EXPRESSION VECTORS, HOST CELLS COMPRISING SUCH VECTORS AND METHODS OF MAKING DESIRED COMPOUNDS USING SUCH CELLS

Abstract: An expression vector having two structural genes that form a synthetic operon expressed under the control of a single regulatory sequence. The operon genes correspond to the structural component of naturally occurring genes whose expression is controlled by distinct separate regulatory sequences. The operon genes code for enzymes in a biosynthetic pathway for producing a desired compound, and at least one of those operon genes is feedback derepres­ sed. The vector is used to transform host cells that are cultured to produce the desired product.

Description: In its various aspects this invention relates to the production of phenylalanine by a biosynthetic pathway. A fundamental limitation of in vivo methods of producing compounds is that organisms often have regulatory mechanisms to limit synthesis to amounts sufficient for the organism's own needs , thus avoiding waste of the organism's raw materials and energy. Often regulation of biosynthesis is effected at the level of gene expression. For example, sufficiency or surfeit of a particular compound causes a cessation in the synthesis of one or more of the enzymes that catalyze reactions in the biosynthetic pathway of that compound. Genetic sequences, located in the vicinity of the structural gene coding for the enzyme, regulate expression of the enzyme. In some cases, more than one kind of regulatory sequence may be associated with a gene. Examples of such regulatory sequences include: promoters, operators , attenuators, antiterminators, ribosome binding sites, and sites for positive effectors. Bernard et al., [1979] Gene 5 :59-76 and Remaut et al. [1981] Gene 15 :81-93 disclose inserting restriction fragments containing Salmonella genes for enzymes in the tryptophan-synthesis pathway into vectors downstream from the phage p L promoter. Expression of the genes is obtained under control of p L . Bernard specifically involves the trp operon genes. Bernard also says that it should be possible to insert a promotorless DNA fragment carrying a gene specifying a measurable enzyme into an EcoRI site in the above-described expression vectors. Renaut involves genes for tryptophan synthetase A. Franklin, (1971) pp. 621-638, The Bacteriophage Lambda ; Cols Spring Harbor Laboratory, Cold Spring Harbor, NY (Hershey Ed.) and Davidson et al. (1974) Mol. Gen. Genet. 130 :9-20 disclose transcription of the tryptophan operon either from the p L promoter of phage lambda or from its naturally occurring promoter. Stryer, Biochemistry , 2 ed. pp. 675-678 (Freeman and Co. San Francisco 1981) discloses that expression of the naturally occurring tryptophan operon of E . coli including trp A and trp B is subject to operator corepression by a complex of tryptophan and the trp R gene product. Expression of that operon is also regulated by a leader peptide attenuation control that depends on the availability of tryptophan for leader peptide synthesis. Tribe, Australian Application 72727/81, discloses a strain of E. coli mutants which have been mutated to produce higher levels of chorismate mutase-prephenate dehydratase (CMPD) and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase than are found in strains with wild-type regulation of those enzymes. Sninsky et al., U.S. Patent 4,374,927, discloses fusing the lac promoter-operator to the structural gene for chloramphenicol acetyltransferase. Expression of that structural gene is regulated by a repress or whose activity is temperature related. Chemical Abstracts (1982) Vol . 97 :180140e (Japanese Appl. 80/154,706), discloses a strain of E. coli containing a trp operon that is resistant to feedback inhibition by a repressor. Sprinson et al., (1976) Acta. Microbiol. Acad. Sci. Hung. 23 :167-170, discloses Salmonella mutants that are resistant to phenylalanine analogs, and which exhibit coordinate derepression of DAHP synthetase ( tyr ) and prephenate dehydrogenase ( aro ). Modrich et al., USSN 527,490 filed 29 August 1983 discloses placing genes for Eco RI endonuclease and methylase downstream from a lambda promoter so as to be expressed by that promoter and thereby take advantage of the control afforded by the lambda repressor. The behavior of these E . coli genes in the reported construction indicated that they may include a naturally occurring internal promoter. Rood et al., (1980) J,. Bacteriology 144 :552, discloses certain plasmids designed for overproduction of enzymes coded for by the tyrosine operon in that the regulatory region of the operon on these plasmids has been altered so that it is not subject to the control of a specific repressor. The authors report that such plasmids give rise to modifications by insertions or deletions that decrease the level of expression of the tyrosine operon. Liu et al., (1983) J. Biol. Chem. 258 :7469-7475, discloses placing the gene coding for guanine-xanthine phosphoribosyltransferase under the control of the lambda phage leftward promoter. DeBoer et al., (1982) PNAS USA 80 :21-25, discloses expression of the enzyme galactokinase under the influence of a hybrid promoter generated from the lac and trp promoters. The hybrid promoter can be repressed by the lac repressor. Jacob et al., (1965) J. of Molecular Biology 13 :704-719 discloses isolation of a mutant having a genetic deletion which results in fusion of a part of the lac operon to a part of the pur operon, forming a new operon which is subject to repressive regulation by purines. Casadaban et al., (1979) proc nat'l Acad. Sci. USA 76 (9):4530-4533 discloses techniques to fuse the lactose operon ( lac ) structural genes minus the lac promoter, to non- lac operon promoters, so that the lac operon is expressed under the control of a non- lac operon promoter. Backman et al., (1976) Proc. Nat'l Acad. Sci. USA 71 :4174 discloses construction of a plasmid in which the promoter of the lac operon is placed adjacent the repressor gone ( c I) of bacteriophage λ, by recombinant techniques. Synthesis of the λ repressor is thus regulated by the lac operator and promoter. By supplying an excess of lac operator sites in comparison to the available lac repressor, synthesis of λ repressor is increased. We describe below use of an expression vector to transform cells to produce a desired compound by a biosynthetic pathway, which vector includes at least two structural genes that are a synthetic operon, meaning that, on the vector construction, they are expressed under the control of a single regulatory sequence and promoter, whereas in naturally occurring constructions, genes corresponding to the operon genes are expressed under the control of distinct separate regulatory sequences. The operon genes code respectively for enzymes in the biosynthetic pathway, and at least one of those genes is feedback derepressed. The natural gene corresponding to at least one of the operon genes is feedback repressed. By "feedback repressed", we mean that expression of the natural gene is subject to repression by a control substance that is a pathway intermediate or product. The corresponding operon gene is "derepressed", meaning that the vector promoter is foreign to the repressed natural gene and expresses operon genes without repression from the pathway intermediate or product. By "foreign" we mean that the promoter does not naturally occur in position to initiate transcription of the corresponding natural gene. Thus, the vector enables uninhibited expression of the operon genes in the presence of all biosynthesis pathway intermediates as well as the pathway product. In the detailed description below, the desired compound is phenylalanine. According to a first aspect of this invention, we provide a host cell engineered to produce phenylalanine by a biosynthetic pathway; said cell being characterised in comprising an expression vector including a synthetic construction comprising a phe A structural gene, and a regulatory DNA sequence positioned to govern the expression of said phe A structural gene, said regulatory DNA being foreign to said phe A structural gene and being effective in said host cell to cause expression of said phe A structural gene in the presence of phenylalanine or intermediates on said biosynthetic pathway. The vector promoter is most preferably the lac promoter, the aro F promoter, or the tet promoter of pBR322. Other preferred promoters include the aro H promoter and the phage m13 gene II promoter. The said structural gene is phe A; the vector may further comprise aro F or aro H as structural gene(s). In a second and alternative aspect, the invention provides a method of making phenylalanine by a biosynthetic pathway, characterised in comprising the steps of: transforming a host cell with an expression vector including a synthetic construction comprising a phe A structural gene, and a regulatory DNA sequence positioned to govern the expression of said phe A structural gene, said regulatory DNA being foreign to said phe A structural gene and being effective in said host cell to cause expression of said phe A structural gene in the presence of phenylalanine or intermediates on said biosynthetic pathway; culturing said cell in a culture medium; and recovering phenylalanine from said medium. Preferably, the phe A gene is derived from a naturally occurring phe A gene by removing the naturally occurring regulatory sequence from the structural gene. The preferred regulatory sequences are those described above. Preferably, the cells are E. coli . The invention enables production of phenylalanine by the above described engineers cells, which avoid normal synthesis-inhibiting regulatory mechanisms, and therefore does not curtail synthesis in response to elevated levels of the product. In many applications, as the product builds up, it is released to the supernatant milieu, thus making recovery of the biochemical more convenient. The engineered vector is able to override natural regulatory modes, e.g., leader/attenuator regulation and operator/repressor regulation. Specifically, exogenous regulatory DNA can effectively control gene expression of gene products that are key enzymes in the pathway, and this finding his broad applicability to other promoters such as phage m13 gene II promoter and the tet promoter of pBR322. Finally, the inclusion of several pathway genes in the same synthetic operon is efficient, simplifies transformation, and reduces the total number of genetic elements necessary, thus reducing the complexity of the genome in the engineered organism. These factors support a higher level of gene expression, and thereby increase the rate of the rate-limiting step in the pathway, as well as the rate of other pathway steps catalyzed by operon gene products. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof. We first briefly describe the drawings. Figs. 1-4 are diagrams representing respectively steps in the engineering of plasmids pKB663, pKB712, pKB750, and pKB766. Expression Vectors The desired product compound is synthesized by cells via a synthesis pathway with steps that are targets of gene-expression regulation. That is, regulation at a step in the synthesis pathway is accomplished by varying the level of expression of the gene for an enzyme to vary the amount of enzyme in the cell, and such regulation involves DNA sequences adjacent to the regulated gene. Replacement of such regulatory DNA sequences with heterologous regulatory sequences, by recombinant DNA techniques, is used to circumvent the normal regulation of a biosynthetic pathway and augment the production of a biochemical beyond the point where normal regulation would have halted its further production. The pathway steps selected for derepression should be rate-limiting ones so that successful derepression will show a gain in product yield. The dual-mechanism-repressed enzymes are the specific targets. In order to deactivate both of the repression mechanisms, it is preferable to remove the regulatory DNA that naturally occurs with the structural gene whose expression is being derepressed and to add a foreign promoter (i.e. one that in naturally occurring structures does not govern expression of the gene). In the preferred embodiments comprising a synthetic operon, the promoter may be foreign to both of the genes, or it may be foreign only to the gene selected for derepression. The two synthetic operon genes should be close to each other, but there may be short, or even quite long, sequences between them, as long as there are no signals to stop transcription between the genes. Each synthetic operon gene corresponds to the structural component of a naturally occurring gene. By the term "corresponds to" we mean that the operon gene is either identical or sufficiently related to the naturally occurring structural gene that the operon gene codes for an enzyme that catalyzes the reaction catalyzed by the natural gene product. Choice of the other operon component offers an opportunity to engineer a construction that highly expresses another pathway enzyme. One preferred choice is another rate-critical pathway step. The other operon component also may be selected to simplify the cloning procedure by selecting a gene with regulatory material that is used on the vector. In that case, the 3' end of the operon may be the derepressed structural gene, and the 5' end may be the other member of the operon with its naturally occurring regulatory sequence. If a promoter is used that is exogenous or foreign to both members of the operon, the members can be linked in either order. Additional structural genes may be added to the operon, and more than one of them may be feedback repressed in its naturally occurring structure, so that it also is derepressed by insertion in the operon. The following three examples of preferred vectors pKB663, pKB712, and pKB750 are illustrative. Each vector is useful in synthesizing the amino acid L-phenylalanine by derepressing expression of the phe A gene of E. coli , which is naturally regulated in response to available phenylalanine. The product of phe A, the dual-function enzyme chorismate mutase-prephenate dehydratase, effects the penultimate steps in phenylalanine biosynthesis. Regulation of phe A expression occurs both by a repressor/operator system and by a leader peptide/attenuator system. When sufficient or excess phenylalanine is available, expression of phe A is turned off, and phenylalanine synthesis stops. The DNA for the phe A-associated promoter/operator and leader peptide/attenuator is replaced with DNA from another promoter. Cells harboring such a construct do not halt expression of phe A nor stop phenylalanine biosynthesis in response to elevated phenylalanine. In consequence, such cells release phenylalanine into the supernatant milieu. Construction of pKB663. One expression vector for producing phenylalanine is illustrated in Fig. 1. 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 et al. Proceedings of the National Academy of Sciences 75 :4271-4274 [1978]) by digestion with endonucleases Stu I and Bgl II. The next step involves plasmid pKB430, which is a derivative of pBR322 that carries a 95 bp Alu I generated DNA fragment containing the lactose operon promoter-operator cloned in the endonuclease Pvu II cleavage site of pBR322 such that a new Pvu II site is created at the border of the lac promoter and the pBR322 sequences, and lac transcription proceeds across this Pvu II site toward the tet region. 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 Pvu II and Bam HI cleavage sites, yielding pKB663. In addition to the phe A gene, pK8663 also carries a gene which determines resistance to β-lactam antibiotics. Construction of pKB712. Fig. 2 depicts the construction of pKB712 from two components: The first component is the lac operon- phe A fusion described above with respect to pKB663. The Ban I site just past the end of the phe A gene is converted to an Eco RI site by abutting to a filled in Eco RI site. Specifically, pKB663 is treated by inserting a Hind III linker at Tth lllI site 5' to the lac promoter. The second component is the aro F structural gene; specifically aro F, contained on pKB45 is subcloned into pBR322 as an Eco RV to Pvu II fragment, yielding pKB648. The aro F gene is preceded (5') by a Eco RV site which is cleaved and exonucleolytically resected, followed by insertion of an Eco RI linker positioned about 20-bp upstream from the aro F gene's ribosome binding site. Near the 3' end of aro F Is a Pvu II site which can be joined to the Pvu II site of pBR322. The TthlllI site 3' to the gene thus cloned is changed to a Hind III by a linker. The above two components are joined at their Eco RI ends and cloned In the Hind III site of pBR322. In the pKB712, the two structural genes are joined at a point approximately eight nucleotides after the first stop codon at the end of phe A and approximately 20 nucleotides upstream from the ribosome binding site of the aro F gene. Construction of pKB750. pKB750 differs from all of the above-described expression vectors in that it is expressed under the control of the aro F promoter, not the lac promoter. pKB750 is constructed as shown in Fig. 3 from two fragments. The first fragment is an aro F gene with its naturally associated promoter, obtained by from pKB45 via pKB648; the segment begins with an EcoRV site 5' to the gene and ends with a Kpn I site positioned next to the Pvu II site at the end of aro F. The second fragment is a phe A gene bounded by a Kpn I site 5' to the gene and an Eco RI site 3' to the gene. The Kpn I site is created by cleavage at the Stu I Side 5' to phe A (e.g. on pKB45 or a suitable derivative) and exonucleolytic resection towards phe A; Kpn I linkers are then added. The Eco RI site is created by abutting a Ban I site to a filled in Eco RI site. These fragments are joined at their Kpn I sites and cloned into pBR322. Construction of pKB766. The phe A gene, or a synthetic operon that included phe A can be expressed from any of a number of exogenous promoters that are not regulated by phe A synthesis pathway compounds. Suitable multipurpose cloning vehicles for making those constructions are pKB707 or pKB766, illustrated in Fig. 4. pKB766 is constructed starting with the Kpn I - Eco RI phe A-containing fragment illustrated in Fig. 3 cloned in vector pKB678 to yield pKB707. The Kpn I site of pKB707 is readily converted to a Bgl II site by means of a linker yielding pKB766. pKB707 and pKB766 contain unique cloning sites 5' to the phe A gene. Insertion of any number of promoter-carrying DNA fragments in the Kpn I site of pKB707 or the Bgl II site of pKB766 results in the synthesis of chorismate mutase-prephenate dehydratase in a manner unregulated by phenylalanine. For example, the gene II promoter from bacteriophage m13 and each of two promoters from the tet region of pBR322 [between the Eco RI site at position 1 and the Ban I site at position 76] are each cloned onto DNA fragments having sticky ends compatible with Bgl II sticky ends and are then cloned into the Bgl II site of pKB766. E. coli are transformed and cultured as described below. Phenylalanine production is scored by ability to crossfeed a phenylalanine auxotroph. Although strains carrying pKB766 cannot crossfeed phenylalanine auxotrophs, all derivatives of pKB766 which carried a promoter were able to produce and secrete phenylalanine and thereby crossfeed a phenylalanine auxotroph. Thus, the promoter-carrying fragments are responsible for the observed phenylalanine secretion. Production of Phenylalanine . E. coli K12 strain YMC9, which has been deposited with the American Type Culture Collection and has an accession number ATCC 33927 (Backman et al., Proceedings of the National Academy of Sciences USA 78 [1981], 3743-3747), is transformed with pKB663, yielding YMC9/pKB663. Plasmid pKB663 is available from strain YMC9/pKB663, which has been deposited with the American Type Culture Collection and has an accession number ATCC 39462. YMC9/pKB663 is cultured at 37 o C in M9 salts plus 4 mg/ml sodium acetate and 1 µg/ml thiamine, and culture turbidity is monitored using a Klett-Summerson Colorimeter (green filter). During growth of this strain on acetate, no detectable phenylalanine appears in the supernatant. When the culture density reaches either 45 or 148 Klett units, the cells are collected by filtration, resuspended in an equal volume of fresh medium, and glucose is added (final concentration: 4 mg/ml). Incubation is continued for 10 to 15 hours, at which point phenylalanine content in the supernatant is determined. Alternatively, pKB663 is used to transform derivatives of YMC9 designated KB285 and KB280 which are described in our co-pending European Patent Application, hereby incorporated by reference, entitled "Cells and their Use in Methods for Producing Product Compounds" and filed the same day as this application. KB280 and KB285 are deposited with the American Type Culture Collection with accession numbers ATCC 39461 and ATCC 39463, respectively. KB285/pKB663 or KB285/pKB663 may be used to synthesize L-phenylalanine as described in that application. Phenylalanine concentration is determined micro-biologically using phenylalanine assay medium (Difco) and Pediococcu s acidilactici ATCC 8042 (Difco Manual, 1953). As described above for pKB663, pKB712 and pKB750 can be used to transform a host such as E. coli YMC9 or derivatives thereof to yield a strain that secretes phenylalanine into the supernatant melieu. Expression of phe A is directed by the foreign promoter, and production of phenylalanine is enhanced when the phe A structural gene is introduced as part of a synthetic operon, rather than as a single deregulated structural gene. Vectors pKB663, pKB712, pKB750, and pKB766 have been deposited by applicant with the American Type Culture Collection in Rockville, Maryland and are designated by accession numbers 39,462, 39,856, 39,857 and 39,858, respectively. Other arrangements are possible within the scope of this invention. For example, derivatives of vectors pKB663, pKB712, and pKB750 may be used as expression vectors; or derivatives of pKB766 may be used as a precursor to an expression vector into which a suitable foreign promoter is inserted. By the term "derivative" we mean naturally occurring or engineered variations of the vector that conserve the desired functions.

Claims: A host cell engineered to produce phenylalanine by a biosynthetic pathway; said cell being characterised in comprising an expression vector including a synthetic construction comprising a phe A structural gene, and a regulatory DNA sequence positioned to govern the expression of said phe A structural gene, said regulatory DNA being foreign to said phe A structural gene and being effective in said host cell to cause expression of said phe A structural gene in the presence of phenylalanine or intermediates on said biosynthetic pathway. A cell according to Claim 1, further characterised in that the phe A structural gene is an E. coli gene. A cell according to Claim 1, further characterised in that said vector further comprises either the aro F or the aro H structural gene positioned so that expression of said aro F or aro H structural gene is governed by said regulatory DNA sequence. A cell according to Claim 1, further characterised in that said regulatory sequence comprises the lac promoter, the aro F promoter, the aro H promoter, the m13 gene II promoter, or the tet promoter of pBR322. A cell according to Claim 1, further characterised in that said vector is selected from: a) pKB663 (ATCC 39462), and derivatives thereof, namely vectors comprising the phe A structural gene and associated lac promoter of pKB663; and b) synthetic constructions comprising pKB766 (ATCC 39858) and said regulatory DNA sequence. A cell according to Claim 1, further characterised in that said cell further comprises either the aro F or the aroH structural gene and a regulatory sequence comprising a promoter positioned to control expression of said aroF or aro H structural gene, said regulatory sequence being effective to cause expression of said aro F or aro H structural gene in the presence of phenylalanine. A cell according to any preceding claim, further characterized in that said cell is an E. coli . A cell according to Claim 7, further characterised in that said cell is E. coli YMC9 transformed with said vector. A method of making phenylalanine comprising culturing a cell according to any of Claims 6, 7 or 8, in a culture medium and recovering the phenylalanine from the medium. A method of making phenylalanine by a biosynthetic pathway, characterised in comprising the steps of: transforming a host cell with an expression vector including a synthetic construction comprising a phe A structural gene, and a regulatory DNA sequence positioned to govern the expression of said phe A structural gene, said regulatory DNA being foreign to said phe A structural gene and being effective in said host cell to cause expression of said phe A structural gene in the presence of phenylalanine or intermediates on said biosynthetic pathway; culturing said cell in a culture medium; and recovering phenylalanine from said medium. A method according to Claim 10, further characterised in that the phe A structural gene is an E. coli gene. A method according to Claim 10, further characterised in that said vector further comprises either the aro F or the aro H structural gene positioned so that expression of said aro F or aro H structural gene is governed by said regulatory DNA sequence. A method according to Claim 10, further characterised in that said regulatory sequence comprises the lac promoter, the aro F promoter, the aro H promoter, the m13 gene II promoter, or the tet promoter of pBR322. A method according to Claim 10 further characterised in that said vector is selected from: a) pKB663 (ATCC 39462), and derivatives thereof, namely vectors comprising the phe A structural gene and associated lac promoter of pKB663; and b) synthetic constructions comprising pKB766 (ATCC 39858) and said regulatory DNA sequence. A method according to Claim 10, further characterised in that said cell further comprises either the aro F or the aro H structural gene and a regulatory sequence comprising a promoter positioned to control expression of said aro F or aro H structural gene, said regulatory sequence being effective to cause expression of said aro F or aro H structural gene in the presence of phenylalanine. A method according to any of Claims 10 to 15, further characterised in that said host cell is an E. coli . A method according to Claim 16, further characterised in that said host cell is E. coli YMC9 transformed with said vector. An expression vector comprising pKB663 (ATCC 39462) or a derivative thereof, namely vectors comprising the phe A structural gene and associated lac promoter of pKB663. An expression vector comprising pKB766 (ATCC 39858) and a regulatory DNA sequence positioned to govern the expression of the phe A structural gene of pKB766, said regulatory DNA being foreign to said phe A structure gene.

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