A dna fragment encoding the 3-phosphoglyceraldehyde (fgd), fgd, the method of obtaining serine

 

(57) Abstract:

The invention is directed to DNA encoding 3-phosphoglyceraldehyde (FGD) with low sensitivity to inhibition by serine compared with FGD wild type. FGD wild-type presents microbial or yeast FGD. By genetic engineering also created DNA encoding a FGD with changes in 25% From the end of the FGD wild type, preferably in a 52 amino acids of the C-end. The invention provides for obtaining pure serine and serine-containing compounds. 3 C. p. F.-ly, 3 ill., table 2.

I. Background of invention.

The present invention relates to the General field of serine biosynthesis and related serine compounds, in particular tryptophan, as well as to products and methods used in this biosynthesis.

Serine is the primary intermediate compound in the biosynthesis of a wide variety of cellular metabolites, including such economically important compounds like choline, glycine, cysteine and tryptophan. In addition, serine is the only source of carbon and is responsible for 60 - 75% of consumption by the cell C1-links during the formation of 5,10-methylentetrahydrofolate from tetrahydrofolate. Such C1-links and the ins and some pyrimidines (e.g., thymidine and hydroxymethylcytosine).

The scheme of the biosynthesis of serine, shown in Fig.1, typically implemented in a variety of tissues and organisms. The first stage of this scheme is the conversion of 3-phospho-D-glyceric acid (PGA) under the action of the enzyme 3-phosphoglyceraldehyde (FGD) 3-phosphohydroxyamino acid (FPC). The gene encoding FGD, was cloned and sequenced, was also installed amino acid sequence of subunit FGD. Tobey and Grant, J. Biol. Chem. 261, 12179-12183 (1980).

In prokaryotes (in particular, bacteria and microorganisms, such as yeast, but not in higher eukaryotes activity FGD inhibited wild-type contained in the cells by serine. The kinetics of this inhibition, which reportedly runs an allosteric manner. Tobey and Grant, J. Biol. Chem. 261, 12179-12183 (1986); Dubrow and Pizer, J. Biol. Chem. 252, 1527-1551 (1977); Mc Kitrick and Pizer, J. Bacteriol 141, 235-245 (1980).

Tosa and Pizer (J. Bacteriol, 106, 972-982 (1971) researched the effect is usually toxic analogue of serine (L-serine-hydroxamate) on the strain of E. coli. The selection on the culture medium containing the analogue, has led to resistant serine mutants. Some mutants, as shown, have a modification in the enzyme e with reduced susceptibility to serine (see J. Bacteriol, 106, 972-982 (1971), Fig.5, table 6 and page 973, lower left column, page 977, lower left column).

II. Summary of the invention.

One of its aspects the invention is directed to DNA encoding 3-phosphoglyceraldehyde (FGD) with low sensitivity to inhibition by serine compared with FGD wild-type, i.e., DNA encoding FGD with something useful for the biosynthesis of enzyme activity and keep this activity at higher content of serine in contrast to the (unmodified) FGD wild-type.

In the recommended embodiments of the invention FGD wild-type presents microbial FGD or yeast FGD. It is also recommended that created genetic engineering DNA encoding a FGD with changes in 25% C-Terminus of FGD wild type, preferably in a 52-amino acid C-Terminus. For example, the created genetic engineering DNA can encode FGD with deletion of part or all of the C-Terminus. It is also recommended that created genetic engineering DNA encodes FGD with box (for example, between VAL363 and ASN364 or between ALA392 and CLN394) in addition to the above deletions, or in the form of independent changes.

The invention also aims to: a) Fsctor, consisting of established genetic engineering and DNA regulatory DNA located and oriented for the expression of recombinant DNA in the host's system of expression; (C) cells expressing containing such vectors, and (d) ways to get serine or originating from serine compounds by culturing such cells. With regard to the above paragraph (c), the recommended cells with a deletion of the SerA of wild-type.

And yet another embodiment of the invention is directed to cellular engineering (e.g., includes recombinant genetic constructs) with the aim of obtaining FGD-encoding mRNA transcript with a modified 3'-end, and that this transcript is translated by the cell with the formation of FGD with low sensitivity to inhibition by serine compared with FGD wild-type.

The invention provides rosregulirovanie important for the biosynthesis of the point of regulation thereby strengthening the formation of numerous compounds in a downward direction from this point, including, in particular, serine and originating from serine compounds, for example, tryptophan. Other originating from serine cellular metabolites (i.e., for which in their biosynthesis serine is p is to be placed, for example, methionine, monophosphate inosine, purine and some pyrimidines (e.g.: thymidine and hydroxymethylcytosine).

III. Description recommended embodiments of the invention.

A. Fig. 1 - 3.

Fig. 1 reflects the stages of the biosynthesis of L-serine from glucose.

In Fig. 2 shows the sequence of E. coli SerA gene according to Tobey and Grant (see above) and amino acid sequence, derived on the basis of the gene.

Fig. 3 reflects bioperene L-serine and tetrahydrofolate in glycine and N5N10-methylentetrahydrofolate.

B. Establishment is not sensitive to serine FGD.

1. The creation of genetic engineering designs.

Featured embodiment of the invention is directed to the biosynthesis of serine and related serine compounds, for example, the above compounds derived from serine in the biosynthesis. The first stage in the biosynthesis of such compounds, as shown below, is to create not sensitive to serine FGD.

We have found that in the FGD, there are specific associated with serine feedback domain, and this domain can be changed with a decrease in the sensitivity to serine, but by maintaining the level of useful activity FGD. On is used as a reference in the subsequent discussion. The sequence of Fig. 2 includes 410 amino acids (including the initial Mel, split-off in the Mature protein). Domain FGD, can reduce the sensitivity to serine without breaking FGD activity is within 25% of the C-Terminus of the molecule, most preferably in the range of 50 C-terminal residues.

Examples of modifications FGD covered by the scope of the invention include deletions of some or all 42 C-terminal amino acids, as well as insertion or replacement within this area, decreasing sensitivity to serine while maintaining the efficiency of FGD. For example, insertion of amino acid residues between Val 363 and Asn 364 leads to higher values of Kifor DGF compared with the wild type when saving activity FGD.

More noticeable increasing values of Kioccurs when the deletion of some or all of the C-terminal amino acid residues. For example, deletions of C-terminal residues GTIRARLLY and replacement on ASLD leads to increasing values of Kiseveral orders of magnitude while maintaining the level of useful activity FGD. Examples of other inserts in the scope of the invention include insertion between Ala392 and Gln394.

Other useful modifications include deletions of the C-Terminus in addition to the inserts and the option is, who may be constructed by genetic engineering methods, including modifying the 3'end of the coding region, which encodes the C-terminal amino acids, with subsequent transformation of the strain-master broker for the purpose of expression of the modified FGD enzyme.

The following methods are searched enzymes with altered affinity to serine (Kiand FGD activity (see below).

2. Search created genetic engineering structures.

In search of genetic constructs derived above methods, the following methods of analysis of FGD activity and sensitivity to serine.

Although not having to invent special significance, analysis of FGD activity nevertheless necessary to establish the degree of sensitivity to serine the modified enzyme. As is well known to specialists, the enzymatic activity is a function of the total number of enzyme molecules and catalytic activity of each molecule. Thus, when comparing the catalytic activity associated feedback options FGD need to take measures to adequately control the relative number of molecules of FGD in the samples for which compares the relative the bottom to determine unambiguously the level of gene expression in cells transformed truncated SerA genes (due to their reduced survival), the most appropriate approach to the comparison of FGD activity generated by different constructs and wild type, is integration at the chromosomal level modified SerA gene that contains standard regulatory elements, in a single copy with subsequent collection of transformants and the determination of the relative catalytic activity in comparison with FGD from wild-type cells.

Can be used any method suitable for determining FGD activity. FGD activity can be determined by measuring both direct and reverse reactions by the method of Mc Kitrick Iohn C. and Lewis I. Pizer, J. Bacteriol 141, 235-245 (1980).

Enzymatic analysis, above, is suitable for determining the sensitivity to serine any FGD enzymes, including enzymes with chemically modified C-end. The analysis is performed in the presence of different concentrations of serine. Catalytic activity in the presence of serine compared with the catalytic activity in the absence of serine, and then calculate the value of Ki.

In most cases, it is recommended to reduce the sensitivity to serine without significant changes FGD is, however, 3-phosphoglyceraldehyde, listed in table 1.

Other designs with modified 3'-ends are also covered by the scope of the present invention, since it is so easy to get tested design and transform the cell by the method of the invention and to analyze the inhibition of serine FGD activity.

Any vector, which leads to the expression of FGD protein with a lack of sensitivity to inhibition by serine, acceptable for the present invention. In General, however, in the absence of sensitivity to serine should avoid high levels of expression do not have feedback FGD, because of the resulting large cytoplasmically number of serine or originating from serine metabolites can be toxic to cells. Thus, any design, coding inhibited by the type of feedback FGD with normal catalytic activity and expression level similar to the level of a native gene, resulting transformation is likely to show high levels of expression of FGD with decreasing cell survival. The toxicity of a large number of generated serine can actually lead to mutants with reduced expression of FGD is I useful in the initial search, however, it is recommended to integrate SerA constructs in single copies in the genome for chromosomal level. In addition, chromosomal integration, as shown below, facilitates the determination of the activity of deletional type of feedback FGD. Thus, in most cases, when it is expected or desirable high catalytic activity, it is recommended to use vectors suitable for odnostadiinoi chromosomal integration. Experts know many such vectors and strategy of their application. Cm. for example, Backman, application for U.S. patent N 07/091837, filed September 1, 1987, which is introduced here as a reference. Can be obtained vectors and constructs to successfully transform and Express the enzyme in an appropriate host with the aim of obtaining the target product. Ways to accomplish this are well known in the art and the present invention does not play a decisive role. In addition to the modified FGD coding DNA expression vector should contain various other elements, described below.

First, present on the vector coding sequences should be accompanied by appropriate regulatory elements required for the Azania ribosomes and the termination sequence. In most cases, native SerA regulatory sequence will be the recommended source of catalytically active part of the molecule, although it is recognized that can be used for many other regulatory sequences, both known and those yet to be discovered.

Secondly, it is recommended that the vector was present sequence encoding a selective marker, and/or genes reporters together with appropriate regulatory elements. The expression of such selective markers is useful in the identification of transformants. Appropriate selective marker genes include genes encoding ampicillin, tetracycline and chloramphenicol.

Third, the desirability of starting point of replication in the plasmid vector in a greater degree depends on the desirability of maintaining genes in the chromosomes or outside the chromosomes. For specialists obvious that strategy, due to the absence of the replication source can be used for promotion of integration into the chromosome. See, for example, U.S. patent 4743546 (Backman and others), which is introduced here as a reference.

After the construction of the expression vector is completed, stitely to serine FGD protein. In most cases, it is useful to use the cells for which it is known inhibition of endogenous FGD protein serine, and in which endogenous SerA gene was deleted and replaced with the mutated gene of the invention. Such cellular systems are applicable for overproductive related serine metabolites. Cells obviously containing sensitive to serine proteins include prokaryotes and yeast.

The following examples illustrate but do not limit the invention.

Example 1.

The design of the SerA gene alleles encoding stable feedback type 3-phosphoglyceraldehyde.

E. coli K12 SerA allocated in the form of a fragment of 6.4, etc. acting on the product of partial hydrolysis in the presence of Sau3A, cloned in BcII site plasmids pTR264. Cm. Backmann and others , application for U.S. patent N 07/285128 filed 16.12.88 and Roberts and others , Gene 12, 123 (1980). This plasmid was named pKB1302. Sal I-Sph I fragment 3 p. T. O. from pKB1302 DNA containing SerA gene clone in pUC19 with education pKB1321. pKB1370 form cloning Hind III - Sal I fragment 3, etc., of O., containing SerA gene in pBR322.

The SerA allele encoding stable feedback type 3-phosphoglyceraldehyde form insert Xba I linkers in the restriction sites in the 3'region SerA GE the insertion of linkers gives: a) pKB1507, encoding a truncated 3-phosphoglyceraldehyde; (b) pKB1507 encoding a truncated 3-phosphoglyceraldehyde, and (c) pKB1508, which encodes 3-phosphoglyceraldehyde with the insertion of four amino acid residues.

Hydrolysis pKB1321 in the presence of Pst I give 3'-terminal redundancy of one DNA strand in the position of 1888. Blunt ends form action fragment maple DNA polymerase 1. Linkers are ligated to the blunt ends of the fragments with the formation of plasmid pKB1509 that encodes a 3-phosphoglyceraldehyde with an insertion of two amino acids, and pKB1510 that encodes a truncated 3-phosphoglyceraldehyde. In the hydrolysis product pKB1370 in the presence of Kpn I to form blunt ends by the action of the fragment maple DNA polymerase 1 and after insertion of linkers receive plasmids encoding truncated 3-phosphoglyceraldehyde (pKB1455 and pKB1512) or 3-phosphoglyceraldehyde with an insertion of two amino acid residues (pKB1511). Deletion plasmids pKB1530 and pKB1531 formed by inserting a BamHI - XbaI fragment of 0.8 T. p. O. from pKB1508 or BamHI - XbaI fragment of 0.9 T. p. O. from pKB1509 respectively in BamHI - XbaI fragment of 5.8 T. p. O. plasmids pKB1511.

In the following table 2 summarizes the data obtained for different designs.

For each constrcifor serine are shown in table 1, as well as the relative catalytic activity for three of these structures after chromosomal integration (see below). N/A indicates that the design is not subjected to chromosomal integration, resulting in the level of activity was not standardized.

3. Chemical modification.

For professionals it is clear that the deletion or modification of the C-Terminus in FGD wild type can be carried out enzymatically or chemically, for example, by the action of various carboxypeptidase, including carboxypeptidase Y or ladirovannye and the presence of lactoperoxidase.

4. Application anticonvulsivants mRNA.

On the other hand has the ability to reduce sensitivity to serine in vivo creating FGD-encoded transcripts, truncated at the 3'end, through education anticonvulsivants mRNA comprising a nucleotide sequence complementary to the coding parts of the 3'regions of the native or transformed FGD-coding sequences.

C. Obtain the target compounds.

As shown in Fig. 3, serine is an intermediate connection upon receipt of glycine. Serine is also prompomolmash for the synthesis of methionine, purines (including inosine) and some pyrimidines. Thus, overproduction serine of phosphoglycerate can be used in a wide variety of bacterial production systems, including systems for the production of choline, glycine, cysteine, methionine, tryptophan, and all purines, including monophosphate inosine.

The following typical examples illustrate the invention.

Example 2.

Obtaining strain-master.

Internal relative to the born in the plasmid SerA gene sequence replaces resistant to kanamycin gene. The resulting plasmid is then used for decontamination of the SerA gene in strain-master exchange of alleles according to the following procedure.

SerA region UMS (ATCC 33920) clone of chromosomal DNA is partially hydrolyzed in the presence of Sau 3AI, complementation PC1523 (arg 161, arg F58, Ser A27, pur A54, thr -25, ton A49, re IAI, spo TI) obtained from the Coli genetic storage Center, Jelski University, new Haven, Connecticut, fragment 3, etc., of O., bearing the SerA gene, subcloning in pUC19 to form plasmid, named pKB1321. From this plasmid fragment Sa II - Hind III 3 T. p. O. again clone in pBR322 with the formation of plasmid pKB1370. Kpn I site at 3'-end of the SerA gene is transformed into BamHI m from pUC-4-K AC (pharmacy), containing the gene of Tn903 kanamycin-resistant. This new plasmid denoted as pKB1429. Create a pBR322 derived, named pKB701 (ATCC 39772, see the application for U.S. patent N 06/757019, which is introduced here as a reference), in which MboI and TThIII I, flanking the replication source, in turn Cloned sites. SalI - EcoRI fragment containing SerA::KanR from pKB1429, clone in pKB701 education pKB1438. Plasmid pKB1438 hydrolyzing in the presence of a kpni restriction sites with the removal of the ori region. The larger fragment containing the region encoding resistance to ampicillin, as well as SerA::KanR, completing the cycle and used in the transformation UMS in the presence of CaCl2. After transformation of the host WMS cells are subjected to selection on ampicillin. In terms of selection of resistant to ampicillin clones develop due to the introduction of circular DNA by homologous recombination in the flanking regions of the SerA gene. Growth resistant to ampicillin isolate in the absence of ampicillinbuy selection leads to loss of the gene of resistance to ampicillin due to homologous recombination of repetitive sequences SerA gene. These strains were identified by loss of ability to produce-lactamases by using Onscreen (BRL) according to the manufacturer's instructions. Ducret the global to ampicillin clones require for their growth on minimal medium in the presence of serine and has also resistant to kanamycin. One of these isolates was named KB875.

Example 3.

Chromosomal integration of the modified SerA sequences sharing alleles.

Allele SerA1455 introduced into the chromosome in a manner analogous to the method of administration SerA::KanR, shown in example 2. Briefly the method consists in the following. Fragment (SaII - Hind III) carrying SerA1455 allele, clone in pKB 701. Plasmid beginning removed by hydrolysis in the presence of a kpni restriction sites. For transformation with resistance to ampicillin and education of the said strain using circular DNA. After selective breeding using Amscreen and replicas for kanamycin allocate KB904 (SerA1455), which, as shown, is sensitive to ampicillin and kanamycin KB904. Received SerA1544 alleles can be transferred to producing strains PI transduction. Miller (1972) "Experimental mol. genetics". Cold spring Harbor Press, pp. 201-205.

Example 4.

Chromosomal integration of the modified SerA sequences recD-dependent gene replacement.

For insertion into the chromosome secA1508 alleles used a different approach. Strain KB875 given the state of the third subunit of ectonucleoside Y for JGP101. Plasmid pKB1506 translate in a linear state and used for transformation JGP101 in the serine prototroph by the method Shevell, etc., J. Bacteriol. 170, 3294-3296 (1988) with the formation of IC P103. Allele serA1508 can then be put into producing strains PI transduction. Miller and other "Experimental mol. genetics". Cold Spring Harbor Lab., pages 201-205 (1972).

Collection overproducing metabolites.

For overproductive related serine metabolites can be generated cells producing FGD with reduced sensitivity to serine, and grown in a fermenter in appropriate circumstances, in most cases on the stationary phase. Cells can then be harvested, lysed and target metabolites can be isolated using standard biochemical techniques. Conditions, principles and references to the cultivation of microbes and collection of specific metabolites can be found in Gruege and Cruege (1982) (Biotechnology. Handbook of industrial Microbiology) and Herrman and Somerville (1983) (Amino acids. Biosynthesis and genetic regulation), which entered here as references.

1. A DNA fragment encoding the 3-phosphoglyceraldehyde (FGD) with reduced sensitivity to inhibition by serine at reevecarney in table 1.

2. Catalytically active FGD with low sensitivity to inhibition by serine obtained by cultivation of a strain of E. coli, transformed with the expression vector containing the DNA fragment under item 1.

3. The method of obtaining serine by cultivation of the producer strain, characterized in that as a producer uses a strain of E. coli, transformed with a DNA fragment coding for FGD with 25% change in the s-end of FGD wild type.

 

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