JPS6192565A - Biosynthesis and cell therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION The present invention is based on pending U.S. Pat.
September 24, 1984), and the above patent application is also a pending U.S. Pat. No. 511,019.
This is one horse that is a partial continuation of No. 0 (dated October 7, 1983).

The present invention is directed to the in vivo production of compounds.

A fundamental constraint on in vivo production methods of compounds is that organisms often have a regulation 'F341Q that limits the synthesis of compounds to amounts sufficient for the organism's own needs, thereby avoiding wasting the organism's raw materials and energy. That's true. Regulation of biosynthesis is carried out by feedback inhibition of enzymes in the biosynthetic pathway, such that when the concentration of an inhibitory compound reaches a certain level, the catalytic activity of the enzyme is substantially reduced (said inhibitory compound is the target compound). or its pre-metabolic precursor).

Various ways to avoid feedback inhibition are known.

European patent (EP) of Synenki et al.
No. 077196 discloses the isolation of a gene encoding DAHP synthase that resists feedback inhibition by aromatic amino acids. This gene is isolated from a mutant strain of E. coli that produces DAHP synthase in the presence of relatively high concentrations of aromatic amino acids.

Analogs of the desired product inhibit the enzymes used in the biosynthesis of the product, as well as the product itself causing feedback inhibition, so the analogue is resistant to It is also known to isolate mutants. Organisms that are not killed by the analog are freed not only from the target product but also from feedback inhibition by the analog. See, eg, Sochida US Pat. No. 4,407,952. This discloses an E. coli mutant strain that resists feedback inhibition by phenylalanine and phenylalanine analogs.

Regulation of biosynthesis also occurs at the gene expression level. For example, a sufficient amount or excess of a particular compound inhibits the expression of a gene encoding one or more enzymes that catalyze a reaction in the biosynthetic pathway of that compound. A gene sequence located near the structural gene that encodes an enzyme regulates the expression of the enzyme. In some cases more than one regulatory sequence is associated with one gene. Examples of such regulatory sequences include promoters, operators, attenuators, antiterminators, liposome binding sites, and positive effector terminal sites.

Unique methods of circumventing feedback control of direct gene transfer are known and described in the above-mentioned cross-referenced patent applications, which are incorporated herein by reference.

SUMMARY OF THE INVENTION In a first aspect, the invention generally avoids feedback inhibition of an enzyme in the biosynthetic pathway of a compound of interest, which enzyme is the first isoenzyme that is feedback-inhibited by the compound of interest, by: characterized by altering cells to increase production of the compound.

As used herein, does it mean that the target compound is "feedback inhibited"? The phenomenon is that once the concentration of the inhibitor reaches a certain level, the catalytic activity of the enzyme is substantially reduced.
(the inhibitor is either the target compound or a metabolic precursor in the biosynthetic pathway of the compound). The above expression does not include inhibition of the enzyme by compounds that are on offshoots of the biosynthetic pathway leading to the synthesis of compounds other than the target compound. To avoid feedback inhibition of the first isozyme, compound-producing cells are transformed with a gene encoding a second isozyme that is not substantially inhibited at the humidity level of the inhibitor.

The gene for this second isozyme is not transcriptionally regulated in response to the concentration of the inhibitor, and thus the second isozyme is produced in sufficient quantities to increase production of the target compound.

In a preferred embodiment of the first aspect, the cell is a member of Canis coliformis and the compound of interest is an inhibitor. If the compound of interest is an aromatic amino acid such as phenylalanine, its isozyme is encoded by the aro gene, eg, the first isozyme is encoded by cLroG and the second isozyme is encoded by arof;'. In another, the compound of interest is threonine, methionine or ricin, and the isozyme catalyzes a step common to the biosynthesis of at least two of these compounds. Transcription of the second isozyme in the presence of the compound of interest is possible by providing sufficient copies of the gene within the cell to outnumber the transcriptional regulatory effectors. Also used are the methods of circumventing transcription control described in the above-mentioned pending patent application.

In a second aspect, the invention is characterized by selecting mutant cells that produce a target compound better than the parental cell population. Ma1 cells contain genes encoding enzymes that catalyze chemical reactions and are subject to feedback inhibition, common to the biosynthesis of both target compounds and second compounds essential for cell growth. The parental cells are cultured in the presence of an enzyme-inhibiting concentration of said inhibitor and a growth-maintaining amount of the second compound is obtained solely as a result of the catalytic activity of the enzyme. Select mutants. Such mutants exhibit release from feedback inhibition.

In a second preferred embodiment, the inhibitor is the compound of interest, the enzyme is an isozyme, and the parental cells are sufficiently effective to provide a growth-maintaining amount of the compound under the selected conditions. It does not have the ability to catalyze that reaction or produce other isozymes. The parent cell is preferably a cell produced by the method described in the first aspect above to increase the production of one of the aromatic or non-aromatic amino acids (threonine, methionine or ricin). If the enzyme is a second isozyme, it is not only inhibited by the second compound, but also to some extent by the same inhibitor that controls the first isozyme (only when the concentration of the inhibitor is much higher). ), the methods described above can be used to generate genes encoding isozymes of the biosynthetic pathway of the compound of interest that are not feedback-inhibited by the compound of interest, second compounds, or their metabolic precursors.

In preferred embodiments of either aspect, the compound of interest is produced by culturing the cells produced as described above in a liquid medium and recovering the compound of interest from the medium.

Therefore, the present invention focuses on the existence of multiple enzymes (isoenzymes) that catalyze the same chemical reaction, but have completely different responses to feedback inhibition by the target product or its metabolic precursors and are functionally different from each other. is used. The resulting organism can perform the reaction in question by using isozymes that are not inhibited by feedback, instead of isozymes that are present in nature and are inhibited by feedback.

Under conditions achieved by avoiding transcriptional regulation as described above, production of the compound will increase as long as sufficient copy numbers of the deregulated isozyme are produced. These genetically engineered cells bypass the normal regulator (lt7) and therefore do not scale back synthesis in response to excess amounts of the product. As the amount increases, the product is released into the surrounding medium, making recovery of the target compound easier.

Other features and advantages of the invention will become apparent from the following description of the preferred embodiments.

The expression vector target product is usually synthesized via a synthetic route that includes at least one step catalyzed by an isoenzyme that is feedback-inhibited, and thus is inhibited by an inhibitor (usually the target
(product) reaches a certain level, the catalytic action of the isozyme is inhibited or greatly reduced.

Such feedback inhibition can be avoided by having the cell perform the reaction step using another isozyme that is not feedback inhibited by the product of interest or its metabolic precursor. Even if unharmed isozymes exist naturally within the cell, their availability is generally insufficient for a variety of reasons, including regulation of gene transcription or inhibition of other substances.

However, if this type of regulation of the isoenzyme can be avoided, and feedback is not inhibited, the amount of product produced increases. The synthetic pathway steps selected to deregulate enzyme 1 inhibition and gene expression will be limited to those steps where successful derepression shows increased yield of product.

The present invention will now be described using vectors useful for the production of L-phenylalanine. An example of a particular isozyme is DAHP synthase, which is encoded by the aro gene. DAHP synthase catalyzes the chemical reactions common to the biosynthesis of all three aromatic amino acids (phenylalanine, tyrosine and tryptophan). ar
The oG gene encodes DAHP intase, which is feedback inhibited by L-phenylalanine. a
Other isozymes encoded by roF and αroI (respectively) are much less sensitive, if at all, to feedback inhibition of L-phenylalanine. Therefore, impairment of L-phenylalanine production is A large amount of aroF or α is also produced.
The roH gene product can be avoided by providing the system in the absence of substances that inhibit the aroF or CLroH gene product in particular. Specifically, the expression vector contains the aroF gene or a variant derived therefrom (described in more detail below). If aroF is used to produce phenylalanine as described below, the ``Controlled Excision''
aroF throughout the production run by using a tyr-host as described in U.S. Patent Application No. 701,091, filed Feb. 13, 1985, by J. It is effective to avoid the presence of tyrosine, which inhibits the gene product.

If that step in the synthetic pathway is further regulated by a DNA sequence that encodes an enzyme and controls the expression level of the gene, the vector also encompasses circumvention of said regulation. One way to circumvent regulation of the expression of the enzyme gene is to circumvent the normal regulation of the biosynthetic pathway by replacing the regulatory DNA sequences with heterologous regulatory sequences using recombinant DNA technology, and The goal is to increase the production of the target compound beyond the point where it ceases to produce any more C. In order to inactivate all possible control mechanisms of gene expression, remove the regulatory DNA that is naturally present with the structural gene (whose expression is derepressed) and remove the exogenous promoter (i.e., the naturally occurring It is preferable to insert something that does not control the expression of the gene into the construct.

In a preferred practical embodiment, this is accomplished by incorporating the gene encoding the deinhibiting enzyme into a synthetic operon, in which the promoter is foreign to all of the operon's structural genes or is derepressed. at least foreign to the gene selected for. Two synthetic operon genes should be close to each other, but there can be short or even fairly long sequences between them, unless there is a signal between them that stops transcription. Each synthetic operon gene corresponds to a structural component of a gene that occurs in nature. "
The expression "corresponding to" means that the operon gene is identical to or closely related to a naturally occurring structural gene, and that the operon gene catalyzes a reaction that is catalyzed by the natural gene product. Selection of other operon components provides the opportunity to genetically engineer constructs that highly express enzymes of alternative synthetic pathways. For one thing, there is another rate-critical
) synthetic route steps are preferred. The cloning process can also be simplified by selecting genes that have other operon components, ie, regulatory substances, used in the vector. In that case, the 3' segment of the operon may be the structural gene that is derepressed, and the 5' segment may be other members of the operon, including naturally occurring regulatory sequences.

If promoters that are exogenous or foreign to both members of the operon are used, the members may be joined in either order. Another structural gene may be added to the operon and one or more of them may be feedback-inhibited in the naturally occurring +74 construct and thus also suppressed by inserting it into the operon. It will be canceled.

Two examples of suitable vectors 71KB 712 and pKB750 are described below.

Each vector not only contains the aroF gene in a form in which it is not subject to feedback transcriptional control, but also because it suppresses the E. coli ph, e A gene (which is regulated in response to available phenylalanine in nature). It is useful for the synthesis of L-phenylalanine because it allows for de-expressed expression. The dual-function enzyme chorismtxte mutase prephenate dehydratase, the product of phe A, performs the penultimate step in phenylalanine biosynthesis. Regulation of pheA expression occurs through both a repressor/operator system and a leader peptide/attenuator system. When sufficient or excess phenylalanine is obtained, pheA expression ceases and phenylalanine synthesis ceases.

The promoter/operator and leader peptide/attenuator DNA associated with phefi is replaced with DNA from another promoter. Cells containing such constructs do not cease expressing pheA or stop its biosynthesis in response to excess phenylalanine. As a result, this type of cell supplies sufficient amounts of phenylalanine to secrete it into the surrounding medium.

Figure 2 shows the production of pKB712 from two components.
c operon-pheAB compound. Plasmid pKB
6G3 is ATCC39462 in American e-type culture collection,! : YMC9 deposited as
/pKB 663 strain. The BanI site just beyond the end of the pheA gene contains the repaired ECQR.
Joins the I site, and in particular converts it into an E co RI site. In detail, the obtained plasmid was treated to produce 1
Insert a Hind@linker into the Tthllll site on the 5' side of the CLC promoter.

The second component was chosen for the reasons stated earlier.
It is an F structural gene. Specifically, aroF contained in pKB45 was converted into EcoRV/Pvu and pBR3 as an l fragment.
22, thereby pK8648
get. The EcoRV site precedes the αγOF gene and is on the 5′ side of the aroF gene), and this EcoRV
The site is excised by echinucunase digestion, and then an ECI)RI clinker is inserted at a position approximately 20 bp upstream from the liposome binding site of the ατoF gene. There is a puu■ site near the 3' end of aroF, and this pvu1 site can be joined to the PτttH site of pBR322. Therefore, the T on the 3' side of the gene to be cloned
The th1111 site is changed to HindM by a linker.

The two components above are joined at their EcoRI ends and 7) cloned into the Hindll site of BR322.

In pKB 712, two structural genes are phaA
It is joined at a position approximately 8 nucleotides after the first stop codon at the end and at a position approximately 20 nucleotides upstream from the liposome binding site of the aroF gene.

pKB750 indicates that it is not a lac promoter (a
It differs from the above expression vectors in that it is expressed under the control of the roF promoter.

As shown in Figure 3, 7) KB750 is made from two fragments.

The first fragment is the αγOF gene with its naturally associated promoter, obtained by passing from pKB45 to pKB648;
Starting from the EcoRV site 5' of the F gene and α
It ends with a KpnI site located next to the Pv1L■ site at the rOF end.

The second fragment is the pheA gene with a Kpn1 site attached to the 5' side of the pheA gene and an ECoRI site attached to the 3' side of the pheA gene. The Kpn1 site is, for example, pKB
5t1L on the 5' side of pheA of 45 or a suitable derivative
One site is cut and excised by exonucleolysis towards pheA, thereby creating itl and then K
pnI') linker is added.

The EcoRI site is an EcoR repaired f3an site.
It is made by joining to the I site.

These fragments were joined at the ■<pn1 site and pBR322
77KB 750 is obtained.

Construction of KB358 The rOF gene product is essentially not inhibited by phenylalanine, which substantially inhibits the aroG gene product. However, when the concentration of phenylalanine increases further due to the aggregation of the above genes (i.
The aroF gene product will lose some of its catalytic activity.

Therefore, the reaction catalyzed by DAHP synthase could possibly be the limiting factor in synthesizing phenylalanine at such a high aK, with mutants of a,rOF not inhibited even at such high concentrations. T! I think it's valid. αro synthesizes all three aromatic amino acids
Substantially dependent on aroF, a type of isozyme,
Using living organisms, it is possible to carry out the selection method described below, which does not include the lethal analogs mentioned in the Background of the Invention.

The parent strain used in this selection method should synthesize all three aromatic amino acids and rely primarily on the aroF gene product. This is accomplished by using any of a number of E. coli strains (eg KB258) that do not have the aro G gene.

αr o H gene is sufficient to proliferate cells A
It may be present since it does not synthesize HP synthetase. If the medium is supplemented with low concentrations of tyrosine (about 20 fnl/l) or phenylalanine at sufficiently high concentrations (at least about 5!J/l) to inhibit the aroF gene product. is intracellular DA
If HP synthase activity is insufficient to supply tyrosine and tryptophan precursors, cell proliferation will cease.

Spontaneous mutations occur in some cells, and the mutation sought is one that releases the αrOF gene product from phenylalanine inhibition. This mutation can be identified because the resulting enzymatic reaction allows the synthesis of tyrosine and tryptophan, thus allowing the mutant to grow.

The parent strain, which relies primarily on aroF for the synthesis of aromatic amino acids, has a
) is cultured in the presence of phenylalanine at a concentration of A naturally occurring mutant that is able to grow at such phenylalanine concentrations is a mutation in aroF (αr
OF) and occurs at least in part as a result of a.
ro F results in an enzyme that is insensitive to feedback inhibition of phenylalanine. The same mutation apparently also eliminates susceptibility to chiro/,/. KB2
One such mutant of 58 is KB358X,! ! It has been given a character and deposited in the American Type Culture Collection K ATCC 53046. This deposited strain was produced 1C under the conditions described below.

KB358 can proliferate in the presence of phenylalanine at a higher concentration than σ0 degrees, which inhibits the proliferation of KB258, but a lag phase is observed before reaching the proliferative phase.

It is also possible to transfer the aroF*-containing genetic material from KB358 to other E. coli strains using known techniques, thereby introducing the aroF advantage into these recipient strains.

Production of Phenylalanine A large number of bacterial strains can be used as production microorganisms for phenylalanine fermentation.

AT to American Type Culture Collection
Escherichia coli K12 strain YMC deposited as CC33927
9 (Proceedings of the National Academy of Sciences by Backman et al.
ational Academy of 5cienc
as) USA78 (1981), 3743-374
7] were transformed with pKB712 or pI (B2S3) and the resulting bacteria were treated with M9 salt, 4 mg/
Culture at 37° C. in the presence of ml glucose and 1 μg/ml thiamine. Incubation continues for ~15 hours OIO, at which point the phenylalanine content in the supernatant is determined.

Plasmid pKB750 or 7) KB712 is
Transforming E. coli strains such as KB285 and I (B280), as described in our co-pending U.S. patent application Ser. It can also be used for pK
B663. is used to transform mutant strains of E. coli such as KB358. KB280 and KB2
85 are ATCC39461 and ATCC39463 respectively in the American Type Culture Collection.
It has been deposited as. The obtained bacteria are used to synthesize L-phenylalanine as described herein.

Phenylalanine concentration is determined by phenylalanine assay ATCC
8042 (Difco Manual, 1953) to quantify biologically.

Vector pKB 663. pKB712. pKB750
Strains ゜pKB766 and KB358 were stored at the American Type Culture Collection, Rockville, Maryland, ATCC 39462゜39856.39, respectively.
857.39858 and 53046. Biotechnica Interna 7, the assignee of this issue, is responsible for replacing these cultures in the event that they die before the expiration of the patent term, and for issuing the above patents to the ATCC. shall be responsible for notifying the public, at which point the deposit will be made available to the general public. Until that point, the above deposits shall be subject to 37 C.
FR Section 1.14 and 35 USC Section 1
It will be available to the Commissioner of Patents subject to 12 conditions.

Other implementations Other implementations are within the scope of the claims.

For example, vector pKB663. Derivatives of pKB712 and pKB750 can be used as expression vectors, and derivatives of pKB766 can be used as expression vector precursors in case a suitable foreign promoter is inserted. The term "derivative" refers to a naturally occurring or genetically engineered variant of a vector that retains the intended function.

The above selection methods and reliance on non-feedback regulated isozymes can be applied to other biosynthetic pathways, such as the lysine, methionine and threonine biosynthetic pathways. Other hosts of the vector vectors described above can also be used.

[Brief explanation of the drawing]

Figures 1 to 4 are plasmid pK and B663゜pK, respectively.
B712. 0 (5 other people) represents the process of creating pKB750 and pKB766 by genetic manipulation.

Claims (25)

[Claims] (1) Feedback inhibition of a first isozyme that catalyzes a reaction in a biosynthetic pathway and whose catalytic activity is substantially inhibited at a concentration exceeding the first concentration level of an inhibitor consisting of a target compound or its metabolic precursor; A method of transforming a cell to increase the production of a compound of interest from said biosynthetic pathway by avoiding a second inhibitor whose catalytic activity is not substantially inhibited at a first concentration level of said inhibitor. A method comprising transforming said cell with DNA comprising a gene encoding an isozyme, and said gene being transcribed in an amount sufficient to enhance production of a compound of interest at said inhibitor concentration. (2) The method according to claim 1, wherein the cell is a member of the Escherichia coli species. (3) Claim 1, in which the inhibitor is the target compound
The method described in section. (4) The method according to claim 1, wherein the target compound is an aromatic amino acid. (5) The method according to claim 4, wherein the target compound is phenylalanine. (6) The method according to claim 1, wherein the first isozyme and the second isozyme are selected from the products of aroF, aroG and aroH, respectively, and the first isozyme is a product of a different gene from the second isozyme. (7) The method of claim 6, wherein the first isozyme is a product of aroG and the second isozyme is a product of aroF or aroH. (8) The method according to claim 7, wherein the second isozyme is a product of aroF. (9) the transcription of the gene is feedback regulated by a transcriptional repression effector, and the transcriptional regulation is controlled by providing the cell with a sufficient number of copies of the gene to outnumber the effector; A method according to claim 1 in which the method of claim 1 is avoided. (10) The target compound is selected from three amino acids, threonine, methionine, and lysine, and the isozyme catalyzes a reaction common to at least two biosynthetic pathways of the amino acids. Method. (11) Cells produced by the method according to claim 1. (12) A cell capable of biosynthesizing a target compound, wherein the biosynthesis consists of a reaction catalyzed by an isozyme that resists feedback inhibition by the target compound or its metabolic precursor, and the isozyme The above-mentioned cell is produced in the cell by a gene that is not feedback-repressed by the target compound or its metabolic precursor, and the cell is produced by transforming a precursor cell using DNA containing the gene. . (13) The cell according to claim 12, wherein the isozyme is a product of the aro gene and the target compound is an aromatic amino acid. (14) The cell according to claim 13, wherein the isozyme is a product of aroF or aroH, and the target compound is phenylalanine. (15) A method for producing a target compound, which comprises culturing the cells according to claim 11 or 12 in a liquid medium and recovering the target compound from the medium. (16) from a parent cell containing a gene encoding a feedback-inhibited enzyme that catalyzes a chemical reaction common to the biosynthesis of the target compound and the biosynthesis of a second compound essential for cell proliferation; A method of selecting mutant cells that produce an increased amount of a compound of interest over the cell's production level, the parental cells being cultured in the presence of an enzyme-inhibiting concentration of an inhibitor; and a growth-maintaining dose of a second Selecting mutant cells capable of growing under conditions such that the compound is obtained as a result of the catalytic activity of the enzyme. (17) The method according to claim 16, wherein the enzyme is inhibited by a target compound. (18) The enzyme is an isozyme that is inhibited at a concentration of the inhibitor above a certain level, and the parent cell lacks the ability to produce a growth-sustaining amount of the second compound by other isozymes at the concentration level of the inhibitor. , the method according to claim 16. (19) The method according to claim 16, wherein the enzyme is DAHP synthase and the target compound is an aromatic amino acid. (20) Claim 19, wherein the target compound is phenylalanine and the enzyme is encoded by aroF.
The method described in section. (21) Claims in which the enzyme is an isozyme that catalyzes a reaction common to the biosynthesis of at least two amino acids among threonine, methionine, and lysine, and the target compound is one of the amino acids. 1st
The method described in Section 6. (22) The enzyme is further feedback-inhibited by a second compound, and the selected mutant cells produce an enzyme that is not feedback-inhibited by the inhibitor or the second compound. the method of. (23) A mutant cell produced by the method according to claim 16. (24) Cell KB358 (ATCC53046) or its derivative strain. (25) A method for producing a target compound, which comprises culturing the cell according to claim 23 or its derivative strain in a medium, and recovering the target compound from the medium.