INTEGRATION OF A GENE INTO A CHROMOSOME AND CONTROLLABLE EXCISION THEREFROM

Abstract: Integrating a gene in a bacterial chromosome and controll­ ing excision of the gene from the chromosome in response to an external stimulus, uses DNA comprising: a) a vector that is incapable of replicating autonomously in the host and that includes the gene (or a site for the insertion of the gene) and an attachment site corresponding to an attach­ ment site on the host chromosome; b) DNA capable of causing the integration of vector a) in the bacterial chromosome and DNA capable of causing the ex­ cision of that vector therefrom; and c) DNA capable of effecting control of excision of vector a) from the bacterial chromosome in response to an externally controlled stimulus.

Description: This invention relates to a method for producing a desired compound in a bacterial cell population and to a set of DNA constructions useful therefor. As explained in more detail below DNA constructions in accordance with this invention are adapted for transfecting a host bacterial cell, for integrating a gene determining a growth-promoting function in the chromosome of the host cell, and for controllably excising the gene therefrom to yield DNA incapable of replicating in the cell. In this way the growth-promoting function is controllably lost from the cell. One instance in which controlled cessation of a cell function is useful involves microbial fermentation of a desired product. To achieve a desired microbial cell population, the cells are allowed to undergo an exponential ("log") phase of cell growth. When a suitable cell population density is reached, it may be advantageous to halt cell-growth so that cellular resources and metabolic energy are not diverted from the production of the desired compound. Ideally, a mechanism to achieve this goal should be absolute, so that some cells in the population will not circumvent the growth control and overgrow the culture. One way to control cell growth is by limiting the availability of one or more nutrients that the cell requires for growth. Once the limited amount of a nutrient is used, cell growth ceases. Cells may be mutated or engineered so that they require a nutrient in order to grow. For example, if the nutrient is a particular amino acid such as tyrosine, cells are mutated or engineered to create a defect in the cell's natural pathway for synthesizing that amino acid. The cells then may be provided the amino acid in an amount selected to allow them to achieve a desired cell population; when the cells reach that population, they become starved for the amino acid and cannot grow. According to a first aspect of the present invention, we provide a method for producing a desired compound in a bacterial cell population, characterised in comprising the steps of : a) transforming a bacterial cell with a vector which is incapable of autonomously replicating in the cell and which contains a gene coding a cell growth-promoting function, and which further contains DNA encoding a bacteriophage attachment site for enzymatic site-specific recombination in, and excision from, the chromosome of the bacterial cell, responsive to a site-specific recombination enzyme system; b) providing in the cell a controlled source of a regulator of the site-specific enzyme system; c) growing the cells under selected conditions regulating said site specific recombination enzyme system to effect integration of said vector in the transformed bacterial cell to a desired cell population; and d) changing the selected condition regulating said site specific recombination enzyme system to effect excision of said gene in the desired cell population. In a second and alternative aspect thereof, the invention provides a set of DNA constructions adapted for transfecting a host bacterial cell, for integrating a gene determining a growth-promoting function in the chromosome of said host bacterial cell, and for controllably excising the gene therefrom to yield a DNA molecule incapable of replicating in said cell, whereby said growth promoting function is controllably lost from said cell, said DNA constructions being characterised in comprising: a) a DNA vector comprising said gene and at least one bacteriophage enzymatic site-specific attachment site corresponding to a comparable site in said host cell, said vector being unable to replicate autonomously in said bacterial cell; b) on the DNA vector or a separate DNA expression vehicle, DNA coding enzymes capable of effecting said site specific attachment, under the control of a promoter that can be regulated in response to environmental conditions; and c) on said vector, or on a separate expression vehicle for transfecting said host cell, DNA expressing a regulator of said promoter. In preferred embodiments: the gene to be integrated and controllably excised codes for a cell-growth function that reduces the yield of a desired compound either by converting the desired compound into other compounds or by shunting intermediates away from the desired compound's biosynthetic pathway. Thus, the gene is integrated in the host cells to improve growth, and then it is excised and lost from the cells to improve compound production. For phenylalanine production, the gene on the excision vector preferably is one or more of tyr A, tyr R or trp E. For tryptophan production, the gene preferably is tna (tryptophanase), trp R, aro P, tyr A or phe A. For proline production, the gene preferably is one coding for proline oxidase. For threonine production, the gene preferably is one coding for an enzyme in the isoleucine biosynthesis pathway. The ppc (phosphoenolpyruvate carboxylase) gene is essential for growth in some strains and provides suitable growth control for producing desired compounds generally. The host bacterial genome lacks the ability, in the absence of the gene of interest, to produce enough of that gene's product to sustain growth. Most preferably, the gene of interest is entirely missing from the host genome, either naturally or by design, and the host genome lacks sufficient homology with any portion of the vector to permit vector integration in the absence of b). Also in preferred embodiments, the DNA of the vector includes elements b) and c) above, and attachment site ( att ) encoded by the DNA is a lambdoid attachment site. The DNA segment may be a natural DNA segment or a segment that is identical to that particular DNA segment or that is a natural, synthetic, or engineered variation of the DNA segment which conserves the specified or desired function. The DNA encoding integration and excision functions is derived from a naturally occurring bacteriophage. The means for controlling excision of the vector is a promoter regulating transcription of an excision enzyme gene, together with a gene that codes for repressor for that promoter, the repressor being subject to external control, such as the lambda c I gene product which loses its repression function at elevated temperatures. The DNA capable of causing vector integration or excision also can be provided by a helper phage, which is itself incapable of integration in the host chromosome. The invention is particularly useful where the organism synthesizes the desired compound from a precursor that also appears on a synthetic pathway that is essential for cell growth. In such organisms, an enzyme catalyzing a biochemical reaction occurring after one of these common precursors could shunt the precursors toward cell growth and away from production of the desired compound. The cell growth pathway is enabled by allowing expression of the enzyme that shunts the precursor into the growth pathway during the log growth phase. By disenabling the expression of genetic material encoding the enzyme for the cell-growth pathway after a suitable cell population density has been reached, the common precursor will be channelled into the synthesis of the desired compound, and cell growth will cease due to the removal of an essential enzyme. If a small number of cells continue to express the genetic material necessary for growth, they might overgrow their disenabled siblings and reduce the product yield. This problem is avoided because, once deleted from the bacterial chromosome, the genetic material coding for the growth function is unable to replicate autonomously. The gene eventually will "dilute out" as the cells continue to divide, and it will be lost from the cells completely. Since regions on the vector that are substantially homologous with the DNA of the host chromosome are avoided, the probability of the vector reintegrating in the host chromosome through a homologous recombination event is extremely low. Thus, integration can only occur by site specific recombination at the desired att site, and the ability to grow and divert important product precursors into cell growth can be disenabled reliably as desired to enhance production of the desired compound. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments. We first describe the drawings. Fig. 1 is a flow chart of the synthesis of the vector xKB803. Figs. 2-10 represent intermediate steps in the construction of the vector. Fig. 11 is a diagram of the vector pKB803. Fig. 12 is a diagram of the excision vector xKB803. Fig. 13 is a diagram of the integration-deficient helper phage, λimm434b104. Fig. 14 is a diagram of the plasmid pKB712. Fig. 15 is a diagram of the plasmid pKB750. Fig. 16 is a flow chart of phenylalanine production. The preferred embodiment described below relates to one specific system useful in microbial production of phenylalanine. Specifically, the system enables controlled expression of the tyr A gene, a critical gene in the tyrosine biosynthesis pathway. Since tyrosine is relatively insoluble, it is difficult to prepare stock solutions suitable for supplementing a medium used for growing tyrosine auxotrophs. Therefore, tyrosine prototrophy in cells to be used for compound producton is a desirable trait for fast growth and efficient culture when log phase growth of the culture is required. However, the tyrosine biosynthesis pathway shares many common metabolic precursors and enzymatic steps with the phenylalanine synthesis pathway, and it is useful to use tyr A⁻ organisms to make phenylalanine because such organisms do not siphon away phenylalanine pathway intermediates and thereby reduce the phenylalanine yield and additionally because tyr A⁻ cells quickly become starved for tyrosine and induce the synthesis of tyrosine precursors which then are used for phenylalanine synthesis. The preferred embodiment described below is only one example of the use of the invention to provide controlled expression of cell-growth functions which may be detrimental once the desired cell population has been achieved and compound production has begun. Those skilled in the art will recognize that there are a great number of possible cell-growth functions that can be controlled using the invention to enhance production of a desired compound. The excision vector comprises DNA from a lysogenic bacteriophage lambda, such as cI₈₅₇S₇, which encodes functions for phage integration/excision and lysogenic control. DNA encoding functions for phage replication, virion formation and host lysis has been deleted. cI₈₅₇S₇ contains a mutant allele of the cI gene which renders the lysogenic control temperature-sensitive, and that aspect of cI₈₅₇S₇ is included in the vector. The vector also includes a structural gene for a protein whose controlled synthesis is desired. Virtually any gene of interest can be cloned into the unique restriction site (XhoI in Fig. 11), and integrated in a host chromosome. Construction of the Vector The vector is constructed by the steps represented in Fig. 1. The appropriate cI₈₅₇S₇ DNA fragment can be obtained by digesting pKB720 (Fig. 2) or eKB101 (ATCC 39855) [both of which are fully described in U.S.S.N. 655,361, filed September 27, 1984, the disclosure of which application is hereby incorporated by reference] with EcoRI and PvuII. The ends of the fragment are converted into KpnI sites by using linkers, and cloned into the KpnI site of pKB742 (Fig. 4) yielding pKB757 (Fig. 5). pKB742 is derived from pBR322 (Fig. 3) [the entire sequence of which is described in Maniatis et al . "Molecular Cloning - A Laboratory Manual", Cold Harbor Laboratory 1982] by removing the DNA coding for tetracycline resistance between EcoRI and TthlllI and replacing it with a KpnI linker. pKB757 is treated with BglII to remove the genes cro , O , and cII, in whole or in part for the reasons described below. The cro gene product binds to the same sites as the c I repressor and regulates gene expression. Principally, it inhibits transcription originating at prm , inhibiting c I transcription. The cro gene product can reduce transcription originating at P L and thereby interfere with the expression of genes ( Xis and Int ) necessary for the excision of the element. The O gene product is required for replication of λ DNA, and should be removed to prevent autonomous replication of the vector. c II acts as an activator of transcription at the promoters of the c I and int genes. Since c II lies between cro and O , it is removed on the fragment also. Removal of c II is also desirable in order to prevent reintegration of an excised element by c II-dependent expression of int . The two largest fragments from the BglII digest of pKB757 are rejoined to yield pKB760 (Fig. 6). Conversion of a unique TthlllI site to a XhoI site by means of a linker results in pKB767 (Fig. 7). It has been found that the rex gene product can precipitate the lysis of the host bacterium under certain conditions. The rex gene, therefore, is removed by replacing the material between the ClaI sites with a XhoI linker, yielding pKB788 (Fig. 8). Further digestion with XhoI and Bal31, and the addition of a XhoI linker yields pKB791 (Fig. 9), in which the XhoI site is located just outside the cI ₈₅₇ gene. The leftward operon containing the genes N, xis , and int is reconstructed on pKB791 by removing the small fragment between the XhoI and BamI sites and replacing it with the XhoI/BamI small fragment of pKB767, yielding pKB800 (Fig. 10). Into the XhoI site of pKB800 is cloned a DNA fragment containing the E . coli tyr A gene, yielding pKB803 (Fig. 11). Digestion of pKB803 with KpnI removes the gene for ampicillin resistance (which can be scored for) and the plasmid replication functions. This yields xKB803. Vectors pKB800 and pKB803 have been deposited with the American Type Culture Collection under the ATCC accession numbers 53019, and 53020. xKB803 is recircularized and transformed into an E . coli strain which is auxotrophic for tyrosine, preferably due to a deletion of the tyr A gene and most preferably due to a deletion of the phe A- tyr A- aro F region of its genome to ensure no homology with the tyr A gene to be transferred from the excision vector. One skilled in the art will be able to isolate an E. coli mutant that is deleted for the above-described operon by standard techniques. Having done so, the mutant is transformed with xKB803 and vector integrants, defined as KB224::xKB803, are scored on the basis of tyrosine prototrophy. Integration of the excision vector can be improved to some extent when the transformed cells are co-infected with an integration-deficient helper phage at a multiplicity of infection of 5-10. This improvement may occur because the excision vector xKB803 cannot express its int gene sufficiently due to its low copy number and its lack of a c II activator gene. The diminished ability of the excision vector to autocatalyze its integration in the host chromosome may actually enhance its rate of loss subsequent to temperature shift; once excised, the vector will not reintegrate. One suitable helper phage is λimm434b104 shown in Fig. 13. Many other helper phages are suitable in that they are not subject to repression by c I encoded on the excision vector and are integration-deficient, but can express the c II and/or int genes sufficiently to improve integration of the excision vector. Other integration deficient phages and their preparation are described in the following: Parkinson et al. (1971) J. Molec. Biol. 56 :369; Parkinson (1971) J. Molec. Biol. 56 :385; and Davis et al. (1971) J. Molec. Biol. 56 :403. Suitable heteroimmune derivatives of these phages are easily prepared by one skilled in the art. Helper-phage-assisted integrants are selected at 30°C as tyr A⁺ bacteria which are immune to lambda infection, sensitive to λimm434b104 (showing that they do not harbor helper phage), and sensitive to ampicillin (showing that they have not acquired all of pKB803). Integrants prepared without the use of a helper-phage are similarly recognized, but without reference to the absence of helper-phage. Derivatives of E . coli which carry the stably integrated excision vector, preferably KB224::xKB803, prepared as described above, can be grown in culture without supplemental tyrosine; however, the cells in such a culture can be expediently converted to tyrosine auxotrophs when the desired cell population has been achieved and enhanced production of product is desired. As long as the culture is grown at 30°C, the temperature-sensitive repressor coded by the cI ₈₅₇ gene maintains the vector in a lysogenic state, and the cells retain the excision vector containing the tyr A gene. Upon shifting the culture to 42°C, the cI₈₅₇ repressor is inactivated and derepression of lambda excision functions occurs. The int and xis genes carried on the excision vector are expressed, and the element excises from the chromosome, taking with it the tyr A gene. Because the vector has no origin of replication, it is lost from the cells within a few generations; the cells become tyr A⁻, and they starve for tyrosine and cease growing. The point at which growth stops is later than, but correlated with, the time at which the temperature shift to 42°C occurs. The cessation of growth can be reversed by the addition of exogenous tyrosine. The production of phenylalanine using the vector is outlined in the flow chart in Fig. 16. Derivatives of E . coli which carry the stably integrated excision vector, preferably KB224::xKB803, prepared as described above, are tyr A⁺, phe A⁻, aro F⁻. The requisite genes for the production of phenylalanine , phe A and aro F, are provided by transforming KB224::xKB803 with a plasmid containing these elements, for example, pKB712, ATCC No. 39856 (Fig. 14) or pKB750, ATCC No. 39857 (Fig. 15) (see our European Patent Applications 85306789.0 and 84306841.2 (EP 0145156) the disclosure of which are hereby incorporated by reference). The transformed bacteria, KB224: :xKB803/pKB712 or KB224::xKB803/pKB750, are now tyr A⁺, phe A⁺, aro F⁺. The culture is maintained at the permissive temperature for log phase growth until the cell population has reached the desired density. Upon shifting the temperature to 42°C, the vector element containing the tyr A gene is excised from the bacterial chromosome and eventually lost from the cells. By eliminating an enzyme which diverts precursor metabolites from the phenylalanine biosynthesis pathway and "turning on" the preceeding steps in the pathway via tyrosine starvation, phenylalanine synthesis proceeds at an enhanced rate. The present invention may be embodied in other ways. For phenylalanine production, excision vectors containing tyr R and/or trp E could also be used. For example, where different genetic complements are desirable at different times, this versatile method of externally controlled change from one genetic complement to another can be applied to other gene systems including multiple gene systems. Other cell-growth related genes can be used to control cell growth in a fermentation process. For example, hosts that are defective in expressing the gene encoding phosphoenolpyruvate carboxylase ( ppc ) such as KB260 (ATCC 33927) are described in our European Patent Application No. 84306842.0 (EP 0140606) the disclosure of which is hereby incorporated by reference. The present invention can be used to insert an effective ppc gene for growth and then to excise it. For producing threonine, genes relating to isoleucine biosynthesis are useful for cell growth and hinder product production. For tryptophan production, the following genes are useful for cell growth and hinder product production: tna (tryptophanase), trp R, aro P, tyr A, and phe A. For proline production, a gene coding for proline oxidase can be included on the excision vector. The above-mentioned genes can be cloned by standard techniques, e.g. from E . coli .

Claims: A method for producing a desired compound in a bacterial cell population, characterised in comprising the steps of : a) transforming a bacterial cell with a vector which is incapable of autonomously replicating in the cell and which contains a gene coding a cell growth-promoting function, and which further contains DNA encoding a bacteriophage attachment site for enzymatic site-specific recombination in, and excision from, the chromosome of the bacterial cell, responsive to a site-specific recombination enzyme system; b) providing in the cell a controlled source of a regulator of the site-specific enzyme system; c) growing the cells under selected conditions regulating said site specific recombination enzyme system to effect integration of said vector in the transformed bacterial cell to a desired cell population; and d) changing the selected condition regulating said site specific recombination enzyme system to effect excision of said gene in the desired cell population. A method according to Claim 1, further characterised in that said cell is transformed to include lambdoid bacteriophage xis genes under the control of a regulatable promoter, and in that the attachment sites are lambdoid attachment sites responsive to the int and xis genes. A method according to Claim 2, further characterised in that said regulatable promoter is the lambdoid P L promoter, and in that said cell further includes a regulatable source of lambdoid CI repressor. A method according to Claim 3, further characterised in that said CI repressor is temperature sensitive, and in that said method includes the step of raising the temperature to inactivate the repressor and allow expression of said xis gene to effect excision of said cell-growth-promoting function. A method according to Claim 1, further characterised in that said cell growth promoting gene enables production of a nutrient for which said cell is otherwise auxotrophic. A method according to Claim 5, characterised in further comprising the step of selecting transformed cells which are prototrophic for the nutrient. A method according to Claim 5 or Claim 6, further characterised in that the nutrient is involved in the biosynthetic pathway of the desired compound. A method according to any preceding claim, further characterised in that said controlled source of said site specific enzyme system is provided on said vector or is provided by a helper phage that is incapable of chromosomal integration in said bacterial cell. A method according to Claim 1, further characterised in that said gene is tyr A, tyr R or trp E and said desired compound is phenylalanine. A method according to Claim 1, further characterised in that said gene is trp R, aro P, tyr A, phe A or tna , and said desired compound is typtophan. A method according to Claim 1, further characterised in that said desired compound is proline and said gene codes proline oxidase. A method according to Claim 1, further characterised in that said gene is ppc . A method according to Claim 1, further characterised in that said cells produce said desired compound via a biosynthetic pathway and in that said gene codes for an enzyme that converts a pathway intermediate to a compound outside the pathway. A method according to Claim 13, further characterised in that the desired compound is phenylalanine and in that the gene codes for an enzyme catalyzing a step in the synthesis of tyrosine or tryptophan. A method according to Claim 13, further characterised in that the desired compound is threonine and in that the gene codes for an enzyme in the isoleucine biosynthesis pathway. A set of DNA constructions adapted for transfecting a host bacterial cell, for integrating a gene determining a growth-promoting function in the chromosome of said host bacterial cell, and for controllably excising the gene therefrom to yield a DNA molecule incapable of replicating in said cell, whereby said growth promoting function is controllably lost from said cell, said DNA constructions being characterised in comprising: a) a DNA vector comprising said gene and at least one bacteriophage enzymatic site-specific attachment site corresponding to a comparable site in said host cell, said vector being unable to replicate autonomously in said bacterial cell; b) on the DNA vector or a separate DNA expression vehicle, DNA coding enzymes capable of effecting said site specific attachment, under the control of a promoter that can be regulated in response to environmental conditions; and c) on said vector, or on a separate expression vehicle for transfecting said host cell, DNA expressing a regulator of said promoter. A set of DNA constructions as defined in Claim 16, further characterised in that said enzymatic site specific attachment sites are lambdoid att sites, said promoter is a lambdoid P L promoter, and said regulator is CI857. A set of DNA constructions as defined in Claim 16, further characterised in being adapted for use in a host for manufacturing phenylalanine and in that said gene is tyr A, tyr R or trp E. A set of DNA constructions as defined in Claim 16, further characterised in being adapted for use in a host for manufacturing tryptophan and in that said gene is trp R, aro P, tyr A, pheA or tna . A set of DNA constructions as defined in Claim 16, further characterised in being adapted for use in a host for manufacturing threonine and in that said gene codes for an enzyme in the isoleucine biosynthesis pathway. A set of DNA constructions as defined in Claim 16, further characterised in being adapted for use in a host for manufacturing proline and in that said gene codes for proline oxidase. A set of DNA constructions as defined in Claim 16, for use in a ppc- host cell, further characterised in that said gene is ppc-. A set of DNA constructions as defined in Claim 16, further characterised in that said vector is pKB803 (ATCC 53020) digested with KpnI to remove the gene for ampicillin resistance and the plasmid replication functions. The plasmid pKB800 (ATCC 53019). The plasmid pKB803 (ATCC 53020).

Inventors: BACKMAN, KEITH C., BALAKRISHNAN, RAMASWAMY
Applicants: BIOTECHNICA INTERNATIONAL, INC., H.J. HEINZ COMPANY
Application year: 1986
Main classification: C12N15
IPC codes: C12N15, C12P13, C12R1