MXPA00009984A - Pyruvate carboxylase overexpression for enhanced production of oxaloacetate-derived biochemicals in microbial cells - Google Patents
Pyruvate carboxylase overexpression for enhanced production of oxaloacetate-derived biochemicals in microbial cellsInfo
- Publication number
- MXPA00009984A MXPA00009984A MXPA/A/2000/009984A MXPA00009984A MXPA00009984A MX PA00009984 A MXPA00009984 A MX PA00009984A MX PA00009984 A MXPA00009984 A MX PA00009984A MX PA00009984 A MXPA00009984 A MX PA00009984A
- Authority
- MX
- Mexico
- Prior art keywords
- cell
- pyruvate carboxylase
- oxaloacetate
- carboxylase
- biochemical
- Prior art date
Links
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Abstract
Metabolic engineering is used to increase the carbon flow toward oxaloacetate to enhance production of bulk biochemicals, such as lysine and succinate, in bacterial fermentations. Carbon flow is redirected by genetically engineering the cells to overexpress the enzyme pyruvate carboxylase.
Description
OVEREXPRESSION OF PIRUVATE CARBOXYLASE FOR THE
IMPROVED PRODUCTION OF BIOCHEMICALS DERIVED FROM
OXALOACETATE IN MICROBIAL CELLS
FIELD AND BACKGROUND OF THE INVENTION
There is an extraordinary commercial potential to produce biochemicals derived from oxaloacetate via aerobic or anaerobic bacterial fermentation processes. Aerobic fermentation processes can be used to produce amino acids derived from oxaloacetate such as asparagine, aspartate, methionine, threonine, isoleucine, and lysine. Lysine, in particular, is of great commercial interest in the world market. The raw material comprises a significant portion of the cost of production of lysine, and therefore, the yield of the process (product generated by substrate consumed) is an important measure of economic performance and viability. The severe metabolic regulation of carbon flux (described later) can limit the yields of the process. The flow of carbon
Ref. 124100 to the oxaloacetate (OAA) remains constant despite the perturbations of the system (J. Vallino et al., Biotechnol. Bioeng., 41, 633-646 (1993)). In a reported fermentation, to maintain this rigid regulation of carbon flux at the desirable low growth rates for lysine production, the cells convert less carbon to oxaloacetate, thereby limiting lysine production (R. Kiss et al. ., Biotechnol. Bioeng., 39, 565-574 (1992)). Therefore, there is an extraordinary opportunity to improve the process by overcoming the metabolic regulation of carbon flux. Anaerobic fermentation processes can be used to produce organic acids derived from oxaloacetate such as malate, fumarate, and succinate. Chemical processes that use petroleum raw materials can also be used, and have historically been more efficient for the production of these organic acids than bacterial fermentations. Succinic acid in particular, and its derivatives, has greater potential for use as specialty chemicals. They can be used advantageously in various applications in the food, pharmaceutical and cosmetics industries, and can also serve as starting materials in the production of useful chemicals such as 1,4-bu t andiol and tetrahydrofuran (L. Schilling, FEMS Microbiol. REV., 16, 101-110 (1995)). The anaerobic rumen bacterium has been considered for use in the production of succinic acid via bacterial fermentation processes, but this bacterium tends to cause lysis during fermentation. More recently, An ae io rob io sp iri 1 l um succinic ip rodu cen seroerobia has been used, which is more robust and produces higher levels of succinate (R. Datta, U.S. Patent 5,143,833 (1992); Datta et al., Eur. Pat. Appl. 405707 (1990)). Commercial fermentation processes use carbohydrates derived from crops to produce volumetric biochemicals. Glucose, a common carbohydrate substrate, is usually metabolized via the Embden-Meyerhof-Parnas trajectory (EMP), also known as gl iopathic trajectories, to fos f oenolpi ruva t o
(PEP) and then pyruvate. All organisms derive some energy from the glycolytic structure of glucose, with respect to whether they are growing aerobically or anaerobically. However, beyond these two intermediaries, the pathways or trajectories for carbon metabolism are different depending on whether the organisms develop aerobically or anaerobically, and the deaths of PEP and pyruvate depend on the particular organism involved as well as the conditions under which the metabolism is occurring. In aerobic metabolism, the carbon atoms of glucose are completely oxidized to carbon dioxide in a cyclic process known as the tricarboxylic acid (TCA) cycle or, sometimes,, the citric acid cycle, or Krebs cycle. The TCA cycle which is when the oxaloacetate is combined with acetyl-CoA to form citrate. The complete oxidation of glucose during the TCA cycle finally releases significantly more energy from a single or simple molecule of glucose that is extracted during glycolysis alone. In addition to supplying the TCA cycle in aerobic fermentations, oxaloacetate also serves as an important precursor for the synthesis of the amino acids asparagine, aspartate, methionine, threonine, isoleucine and lysine. This aerobic trajectory is shown in Figure 1 for Es ch e r i ch i a c o l i, the microorganisms most commonly studied. Anaerobic organisms, on the other hand, do not completely oxidize glucose. In contrast, pyruvate and oxaloacetate are used as acceptor molecules in the reoxidation of reduced cofactors (NADH) generated in the EMP path. This leads to the generation and accumulation of reduced biochemists such as acetate, lactate, ethanol, formate and succinate. This anaerobic trajectory for E. c or l i is shown in Figure 2. The intermediaries of the TCA cycle are also used in the biosynthesis of many important cellular compounds. For example, otee toglut is used for biomes to internalize the amino acids glutamate, glutamine, arginine, and proline, and succinyl-CoA is used for bi or s int e t i za r porphyrins. Under anaerobic conditions, these important intermediaries are still necessary. As a result, succ ini 1 -CoA, for example, is made under anaerobic conditions from oxaloacetate in a reverse reaction;
that is, the TCA cycle works again from the oxaloacetate to succinyl -CoA. The oxaloacetate that is used for the biosynthesis of these compounds must be filled if the TCA cycle is to continue the decrease and the metabolic functionality is to be maintained. Many organisms that have developed this way are known as "anaplerotic trajectories" that regenerate intermediaries to recruit in the TCA cycle. Among the important reactions performed by this filling or supply are those in which the oxaloacetate is formed from PEP or pyruvate. These trajectories that re-supply intermediates in the TCA cycle can be used either during aerobic or anaerobic metabolism. PEP occupies a central position, or node, in carbohydrate metabolism. As the final intermediate in glycolysis, and therefore the intermediate precursor in the formation of pyruvate via the action of the enzyme pyruvate kinase, it serves as a source of energy. Additionally, the PEP can fill intermediaries in the TCA cycle via the anaplerotic action of the enzyme PEP carboxylase, which converts PEP directly into the TCA intermediate oxaloacetate. PEP is also frequently a co-substrate for glucose uptake in the cell via the phosphotransferase system (PTS) and is used for intimate and aromatic amino acids. In many organisms, the TCA cycle intermediates can be regenerated directly from pyruvate. For example, pyruvate carboxylase (PYC), which is found in some bacteria but not in E. c or l i, regulates the formation of oxaloacetate by the carboxylation of pyruvate using carboxyben t ina. As might be expected, the division of PEP is rigidly regulated by the cellular control mechanisms, causing a metabolic "paralysis" which limits the amount and direction of the carbon flow through this union or junction. Enzyme-mediated conversions that occur between PEP, pyruvate and oxaloacetate are shown in Figure 3. Intermediates of the TCA cycle can also be regenerated in some plants and microorganisms from acetyl-CoA via that which is known as the "derivation". of glyoxylate "," glyoxylate step "or glyoxylate cycle (Figure 4). This trajectory allows organisms to grow on 2-carbon substrates to fill their oxaloacetate. Examples of 2-carbon substrates include acetate and other fatty acids as well as long-chain n-alkanes. These substrates do not provide a 3-carbon intermediate such as PEP which can be carboxylated to form oxaloacetate. In the derivation of glyoxylate, the isocitrate of the TCA cycle is cleaved into glyoxylate and succinate by the enzyme isocitrate lyase. The liberated glyoxylate is combined with acetyl-CoA to form malate through the action of the enzyme malate synthase. Both succinate and malate generate oxaloacetate through the TCA cycle. . The expression of the genes encoding glyoxylate passage enzymes is tightly controlled, and normally these genes are repressed when 3-carbon compounds are available. Jan . coli, for example, the genes encoding the glyoxylate passage enzymes are located on the aceBAK operon and are controlled by several transcriptional regulators: icIR (A. Sunnarborg et al., J. Bacteriol., 163, 2642-2649 (1990 )), fadR (W. Nunn et al., J.
Bacteriol. , 148, 83-90 (1981)), fr u R (A. Chia et al., J_ ^ Bacteriol., 171, 2424-2434 (1989)), and arcAB (S. luchi et al., J. Bacteriol. ., 171, 868-873 (1989), S. luchi et al., Proc. Nati, Acad. Sci. USA, 85, 1888-1892 (1988)). Glyoxylate step enzymes are not expressed when E. c or l i is developed on glucose, glycerol, or pyruvate as a carbon source. The derivation of glyoxylate is induced by fatty acids such as acetate (Kornberg, Biochem 99, 1-11 (1966)). Several metabolic engineering strategies have been sold, with little success, in an effort to overcome the rigidity of the network surrounding carbon metabolism. For example, overexpression of the native enzyme PEP carboxylase in E. co l i was shown to increase the carbon flux towards oxaloacetate (C. Millard et al., Appl. Environ, Microbiol., 62, 1808-1810 (1996); W. Farmer et al, Appl. Env. Microbiol. , 63, 3205-3210 (1997)); however, such genetic manipulations also cause a decrease in glucose uptake (P. Chao et al., Appl. Env. Microbiol., 59, 4261-4265 (1993)), since PEP is a co-substrate required to transport glucose via the phosphotransferase system. An attempt to improve lysine biosynthesis in Corynebacterium glutamicum by overexpression of PEP carboxylase was probably not successful (J. Cremer et al., Appl. Env. Microbiol., 57, 1746-1752 (1991)). In another approach to deviate the carbon flux towards oxaloacetate, the glyoxylate derivative in E. coli was not repressed, leaving one of the transcriptional regulators, fR, out of combat. Only a slight increase in biochemicals derived from oxaloacetate was observed (W. Farmer et al., Appl. Environ. Microbiol., 63, 3205-3210 (1997)). In a different approach, the enzyme enzyme from Ascaris suum was overproduced in the E. coli mutant that was deficient for the enzymes that convert pyruvate to lactate, acetyl-CoA, and format. This causes pyruvate to be converted to malate that increases succinate production (see Fig. 2). However, this approach is problematic, since the mutant strain in question can not grow under the strict anaerobic conditions which are required for the optimal fermentation of glucose to organic acids (L. Stols et al., Appl. Biochem. Biotechnol. , 63-65, 153-158 (1997)). A metabolic engineering approach that successfully overcomes the network stiffness that characterizes carbon metabolism and diverts more carbon towards oxaloacetate, which increases the performance of biochemicals derived from oxaloacetate by amount of glucose added, could represent a significant advance and advance in the expected field for a long time.
DESCRIPTION OF THE INVENTION
The present invention employs a unique metabolic engineering approach which overcomes a metabolic limitation that the use of cells regulates the synthesis of biochemical oxaloacetate. The invention uses metabolic engineering to divert more carbon from pyruvate to oxaloacetate using the enzyme pyruvate carboxylase. This feeding can be done by introducing a natural (i.e., endogenous) and / or foreign or foreign (i.e. heterologous) nucleic acid fragment which encodes a pyruvate carboxylase in a host cell, so that a functional pyruvate carboxylase is overproduced in the cell. Alternatively, the DNA of a cell that endogenously expresses a pyruvate carboxylase can be mutated to alter the transcription of the natural pyruvate carboxylase gene so as to cause the overproduction of the natural enzyme. For example, a mutated chromosome can be obtained by employing either chemical mutagenesis or transposition and then selecting mutants with improved pyruvate carboxylase activity using methods that are well known in the art. The overexpression of pyruvate carboxylase causes the carbon flux to be deviated preferentially towards the oxaloacetate and thus increases the production of biochemicals which are bios intiated from oxaloacetate as a metabolic precursor.
Accordingly, the present invention provides a metabolically engineered cell that overexpresses pyruvate carboxylase. The overexpression of pyruvate carboxylase is preferably effected by transforming the cell with a DNA fragment encoding a pyruvate carboxylase which is derived from an organism which endogenously expresses pyruvate carboxylase, such as Rhi zobium etli or Pseudomonas fluorescens. Optionally, the metabolically engineered cell of the invention overexpresses PEP carboxylase in addition to pyruvate carboxylase. Also optionally, the cell engineered by metabolic engineering does not express a detectable level of PEP carboxylase. In a particularly preferred embodiment of the invention, the metabolically engineered cell is a C cell, Glutamicum, E. coli, Brevibacterium flavum, or Breviba c ter ium lactofermentum which expresses a heterologous pyruvate carboxylase. The invention also includes a method for making a metabolically engineered cell that involves the transformation of a cell with a nucleic acid fragment containing a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity, to produce an engineered cell. Metabolic that expresses pyruvate carboxylase. The method optionally includes the co-transformation of the cell with a nucleic acid fragment containing a nucleotide sequence encoding an enzyme having the PEP carboxylase activity so that cells metabolically engineered also overexpress the PEP carboxylase . Also included in the invention is a method for producing a biochemical derived from oxaloacetate which includes providing a cell that the biochemical produces; transforming the cell with a nucleic acid fragment containing a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity; expressing the enzyme in the cell to cause increased biochemical production; and isolate the biochemist from the cell. Preferred biochemists having oxaloacetate as a metabolic precursor include, but are not limited to, amino acids such as lysine, asparagine, aspartate, methionine, threonine, and isoleucine; organic acids such as succinate, malate and fumarate; pyrimidine nucleotides; and porphyrins. The invention further includes a nucleic acid fragment isolated from P. fluorescens which contains a nucleotide sequence encoding a pyruvate carboxylase enzyme, preferably the a4β4 pyruvate carboxylase enzyme produced by P. fluorescens.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. The aerobic trajectory in E. coli showing glycolysis, the TCA cycle, and the biosynthesis of biochemicals derived from oxaloacetate; interrupted lines mean that multiple steps are required for compound biosynthesis while solid lines mean a one-step conversion; the participation of PEP in glucose absorption is shown by a thin line: the trajectory as shown is not stoichiometric, nor does it include cofactors.
Figure 2. The anaerobic trajectory in E. c or l i showing the glycolysis and biosynthesis of selected biochemical oxaloacetate derivatives; the participation of PEP in the absorption of glucose is shown by the interrupted line; the trajectory as shown is not stoichiometric, nor does it include all cofactors. Figure 3. Biological pathways that directly regulate the intracellular levels of oxaloacetate; not all organisms contain all of these enzymes; E. c or l i, for example, do not contain pyruvate carboxylase. Figure 4. The TCA cycle, which shows the entry into the cycle of intermediaries with 3 carbons and also includes the derivation of glyoxylate for intermediates of 2 carbons
(darker arrows). Figure 5. Kinetic analysis of pyruvate carboxylase activities for MG1655 pUC18 (Q) and MG1655 pUC18-pyc (•) with respect to pyruvate. Figure 6. Effects to increase aspartate concentrations on pyruvate carboxylase activity.
Figure 7. 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 (0). Figure 8. Growth of a strain E. c or l i without expression of pp c that contains either pUC18 or the pUC18-pyc construct in the minimum medium that uses glucose as a single carbon source. Figure 9. Effect of nicotinamide nucleotides on the activity of pyruvate carboxylase: NADH (O), NAD + (Ü), NADPH (?) And NADP + (0). Figure 10. Growth configuration and selected fermentation products of wild-type strain (MG1655) under strict or severe anaerobic conditions in a limited glucose medium (10 g / L); concentrations of glucose (•), succinate (B), lactate (O), format (D) and dry cell mass (?) were measured. Figure 11. Growth configuration and fermentation products selected from wild-type strain with expression vector
(MG1655 / pUC18) / cloning of pUC18 under stringent anaerobic conditions in a limited glucose medium (10 g / L); concentrations of glucose (•), succinate (U), lactate (O), format (D) and dry cell mass (?) were measured. Figure 12. Growth or culture configuration and fermentation products selected from wild type strain with pyc gene (MG1655 / pUCl 8-pyc) under strict anaerobic conditions in a limited glucose medium (10 g / L); concentrations of glucose (•), succinate (M), lactate (O), format (D) and dry cell mass (?) were measured. Figure 13. Growth or culture configuration and production of threonine in the strain producing the ßIM-4 threonine (ATCC 21277) containing either pTrc99A or pTrc99A-pyc under strict aerobic conditions in a limited glucose medium (30 g / L ); optical density in the strain containing pTrc99A (O), optical density in the strain containing pTrc99A-pyc (D), concentrations of threonine in the strain containing pTrc99A (•), and concentrations of threonine in the strain containing pTrc99A- pyc (M) were measured.
DETAILED DESCRIPTION OF THE INVENTION Metabolic engineering genetically involves the overexpression of particular enzymes at critical points in a metabolic pathway, and / or blocking the synthesis of other enzymes, to overcome or avoid the "paralyzed" metabolic. The goal of metabolic engineering is to optimize the value and conversion of a substrate into a desired product. The present invention employs a unique metabolic engineering approach which overcomes a metabolic limitation that uses cells to regulate the synthesis of biochemical oxaloacetate. Specifically, the cells of the present invention are engineered to overexpress a functional pyruvate carboxylase, which results in increased levels of oxaloacetate. Genetically engineered cells are referred to herein as "metabolically engineered" cells when genetic engineering is aimed at disrupting or altering a metabolic pathway so as to cause a change in carbon metabolism. An enzyme is "overexpressed" in a cell engineered by metabolic engineering when the enzyme is expressed in the metabolically engineered 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 the enzyme in the cell is considered an "overexpression" of this enzyme for purposes of the present invention. Many organisms can synthesize oxaloacetate from either PEP via the enzyme PEP carboxylase, or from pyruvate via the biotin-dependent pyruvate carboxylase enzyme. The representatives of this class of organisms include C. Glutamicun, R. etli, P. fluorescens, Pseudomonas ci t roñe 1 lo 1 i s,
Azotobacter vinelandii, Asperg i 1 lus n idulans, and rat liver cells. Other organisms can not synthesize oxaloacetate directly from pyruvate because they lack the enzyme pyruvate carboxylase. E. coli, Fibrobacter succinogenes, and Ruminococcus flaviens are representative of this class of organisms. In any case, the closeness of the metabolic engineering of the present invention can be used to redirect carbon to oxaloacetate and, as a result, improves the production of biochemicals which use oxaloacetate as a metabolic precursor. The cell that is metabolically engineered according to the invention is not limited in any way to any particular type or cell type. It can be a eukaryotic cell or a prokaryotic cell; it may include, but is not limited to, a human, animal, plant, insect, yeast, protozoan, bacterial, or antibacterial cell. Preferably, the cell is a microbial cell, more preferably a bacterial cell. Advantageously, the bacterial cell can be an E. coli cell, C. glutamicum. B. flavum or B. lactofermentum; these strains are commonly used in the industry to make amino acids which can be derived from oxaloacetate using bacterial fermentation processes. The strains of E. mutant coli are being commonly considered for the commercial synthesis of succinate via anaerobic fermentation (L. Stols et al., Appl. Environ Microbiol., 63, 2695-2701 (1997); L. Stols et al APP1 Biochem. Biotech. , 63, 153-158 (1997)), although A. succini ciproducen s has been considered in the past. Rhizopus fungi have now been considered for producing fumarate via aerobic fermentations (N. Cao, Appl. Biochem. Biotechnol., 63, 387-394 (1997), J. Du et al., Appl. Biochem. Biotech., 63, 541-556 (1997)). The bacterium lacking endogenous pyruvate carboxylase, such as E. coli, Fibrobacter succinogenes, and R. f lac ians, can be used in the metabolic engineering strategy described by the invention. Optionally, the cell engineered by metabolic engineering has been engineered to interrupt, block, attenuate or inactivate one or more metabolic pathways that entrain carbon away from the oxaloacetate. For example, alanine and valine typically can be bios intiated directly from pyruvate, and by inactivating the enzymes involved in the synthesis of either or both of these amino acids, the production of oxaloacetate can be increased. Thus, the metabolically engineered cell of the invention can be an alanine and / or an auxotrophic valine, more preferably an alanine C. gl u t a m i c um and / or an auxotrophic valine. Similarly, the metabolically engineered cell can be subjected to some technique to reduce or eliminate the production of PEP carboxykinase, which catalyzes the formation of PEP from the oxaloacetate (the inversion of the reaction catalyzed by PEP carboxylase). The prevention or reduction of the expression of a functional carboxykinase PEP will result in more carbon derived from oxaloacetate and consequently the amino acids and organic acids bios int et i zados of the same. Another alternative involves interfering with the metabolic path used to produce acetate from acetyl CoA. The interruption of this trajectory will result in higher levels of acetyl CoA, which can then indirectly result in increased amounts of oxaloacetate. Further, where the pyruvate carboxylase enzyme that is expressed in the metabolically engineered cell is that which is activated by acetyl CoA (see below), the higher levels of acetyl CoA in these mutants leads to the increased activity of the enzyme, causing the additional carbon to flow from pyruvate to oxaloacetate. Thus, acetate mutants "are cells treated by metabolic engineering, preferred. The pyruvate carboxylase expressed by the metabolically engineered cell 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 genes encoding a pyruvate carboxylase contains a heterologous pyruvate carboxylase. The heterologous nucleic acid fragment may or may not be integrated into the host genome. The term "pyruvate carboxylase" means a molecule having pyruvate carboxylase activity, ie capable of catalyzing the carboxylation of pyruvate to produce oxaloacetate. The term "pyruvate carboxylase" thus includes pyruvate carboxylase enzymes that are present naturally, together with fragments, derivatives, or other chemical, enzymatic or structural modifications thereof, including enzymes encoded by insertion, deletion or mutant sites of genes of pyruvate carboxylase that are naturally present, while the pyruvate carboxylase activity is retained. The 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 Saccha romyces 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. Myers et al., Biochemistry, 22, 5090-5096 (1983)); pyruvate carboxylase from Schizosaccharomyces pombe (Gen bank D78170); pyruvate carboxylase of R. et 1 i
(GenBank U51439; M. Dunn et al., J. Bacteriol.,
178, 5960-5070 (1996)); Pyruvate carboxylase from Rattus norvegicus (GenBank U81515; S. Jitrapakdee et al., J. Biol. Chem., 272, 20522-20530 (1997)); pyruvate carboxylase from Bacillus stear or thermophilus (GenBank D83706, H. Kondo, Gene, 191, 47-50 (1997), S. Libor, Biochemistry, 1_8_, 3647-3653 (1979)); pyruvate carboxylase of P.
fluorescens (R. Silvia et al., J. Gen. Microbiol.93, 75-81 (1976); and pyruvate carboxylase from C. glutamicum (GenBank Y09548) Preferably, pyruvate carboxylase expressed by metabolically engineered cells is derived from R. etli or P. fluorescens Pyruvate carboxylase in R. etli is encoded by the pyc gene (M. Dunn et al., J. Bacteriol., 178, 5960-5970 (1996).) The enzyme R. etli is classified as an a4 pyruvate carboxylase, which is inhibited by aspartate and requires acetyl CoA for activation Elements of this class of pyruvate carboxylases will not appear particularly well suited for use in the present invention, since they redirect the flow of carbon from pyruvate to oxaloacetate could be expected to cause reduced production of acetyl CoA, and increased aspartate production, both of which will decrease the activity of pyruvate carboxylase, however, the expression of pyruvate carboxylase R. etl In a bacterial host, it is shown here that it is effective to increase the production of oxaloacetate and its downstream metabolites (see Examples I and II). In addition, this can be done without adversely affecting the uptake of glucose by the host (see Example III) which has been an obstacle in previous efforts to divert carbon to oxaloacetate by overexpression of PEP carboxylase (P. Chao et al., Appl. Env. Microbiol., 59, 4261-4265 (1993)). In a particularly preferred embodiment, the metabolically engineered cell expresses an a4β4 pyruvate carboxylase. The elements of this class of 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. 1 ' . Fl u o re s c is a known organism to express a pyruvate carboxylase a4ß4. The metabolically engineered cell of the invention is, therefore, preferably one that has been transformed with a nucleic acid fragment isolated from P. fl uo re s cen s which contains a nucleotide sequence encoding a pyruvate carboxylase expressed herein, more preferably the isolated pyruvate carboxylase and is described in R. Silvia et al., J. Gen
Microbiol. , 93, 75-81 (1976), which is incorporated herein by reference, in its entirety. Accordingly, the invention also includes a fragment of nucleic acid isolated from P. fl uo re s cen s which includes a nucleotide sequence encoding a pyruvate carboxylase, most preferably a nucleotide sequence encoding pyruvate carboxylase isolated and described in R. Silvia et al., J. Gen. Microbiol. , 93, 75-81 (1976). The metabolically engineered cell of the invention is made by transforming a host cell with a nucleic acid fragment comprising a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity. Transformation methods for animal, plant and bacterial cells are well known in the art. Bacterial transformation methods include electroporation and chemical modifications. The transformation produces a cell metabolically engineered that overexpresses pyruvate carboxylase. The preferred cells and pyruvate carboxylase enzymes are as described above in relation to the metabolically engineered cell of the invention. Optionally, the cells are further transformed with a nucleic acid fragment comprising a nucleotide sequence encoding an enzyme having PEP carboxylase activity to produce a metabolically engineered cell that also overexpresses pyruvate carboxylase, also as described above. The invention is broadly understood because it includes methods for making various embodiments of the cells treated by metabolic engineering of the invention described herein. Preferably, the nucleic acid fragment is introduced into the cell using a vector, although "naked DNA" can also be used. The fragment of the nucleic acid may be circular or linear, single-stranded or double-stranded, and may be DNA, RNA, or any modification or combination thereof. The vector can be a plasmid, a viral vector or a cosmid. The selection of a plasmid vector or skeleton depends on a variety of desired characteristics in the resulting construct, such as a selection marker, plasmid reproduction rate, and the like. Plasmids suitable for expression in E. c or l i for example, include pUC (X), pKK223-3, pKK233-2, pTrc99A, and pET- (X) where (X) denotes a vector family in which numerous constructs are available. The 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. The pET- (X) vectors can be obtained from Promega (Madison, WT) Stratagene (La Jolla, CA) and Novagen (Madison, WT). To facilitate replication within a host cell, the vector preferably includes an origin of replication (known as an "ori") or replicon. For example, CoIEl and P15A replicons are commonly used in plasmids that are to be propagated in E. c or l i. The nucleic acid fragment used to transform the cell according to the invention, optionally can include a promoter sequence operably linked to the nucleotide sequence encoding the enzyme to be expressed in the host cell. A promoter is a fragment of DNA which causes the transcription of the genetic material. Transcription is the formation of an RNA chain according to the genetic information contained in the DNA. The invention is not limited by the use of any particular promoter, and a wide variety is known. The promoters act as regulatory signals that bind the RNA polymerase in a cell to initiate the transcription of a downstream
(3 'address) that encodes the sequence. A promoter is "operably linked" to a nucleic acid sequence if it is, or can be used to, control or regulate the transcription of this nucleic acid sequence. The promoter used in the invention can be a constituent or an inducible promoter. It may be, but it does not need to be, heterologous with respect to the host cell. Preferred promoters for bacterial transformation include l a c, l a c UVS, t a c, t r c, T7, SP6 and a ra. The fragment of the nucleic acid used to transform the host cell can optionally include a Shine Dalgarno site (eg, a ribosome binding site) and a start site (eg, the ATG codon) to initiate the translation or translation of the message transcribed to produce the enzyme. A termination sequence for the final translation may also optionally be included. A terminator sequence is typically a codon for which there is no corresponding aminoacet i 1 -tRNA, thus terminating polypeptide synthesis. The nucleic acid fragment used to transform the host cell may optionally include a transcription termination sequence. Terminators rmB, which is an extension of DNA containing two terminators, TI 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 encodes a gene product, usually an enzyme, which inactivates or otherwise detects or is detected by a compound in the culture medium. For example, the inclusion of a marker sequence can render the transformed cell resistant to an antibiotic, or it can confer the specific metabolism of the compound on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol and tetracycline. In a preferred embodiment, the host cell, preferably E. coli, C. glutamicum, B. flavum or B. lactofermentum, is transformed with a nucleic acid fragment comprising a pyruvate carboxylase gene, preferably a gene that is isolated from R. etli or P. fluorescens, most preferably the gene pyc of R. etli, so that the gene is transcribed and expressed in the host cell to cause the increased production of oxaloacetate and, consequently, the increased production of the metabolite of interest current below, relative to a comparable natural type cell . The metabolically engineered cell of the invention overexpresses pyruvate carboxylase. Established otherwise, the metabolically engineered cell expresses pyruvate carboxylase at a level greater 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 skilled in the art and occurs under comparable growth conditions. For example, the activity of pyruvate carboxylase can be quantified and compared using the method of Payne and Morris (J. Gen. Microbiol., 59, 97-101 (1969)). The metabolically engineered cell overexpressing pyruvate carboxylase will produce a higher activity than a wild-type cell in this assay. In addition, or alternatively, 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 the pools. of detection which are commercially available from, for example, Pierce Chemical Company (Rockford, IL), Sigma Chemical Company (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN) to visualize biotinylated proteins on Western blots. In some suitable host cells, the expression of pyruvate carboxylase in the wild-type cell, without engineering treatment, can be subsequently detectable levels. Optionally, the metabolically engineered cell of the invention also overexpresses PEP carboxylase. In other words, the metabolically engineered cell optionally expresses the PEP carboxylase at a level higher than the level of PEP carboxylase expressed in a comparable wild-type cell. Again, this comparison can be made in any number of ways by one skilled in the art and occurs under comparable culture conditions. For example, the activity of PEP carboxylase can be tested, quantified and compared. In one assay, PEP carboxylase activity is measured in the absence of ATP using PEP instead of pyruvate as the substrate, observing the appearance of CoA-dependent robenzoate formation at 412 nm (see Example III). The metabolically engineered cell that overexpresses PEP carboxylase will produce greater PEP carboxylase activity than a wild-type cell. In addition, or alternatively, the amount of PEP carboxylase can be quantified and compared by preparing the protein extracts from the cells, subjecting them to SDS-PAGE, transferring them to a Western blot, then detecting the PEP protein carboxylase using PEP antibodies in the cells. conjunction with detection kits available from Pierce Chemical Company (Rockford IL), Sigma Chemical Company (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN) to visualize the antigen-antibody complexes on Western blots. In a preferred embodiment, the metabolically engineered cell expresses PEP carboxylase derived from a cyanobacteria, most preferably An a cys t i s n i du l a n s. The invention also includes a method for producing a biochemical derivative of oxaloacetate by improving or increasing the production of biochemicals in a cell which is, prior to transformation as described herein, capable of interacting with the biochemist. The cell is transformed with a nucleic acid fragment comprising a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity, the enzyme is expressed in the cell so as to cause increased production of the biochemical relative to a wild type cell , comparable, and the biochemical is isolated from the cell according to known methods. Biochemists can be isolated from cells treated by metabolic engineering using protocols, methods and techniques that are well known in the art. For example, succinic acid can be isolated by elect rodiáli sis (D. Glassner, U.S. Patent No. 5,143,833 (1992)) or by precipitation as calcium succinate (R. Datta, U.S. Patent No. 5,143,833 (1992)); Malic acid can be isolated by reading t rodiáli s i s
(R. Sieipenbus ch, North American Patent, No.
4,874,700 (1989)); lysine can be isolated by adsorption / inverted osmosis (T. Kanedo et al., US Patent No. 4,601,829 (1986)). Preferred host cells, biochemical oxaloacetate derivatives, and pyruvate carboxylase enzymes are as described herein. The biochemicals that are produced or are produced in, and are isolated from, the cells treated by metabolic engineering according to the method of the invention, are those that are or can be derived from the oxaloacetate (i.e. , with respect to this oxaloacetate is a metabolic precursor). These biochemical oxaloacetate derivatives include, but are not limited to, amino acids such as lysine, asparagine, aspartate, methionine, threonine, arginine, glutamate, glutamine, proline and isoleucine; organic acids such as succinate, malate, citrate, isocitrate, α-ketoglutarate, succinyl-CoA and fumarate; pyrimidine nucleotides; and porphyrins such as cytochromes, hemoglobins, chlorophylls, and the like. It is understood that the terms used herein describe acids (eg, the terms succinate, aspartate, glutamate, malate, fumarate, and the like) are not intended to denote any particular ionization state of the acid, and are understood to include both protonated forms and deprotetions of the compound. For example, the terms aspartate and aspartic acid refer to the same compound and are used interchangeably, as well as succinate and succinic acid, malate and malic acid, fumarate and fumaric acid, and so on. As is well known in the art, the state of protonation of the acid depends on the pKa of the acid group and the pH of the medium. At a neutral pH, the acids described herein are typically disproportionated. In a particularly preferred method, lysine and succinate are produced in and are obtained from a metabolically engineered bacterial cell expressing pyruvate carboxylase, preferably pyruvate carboxylase derived from either R. e t l i o P. fl u o r e s c e n s. The method of the invention is to be broadly understood because it includes the production and isolation of any or all of the oxaloacetate-derived biochemicals recovered or recoverable from metabolically engineered cells of the invention, with respect to whether the biochemicals are actually synthesized to from the oxaloacetate according to the metabolic trajectories shown in Figures 1-3 or any other currently known metabolic trajectories. The advantages of the invention are illustrated by the following examples. However, the particular materials and amounts thereof cited in these examples, as well as other conditions and details, are being interpreted for broad application in the art and should not be constructed to unduly restrict or limit the invention in any way.
Example 1. Expression of the Pyruvate Enzyme Carboxylase R. e t l i Allow E. c or l i Convert Pyruvate to Oxaloacetate
Materials and Methods Bacterial strains, plasmids and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Strains of E. c or l i were grown in cultures of LB Miller (rich) or minimal mean M9 (J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972)). Strains carrying a plasmid were supplemented with the aprotic antibiotic to detect the marker gene; ampicillin was used at 100 μg / ml in rich medium and 50 μg / ml in the minimum medium, while chloramphenicol was used at 20 μg / ml in rich medium and 10 μg / ml in the minimum medium. When isopropyl β-D-t iogalactopiran ida (IPTG) was used to induce the pUC18-pyc construct, it was added at a final concentration of 1 mM.
TABLE 1: Strains and Plasmids
Strains Genotype Reference or source MC1061 araD139? (AraABOIC- M. Casadaban et al., J. Mol. Leu) 1619? (2ac) 74 gaJU Biol., 138, 179-207 (1980) galK rpsL hsr hs * ALS225 MC1061 F 'Ladq ^ + Y + A + t E. Altman, University of Georgia
MG1665 wt M. Guyer et al., Quant. Biol., Cold Spring Harbor Symp. , 45, 135-140 (1980) JCL 1242? (ArgF-lac) U169 ppcnKn P. Chao er al., Appl. Env. Microbiol., 59, 4261-4265 (1993)
Plasmids Characteristics Reference or source Relevant pUC18 Amp (R), ColEl ori J. Norrander et al., Gene. , 26, 101-106 (1983) pPCl Tet (R), pyc M. Dunn et al., J. Bacteriol., 178, 5960-5970 (1996) pUC18-p and Amp (R), pyc regulated by this Example Plac , ColEl ori pBAll Cam (R), £ > irA, P15A D. Barker et al. , J. Mol. ori Biol. , 146, 469-492 (1981)
Construction of pUC18-pyc. The R gene. e t l i pyc, which encodes pyruvate carboxylase, were amplified using the polymerase chain reaction (PCR). The Pfu polymerase (Stratagene, La Jolla, CA) was used in place of the Taq polymerase and the plasmid pPCI served as the DNA annealing. The primers were designated based on the published pyc gene sequence (M. Dunn et al., J. Bacteriol., 178, 5960-5970 (1996)) to convert the initiation signals of the translation or translation of pyc to join with those of the lacZ gene. These primers also introduced a Kp n I restriction site (GGTACC) at the start of the amplified fragment and a BglII restriction (AGATCT) at the end of the amplified fragment; the forward primer 5'TAC TAT GGT ACC TTA GGA AAC AGC TAT_ GCC CAT ATC CAA GAT ACT CGT T 3 '(SEQ ID NO: l), reverse primer 5' ATT CGT ACT CAG GAT CTG AAA GAT CTA ACÁ GCC TGA CTT TAC ACA ATC G 3 '(SEQ ID NO: 2) (the Kpn l, Shine Dalgarno, ATG bootstrap, and B gl II sites are underlined). The resulting 3.5 kb fragment was isolated gel, restricted with K p n I and BglII and then ligated to the pUCld gel isolated DNA which has been restricted with Kp n I and Bamti I to form the pUC18-pyc construct. This construction or construct, identified as "ALS225 pUC18-pyc Co-plasmid in E. co li", was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., 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 Spotting. Extracts of heat denatured cells were separated on 10% of SDS-PAGE gels as per Altman et al. (J. Bact., 155, 1130-1137 (1983)) and Western blots were performed as per Carroll and Gherardini (Infect, Immun., 64, 392-398 (1996)). The ALS225 E cells. c or l i containing either pUCld or pUC18-pyc were cultured at mid-period in the rich medium at 37 ° C both in the presence and absence of IPTG. Because ALS225 contains l a cq l in F ', the significant induction of the pUC18-pyc construct should not occur unless IPTG is added. The protein extracts were prepared, subjected to SDS PAGE, and Western spotting. Proteins that had been biotinylated i n vi were then detected using the Sigma-Biot protein detection assembly (Sigma Chemical Corp., St Louis, MO). The manufacturer's instructions were followed except that during the development of the Western blots the biotinization step of the protein was omitted, thus allowing the detection of only those proteins which have been biotinylated. Pyruvate carboxylase (PC) Enzyme assay. For measurements of pyruvate carboxylase activity, 100 mL of medium-long phase culture were collected by configuration to
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 resuspended in 4 mL of 100 mM Tris-Cl
(pH 8.0) and were subsequently subjected to cell fracture by sonication. The cell debris was removed by centrifugation at 20,000 x g for 15 minutes at 4 ° C. Pyruvate carboxylase activity was measured by the method of Payne and Morris (J. Gen. Microbiol., 59, 97-101 (1969)). In this test the oxaloacetate produced by pyruvate carboxylase was converted to citrate by the addition of citrate synthase in the presence of acetyl CoA and 5,5-di-thiobis (2-nit ro-benzoate) (DTNB) (Aldrich Chemical Co. .); the enzyme pyruvate carboxylase homot e rame ra of R. e t l i requires the acetyl A coenzyme for activation. The rate of increase in absorbance at 412 nm was observed due to the presence of the CoA-dependent formation of the 5-thio-2-ni tr oben zoa to, first after the addition of pyruvate and then after the addition of ATP . The difference between these two speeds was taken as the ATP-dependent pyruvate carboxylase activity. The concentration of the reaction components per milliliter of mixture was as follows: 100 mM Tris-Cl (pH 8.0), 5 mM MgCl2'H20, 50 mM Na HC03, 0.1 mM acetyl CoA, 0.25 mM DTNB, and 5 units ( U) of citrate synthase. Pyruvate, ATP, ADP, or aspartate, were added as specified in the Results section, later. The reaction was started by adding 50 μl of cell extract. One unit of pyruvate carboxylase activity corresponds to the formation of 1 μmol of 5-thio-2-ni t robenzoat or per mg of protein per minute.
All enzyme assays were performed in triplicate and a standard error of less than 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)).
RESULTS
Expression of the enzyme pyruvate carboxylase R. etli in E. coli. The R. etli pyc gene, which encodes pyruvate carboxylase, was amplified by PCR from pPCl and subcloned into the expression / cloning vector of pUC18 as described above. Because the signals of initiation of translation or translation of the R. etli pyc gene were not optimal (pyc of R. etli uses the start codon of rare TTA as well as also a short spacing distance between Shine Dalgarno and the start codon), translation or translation start signals were converted to bind to that of the lacZ gene which can be expressed at high levels in E. coli using a variety of expression vectors. When the induced cell extracts of the pUC18-pyc construct were tested via western blots developed to detect biotinylated proteins, a band of approximately 120 kD was detected. This value is consistent with the size assessment previously reported for the pyruvate carboxylase enzyme of R. e t l i (M. Dunn et al., J. Bacteriol., 178, 5960-5970 (1996)). By comparing serial dilutions of pyruvate carboxylase which was expressed from the construction of pUC18-pyc with the commercially obtained purified pyruvate carboxylase enzyme, it was determined that, under the fully induced conditions of pyruvate carboxylase from R. e t l i was expressed at 1% of the total cellular protein in E. c or l i. Biotin and biotin haloenzyme synthase effects on the expression of R pyruvate carboxylase. e t l i biotinylated in E. c or l i. Pyruvate carboxylase is a biotin-dependent enzyme, and regulates the formation of oxaloacetate by a two-step pyruvate carboxylation. In the first reaction step, the biotin is carboxylated with ATP and bicarbonate as substrates, whereas in the second reaction the carboxyl group of the carboxy anhydrin is transferred to pyruvate. All pyruvate carboxylases studied to date have been found to be biotin dependent and exist as multimeric proteins, but the size and structure of the associated subunits can vary considerably. Pyruvate carboxylases from different bacteria have been shown to form a, or a4ß4 structures with the size of the subunit varying from 65 to 130 kD. In all cases, however, the α-subunit of the pyruvate carboxylase enzyme has been shown to contain three catalytic fields - a biotin carboxylase field, a t ranscarboxy sa field, and a biotin carboxyl carrier protein field - which They work collectively to catalyze two-step pyruvate to oxaloacetate conversions. In the first step, a prosthetic group of biotin bound to a lysine residue is treated with carboxylate with ATP and HCO "3, while in the second step, the carboxyl group is transferred to pyruvate.The biot initiation of pyruvate carboxylase occurs after translation or translation and is catalyzed by the enzyme halotenzyme biotin synthase In this experiment, E. coli cells containing the pUC18-pyc construct were cultured under induction conditions in minimal defined medium which either does not contain biotin added, or biotin added at 50 or 100 ng / mL Specifically, MG1655 pUC18-pyc cells were grown half-grown at 37 ° C in the M9 medium containing varying amounts of biotin.The protein extracts were prepared, subjected to SDS PAGE, already stained Western The proteins which have been biotinylated in vi were then detected using the Sigma-Blot protein detection equipment, as described above. The MG1655 was used in this experiment because its growth was significantly faster than that of ALS225 in a minimal medium. Because the MG1655 does not contain lacIP1, the maximum expression of pyruvate carboxylase can be achieved without the addition of IPTG. The amount of biotinylated pyruvate carboxylase that was presented in each sample was quantified using a Stratagene Eagle Eye II Still Video. The biotope of pyruvate carboxylase that was expressed from the pUC18-pyc construct was clearly affected by biotin levels. Cells that have produced all of their n ovo biotin significantly expressed lower amounts of biotinylated protein. The addition of biotin at a final concentration of 50 ng / mL was sufficient to biotinylate all the pyruvate carboxylase that was expressed via the pUC18-pyc construct. Since the post-translational biotinylation of pyruvate carboxylase is carried out by the enzyme holoenzyme biotin synthase, the effect of the holoenzyme biotin synthase on biotinization of pyruvate carboxylase was investigated. This analysis was accompanied by the introduction of multicopies of plasmid pBAll (which contains the b i rR gene encoding the holoenzyme biotin synthase) in E cells. col i that also harbor the pUC18-pyc construct; pBAll is a pACYC184 derivative and is also compatible with pUC18-pyc. The effects of the excess enzyme holoenzyme biotin synthase were examined in a rich medium where biotin could also be present in excess. Specifically, ALS225 cells containing pUC18-pyc, or pBAll, were grown in medium-log at 37 ° C, in a rich medium containing IPTG. Protein extracts were prepared, subjected to SDS PAGE, and Western blotting, and proteins which have been biotinylated vi nally, were then detected using the Sigma-Blot protein detection equipment as described above. Barker et al. (J. Mol. Biol., 469-492 (1981)) has shown that pBAll causes a 12-fold increase in levels of the enzyme holoenzyme biotin synthase. The amount of biotinylated pyruvate carboxylase that was presented in each sample was quantified using a Stratagene Eagle Eye II Still Video System. Extracts of proteins prepared from the cells, which contain either pUC18-pyc alone or both pUC18-pyc and pBAll, provided equal amounts of the biotinylated pyruvate carboxylase protein. This result suggests that a single chromosomal copy of bi rA is sufficient to biotinylate all the pyruvate carboxylase that is expressed when biotin is present in excess. Pyruvate carboxylase of R. e t l i can convert pyruvate to oxaloacetate in E. c or l i.
To confirm that the expressed pyruvate carboxylase protein was enzymatically active in E. c or l i, the coupled enzyme assay developed by Payne and Morris was used to assess the activity of pyruvate carboxylase (J. Payne et al., J. Gen. Microbiol., 59, 97-101 (1969)). Cell extracts containing the pUC18-pyc induced construct (MG1655 pUC18-pyc), were tested for their pyruvate carboxylase activity using varying amounts of pyruvate, and compared with the controls containing the pUC18 construct (MG1655 pUC18). The ATP was added at a final concentration of 5 mM to the reaction mixture and the pyruvate carboxylase activity was determined in the presence of increased amounts of pyruvate. Figure 5 shows that the cells of E. c or l i that harbor the pUC18-pyc construct will be able to unnecessarily convert pyruvate to oxaloacetate and that the activity of pyruvate carboxylase was observed after the kinetics of Mi chae 1 i-Ment en. A Linewea-Burke design of these data revealed that the saturation constant (Km) for the expressed pyruvate carboxylase was 0.249 mM with respect to pyruvate. This value is in excellent agreement with other pyruvate 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. Biochem. Biophys., 164, 641-654 (1974)). It is well documented that pyruvate carboxylase a4 enzymes can be inhibited by either aspartate or adenosine diphosphate (ADP). Aspartate is the first amino acid that is synthesized from oxaloacetate and ADP is released when pyruvate carboxylase converts pyruvate to oxaloacetate. Pyruvate carboxylase activity in the presence of each of these inhibitors was evaluated using extracts of MG1655 cells containing the pUC18-pyc construct. The effect of aspartate was analyzed by the addition of ATP and pyruvate to the reaction mixture, at final concentrations of 5 mM and 6 mM respectively, afterwards, determining the activity of pyruvate carboxylase in the presence of increased amounts of aspartate. Figure 6 shows the pyruvate carboxylase activity that was obtained in the presence of different concentrations of aspartate. As expected, the activity of pyruvate carboxylase 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 the addition of pyruvate to the reaction mixture at a final concentration of 5 mM, then the activity of pyruvate carboxylase was determined in the presence of increased amounts of ATP. Figure 7 shows that ADP severely affected the observed pyruvate carboxylase activity and acted as a competitive inhibitor of ATP. A Lineweaver-Bur ke design of these data revealed that the saturation constant (Km) for the expressed pyruvate 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 pyruvate 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. Biochem. Biophys., 164, 641-654 (1974)). To show that the expression of pyruvate carboxylase R. e t l i in E. coli can correctly divert the carbon flux of pyruvate to oxaloacetate, it was tested if the pUC18-pyc construct could allow an E. coli strain, which contains a null allele pp c (the pp c encodes the PEP caboxylase) to grow in a minimum glucose medium. Because E. coli lacks pyruvate carboxylase and is thus only able to synthesize PEP oxaloacetate, (see Figure 3), E. coli strains which contain an interrupted ppc gene can not grow in a minimal medium, which uses glucose as the sole carbon source (P. Chao et al., App. Env. Microbiol., 59, 4261-4265 (1993)). The cell line used for this experiment was JCL1242 (ppc:: kan), which contains a kanamycin-resistant cassette that has been inserted into the ppc gene and also does not express the enzyme PEP carboxylase. JCL1242 cells containing either pUC18 or the pUC18-pyc construct were fragmented into thiamine ampicillin IPTG plates with minimal M9 glucose and incubated at 37 ° C for 48 hours. As shown in Figure 8, the E cells. coli, which contain both the null ppc allele and the pUC18-pyc construct, were able to grow on minimal glucose plates. This complementation demonstrates that a branching point can be created at the level of pyruvate, which results in the redirection of the carbon flux to oxaloacetate, and clearly shows that pyruvate carboxylase is able to divert the carbon flux of pyruvate to oxaloacetate in E. coli.
Example II The Expression of Pyruvate Carboxylase of R. etli Causes the Increased Production of Succinate in E. coli.
MATERIALS AND METHODS Bacterial strains and plasmids. The E. coli strains used in this study are listed in Table 2. The mutant lactate dehydrogenase strain designated RE02 was derived from. MG1655 by the transduction of phage Pl using the strain E. coli NZN111 (P. Bunch et al., Microbiol., 143, 187-195 (1997)).
Table 2: Strains and plasmids used
The pyc gene of R. etli was originally cloned under the control of the lac promoter (Example 1). Because this promoter is subject to catabolic repression in the presence of glucose, a 3.5 kb Xbal-Kpnl fragment of pUC18-pyc was ligated into the expression vector pTrc99A, which has been digested with Xbal and Kpnl. The new plasmid was designated pTrc99A-pyc. This plasmid, identified as "Plasmid in E. coli ALS225 pTrc99-A-pyc", was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA, 20110-2209, USA, and was assigned ATCC number 207112. The deposit was received by the ATCC on February 16, 1999. In this new construct, the transcription of the pyc gene is under the control of the artificial promoter trcy and is not subject to catabolic repression in the presence of the glucose Conditions of medium and growth. For the construction of the strain, the strains E. c or l i were grown aerobically in a Luria-Bertani medium (LB). The anaerobic fermentations were carried out in 100 mL serum bottles with 50 mL of LB medium supplemented with 20 g / L of glucose and 40 g / L of MgC03. The fermentations were finished at 24 hours to the point at which the values of all the fermentations were approximately pH 6.7, and the glucose was used completely. For strains containing the plasmid, either ampicillin or carbenicillin was added to introduce the selective pressure during fermentation. Each of these antibiotics was initially introduced at 100 μg / mL. In a series of experiments, no additional antibiotic was added during the fermentation, while in a second series of experiments, an additional 50 μg / mL was added at 7 hours and 14 hours. Pyruvate carboxylase was induced by the addition of 1 mM of IPTG. For enzyme assays, the cells were grown in an LB medium supplemented with 20 g / L glucose and buffered with 3.2 g / L Na 2 CO 3. Analysis of the fermentation product and enzyme assay. Glucose, succinate, acetate, formate, lactate, pyruvate and ethanol were analyzed by high pressure liquid chromatography (HPLC) using a Coregel 64-H ion exclusion column (Interactive Chorma t ography, San José, CA) and a Differential refractive index detector (Model 410, Waters, Milford, MA). The eluent was 4 mN H2SO4 and the column was maintained at 60 ° C. For measurements of enzymatic activity, 50 mL of the medium-log phase culture was harvested by centrifugation (10000 xg for 10 minutes at 4 ° C) and washed with 10 mL of 100 mM Tris-HCl buffer (pH 8.0 ). The cells were then resuspended in 2 mL of 100 mL of Tris-HCl buffer and subjected to cell disruption by sonication. Cell debris was removed by centrifugation (20000 x g for 15 minutes at 4 ° C). The activity of pyruvate carboxylase was measured (J. Payne et al., J. Gen. Microbiol 59, 97-101 (1969), see also Example 1), and the endogenous activities of PEP carboxylase (K. Terada et al. ., J. Biochem., 109, 49-54 (1991)), malate dehydrogenase and lactate dehydrogenase (P. Bunch et al., Microbiol., 143, 187-195 (1997)). The total protein in the cell extract was determined using the Lowry method.
RESULTS Table 3 shows that the activity of pyruvate carboxylase could be suppressed when the pTrc99-A-pyc construct is introduced into either the wild type cells (MG1655) or the wild-type cells which contain a null mutation Idh - (RE02 ). The presence of IPTG did not significantly affect the expression of other important metabolic enzymes, such as PEP carboxylase, lactate dehydrogenase and malate dehydrogenase.
or (-p < -p
TABLE 3: Enzymatic activity in exponential phase cultures
s > To stimulate the effect of the expression of pyruvate carboxylase in the distribution of the final products of the fermentation, several fermentations were performed in 50 mL serum bottles (see Table 4).
C-p O Cp Cp
TABLE 4: Effect of pyruvate carboxylase in the distribution of the product from the fermentation of glucose of E. coli
(You
The antibiotics were added either once at 0 hours at a concentration of 100 ug / L (lx) or added at 0 hours at a concentration of 100 ug / mL and again at 7 hours and 14 hours at 50 ug / hour. mL (3x). The values are the main ones of three replies and standard deviations shown in parentheses. To calculate the pure yield of each product per gram of glucose consumed, the concentration of the final product was divided by 20 g / L of glucose.
As shown in Table 4, the expression of pyruvate carboxylase caused a significant increase in succinate production in both MG1655 (wild type) and RE02 (Idh -). With MG1655, the induction of pyruvate carboxylase increased the succinate production 2.7 parts from 1.57 g / L in the control strain to 4.36 g / L, thus making succinate the major product of glucose fermentation. This increase in succinate was accompanied by decreased lactate and formate formation, indicating that the carbon was diverted away from the lactate towards succinate formation. A similar carbon shift from lactate to succinate was previously achieved by over-expression of native PEP carboxylase (C. Millard et al., App. Environ, Microbiol., 62, 1808-1810 (1996)). Table 4 also shows that ampicillin and carbenicillin were equally effective in maintaining sufficient selective pressure, and that the addition of more than either antibiotic, during fermentation, did not further increase the production of succinate. This evidence indicates that an initial dose (100 μg / mL) is sufficient to maintain the selective pressure through fermentation, a result of which may be due to the relatively high final pH (6.8) observed in the fermentation studies against the Final pH (6.0) observed in previous studies (C. Millard et al., App. Environ Microbiol, 62, 1808-1810 (1996)). Because the introduction of pyruvate carboxylase in E. co li was thus successful in the direction of more carbon to the succinate branching, we have also been interested in the determination of whether the additional carbon can be directed to the succinate by the elimination of the lactate dehydrogenase, since this enzyme also competes for pyruvate. Table 4 compares the results of the fermentations using the strain RE02 (Idh -) with or without the plasmid pTrc99A-pyc. Compared with the wild-type strain (MG1655), strain RE02 showed no significant change in succinate production. Instead, fermentations with strain RE02, whether they contained plasmid pTrc99A-pyc or not, resulted in increased formate, acetate and ethanol production, accompanied by pyruvate secretion. The fact that pyruvate was secreted in the fermentation broth indicated that the ratio of glycolysis was greater than the pyruvate utilization ratio. The observed increase in formate concentrations in the Idh - mutant may be caused by the accumulation of pyruvate, a compound which is known to exert a positive allosteric effect on the formate lyase of pyruvate (G. Savers et al., J. Bacteriol., 170., 5330-5336 (1988) With RE02, the induction of pyruvate carboxylase increased the production of suctioned 1.7 parts from 1.73 g / L in the control strain to 2.92 g / L. The increase in succinate obtained in the Idh - mutant strains was significantly lower than that obtained in the wild type strain (MG1655) .A possible explanation for this observation may be that the activity of pyruvate carboxylase was inhibited by a cellular compound which accumulated in the Idh mutants - during glycolysis, two moles of nicotinamide adenine dinucleotide (NADH) were generated per mole of glucose. NADH is then oxidized during the formation of ethanol, lactate and succinate, under anaerobic conditions. The inability of Idh mutants - to consume NADH through lactate formation, can put stress on the oxidant capacity of these strains, leading to an accumulation of NADH. Indeed, this reduced cofactor has previously been shown to inhibit pyruvate carboxylase isolated from Sa c ch a romyce s ce re vi siae (J. Cazzulo et al., Biochem., J., 112, 755-762 (1969 )). In order to clarify whether such oxidation stress may be the cause of the attenuated benefit that was observed when pyruvate carboxylase was expressed in the Idh- mutants, we investigated the effect of both oxidized and reduced nitotinamide adenine dinucleotides (NADH / NAD +) in the activity of pyruvate carboxylase. Enzyme assays were conducted with the cell-free crude extract obtained from MG1655 pTrc99A-pyc. All tests were conducted in triplicate, and the average values are shown in Figure 9. The standard deviation was not greater than 5% for all data points. NADH inhibited pyruvate carboxylase, while NAD +, NADP + and NADPH did not. The lower succinate was increased with the mutant RE02, the mutant Idh - is therefore hydrolyzed as a result of the accumulation of intracellular NADH, a cofactor which appears to inhibit pyruvate carboxylase activity.
Example III The Expression of R. e t l i of Piruvato
Carboxylase Does Not Affect Glucose Uptake in
E. c o l i in Anaerobic Fermentation
METHODS
Microorganisms and Plasmids. Strain E coli MG 1655 (wild type F "? ~; M. Guyer et al., Quant. Biol., Cold Spring Harbor Symp., 45, 135-140 (1980); see also Example 1) and the pUC18-pyc plasmid , which contains the pyc gene of R. etli (see Example 1) Medium and fermentation All 2.0 L fermentations were carried out in fe rment adore s of upper platforms New Brunswick BioFlo III 2.5 L (New Brunswick Scientific , Edison, NJ) in Lur ia -Be rt an i (LB) supplemented with glucose, 10 g / L; Na2PH04 * 7H20, 3 g / L; KH2P04, 1.5 g / L; NH4C1, 1 g / L; MgSO4; 7H20, 0.25 g / L, and CaCl2 * 2H20, 0.02 g / L. The fermenters were inoculated with 50 mL of culture that were grown anaerobically.The fermenters were operated at 150 rpm, 0% oxygen saturation. (Ingold polarophic oxygen sensor, New Brunswick Scientific, Edison, NJ), 37 ° C, and pH 6.4, which was controlled with 10% NaOH The anaerobic conditions were maintained by the sudden flow of the separating head of the ferment with carbon dioxide free of oxygen. When necessary, the medium was supplemented with an initial concentration of 100 μg / mL of ampicillin, previously shown to be sufficient to maintain the selective pressure (E jus I). Analytical methods. Cell growth was monitored by measuring the optical density (OD) (spectrophotometer DU-650, Buckman Instruments, San Jose, CA) at 600 nm. This optical density was correlated with the dry cell mass using a dry cell mass calibration curve (g / L) = 0.48 x OD. The fermentation and glucose products were analyzed by high pressure liquid chromatography, using a Coregel 64-H ion exclusion column (Interactive Chromatography, San Jose, CA) as described in Example II. The activity of pyruvate carboxylase and the endogenous activity of PEP carboxylase was measured by growing each clone separately in 160 mL serum bottles under stringent anaerobic conditions. The cultures were harvested in medium-logarithmic growth, washed and subjected to cell breakage by sonication. Cell debris was removed by centrifugation (20000 x g for 15 minutes at 4 ° C). The pyruvate 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 pyruvate as the substrate, with the appearance of the formation of ioni t CoA-dependent robenzoate at 412 nm monitored. The total protein in the cell extract was determined using the Lowry method.
RESULTS
The E co li MG1655 grew anaerobic with 10 g / L of glucose as energy and a carbon source to produce the final products shown in Figure 2. The participation of fos foenolpiruvat or in the uptake of glucose is shown by the line faded The biochemical path is not stoichiometric or all cofactors are shown. Figure 10 shows the concentrations of dry cell mass, succinate, lactate, formate and glucose with time in a typical 2 liter fermentation of this wild-type strain. Figure 11 shows these concentrations with time in the fermentation of this wild-type strain with the cloning / expression vector pUC18. After full utilization of glucose, the average final succinate concentration for the wild-type strain was 1.18 g / L, while for the wild-type strain with the pUCld vector the final concentration of succinate was 1.00 g / L. L. For these fermentations, the average concentration of final lactate was 2.33 g / L for the wild type strain and 2.27 g / L for the same strain with pUCl 8. Figure 12 shows the concentrations with time of the dry cell mass, succinate , lactate, formate and glucose in a fermentation of the strain containing the pUC18-pyc plasmid. The figure shows that the expression of pyruvate carboxylase causes a substantial increase in the final concentration of succinate 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 gL. These concentrations correspond to a 50% increase in succinate and approximately a 20% decrease in lactate concentration, indicating that carbon was diverted from lactate to succinate formation in the presence of pyruvate caboxylate. The activities of PEP carboxylase and pyruvate carboxylase were tested in free extracts of wild-type cells and strains containing the plasmid, and these results are shown in Table 5. In the wild-type strain and the strain carrying the vector there pyruvate carboxylase activity was detected, while this activity was detected in clone MG1655 / pUC18-pyc. The activity of PEP carboxylase was observed in all three strains.
Table 5. Enzymatic activity in medium-logarithmic growth culture
To determine the ratios of glucose consumption, succinate production, and production of cell mass during fermentations, each series of concentration data was returned to a fifth polynomial order. (These measurement curves are shown in Figures 10-12 with the measured concentrations). By taking the first derivative of this function with respect to time, an equation result provides these relationships, as functions of time. This procedure is analogous to the previous methods (E. Papoutsakis et al., Biotechnol Bioeng, 27, 56-66 (1985), K. Reardon et al., Biot e chnol.
Prog, 3, 153-167 (1987)) used to calculate metabolic fluxes. In the case of fermentations with both pyruvate carboxylase and PEP carboxylase present, however, the flow analysis can not be completed due to a mathematical singularity at the PEP / pyruvate nodes (S: Park et al., Biotechnol. Bioeng, 55 , 864-879 (1997)). However, using this range, glucose uptake and succinate ratios and cell mass production can be determined. Table 6 shows the results of calculating the ratios of glucose and succinate uptake and the production of cell mass in a strain E. co l i wild-type (MG1655), the wild-type strain with the cloning / expression vector pUC18 (MG1655 / pUC 18) and the wild-type strain with MG1655 / pUC 18-p and c. All units are g / Lh, and the values in parentheses represent the standard deviation of the measurements.
Cp O n cp TABLE 6. Relationships of glucose uptake, succinate production, and cell production
As these results demonstrate, the addition of the cloning vector or the vector with the pyc gene has no significant effect on the average glucose uptake during the final 4 hours of the fermentations. Indeed, the presence of the pyc gene currently increases the maximum uptake of glucose up to about 14% from 2.17% g / Lh to 2.47 g / Lh. The presence of the cloning vector pUC18 slightly reduces the succinate production ratios. As expected from the data shown in Figure 12, expression of the pyc gene resulted in an 82% increase in succinate production at the time of maximal glucose uptake, and a 68% increase in the ratio of the succinate production during the final 4 hours of fermentation. The maximum cell growth ratio (which occurred at 4-5 hours for each of the fermentations) was 0.213 g / Lh in the wild-type strain, but decreased in the presence of pUC18 (0.169 g / Lh) or pUC18-pyc (0.199 g / lH). Similarly, the total cell yield was 0.0946 g of dried cells / g of glucose consumed for the wild type, but 0.0895 g / g for the wild type with pUC18 and 0.0882 g / g for the wild-type strain with pUC18- pyc. This decrease in biomass can be due to the consumption or wear of one mole of energy unit (ATP) per mole of pyruvate converted to oxaloacetate by pyruvate carboxylase and the increased demands of protein synthesis in strains containing the plasmid. A specific cell growth relationship can not be calculated since the growth of this strain shows the logarithmic growth only for the first few hours of growth. In summary, the expression of pyruvate carboxylase of R. e t l i in E. c i l causes a significant increase in succinate production at the expense of lactate production without affecting glucose uptake. This result has dramatic ramifications for bacterial fermentation procedures, which are used to produce biochemical oxaloacetic derivatives. Because the overexpression of pyruvate carboxylase causes increased production of oxaloacetic-derived biochemists without affecting glucose uptake, this technology can be advantageously employed in fermentation processes in order to obtain more product per amount of glucose consumed or of entry. Example VI. The Expression of R. e t l i of Pyruvate Carboxylase Causes Increased Production in E. c o l i
MATERIALS AND METHODS
Strains and bacterial plasmids. The strain producing ßIM-4 threonine (ATCC 21277) was used in this study (Shiio and Nakamori, Agr. Biol. Chem., 33, 1152-1160 (1969), I. Shiio et al., US Pat. 3,580,810 (1971)). This strain was transformed with either pTRc99A-pyc (see Example II) or pTRc99A (E. Amann et al., Gene, 69, 301-315 (1988).) Media and growth conditions Aerobic fermentations were carried out in Bioflow II fermentors with a volume of 2.0 L The medium used for this fermentation contains (per liter): glucose, 30.0 g, (NH4) 2S04 10.0 g, FeS04 »H20, 10.0 mg, MnS04« H20, 5.5 mg / L; L-proline, 300 mg, L-isoleucine, 100 mg, L-methionine, 100 mg, MgSO4 * 7H20, 1 g, KH2P04, 1 g, CaCO3, 20 g, thiamine «HCl, 1 mg, d-biotin, 1 mg In order to maintain the selective pressure for strains carrying the plasmid, the medium is initially supplemented with 50 mg / L ampicillin.Also, the IPTG was added at a final concentration of 1 mmol / L to 2 hours to fermentations carried out with any of these strains s Fermentation product analysis Cell growth was determined by measuring the optical density at 550 nm of a 1:21 dilution of the sample at 0. 1M HCl Glucose, acetic acid and other organic acids were analyzed by high pressure liquid chromatography as previously described (Eiteman and Chastain, 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-aldialdehyde derivatization method (D. Hill, et al., Anal. Chem., 51, 1338-1341 (1979); V. Svedas et al. , Anal. Biochem., 101, 188-195 (1980)).
RESULTS The strain producing ßIM-4 threonine (ATCC 21277), which harbors either the control plasmid pTrc99A or the plasmid pTrc99A-pyc, which over produces pyruvate carboxylase, was grown aerobically with 30 g / L glucose as energy and carbon source and the production of threonine was measured. As shown in Figure 13, the overproduction of pyruvate carboxylase caused a significant increase in the production of threonine in strain E. c or l i that produces threonine. At 17 hours, when the initial intake glucose or consumption was consumed, a thunine concentration 0.57 g / L was detected in the parental strain harboring the control plasmid pTrc99A, while a threonine concentration of 1.37 g / L was detected. was detected in the parental strain harboring the pTrc99A-pyc plasmid. Since the final OD550 of both cultures was within 10% of each other at the end of the fermentation, the 240% increase in the concentration of threonine that caused the overproduction of pyruvate carboxylase can be considered significant. As in our anaerobic fermentation studies (see Example III), we found that glucose uptake was not adversely affected by the overproduction of pyruvate carboxylase.
Example V. Increased Lysine Synthesis by C. glutamicum
C. glutamicum, has been widely the preferred microorganism for the enzymatic production of lysine in the biochemical industry. Strains that originate naturally from C. glutamicum make more of the oxaloacetate-derived amino acids than many other known microbes. See Kirk et al., Encyclopedia of Chemical Technology, 4th Ed., Vol. 2, pp. 534-570 (1992). The strains that are commercially used to make lysine are typically those in which all the biotics branch out after the oxaloacetate, which make any amino acid instead of the plants that have been hit, thus maximizing the biosynthesis of lysine. The enzyme pyruvate carboxylase has only recently been found in C.
glutamicum, and do not appear to be highly expressed when C. glutamicum is grown in a medium, which uses glucose as the carbon source (P. Pe t er s - endi sh et al., Microbiology (Reading), 143 1095-1103 (1997); M. Kofflas et al., GenBank submission number AF038548 (admitted on December 14, 1997). Although it contains its own endogenous pyruvate carboxylase, a more convenient way to over express this enzyme in C. gl tamicum is to insert the pyc foreign gene of R.etli. Accordingly, the current construct of pUC18 as described in Examples I and II, will be transferred into C. glutamicum using the Pexo launcher vector (G. Eikmanns et al., Gene, 102, 93098 (1991)). Overexpression of pyruvate carboxylase in Corynebacterium glutamicum can also be achieved using the gene encoding pyruvate carboxylase from P. fluorescens. Carbon is expected to be diverted to lysine in an aerobic fermentation and increased lysine yield.
Example VI. Increased Lysine Synthesis by C. glutamicum Auxotrofas
Recent evidence shows that acetate, valine and alanine each accumulate in the latter stages of lysine synthesis in C. glutamicum (J. Vallino et al., Biotechnol.bioeng., 41, 633-646 (1993)). Since each of these products is derived directly from pyruvate, this observation suggests that there is a bottleneck in the path to pyruvate (see Figure 1). C. glutamicum has been designed in accordance with the invention to over-express pyruvate carboxylase, it already has an additional means of consuming pyruvate, and still, more carbon can be diverted to lysine if one or more of these pathways are blocked. The alanine and valine auxotrophs and the mutants of C. glutamicum acetate can be designed to over express the carboxylase pyruvate according to the invention, in addition, to increase the yield of lysine.
Example VII. Increased Synthesis of Threonine in C. glutamicum C. glutamicum can also be used to produce threonine, however, the strains that are used for the synthesis of threonine are different from the strains that are used for the synthesis of lysine. In threonine-producing strains, all biotic branches after oxaloacetate, which make any amino acid in the place of the threonine that has been hit, maximize threonine biosynthesis. Because the difference between the strains that produce threonines and those that produce lysine occur after the oxaloacetate knot, the metabolic engineering technology of the invention can also be applied to strains that produce threonine from C. glutamicum to increase the synthesis of threonine. The synthesis of threonine is further increased in an auxotrophic C. glutamicum as described above with Example VI, which refers to the synthesis of lysine in C. glutamicum.
Example VIII. Increase in Biochemical Production Using Pyruvate Carboxylase from Pfluores cen s One of the main reasons in the metabolic network. responsible for the regulation of intracellular levels of oxaloacetate, is also tightly controlled due to the fact that the key enzymes which are involved in this process are both positively and negatively regulated. In many organisms such as R. etli, pyruvate carboxylase requires the positive effector of the acetyl coenzyme A molecule for this activation and is recruited due to the inhibition of feedback by aspartate (P. Attwood, Int. J. Biochem., Cell. Biol., 27, 231-249 (1995), M. Dunn et al., J. Bacteriol., 178, 5960-5970 (1996)). The benefits obtained from the overproduction of R. The pyruvate carboxylase is then limited by the fact that the deviation of carbon from pyruvate to oxaloacetate reduces either the levels of acetyl coenzyme A and increases the aspartate levels. Pyruvate carboxylase of P. However, it does not require acetyl coenzyme A for its activation and is not affected by the inhibition of the feedback caused by aspartate (R. Silvia et al., J.
Gen. Microbiol., 93, 75-81 (1976)): The pyruvate carboxylase from P. fluorescens over produced may allow even more carbon flux to be diverted to the oxaloacetate. Because the genes encoding pyruvate carboxylase in the bacterium appear to be highly homologous, the P. fluorescens pyc gene can be easily isolated from a genomic library using probes which have been prepared from the R. etli gene. The gene for pyruvate carboxylase in P. fluorescens will thus be identified, isolated and cloned into an expression vector using standard genetic engineering technique. Alternatively, the pyruvate carboxylase enzyme can be isolated and purified from P. fluorescens after the activity of pyruvate carboxylase (as described in the Examples above) and can also be assayed by the biotinylated protein using stained esterns. The N-terminal amino acid sequence of the purified proteins was determined, then a degenerate oligonucleotide probe is made, which is used to isolate the pyruvate carboxylase-encoding gene from a genomic library that has been prepared from P.
fluorescens. - The pyc clone thus obtained is sequenced. From the sequence data, the oligonucleotide primers were designed in such a way that cloning of this gene into an expression vector is allowed so that the pyruvate carboxylase can be over produced in the host cell. Any method can be used to provide a vector encoding the P. fluorescens pyc gene, which is then used to transform the host E. coli or C. glutamicum cell. The pyruvate carboxylase of P. fluorescens is expressed in the host cell, and the biochemical production is increased as described in the preceding examples.
EXAMPLE IX Increase in Biochemical Production by Overexpression of Both Pyruvate Carboxylase and PEP carboxylase
Many PEP organisms can be carboxylated to oxaloacetate via PEP carboxylase or can be converted to pyruvate by pyruvate kinase (I. Shiio et al., J. Biochem., 48., 110-120 (1960); M. Jetten et al., Appl. Microbiol. Biotechnol., 41, 47-52 (1994)). A possible strategy that was tested to increase the carbon flux towards oxaloacetate in C. gl u t a m i c um was to block the carbon flux from PEP to pyruvate (see Figure 3). However, the production of lysine by the pyruvate kinase mutants was 40% less than by an original strain, indicating that pyruvate is essential for the production of high level lysine (M. Gluber et al., Appl. Microbiol. Bitechno ., 60, 47-52 (1994)). The carbon flux towards the oxaloacetate can be increased by the over-expression of PEP carboxylase in conjunction with the expressed pyruvate carboxylase without the concomitant blocking of the carbon flux from PEP to pyruvate or affecting glucose uptake. In heterotrophs such as C. glut am icum, however, PEP carboxylase requires acetyl CoA for its activation, and is inhibited by aspartate (S. Mike et al., Annals NY Acad. Sci., 272, 12 -29 (1993)); Here the amplification of the C. gl u t am i c um genes from PEP carboxylase has not resulted in increased lysine yield (J. Kremer et al., App. Environ.
Microbiol. , 57, 1746-1752 (1991)). PEP carboxylase isolated from the cyanobactia Anacys tis nidulans, however, does not require acetyl CoA for activation or is inhibited by aspartate (M. Utter et al., Enzymens, 6, 117-135 (1972 )). Therefore, this heterologous enzyme can be used to increase the carbon flux to the oxaloacetate in C. glutamicum. The genes encoding PEP carboxylase in A. nidulans have been isolated and cloned (T. Kodaki et al., J. Bioche,., 97, 533-539 (1985)).
Example X. Increase of Biochemical Production by Breakdown of the pck Gene Coding the PEP Carboxylase in Conjunction with the Pyruvate Carboxylase Over Expressed
Some carbon which is diverted to the oxaloacetate via the pyruvate carboxylase over produced, is similarly converted back to PEP due to the presence of PEP carboxykinase. More carbon can be diverted to the oxaloacetate in these systems if the host cell contains a broken pck gene, such as an E strain. co l i, which contains a null allele p ck (for example A. Goldie, J. Bacteriol., 141, 1115-1121 (1980)).
The complete description of all patents, patent documents and publications cited herein are incorporated by reference. The detailed description and examples mentioned above have been given for clarity of understanding only. They are not unnecessary limitations to be understood from these. The invention is not limited to the exact details shown and described, variations obvious to one skilled in the art will be included within the invention defined by the claims.
LIST OF SEQUENCES < 110 > The University of Georgia Research Foundation, Inc
< 120 > OVEREXPRESSION OF PIRUVATE CARBOXYLASE FOR IMPROVED PRODUCTION OF BIOCHEMICAL DERIVATIVES OF MICROBIAL CELL OXALOACETATE
< 130 > 235,00030201
< 140 > Not assigned < 141 > 1999-04-13
< 150 > 60 / 081,598 < 151 > 1998-04-13
< 160 > 2
< 170 > Patentln Ver. 2.0
< 210 > 1 < 211 > 49 < 212 > DNA < 213 > Artificial sequence
< 220 > < 223 > Description of the Artificial Sequence: forward primer < 400 > 1 tactatggta ccttaggaaa cagctatgcc catatccaag atactcgtt 49
< 210 > 2 < 211 > 49 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of the Artificial Sequence: inverted primer
< 400 > 2 attcgtactc aggatctgaa agatctaaca gcctgacttt acacaatcg 49
It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property.
Claims (50)
1. A cell treated by metabolic engineering, characterized in that it expresses a heterologous pyruvate carboxylase.
2. The cell treated by metabolic engineering according to claim 1, characterized in that it is a bacterial cell.
3. The bacterial cell according to claim 2, characterized in that it is selected from the group consisting of a Corynebacterium glutamicum cell, a cell Escherichia coli, a Brevibacterium flavum cell and a Brevibacterium lactofermentum cell. .
4. The bacterial cell according to claim 3, characterized in that it is a C. glutamicum cell.
5. The C. glutamicum cell according to claim 4, characterized in that it has at least one of the mutations selected from the group consisting of alanine-, valine- and acetate ".
6. The bacterial cell according to claim 3, characterized in that it is an E. coli cell.
7. The metabolically engineered cell according to claim 1, characterized in that it expresses pyruvate carboxylase at a level higher than the level of pyruvate carboxylase expressed in a comparable wild-type cell.
8. The cell treated by metabolic engineering according to claim 1, characterized in that it expresses a pyruvate carboxylase derived from Rhizobium etli.
9. The cell treated by metabolic engineering according to claim 8, characterized in that it has been transformed with a pyc gene of R. etli.
10. The cell treated by metabolic engineering according to claim 1, characterized in that it expresses a pyruvate carboxylase derived from Pseudomonas fluorescens.
11. The cell treated by metabolic engineering according to claim 10, characterized in that it has been transformed with a pyc gene of P. fluorescens.
12. The cell treated by metabolic engineering in accordance with the claim 1, characterized in that a comparable natural-type cell lacks an endogenous pyruvate carboxylase.
13. The metabolically engineered cell according to claim 1, characterized in that it further comprises PEP carboxylase, wherein the cell engineered by metabolic engineering overexpresses PEP carboxylase. !
14. The cell metabolically engineered according to claim 13, characterized in that it expresses PEP carboxylase derived from An a cys t i s n i du l a n s.
15. The metabolically engineered cell according to claim 1, characterized in that it further comprises PEP carboxykinase, wherein the metabolically engineered cell expresses PEP carboxykinase at a level lower than the level of PEP carboxykinase expressed in the comparable wild-type cell.
The cell treated by metabolic engineering according to claim 15, characterized in that it does not express a detectable level of PEP carboxykinase.
17. A C cell, gl u t am i c um, characterized in that it comprises a heterologous pi ruva t or carboxylase.
18. One cell E. co l i characterized in that it comprises pyruvate carboxylase.
19. A method for producing a metabolically engineered cell, characterized in that it expresses a heterologous pyruvate carboxylase comprising transforming a cell with a nucleic acid fragment comprising a nucleotide sequence encoding a heterologous enzyme having pyruvate carboxylase activity to produce a treated cell by metabolic engineering than that which expresses a heterologous pyruvate carboxylase.
20. The method according to claim 19, characterized in that the cell that is transformed is a bacterial cell.
21. The method according to claim 20, characterized in that the bacterial cell is selected from the group consisting of a C. glutamicum cell, an E. coli cell, a B. flavum cell and a B cell. The ctofermen t um.
22. The method according to claim 21, characterized in that the bacterial cell is a C. glutamicum cell.
23. The method according to claim 21, characterized in that the bacterial cell is an E. coli cell.
24. The method according to the rei indication 19, characterized in that the nucleic acid fragment comprises a nucleotide sequence selected from the group consisting of an R gene. e t l i which encodes pyruvate carboxylase and a P. fl u o re s c gene encoding pyruvate carboxylase.
25. In addition, it comprises transforming the cell with a nucleic acid fragment comprising a nucleotide sequence encoding the PEP carboxylase so that the metabolically engineered cell overexpresses the PEP carboxylase.
26. The method according to claim 19, characterized in that the cell metabolically engineered does not express a detectable level of PEP carboxykinase.
27. The method according to claim 19, characterized in that the metabolically engineered cell expresses pyruvate carboxylase at a level higher than the pyruvate carboxylase level expressed in a comparable wild-type cell.
28. A method for producing a metabolically engineered cell, characterized in that it comprises mutation of a gene of a cell, the gene encoding an enzyme having pyruvate carboxylase activity, to produce a metabolically engineered cell overexpressing pyruvate carboxylase.
29. The method according to claim 28, characterized in that the cell is a bacterial cell.
30. The method according to claim 29 characterized in that the cell is a C. gl u t ami cum cell.
31. A method for producing a biochemical oxaloacetate derivative, characterized in that it comprises: (a) providing a cell that the biochemical produces; (b) transforming the cell with a nucleic acid fragment comprising a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity; (c) expressing the enzyme in the cell to cause increased biochemical production; and (d) isolating the biochemical from the cell.
32. The method according to claim 31, characterized in that step (a) comprises providing a bacterial cell that produces the biochemical.
33. The method according to claim 32, characterized in that the bacterial cell is selected from the group consisting of a cell C. gl u t a m i c um, an E cell. co l i, a B. flavum cell and a B. lactofermentum
34. The method according to claim 33, characterized in that the bacterial cell is an E. coli cell.
35. The method according to claim 33, characterized in that the bacterial cell is the C. gl tamicum cell.
36. The method according to claim 31, characterized in that the nucleic acid fragment comprises a nucleotide sequence selected from the group consisting of a R. etli gene encoding pyruvate carboxylase and a P. fluorescens gene encoding pyruvate carboxylase.
37. The method according to claim 31, characterized in that the biochemical derived from the oxaloacetate is selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pyrimidine nucleotide.
38. The method according to claim 31, characterized in that the biochemical derivative of oxaloacetate is selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, methionine, threonine, proline, isoleucine, lysine, malate, fumarate, succinate, citrate , isocitrate, otee toglutarat oy succini 1 -CoA.
39. The method according to claim 38, characterized in that the biochemical oxaloacetate derivative is lysine.
40. The method according to claim 38, characterized in that the biochemical oxaloacetate derivative is succinate.
41. The method according to claim 38, characterized in that the biochemical derivative of oxaloacetate is threonine.
42. A method for producing a biochemical oxaloacetate derivative characterized in that it comprises: (a) providing a cell that the biochemical produces; (b) mutating a gene from a cell, the gene encoding an enzyme having pyruvate carboxylase activity; (c) overexpressing the enzyme of the mutated gene to cause increased biochemical production; and (d) isolating the biochemical from the cell.
43. The method according to claim 42, characterized in that step (a) comprises providing a bacterial cell that produces the biochemistry.
44. The method according to claim 43, characterized in that the bacterial cell is a C cell. gl u t am i c um.
45. The method according to claim 42, characterized in that the biochemical oxaloacetate derivative is selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pi rimidine nucleotide.
46. The method according to claim 45, characterized in that the biochemical oxaloacetate derivative is selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, methionine, threonine, proline, isoleucine, lysine, malate, fumarate, succinate, citrate , isocitrate, otee toglut for toy succini 1 -CoA.
47. The method according to claim 46, characterized in that the biochemical derivative of oxaloacetate is lysine
48. The method according to claim 46, characterized in that the biochemical oxaloacetate derivative is succinate.
49. The method according to claim 46, characterized in that the biochemical oxaloacetate derivative is threonine.
50. A fragment of the nucleic acid isolated from P. fl u or ress comprising a nucleotide sequence encoding a pyruvate carboxylase.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US60/081,598 | 1998-04-13 | ||
US60/082,850 | 1998-04-23 |
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MXPA00009984A true MXPA00009984A (en) | 2002-06-05 |
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