CELLS AND THEIR USE IN METHODS FOR PRODUCING PRODUCT COMPOUNDS

Abstract: Cells which are: 1) capable of converting a raw material to a product compound not produced in the glycolytic path­ way via a first bioconversion pathway which does not produce a tricarboxylic acid cycle (TAC) intermediate; 2) capable of producing a net increase in a TAC intermediate from a first carbon source via a second bioconversion path­ way inhibitable by an inhibiting compound produced in the glycolytic pathway; and 3) incapable of producing a net increase in a TAC intermediate other than via the second bioconversion pathway. The cells are used in a method to produce the product compound.

Description: This invention relates to producing a desired compound and to cells used in such production. In conducting fermentation, the cell mass should be adequate to convert raw material into product, but product yields will be reduced if raw material is squandered in producing a cell mass substantially in excess of the amount needed for a reasonable rate of raw material conversion. Accordingly, it is desirable to control cell mass, for example by controlling nutrients in the cell medium. At the same time, the method used to control cell mass should not adversely affect the ability of the cell structure to produce the desired fermentation product. According to a first aspect of the present invention, there is provided a method of producing a product compound using cells to convert a raw material into said product compound, said product compound being a compound that is not part of the glycolytic pathway, the method comprising the steps of: selecting as cells to accomplish said conversion cells that: (a) are defective in their ability to replenish tricarboxylic acid cycle (TAC) intermediates from glycolytic pathway products and b) have been engineered to convert, or enhance conversion of, said raw material to said product compound via a biosynthesis pathway that is free from any TAC intermediates; culturing the selected cells in a culture medium; and recovering said product compound from said medium. As used herein, "glycolytic pathway products" means the products in the Embden-Meyerhof pathway sequence: glucose; glucose-6-phosphate; fructose-6-phosphate; fructose-1,6-diphosphate; dihydroxyacetone-phosphate; glyceraldehyde-3-phosphate; 1,3-diphosphoglycerate; 3-phosphoglycerate; 2-phosphoglycerate; phosphoenolpyruvate (PEP); pyruvate; acetaldehyde; and acetate. The TAC intermediates are succinate, oxalacetate, malate, fumarate, 2-keto-glutarate, isocitrate, and citrate. In a second and alternative aspect of this invention, that is provided a cell characterised by permanent incapacity to form oxalacetate from glycolytic pathway products, said cell being engineered to enhance or to enable production of a desired product compound via a bioconversion pathway that is free from any tricarboxylic acid cycle (TAC) intermediates, said product compound being a compound that is not part of the glycolytic pathway. In preferred cells, there is a vector comprising DNA encoding an enzyme that catalyzes a reaction in the said bioconversion pathway and the cells are capable of replacing TAC intermediates solely via the glyoxalate shunt, which comprises reactions catalyzed by isocitrate lyase and malate synthase, and is subject to inhibition. In preferred arrangements, the cells are just cultured to a desired cell mass under growth medium conditions such that cells can replace TAC intermediates in sufficient amounts to support growth of the cells, and are thereafter cultured under product-production medium conditions such that cell growth is substantially inhibited due to inadequate replacement of TAC intermediates. In this way we provide a convenient method of controlling the extent of the cell mass. The method is not dependent on limiting any nutrient that is required for effecting the conversion of raw material to end product, and the method channels the cell's metabolism away from growth pathways, thereby increasing the efficiency of conversion of raw material to end product. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims. The single Fig 1 of the drawings is a flow diagram showing various bioconversion pathways used in growth and fermentation. The Microbial Cells In the preferred embodiment, the metabolic characteristics of the cells are illustrated in Fig. 1. In Fig. 1, cells derive energy from carbohydrates by degradation through a variety of pathways to PEP, which enters the tricarboxylic acid pathway. As used in this application, "carbohydrates" means glucose, other saccharides, and polysaccharides which the cell can hydrolyze to saccharides. The TAC includes a catabolic route (shown in solid line in Fig. 1), which produces energy using the four-carbon intermediates quasicatalytically: ie, a highly oxidized four-carbon intermediate is fused to a less oxidized two-carbon intermediate; then two relatively highly oxidized one-carbon molecules (carbon dioxide) are severed leaving a less oxidized four-carbon molecule. Useful energy is derived by oxidizing that four-carbon molecule back to the starting point; ie, oxalacetate. The net effect of this catabolic portion of the TAC is production of energy and carbon dioxide with a net loss of acetate. There is no net consumption of the four carbon TAC intermediates. The TAC has another important function in the cell, however. It provides a pool of biosynthetic precursors for a variety of important cellular components, including amino acids and nucleic acids. Thus, while the four-carbon TAC intermediates are not consumed in the above-described catabolic cycle, these intermediates are siphoned off, particularly during cell growth, to form various cellular components, and a mechanism for replacing these intermediates is needed to support cell growth. Pathways for replacing TAC intermediates are known as anaplerotic (filling-up) pathways. In one such pathway (dashes in Fig. 1), PEP is converted to oxalacetate via the enzyme phosphoenolpyruvate carboxylase. In another such pathway (dots in Fig. 1), called the glyoxalate shunt of the TAC cycle, citrate is converted (via isocitrate) to succinate and glyoxalate by the enzyme isocitrate lyase. Acetate is combined with glyoxalate by the enzyme malate synthase to yield malate. The net result of the glyoxalate shunt is the replenishing of the TAC by a gain of a four-carbon TAC intermediate at the expense of two two-carbon molecules (acetate). A cell that has lost its ability to replenish TAC intermediates from phosphoenolpyruvate can nevertheless grow on acetate, because TAC intermediates are replenished via the glyoxylate shunt. However, the glyoxylate shunt is inhibited by products of the glycolytic pathway upstream from acetate. Thus cells which are capable of growth on acetate alone are inhibited from growth by the addition of glucose. [Kornberg (1966) Biochem. J. 99 :1-11; Krulwich et al. (1976) J. Bateriol. 127 :179-183]. It is possible to develop cell strains whose growth on a non-carbohydrate such as acetate is inhibited by glycolytic pathway substances produced from carbohydrates, and which nevertheless retain their viability and their ability to produce a desired fermentation product in the presence of carbohydrates and/or acetate. As explained below, growth of such cells may be readily controlled, e.g. in a fermentation process. The products thus produced include compounds which: 1) are not produced in the glycolytic pathway; and 2) are produced by a pathway which does not produce a net increase of a TAC intermediate. The cells may be formed by modifying a parent cell to enable (or enhance the effectiveness of) a pathway for forming those product compounds, and modifying parent cells to impair their ability to form phosphoenolpyruvate carboxylase, and thus their ability to convert PEP to oxalacetate. Suitable cell lines include those belonging to the following genera: Bacillus, Streptomyces, Escherichia, Klebsiella, Salmonella, and Saccharomyces, and particularly strains derived from wild-type strains capable of converting carbohydrates to TAC intermediates. A particularly preferred species is E . coli , and strains thereof described below which are derived from the K12 strain YMC9, and which lack the ability to synthesize phosphoenolpyruvate carboxylase (ppc), an enzyme necessary for conversion of glucose to TAC intermediates, by virtue of lesions in the ppc gene. A. KB260 Strain KB260, a strain which demonstrates control of cell-mass growth, is constructed as follows. Strain YMC9 (ATCC accession No. 33927) is mutagenized with the mu d ( amp lac )1 random insertional mutator element according to the procedure of Casadaban and Cohen (Casadaben, M.J., and Cohen, S.N. Proceedings of the National Academy of Sciences 76 :4530-4533 [1979]). Cells which have been mutated re selected by growth in the presence of ampicillin, since acquisition of the mutator element results in resistance to β -lactam antibiotics. A large pool of random mutants is selectively enriched for mutants unable to synthesize arginine by growing the pooled mutants in M9 minimal salts supplemented with 2mg/ml glucose with 1 µg/ml thiamine and 40 µg/ml L-arginine at 30°C until the culture is growing logarithmically, next collecting and washing the cells by filtration, resuspending the cells in M9 minimal salts with 2mg/ml glucose and 1 µg/ml thiamine, incubating the cells at 30°C for 1 to 1.5 hours, adding D-cycloserine (final concentration: 0.002M), and collecting the viable cells which remain after lysis of the culture. Among these cells are mutants of YMC9 which cannot grow in the absence of exogenous arginine (or a biosynthetic precursor of arginine). Typical of such mutants is strain KB243 which is capable of growth when the medium is supplemented with arginine, or with either ornithine or citrulline, which are precursors of arginine. Strain KB243 is plated on agar plates containing M9 salts, 2mg/ml glucose, 1 µg/ml thiamine, 40 µg/ml L-arginine, and 0.5mg/ml succinate and incubated at 42°C. This results in the death of most cells on the plate because of lethal functions on the mud( amp lac )1 element. Among the surviving bacteria are deletion mutants which had lost portions of the mu d ( amp lac ) 1 element and surrounding DNA. An example of such a strain is KB244, which has lost genetic information for resistance to β -lactam antibiotics and is incapable of growth when provided with ornithine or citrulline instead of arginine. This last property identifies the lesions in the arginine biosynthetic pathway as falling in the genes of the arg ECBH cluster, which is linked to the ppc gene on the E . coli chromosome (Bachmann, B.J. Microbiological Reviews 47:180-230 [1983]). Strain KB244 is transduced to arginine prototrophy with Pl bacteriophage grown on E . coli strain DL10 (LeMaster, D.M. and Cronan, Jr., J.F. Journal of Biological Chemistry 257:1224-1230 [1982]) which has an unmutated arg ECBH cluster but carries a mutation in the ppc gene. Isolates are examined for growth on M9 minimal salts plus 2mg/ml glucose and 1 µg/ml thiamine, plus or minus 0.5mg/ml succinate. Strains which acquire a ppc ⁻ allele are unable to grow without the succinate. Typical of the ppc ⁻ transductants obtained is KB260. The mutation in the KB260 gene responsible for affecting phosphoenolpyruvate carboxylase (PPC) synthesis is a point mutation, and, therefore, there is the possibility of reversion, resulting in acquisition of the ability to synthesize PPC. Other strains are described below in which the loss of PPC synthesis is attributable to a type of mutation that is less susceptible to reversion. B. KB280 and KB285 Strains KB280 and KB285, which demonstrate control of cell-mass growth in a fermentation process, are constructed as follows. Strain YMC9 is mutagenized with the mud ( amp lac )1 insertional mutator element, as above. A pool of mutated cells is enriched for ppc ⁻ mutants by growing them on M9 minimal salts with 2mg/ml glucose, 1 µg/ml thiamine, and 0.5mg/ml succinate at 30°C until the culture is growing logarithmically, collecting and washing the cells by filtration, resuspending the cells in M9 minimal salts plus 2mg/ml glucose, and 1µg/ml thiamine incubating the cells at 30°C for 1 to 1.5 hours, adding D-cycloserine (final concentration: 0.002M) and collecting the viable cells which remain after lysis of the culture. Among such cells are mutants which require exogenous succinate for growth in the presence of glucose. Typical of such strains are KB279 and KB284. Cells of each strain are plated on agar plates containing M9 salts, 2mg/ml glucose, 1 µg/ml thiamine, 0.5mg/ml succinate, and 40 µg/ml L-arginine and incubated at 42°C. Among survivors are bacteria which have lost portions of the mu d ( amp lac )1 element and surrounding DNA. Such strains are no longer resistant to β -lactam antibiotics, and certain isolates have acquired a requirement for exogenous arginine for growth. This property confirms that the lesions in KB279 and KB284 are in fact in the ppc gene. Isolates KB280 (from KB279) and KB285 (from KB284) are saved. Each has lost its progenitor's resistance to β -lactam antibiotics, but neither has acquired a defect in arginine biosynthesis. KB280 and KB285 have been deposited with the American Type Culture Collection and have accession numbers ATCC 39461 and ATCC 39463, respectively. Controlling Cell-Mass . The strains described above are cultured to a predetermined cell mass, and then further cell mass growth is readily controlled. Specifically, the strain is cultivated on a carbohydrate-free medium. If carbohydrates are present initially, they will be metabolized to inhibiting compounds which will stop growth. Eventually, the carbohydrates and glycolytic intermediates may be depleted by various metabolic pathways, and growth can begin. Once desired cell mass is achieved (e.g. after about eight hours) a carbohydrate is added to inhibit continued cell growth by inhibiting the non-glucose-to-TAC pathway, thus depriving the cell of its only source of TAC cycle intermediates needed for cell growth. The amount of carbohydrate added will depend largely on whether the carbohydrate serves solely as a generator of inhibiting substance, or whether it also serves as the raw material for production of the desired compound. About 2 to 4 mg/ml of glucose is satisfactory to inhibit cell growth. More glucose is added (either at the outset of the fermentation phase or continuously during that phase) when glucose serves as the raw material for fermentation in order to maintain sufficient raw material levels to drive fermentation pathway sections. As specifically related to the above-described strains of E . coli , strain KB260 is inoculated into a culture containing 20 ml of M9 salts, 4mg/ml sodium acetate, 1 µg/ml thiamine and grown at 37°C. Culture turbidity is monitored with a Klett-Summerson Colorimeter. When the turbidity reaches 40-50 Klett units (green filter), glucose (final concentration: 4mg/ml) is added to the KB260 culture, and culture turbidity of all cultures is monitored for another nineteen hours. The number of viable cells in the KB260 culture is also determined four hours and nineteen hours after the addition of glucose. The growth rate of KB260 is like that of its parent YMC9 when grown entirely on acetate, but KB260 and YMC9 respond dramatically differently to the addition of glucose. YMC9 increases its growth rate and reaches a higher saturation density with glucose than either YMC9 or KB260 on acetate alone. KB260 in contrast, ceases accumulation of cell mass shortly after the addition of glucose, and fails to accumulate new cell mass over intermediate to long times. Unlike many other schemes in which an added substance halts cell growth, however, the addition of glucose to the KB260 culture does not significantly impair the viability of the cells. Producing The Desired Product . Depending on the desired product and the capability of the parent cells, strains may require modification, such as by the addition of a plasmid with DNA coding for an enzyme catalyzing a reaction in the product fermentation pathway. For example, to produce L-phenylalanine (a component of a non-sugar sweetener useful in a wide variety of food products) the strains described above are transformed with a plasmid pKB663 described below. A fragment of DNA carrying the phe A gene of E . coli but not its associated promoter, operator, leader peptide, or attenuator is prepared from plasmid pKB45 (Zurawski, G., et al. Proceedings of the National Academy of Sciences 75 :4271-4274 [1978]) by digestion with endonucleases StuI and BglII. Plasmid pKB430 is a derivative of pBR322 which carries a lactose operon promoter-operator abutting an endonuclease PvuII cleavage site. The phe A containing DNA fragment is cloned by standard techniques (Bolivar, F. and Backman, K. Methods in Enzymology, Vol. 68 [1980]) in pKB430 between the PvuII and BamHI cleavage sites, yielding pKB663. On pKB663, expression of phe A is directed by the lac promoter and is not regulated in response to accumulation of L-phenylalanine, as is the phe A gene when associated with its normal regulatory elements. pKB663 also carries a gene which determines resistance to β -lactam antibiotics such as ampicillin. Plasmid pKB663 is available from YMC9/pKB663 which is deposited with the American Type Culture Collection and has accession number ATCC 39462. Strains YMC9, KB280, and KB285 are transformed with pKB663 (see Bolivar, F. and Backman, K. Methods in Enzymology, Vol. 68 [1980]) yielding YMC9/pKB663, KB280/pKB663, and KB285/pKB663. Due to their tendency to shed their plasmids, these strains are maintained on agar plates containing M9 salts, 2mg/ml glucose, 0.5mg/ml succinate, 1 µg/ml thiamine, and 100 µg/ml ampicillin. Cultures of YMC9/pKB663, KB280/pKB663, and KB285/pKB663 are grown at 37°C in 20ml of M9 salts, 4mg/ml sodium acetate, 1µg/ml thiamine to a density of either 45 to 60 or 130 to 150 Klett units (green filter), at which time the culture is collected by filtration, the cells are resuspended in an equal volume of fresh medium, and glucose is added to a final concentration of 4mg/ml. The pattern of cell growth thereafter resembles YMC9 (described above) (for YMC9/pKB663) or KB260 (also described above) (for KB280/pKB663 and KB285/pKB663) following glucose addition. Incubation is continued at 37°C for 10 to 15 hours, at which point culture density, cell viability, and L-phenylalanine content of the medium are determined. Additional information concerning the method of modifying YMC9 and the method of culturing the modified cells to produce L-phenylalanine is described in our co-pending European Patent Application No. 84306841.2 (Publication No. EPO145156), the disclosure of which is hereby incorporated by reference. Other arrangements are possible within the scope of this invention. For example, the cells used may be microbial cells or cell tissue from multi-celled organisms.

Claims: A method of producing a product compound using cells to convert a raw material into said product compound, said product compound being a compound that is not part of the glycolytic pathway, the method comprising the steps of: selecting as cells to accomplish said conversion cells that: (a) are defective in their ability to replenish tricarboxylic acid cycle (TAC) intermediates from glycolytic pathway products and b) have been engineered to convert, or enhance conversion of, said raw material to said product compound via a biosynthesis pathway that is free from any TAC intermediates; culturing the selected cells in a culture medium; and recovering said product compound from said medium. A method according to Claim 1, wherein said cells are ppc-. A method according to Claim 1, wherein said cells are capable of replacing TAC intermediates solely via the glyoxalate shunt, which is subject to inhibition. A method according to any preceding Claim, wherein said product compound is produced under conditions effectively preventing replacement of TAC intermediates. A method according to any preceding Claim, comprising (a) first culturing said cells to a desired cell mass under growth medium conditions such that said cells can replace TAC intermediates in sufficient amounts to support growth of said cells, and (b) thereafter culturing said cells under product-production medium conditions such that cell growth is substantially inhibited due to inadequate replacement of TAC intermediates. A method according to any preceding Claim, wherein said cells are permanently incapable of forming oxalacetate from glycolytic pathway products, and are capable of converting acetate or acetic acid to succinate or malate via the glyoxalate shunt, in the absence of an inhibiting substance. A method according to Claim 6, wherein said inhibiting substance is glucose or a compound derived from glucose by glycolysis. A method according to each of Claims 5 to 7, wherein said growth medium lacks TAC-replacement-inhibiting levels of glucose. A method according to any preceding Claim, wherein said product compound is an aromatic amino acid. A method according to Claim 9, wherein said product compound is phenylalanine. A method according to any preceding Claim, wherein said cells are microbial cells, preferably microbial cells selected from any of the genus Bacillus, Streptomyces, Escherichia, Klebsiella, Salmonella, and Saccharomyces. A method according to Claim 11, wherein said microbial cells are members of the species Escherichia coli . A method according to Claim 12, wherein said microbial cells are E. coli K12 strain KB280, E . coli K12 strain KB285, or E . coli K12 strain YMC9, which have been engineered as defined in Claim 1. A method according to Claim 1 or Claim 7, wherein said cells comprise a cloning vector to enhance the ability of said cells to produce said product compound. A method according to Claim 14, wherein said cloning vector comprises DNA coding for an enzyme that catalyzes a step in said synthesis pathway. A method according to Claim 14, wherein said cells are enhanced in their ability to produce phenylalanine by the inclusion of a deregulated pheA gene. A cell characterised by permanent incapacity to form oxalacetate from glycolytic pathway products, said cell being engineered to enhance or to enable production of a desired product compound via a bioconversion pathway that is free from any tricarboxylic acid cycle (TAC) intermediates, said product compound being a compound that is not part of the glycolytic pathway. A cell according to Claim 17, wherein said cell is capable of replacing TAC intermediates solely via the glyoxalate shunt, which is subject to inhibition. A cell according to Claim 17 or 18, wherein said product compound is an aromatic amino acid. A cell according to Claim 19, wherein said cell is engineered to include a deregulated pheA gene. A cell according to Claim 17, wherein said cell comprises a vector comprising DNA encoding an enzyme that catalyzes a reaction in said bioconversion pathway. A cell according to Claim 17, wherein said cell is a member of genus Bacillus, Steptomuyces, Escherichia, Klebsiella, Salmonella, or Saccharomyces. A cell according to Claim 22, wherein said cell is E. coli K12 strain KB280 or E. coli strain KB285, which has been engineered as defined in Claim 17. A cell according to Claim 23, wherein said cell is deposited as ATCC Accession No. 39461 or 39463.

Inventors: BACKMAN, KEITH C.
Applicants: BIOTECHNICA INTERNATIONAL, INC., H.J. HEINZ COMPANY
Application year: 1984
Main classification: C12P1
IPC codes: C12P7, C12P19, C12P13, C12N1, C12N15, C12R1, C12P1