Morris (J. Gen. Microbiol..
  • concentration of reaction components per miUiliter of mixture was as follows: 100 mM Tris-Cl (pH 8.0), 5 mM MgCl 2 H 2 O, 50 mM NaHCO 3 , 0.1 mM acetyl CoA, 0.25 mM DTNB, and 5 units (U) of citrate synthase. Pyruvate, ATP,
  • ADP or aspartate
  • the reaction was started by adding 50 ⁇ l of cell extract.
  • One unit of pyruvate carboxylase activity corresponds to the formation of 1 ⁇ mol of 5-thio-2- nitrobenzoate per mg of protein per minute. All enzyme assays were performed in triplicate and a standard error of less then 10% was observed.
  • the total protein in the cell extracts was determined by the Lowry method (O. Lowry et al., J. Biol. Chem.. 193. 265-275 (1951)).
  • the R. etli pyc gene which encodes pyruvate carboxylase, was PCR amplified from pPCl and subcloned into the pUC18 cloning/expression vector as described above. Because the translational start signals of the R. etli pyc gene were nonoptimal (pyc from R. etli uses the rare TTA start codon as well as a short spacing distance between the Shine Dalgamo and the start codon), the translational start signals were converted to match that of the lacZ gene which can be expressed at high levels in E. coli using a variety of expression vectors.
  • Biotin and biotin holoenzyme synthase on the expression of biotinylated R. etli pyruvate carboxylase in E. coli.
  • Pyruvate carboxylase is a biotin-dependent enzyme, and mediates the formation of oxaloacetate by a two- step carboxylation of pyruvate. In the first reaction step, biotin is carboxylated with ATP and bicarbonate as substrates, while in the second reaction the carboxyl group from carboxybiotin is transferred to pyruvate.
  • the subunit of the pyruvate carboxylase enzyme has been shown to contain three catalytic domains - a biotin carboxylase domain, a transcarboxylase domain, and a biotin carboxyl carrier protein domain - which work collectively to catalyze the two-step conversion of pyruvate to oxaloacetate.
  • a biotin prosthetic group linked to a lysine residue is carboxylated with ATP and HCO 3 ⁇
  • the carboxyl group is transferred to pyruvate.
  • biotinylation of pyruvate carboxylase occurs post-translationally and is catalyzed by the enzyme biotin holoenzyme synthase.
  • E. coli cells containing the pUC18-p "c construct were grown under inducing conditions in minimal defined media which either contained no added biotin, or biotin added at 50 or 100 ng mL.
  • MG1655 pUC18- ⁇ yc cells were grown to mid-log at 37°C in M9 media that contained varying amounts of biotin. Protein extracts were prepared, subjected to SDS PAGE, and Western blotted.
  • Biotinylation of pyruvate carboxylase was carried out by the enzyme biotin holoenzyme synthase, the effect of excess biotin holoenzyme synthase on the biotinylation of pyruvate carboxylase was investigated.
  • the effect of aspartate was analyzed by adding ATP and pymvate to the reaction mixture to final concentrations of 5 mM and 6 mM, respectively, then determining pymvate carboxylase activity in the presence of increasing amounts of aspartate.
  • Fig. 3 shows the pymvate carboxylase activity that was obtained in the presence of different concentrations of aspartate.
  • the pyruvate carboxylase activity was inhibited by aspartate and the specific activity decreased to approximately 43% in the presence of 8 mM aspartate.
  • the effect of ADP was analyzed by adding pyruvate to the reaction mixture to a final concentration of 5 mM, then determining pyruvate carboxylase activity in the presence of increasing amounts of ATP.
  • Fig. 4 shows that ADP severely affected the observed pymvate carboxylase activity and acted as a competitive inhibitor of ATP.
  • a Lineweaver-Burke plot of these data revealed that the saturation constant (Kêt for expressed pymvate carboxylase was 0.193 mM with respect to ATP and that the inhibition constant for ADP was 0.142 mM. Again, these values were in excellent agreement with other pymvate carboxylase enzymes that have been studied H. Feir et al., Can. J. Biochem.. 47, 698-710 (1969); H. Modak et al., Microbiol.. 141. 2619-2628 (1995); M. Scrutton et al., Arch.
  • E. coli lacks pymvate carboxylase and thus is only able to synthesize oxaloacetate from PEP
  • E. coli strains which contain a disrupted #pc gene can not grow on minimal media which utilizes glucose as the sole carbon source (P. Chao et al., Appl. Env. Microbiol.. 59, 4261-4265 (1993)).
  • the cell line used for this experiment was JCL1242 (ppc :ka ), which contains a kanamycin resistant cassette that has been inserted into the ppc gene and thus does not express the PEP carboxylase enzyme.
  • JCL1242 cells containing either pUC18 or the pUCl S-pyc construct were patched onto minimal M9 glucose thiamine ampicillin EPTG plates and incubated at 37 C for 48 hours.
  • E. coli cells which contain both the ppc null allele and the pUC 18-pyc constmct were able to grow on minimal glucose plates.
  • This complementation demonstrates that a branch point can be created at the level of pymvate which results in the rerouting of carbon flow towards oxaloacetate, and clearly shows that pymvate carboxylase is able to divert carbon flow from pymvate to oxaloacetate in E. coli.
  • Example II. Expression of R. etli Pyruvate Carboxylase causes Increased Succinate Production in E. coli
  • E. coli strains used in this study are listed in Table 2.
  • the lactate dehydrogenase mutant strain designated RE02 was derived from MG1655 by PI phage transduction using E. coli strain NZNl 11 (P. Bunch et al., Microbiol.. 143. 187-195 (1997)).
  • This plasmid identified as "Plasmid in E. coli ALS225 pTrc99A-pyc", was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA, 20110-2209, USA, and assigned ATCC number 207112. The deposit was received by the ATCC on February 16, 1999.
  • ATCC American Type Culture Collection
  • the transcription of the pyc gene is under the control of artificial trc promoter and thus is not subjected to catabolic repression in the presence of glucose.
  • E. coli strains were grown aerobically in Luria-Bertani (LB) medium. Anaerobic fermentations were carried out in 100 mL serum bottles with 50 mL LB medium supplemented with 20 g/L glucose and 40 g/L MgCO 3 . The fermentations were terminated at 24 hours at which point the pH values of all fermentations were approximately pH 6.7, and glucose was completely utilized.
  • LB Luria-Bertani
  • Table 3 shows that pymvate carboxylase activity could be detected when the pTrc99A-/ryc constmct was introduced into either wild type cells (MG1655) or wild type cells which contained a Idhr null mutation (RE02).
  • EPTG did not significantly affect the expression of other important metabolic enzymes such as PEP carboxylase, lactate dehydrogenase and malate dehydrogenase.
  • Antibiotics either added once at 0 hours at a concentration of 100 ⁇ g/mL (lx) or added at 0 hours at a concentration of 100 ⁇ g/mL and again at 7 hours and
  • NADH neuropeptide deficiency deficiency
  • Idhr mutants are generated per mole of glucose. NADH is then oxidized during the formation of ethanol, lactate and succinate under anaerobic conditions. The inability of the Idhr mutants to consume NADH through lactate formation may put stress on the oxidizing capacity of these strains, leading to an accumulation of NADH. Indeed, this reduced cofactor has previously been shown to inhibit a pymvate carboxylase isolated from Saccharomyces cerevisiae (J. Cazzulo et al., Biochem. J.. 112. 755-762 (1969)).
  • NADH inhibited pymvate carboxylase whereas NAD + , NADP + and NADPH did not.
  • the lower succinate enhancement with RE02 the Idhr mutant is therefore hypothesized to result from an accumulation of mtracellular NADH, a cofactor which appears to inhibit pymvate carboxylase activity.
  • Example III Expression of R. etli Pyruvate Carboxylase Does Not Affect Glucose Uptake in E. coli in Anaerobic Fermentation
  • E. coli strain MG1655 wild type F ⁇ ; M. Guyer et al., Quant. Biol.. Cold Spring Harbor Svmp.. 45, 135-140 (1980); see also Example I
  • the plasmid p ⁇ JC18-pyc which contains the pyc gene from R. etli (see Example I).
  • L New Brunswick BioFlo III bench top fermenters (New Brunswick Scientific, Edison, NT) in Luria-Bertani (LB) supplemented with glucose, 10 g L; Na 2 PHO 4 -7H 2 O, 3 g/L; KH 2 PO 4 , 1.5 g/L; NH 4 C1, 1 g/L; MgSO 4 -7H 2 O, 0.25 g/L; and CaCl 2 -2H 2 O, 0.02 g/L.
  • the fermenters were inoculated with 50 mL of anaerobically grown culture. The fermenters were operated at 150 rpm, 0% oxygen saturation (Ingold polarographic oxygen sensor, New Brunswick Scientific, Edison, NT), 37°C, and pH 6.4, which was controlled with 10% NaOH.
  • Anaerobic conditions were maintained by flushing the headspace of the fermenter with oxygen-free carbon dioxide.
  • the media was supplemented with an initial concentration of 100 ⁇ g/mL ampicillin, previously shown to be sufficient to maintain the selective pressure (Example II).
  • OD optical density
  • Glucose and fermentation products were analyzed by high-pressure liquid chromatography using Coregel 64-H ion-exclusion column (Interactive Chromatography, San Jose, CA) as described in Example II.
  • the activity of pymvate carboxylase and the endogenous activity of PEP carboxylase was measured by growing each strain and clone separately in 160 mL serum bottles under strict anaerobic conditions. Cultures were harvested in mid-logarithmic growth, washed and subjected to cell disruption by sonication. Cell debris were removed by centrifugation (20000 g for 15 minutes at 4°C). Pymvate carboxylase activity was measured as previously described (Payne and Morris, 1969), and the PEP carboxylase activity was measured in the absence of ATP using PEP instead of pymvate as the substrate, with the appearance of
  • RESULTS E. coli MG1655 was grown anaerobically with 10 g/L glucose as energy and carbon source.
  • Fig. 8 shows the dry cell mass, succinate, lactate, formate and glucose concentrations with time in a typical 2-liter fermentation of this wild-type strain.
  • Fig. 9 shows these concentrations with time in a fermentation of this wild-type strain with the cloning/expression vector pUCl ⁇ . After complete glucose utilization, the average final concentration of succinate for the wild-type strain was 1.18 g/L, while for the wild-type strain with the vector pUC18 the final succinate concentration was 1.00 g/L.
  • Fig. 10 shows the concentrations with time of dry cell mass, succinate, lactate, formate and glucose in a fermentation of the strain containing the pUClS-pyc plasmid. This figure shows that the expression of pymvate carboxylase causes a substantial increase in final succinate concentration and a decrease in lactate concentration. Specifically, for the wild-type with pUC18- pyc the average final succinate concentration was 1.77 g/L, while the average final lactate concentration was 1.88 g/L. These concentrations correspond to a 50% increase in succinate and about a 20% decrease in lactate concentration, indicating that carbon was diverted from lactate toward succinate formation in the presence of the pymvate carboxylase.
  • the addition of the cloning vector or the vector with the pyc gene had no significant effect on the average glucose uptake during the final 4 hours of the fermentations. Indeed, the presence of the pyc gene actually increased the maximum glucose uptake about 14% from 2.17 g/Lh to 2.47 g/Lh. The presence of the pUC 18 cloning vector reduced slightly the rates of succinate production. As expected from the data shown in Fig. 10, the expression of the pyc gene resulted in an 82% increase in succinate production at the time of maximum glucose uptake, and a 68% increase in the rate of succinate production during the final 4 hours of the fermentations. The maximum rate of cell growth (which occurred at 4-5 hours for each of the fermentations) was
  • Bacterial strains and plasmids Bacterial strains and plasmids.
  • the threonine-producing strain ⁇ EM-4 (ATCC 21277) was used in this study (I. Shiio et al., Agr. Biol. Chem.. 33,
  • the media used for these fermentation contained (per liter): glucose, 30.0 g; (NH 4 ) 2 SO 4 10.0 g, FeSO 4 H 2 O, 10.0 mg; MnSO 4 H 2 O, 5.5 mg/L; L-proline, 300 mg; L-isoleucine, 100 mg; L-methionine, 100 mg; MgSO 4 -7H 2 O, 1 g; KH 2 PO 4 , 1 g; CaCO 3 , 20 g; thiamine HCl, lmg; d- biotin, 1 mg.
  • Fermentation product analysis Cell growth was determined by measuring optical density at 550 nm of a 1:21 dilution of sample in 0.1M HC1. Glucose, acetic acid and other organic acids were analyzed by high-pressure liquid chromatography as previously described (M. Eiteman et al., Anal. Chim. Acta. 338, 69-75 (1997)) using a Coregel 64-H ion-exclusion column. Threonine was quantified by high-pressure liquid chromatography using the ortho- phthaldialdehyde derivatization method (D. Hill et al., Anal. Chem.. 51, 1338- 1341 (1979); V. Svedas et al. Anal. Biochem.. 101, 188-195 (1980)). RESULTS
  • the threonine-producing strain ⁇ EM-4 (ATCC 21277), harboring either the control plasmid pTrc99A or the plasmid pTrc99A-/?yc which overproduces pymvate carboxylase, was grown aerobically with 30 g/L glucose as energy and carbon source and the production of threonine was measured. As shown in Fig.
  • etli pymvate carboxylase are thus limited by the fact that diverting carbon from pymvate to oxaloacetate both depletes acetyl coenzyme A levels and increases aspartate levels.
  • the pymvate carboxylase from P. fluorescens does not require acetyl coenzyme A for its activation and it is not affected by the feedback inhibition caused by aspartate (R. Silvia et al., J. Gen. Microbiol.. 23, 75-81 (1976)).
  • Overproduced P. fluorescens pymvate carboxylase should aUow even more carbon flow to be diverted towards oxaloacetate.
  • the P. fluorescens pyc gene may be readily isolated from a genomic Ubrary using probes which have been prepared from the R. etli gene.
  • the gene for pymvate carboxylase in P. fluorescens will thus be identified, isolated, and cloned into an expression vector using standard genetic engineering techniques.
  • the pyruvate carboxylase enzyme can be isolated and purified from P. fluorescens by following pyruvate carboxylase activity (as described in the above Examples) and also by assaying for biotinylated protein using Western blots.
  • the N-terminal amino acid sequence of the purified protein is determined, then a degenerate oligonucleotide probe is made which is used to isolate the gene encoding pyruvate carboxylase from a genomic library that has been prepared from P. fluorescens.
  • the pyc clone thus obtained is sequenced.
  • oligonucleotide primers are designed that allow cloning of this gene into an expression vector so that pymvate carboxylase can be overproduced in the host cell. Either method can be used to yield a vector encoding the P. fluorescens pyc gene, which is then used to transform the host E. coli or C. glutamicum cell.
  • Pyruvate carboxylase from P. fluorescens is expressed in the host cell, and biochemical production is enhanced as described in the preceding examples.
  • PEP can be carboxylated to oxaloacetate via PEP carboxylase or it can be converted to pymvate by pymvate kinase (I. Sh o et al., J. Biochem.. 48, 110-120 (1960); M. Jetten et al., Appl. Microbiol. Biotechnol.. 41, 47-52 (1994)).
  • pymvate kinase I. Sh o et al., J. Biochem.. 48, 110-120 (1960); M. Jetten et al., Appl. Microbiol. Biotechnol.. 41, 47-52 (1994)
  • One possible strategy that was tried to increase the carbon flux toward oxaloacetate in C. glutamicum was to block the carbon flux from PEP toward pymvate.
  • lysine production by pymvate kinase mutants was 40% lower than by a parent strain, indicating that pymvate is essential for high-level lysine production (M. Gubler et al., Appl. Microbiol. Biotechnol.. 60.
  • Carbon flux toward oxaloacetate may be increased by overexpressing PEP carboxylase in conjunction with overexpressed pymvate carboxylase without concomitantly blocking carbon flux from PEP to pyruvate or affecting glucose uptake.
  • PEP carboxylase isolated from the cyanobacteria Anacystis nidulans does not require acetyl CoA for activation nor is it inhibited by aspartate (M. Utter et al., Enzymes. 6, 117-135 (1972)). Therefore, this heterologous enzyme can be used to increase the carbon flux towards oxaloacetate in C. glutamicum.
  • the genes encoding PEP carboxylase in A. nidulans have been isolated and cloned
  • E. coli strain ALS226 which is MC1061 / FlacP 1 Z::Tn5 T A + (E. Altman, University of Georgia).
  • the wild-type E. coli strain MG1655, F- ⁇ - (M. Guyer et al., Cold Spring Harbor Symp. Quant. Biol. 45: 135-140 (1980)), was used for all of the other studies.
  • the plasmids used in this work are described in Table 7.
  • primers 5' ATC AGA CTG CAG GAG GTAACA GCTAmAAC AAA GGI GT&ATC CGA CC 3* (S ⁇ Q ⁇ D NO:5) and 5' TAG CAG TGG CAG CTC TGA Q C E TCC ACA ATC JCT GCA ATA AGT CG 3' (SEQ ED NO:6) were used to PCR amplify a 1006 bp fragment from the pXE60 plasmid which contains the wild-type Pseudomonas putida xylE gene isolated from the TOL pWWO plasmid (restriction enzyme sites are indicated with a double underline while the regions of homology to xylE are indicated by with a single underline).
  • the resulting fragment was gel isolated, digested with Pst I and Hind EQ, and then Ugated into the pTrc99A vector which had been digested with
  • a 3504 bp fragment from pTrc99A-/ ⁇ cZthat contained the lacZ gene and the trc promoter was Ugated to a 3178 bp fragment from pACYC184 that contained the cat gene and the ori of replication.
  • the 3054 bp lacZ fragment was prepared by digesting pTrc99A-/ ⁇ cZ with Nar I and Hind III and then filling in with Klenow, while the 3178 bp fragment containing cat and the ori was prepared by digesting pACYCl 84 with Hind Ed. A clone was then selected which transcribed lacZ in the clockwise direction with respect to pAC YC 184.
  • pACYCl 84- y/E a 1365 bp fragment from pTrc99A-ry/E that contained the xylE gene and the trc promoter was Ugated to a 3178 bp fragment from pAC YC 184 that contained the cat gene and the ori of replication.
  • the 1365 bp xylE fragment was prepared by digesting pTrc99A- y/E with Nar I and Hind Ed and then filling in with Klenow, while the 3178 bp fragment containing cat and the ori was prepared by digesting pACYCl 84 with Hind EEL A clone was then selected which transcribed xylE in the clockwise direction with respect to pACYCl 84. Media.
  • EPTG was added at a final concentration of 1 mM as a gratuitous inducer of the lac-based expression vectors that were used in this study.
  • AmpicuTin was added at a final concentration of 100 ⁇ g/ml as selective pressure for plasmids with ColEl repUcons and chloramphenicol was added at a final concentration of 20 ⁇ g/ml as selective pressure for plasmids with P 15 A repUcons.
  • LB MiUer broth was used for shake flask studies. Samples of 2 mL volume were inoculated from a plate and were grown for approximately 12 hours in LB MiUer broth containing ampicillin and chloramphenicol. These samples were diluted 1 :200 into 50 ml of fresh LB MiUer broth containing ampiciUin and chloramphenicol, and allowed to grow until an OD 550 of 0.5 was reached. Cultures were then diluted to an OD 550 of 0.1 using 100 mL of fresh LB MiUer broth containing ampicillin and chloramphenicol. EPTG was added and samples were processed at regular intervals and assayed for either ⁇ -galactosidase or catechol 2,3-dioxygenase activities.
  • the media contained: 30 g/L glucose, 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 5 g/L 1.5 g/L KH 2 PO 4 , 1.0 g/L NH 4 C1, 0.25 g/L MgSO 4 »7H 2 0, 20 mg/L CaCl 2 »2H 2 O, 1.0 mg/L biotin.
  • Samples of 10 mL volume were inoculated from a plate and were grown for approximately 10 hours in the fermentation media containing ampicillin and chloroamphenicol. The 10 mL volume was used to inoculate a 100 mL shake flask containing the identical fresh media.
  • One enzyme unit (EU) of catechol 2,3-dioxygenase is defined as the quantity of enzyme which converts one ⁇ mole of catechol to 2-hydroxymuconic semialdehayde per minute at 30 ° C and pH 8.
  • Catechol has an abso ⁇ tivity of 36 absorbance units/mM for a 1.0 cm Ught path.
  • Pymvate carboxylase activity was measured by the coupled method of J. Payne et al. (J. Gen. Microbiol. 59:97-101 (1969)).
  • One enzyme unit (EU) of pymvate carboxylase is defined as the quantity of enzyme which converts one ⁇ mole of pyruvate to oxaloacetate per minute at 30 °C and pH 8.
  • Glucose and low molecular weight fermentation products were analyzed by high-pressure liquid chromatography using a Coregel 64-H ion- exclusion column as described by Eiteman et al. f Anal. Chim. Acta. 338:69-75 (1997)).
  • the plasmid pTrc99A- ⁇ yc which ove ⁇ roduces pymvate carboxylase
  • the control plasmid pTrc99A were transformed into the E. coli strain MG1655 pACYC-/ ⁇ cZ which ove ⁇ roduces ⁇ -galactosidase.
  • the resulting strains, MG1655 pTrc99A pACYC-/ ⁇ cZand MG1655 pTrc99A-/7yc pAC YC-lacZ were simultaneously grown in rich media and samples were removed for analysis of ⁇ -galactosidase activity. The results are shown in Fig. 12.
  • the plasmid pTrc99A- 7yc which ove ⁇ roduces pyruvate carboxylase and the control plasmid pTrc99A were transformed into the E. coli strain MG1655 pACYC-xylE which ove ⁇ roduces catechol 2,3-dioxygenase.
  • the resulting strains, MG1655 pTrc99A pACYC-xylE and MG1655 pTrc99A-/?yc pACYC-xylE were simultaneously grown in rich media and samples were removed for catechol 2,3-dioxygenase assays. The results are shown in Fig. 13.
  • the activity of ⁇ -galactosidase was therefore 137% greater in the fermentation using the strain with the pyc gene than in the fermentation using the strain without the pyc gene.
  • the presence of pymvate carboxylase was confirmed by an enzyme assay.
  • Fig. 15 shows these results.
  • the strain without pyc consumed most of the glucose after 6 hours of fermentation.
  • the strain v ⁇ thpyc consumed glucose more slowly, with about 7 g/L glucose remaining after 6 hours and about 3 g/L still remaining after 12 hours.
  • the slower glucose consumption rate in the pTrc99A-pyc strain did not result in lower cell growth rate.
  • Example LX Enhanced Yield of a Recombinant Protein in E. coli Expressing Pyruvate Carboxylase Using a Defined Minimal Media.
  • Bacterial strains and plasmids Bacterial strains and plasmids.
  • the host strain was E. coli MGl 655.
  • Table 7 Example VEII describes the plasmids which were used to express ⁇ -galactosidase and pymvate carboxylase in this study. Because both the pTrc99A and pACYCl 84 expression plasmids contain the lac promoter, isopropyl- ⁇ -thiogalactopyranoside
  • EDTA «2H 2 O, 9.6mg; CuCl 2 «2H 2 0, 1.5mg; Na ⁇ oO ⁇ H 2.5mg; CoCl 2 »6H 2 O, 2.5mg; ZnCl 2 -2H 2 O, 5.0mg; glucose, 20g; MgSO 4 »7H 2 O, 0.6g; CaCl 2 «2H 2 O, 70mg; ampicillin, lOOmg; and chloramphenicol, 20mg. Media components were added in the order listed to prevent precipitation of metals. Precultures were grown at 250 rpm and 37°C to an optical density near 1.5.
  • the fermentation media C2 contained (per L): KH 2 PO 4 , 6.00g; (NH 4 ) 2 HPO 4 , 8.00g; citric acid, 2.1g; Fe 2 (SO 4 ) 3 , 62.5mg; H 3 BO 3 , 3.8mg; MnCl 2 »4H 2 O, 18.8mg; disodium EDTA»2H 2 O, 12mg; CuCl 2 »2H 2 O, 1.9mg; Na 2 MoO 4 .2H 2 O, 3.1mg; CoCl 2 .6H 2 O, 3.1mg; Zn(CH 3 COO) 2 «2H 2 O, lOmg; glucose, 30g; MgSO 4 -7H 2 O, 1.5g; CaCl 2 «2H 2 O, 70mg; biotin, lmg; thiamine»HCl, lmg; ampicillin, lOOmg; and chloramphenicol, 20mg.
  • Glucose and acetate were analyzed by high-pressure liquid chromatography as previously described (M. Eiteman et al., Anal. Chem. Acta. 338. 69-70 (1997)) using a Coregel 64-H ion-exclusion column (Interactive Chromatography, San Jose, CA). CO 2 and O 2 were measured continuously in the fermentation off-gas (Ultramat 23 gas analyzer, Siemens, Kunststoff, Germany). Enzyme Assays. Aliquots (1.5 mL) of the samples were thawed and centrifuged (6000 xg for 20 minutes).
  • the cells were washed and resuspended in 1.0M Tris buffer (pH 8.0), ruptured with a French® Pressure Cell (850 psi) and centrifuged (25,000 ⁇ g for 20 min at 4°C).
  • the cell-free extract was analyzed for pymvate carboxylase following the method of Payne and Morris (J. Payne et al., J. Gen. Microbiol.. 59, 97 - 101 (1969)).
  • One unit of pymvate carboxylase activity converts one ⁇ mole of pymvate per minute to oxaloacetate at 30°C and pH 8.
  • ⁇ -galactosidase activity For ⁇ -galactosidase activity, aliquots (1.5 mL) were thawed and diluted to an OD 550 of 0.1 with Luria-Bertani (LB) broth (R. L. Rodriguez et al, Biotechnolog. Bioeng.. 57, 71-78 (1983)). Diluted samples were analyzed for ⁇ - galactosidase activity following the protocol of A. B. Pardee et al.. J. Mol. Biol.. 1, 165-178 (1959). One unit of ⁇ -galactosidase activity produced one nmole of ⁇ -nitrophenol per minute at 30 °C and pH 7.
  • samples were thawed and centrifuged (6000 ⁇ g for 20 minutes at 4°C).
  • the cells from complex media were washed and resuspended in 1.0M Tris buffer (pH 8.0) and then ruptured with a French® Pressure Cell (850 psi) and centrifuged (25,000 x g for 20 minutes at 4°C).
  • Samples from defined media fermentations were disrupted with Bper ETTM Bacterial Protein Extraction Reagent (Pierce). Total cellular protein content was determined for samples taken from defined media fermentations using a BCATM Protein Assay Kit (Pierce).
  • Media C2 contained 30 g/L of glucose and 2.18 g/L of ammonium ion (NH 4 + ).
  • the relatively high concentration of NH 4 + was required since there was no other nitrogen source (such as protein hydrolysate) to provide precursors molecules to derive the amino acids.
  • this medium should most clearly demonstrate any difference caused by adding an additional anaplerotic pathway to E. coli's central metabolism.
  • Figures 18 and 19 display representative fermentation profiles of E. coli using Media C2 for the pyc and pyc * strains, respectively.
  • the pymvate carboxylase activity in these aerobic fermentations was 0.42 EU/ml as opposed to 0.06 EU/ml in the anaerobic fermentation.
  • the fact that pymvate carboxylase activity was seven times higher could explain the effect on specific glucose uptake and specific growth rate. Nonetheless, the end result was a significant increase in the maximum activity of model recombinant protein that could be obtained. Although the average maximum acetate concentration in the fermentations of E.
  • This technology can be readily applied in eukaryotic systems using methodologies analogous to those described in Examples VEH and EX.
  • yeast Saccharomyces cerevisiae two compatible plasmids are constructed. The first plasmid expresses the pymvate carboxylase enzyme while the second plasmid expresses the protein whose expression is to be augmented.
  • multiple plasmids can coexist in the same cell and each plasmid is selected for using a different positive selection pressure.
  • antibiotics are usually used to provide selective pressure for plasmids in bacteria
  • biosynthetic enzymes such as those involved in the synthesis of histidine, leucine, tryptophan, or uracil are usually used to accompUsh this in S. cerevisiae.
  • URA3 genes are routinely used.
  • plasmids are available in yeast that are well suited for the expression of heterologous proteins, such as pG-3 (M. Schena et al., Meth. EnzvmoL. 194. 389-398 (1991)), pRA-6 (T. Nagashima et al., Biosci. Biotech. Biochem.. 58, 1292-1296 (1994)), pTRPl 1 (K. Kitamoto et al., Agric. Biol.
  • the pymvate carboxylase gene and the gene that encodes the protein to be tested are cloned into two compatible yeast expression vectors which contain different selectable biosynthetic enzymes.
  • the two resulting plasmids are then transformed into a yeast recipient cell that is deficient for both of the biosynthetic enzymes that are being used as selectable markers.
  • a yeast cell that ove ⁇ roduces pymvate carboxylase and the protein to be tested is compared to a yeast cell that ove ⁇ roduces the protein to be tested but does not also ove ⁇ roduce pymvate carboxylase.
  • the parental plasmid is substituted for the plasmid that ove ⁇ roduces pymvate carboxylase.
  • the two yeast cells are grown in a minimal defined media which allows for selection of the biosynthetic enzymes that are expressed by the two plasmids.
  • Such media typicaUy contains 6.7 g/L of Difco yeast nitrogen base without amino acids and 20 g/L of glucose. Amino acid and base supplements are then added separately.
  • Various salts can be added to facilitate growth to high densities and/or increase the buffering capacity of the medium.
  • various feeding strategies can be used to facilitate growth to high cell densities.
  • the protein or peptide that is overexpressed can be encoded in a chromosomaUy integrated nucleic acid, although use of a plasmid encoding the polypeptide is preferred.

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Abstract

La présente invention concerne l'amélioration des rendements de protéines qui résulte de l'augmentation de l'activité de la pyruvate carboxylase dans divers systèmes d'expression de protéines.
PCT/US2000/028578 1999-10-13 2000-10-13 Systeme et procedes d'expression de proteines a haut rendement WO2001027258A2 (fr)

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MXPA02003634A MXPA02003634A (es) 1999-10-13 2000-10-13 Metodo y sistema de expresion de proteina de alto rendimiento.
JP2001530463A JP2003511067A (ja) 1999-10-13 2000-10-13 高い収量のタンパク質発現系および方法
CA002387605A CA2387605A1 (fr) 1999-10-13 2000-10-13 Systeme et procedes d'expression de proteines a haut rendement
AU12068/01A AU1206801A (en) 1999-10-13 2000-10-13 High yield protein expression system and methods
BR0014758-3A BR0014758A (pt) 1999-10-13 2000-10-13 Sistemas e métodos para expressão de proteìna com alto rendimento
EP00973568A EP1235903A2 (fr) 1999-10-13 2000-10-13 Systeme et procedes d'expression de proteines a haut rendement
HK03101556.1A HK1052198A1 (zh) 1999-10-13 2003-03-03 超顯示丙酮酸鹽羧酶或丙酮酸鹽磷烯醇丙酮酸鹽羧酶的代謝工程細胞，及產生及使用該等細胞的方法

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WO2002064808A1 (fr) * 2001-02-13 2002-08-22 Cheil Jedang Corporation Procede de production de l-threonine
DE10219714A1 (de) * 2002-05-02 2003-11-27 Holland Sweetener Co Verfahren zur mikrobielien Herstellung von aromatischen Aminosäuren und anderen Metaboliten des aromatischen Aminosäurebiosyntheseweges
WO2005087940A1 (fr) * 2004-03-11 2005-09-22 Wisconsin Alumni Research Foundation Micro-organismes genetiquement modifies presentant un metabolisme modifie
US7303906B2 (en) 2002-09-06 2007-12-04 Wisconsin Alumni Research Foundation Competent bacteria
US7368266B2 (en) 2001-02-13 2008-05-06 Cj Corporation Method for L-threonine production
US8039243B2 (en) 2002-01-23 2011-10-18 Wisconsin Alumni Research Foundation Insertion sequence-free bacteria
US8043842B2 (en) 2002-01-23 2011-10-25 Wisconsin Alumni Research Foundation Bacteria with reduced genome
US8119365B2 (en) 2002-01-23 2012-02-21 Wisconsin Alumni Research Foundation Insertion sequence-free bacteria
EP2434016A2 (fr) 2004-01-16 2012-03-28 Pfenex, Inc. Expression de proteines mammifères dans Pseudomonas fluorescens
US8765408B2 (en) 2002-01-23 2014-07-01 Wisconsin Alumni Research Foundation Prophage element-free bacteria
US10041102B2 (en) 2002-10-08 2018-08-07 Pfenex Inc. Expression of mammalian proteins in Pseudomonas fluorescens
US10689640B2 (en) 2007-04-27 2020-06-23 Pfenex Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins

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EP1584680A1 (fr) * 2004-04-08 2005-10-12 Boehringer Ingelheim Austria GmbH Procédé de fermentation discontinue pour la préparation de l'ADN plasmidique
JP4720114B2 (ja) * 2004-05-20 2011-07-13 三菱化学株式会社 オキザロ酢酸またはオキザロ酢酸誘導体の製造方法
KR20130019457A (ko) 2004-07-26 2013-02-26 다우 글로벌 테크놀로지스 엘엘씨 균주 조작에 의한 개선된 단백질 발현 방법
MX2009011523A (es) 2007-04-27 2009-11-09 Dow Global Technologies Inc Metodo para clasificar rapidamente huespedes microbianos para identificar ciertas cepas con rendimiento y/o calidad mejorados en la expresion de proteinas heterologas.
EP2692911B1 (fr) 2011-03-31 2017-02-22 Kunimine Industries Co., Ltd. Utilisation d'un agent destiné à la recherche des conditions de cristallisation des protéines, et procédé destiné à la recherche des conditions de cristallisation des protéines
JP2015156844A (ja) * 2014-02-25 2015-09-03 花王株式会社 枯草菌変異株及びそれを用いたジピコリン酸の製造方法

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7011961B2 (en) 2001-02-13 2006-03-14 Cheil Jedang Corporation Method for L-threonine production
WO2002064808A1 (fr) * 2001-02-13 2002-08-22 Cheil Jedang Corporation Procede de production de l-threonine
US7368266B2 (en) 2001-02-13 2008-05-06 Cj Corporation Method for L-threonine production
US8039243B2 (en) 2002-01-23 2011-10-18 Wisconsin Alumni Research Foundation Insertion sequence-free bacteria
US8043842B2 (en) 2002-01-23 2011-10-25 Wisconsin Alumni Research Foundation Bacteria with reduced genome
US8119365B2 (en) 2002-01-23 2012-02-21 Wisconsin Alumni Research Foundation Insertion sequence-free bacteria
US8765408B2 (en) 2002-01-23 2014-07-01 Wisconsin Alumni Research Foundation Prophage element-free bacteria
DE10219714A1 (de) * 2002-05-02 2003-11-27 Holland Sweetener Co Verfahren zur mikrobielien Herstellung von aromatischen Aminosäuren und anderen Metaboliten des aromatischen Aminosäurebiosyntheseweges
US7303906B2 (en) 2002-09-06 2007-12-04 Wisconsin Alumni Research Foundation Competent bacteria
US10041102B2 (en) 2002-10-08 2018-08-07 Pfenex Inc. Expression of mammalian proteins in Pseudomonas fluorescens
EP2434016A2 (fr) 2004-01-16 2012-03-28 Pfenex, Inc. Expression de proteines mammifères dans Pseudomonas fluorescens
WO2005087940A1 (fr) * 2004-03-11 2005-09-22 Wisconsin Alumni Research Foundation Micro-organismes genetiquement modifies presentant un metabolisme modifie
US10689640B2 (en) 2007-04-27 2020-06-23 Pfenex Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins

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JP2003511067A (ja) 2003-03-25
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