[Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

[New method of construction of artificial translational-coupled operons in bacterial chromosome].

The new method of translational-coupled operons construction in bacterial chromosome has been developed on the basis of recombineering approach. It includes construction in vitro of the artificial operon with efficiently translated proximal cistron followed by its insertion E. coli chromosome, modification of the operon due to Red-driven insertion of the special "Junction" with excisable selective marker in the intercistronic region of the initial operon and excising the marker. The structure of this Junction has been designed and tested in the present investigation. It consists of: 1) E. coli rplC-rplD intercistronic region for placing the TAA-codon of the proximal operon's gene in the SD-sequence (TAAGGAG) of rplD; 2) Cm(R)-gene flanked by lambdaattL/R-sites in such a fashion that after lambdaInt/Xis-driven excision of the marker the residual lambdaattB-site would not contain the termination codons in frame with ATG of rplD; 3) E. coli trpE-trpD intercistronic region for location of ATG of trpD at the position of initiation codon of the distal gene of original operon. The general design of desired construction provides the conversion of the original two-cistronic operon into three-cistronic operon with translational-coupled genes, where the coupling of the artificial ORF (rplD'-lambdaattB-'trpE) with the proximal gene is occurred due to rplC-rplD intercistronic region and the coupling of this ORF with the distal gene--due to trpE-trpD. The experimental implementation of the described strategy was showed by construction of artificial operon P(tac-aroG4-serA5, where expression optimization of the distal serA5 gene was achieved via construction of three-cistronic operon with translational-coupled genes.

[Gene yddG of Escherichia coli encoding the putative exporter of aromatic amino acids: constitutive transcription and dependence of the expression level on the cell growth rate].

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of oversynthesis of aromatic amino acids, YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P(yddG) promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with sigma70 or sigma(S) subunits. By constructing a gene of the hybrid protein YddG'-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of beta-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity reached approximately 3 to 4% of the level of LacZ in E. coli wild-type cells under induction of the lac operon). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (> 1 mM) in the culture medium.

[Tuning of expression level of the genes of interest located in the bacterial chromosome].

The new method of construction of the set of E. coli clones, differing in the promoter strength upstream the gene of interest, has been developed and tested using native E. coli MG 1655 lacZ gene as the reporter. This method includes the construction of the promoter-carrying DNA fragment obtained by PCR with consensus P(tac) as a template and the primers that lead to randomization of 4 central nucleotides in the promoter "-35"-region, linking the obtained fragments with the selective marker (Cm(R)) followed by Red-driven integration of the resulted DNA fragments directly in E. coli MG1655 chromosome instead the native lacI-gene and promoter/operator region of lac-operon. Due to direct determination of LacZ-activity in the independently obtained clones-integrants, we have found 14 new promoters (from 44 = 256 possible variants) that differ in their strength up to 100 fold (LacZ-activity in the corresponding strains smoothly varies from 10(2) for the weakest tested promoter up to 10(4) Miller U detected for the initial P(tac)). Sequencing of obtained promoters revealed that randomization of three positions in the "-35"-region is sufficient to obtain representative promoter library that would decrease the total number of potential promoter variants from 256 up to 64. It seems probable that exploiting of the developed method leading to one-step construction the library of clones with varied expression of gene/operon of interest could be useful tool in the modem metabolic engineering for optimization of genes expression.

A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator.

A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.

Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1 beta synthesis in Escherichia coli.

A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and a low Cop at 28 degrees C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42 degrees C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-1 beta (hIL-1 beta) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1 beta (re-hIL-1 beta) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the water-soluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1 beta. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg of re-hIL-1 beta/g of wet cells. The re-hIL-1 beta specific activity was about 2 x 10(8) units/mg, coinciding with that of the authentic hIL-1 beta.

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells.

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

[Creation of artificial hybrid operons with partially overlapping genes to achieve an expression of heterologous genes in Escherichia coli cells]. / Sozdanie "iskusstvennykh gibridnykh operonov s chastichno perekryvaiushchimisia genami" dlia dostizheniia ékspressii geterologichnykh genov v kletkakh Escherichia coli.

A new method of optimization of foreign gene expression in E. coli, based on the construction of hybrid operons with partially overlapping genes is described. The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis. Such an approach has provided the synthesis of human interferon alpha F in E. coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture. The reinitiation of the distal gene translation is shown to take place in the intercistronic region. Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter. The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E. coli trpE-trpD genes are used for translational coupling.

[Effectiveness of expression of chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli. III. Relative effectiveness of cloned promoters of Escherichia coli and coliphages]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. III. Izuchenie otnositel'noi éffektivnosti klonirovannykh promotorov Escherichia coli i kolifagov.

The study on the rate of initiation of model gene cat transcription under the control of E. coli (Plac UV5, Ptrp, Pcat, Ptac), phage lambda (PL, PR), phi X174 (PD) promotors was carried out by means of hybridization of pulse labelled in vivo mRNA with the DNA coding parts. The presence of gene bla(Apr) with its own constitutive promoter in all the recombinants permitted to use the level of appropriate mRNA in the cell as an internal standard. This method allowed to evaluate the true efficiency of the promoters in question. The strength of the promoters studied is shown to be equal within the limit of one order value.

[Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulator regions in Escherichia coli cells. II. Molecular cloning of promoters]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. II. Molekuliarnoe klonirovanie promotorov.

The trpOP, lacUV5, tacOP, PR and PL-promotors were cloned in the previously obtained pML4 vector plasmid. The expression of structural gene cat was studied by the chloramphenicolacetyltransferase determination in cell extracts. The level of protein synthesis by appropriate recombinant plasmids was analysed in vivo and in vitro. It was shown that the efficiency of the gene expression is determined by both the "strength" of the promotors and mRNA translation specificity. The obtained collection of the plasmids might be used for the determination of the promotor strength by the hybridization of pulse-labeled mRNA with DNA and an effective expression of the genes by means of "hybrid protein gene" and "hybrid operon" constructions.

[Effectiveness of translation coupling in hybrid operons]. / Issledovanie éffektivnosti transliatsionnogo sopriazheniia v gibridnykh operonakh.

The possibility of creating artificial overlappons was studied on the model of two genes, that coding for the N-terminal part of lambda cro protein and the cat of E. coli. To test the dependence of translational coupling efficiency on the intercistronic region a series of recombinant DNA molecules carrying different hybrid operons with partially overlapping genes was constructed. The translational efficiency of the distal to the promoter gene was shown to depend on the intercistronic region structure: overlapping of the AUG codon with the terminating one of the proximal gene in the UGAUG manner is optimal for the translational coupling, and the displacement of AUG at several nucleotides in both directions decreases the translational reinitiation efficiency for the ribosomes, that have synthesised the first gene product.

[Study of the kinetics of the reaction reuniting DNA fragments catalyzed by DNA-ligase. Case of homogeneous molecules]. / Isledovanie kinetiki reaktsii vossoedineniia fragementov DNK, kataliziruemoi DNK-ligazoi. Sluchai gomogennykh molekul.

The kinetics of different DNA forms formation were studied during the ligation reaction. Reaction was carried out using as a model BamH1 DNA fragments of plasmid pBR322 and DNA-ligase of bacterophage T4. The products of the reaction were separated with 1% agarose gel, followed by DNA quantitation in the corresponding bands by scanning fluorimeter. The kinetics of the ligation reaction were measured in the range of DNA concentrations from 1 to 100 micrograms/ml. The system of differential equations, that described the kinetics of the ligation reaction, was obtained. This has no analytical solution, but may be approximately calculated with a computer. The analysis of kinetic equations was made in order to optimize the ligation reaction. The optimal DNA concentrations were found allowing the maximal yield of multimer circular DNA forms. The simple formula for calculatng these concentrations were developed. The yield of circular DNA multimers might be increased if the reaction is carried out at two stages: initially at high DNA concentration for linear multimers formation followed then a lower concentration after appropriated dilution. The formula for determiantion of the dilution time is presented. The data obtained allow to optimize the reaction conditions providing the recombinant DNA molecules formation.

[Kinetics of the reactions joining DNA fragments, catalyzed by DNA-ligase. II. Heterogeneous mixture of fragments. Optimization of the reaction conditions]. / Issledovanie kinetiki reaktsii vossoedineniia fragmentov DNK, kataliziruemoi DNK-ligazoi. II. Geterogennaia smes' fragmentov. Optimizatsiia uslovii reaktsii ligirovaniia.

The system of kinetic equations was proposed describing the joining of heterogeneous DNA fragments by DNA-ligase. The DNA fragments were considered as flexible rods and characterized only by molecular weights and concentrations. The program for calculating the concentrations of all circular and linear DNA forms is described. The program has been written in FORTRAN IV and calculations were made with EC 10--10. For the maximal yield of the multimeric circular DNA molecules it is recommended to begin the ligation at a high DNA concentration and then after a certain fixed time to dilute the reaction mixture. The optimum time intervals and ratios of fragment concentrations were calculated. The yield of different ligation products has been determined. The calculations of optimum reaction conditions were carried out for the construction of hybrid molecules containing DNA fragments with or without selective genetic markers.

[Interaction of RNA polymerase with hybrid plasmids carrying Escherichia coli threonine operon]. / Osobennosti vzaimodeistviia RNK-polimerazy s gibridnymi plazmidami, nesushchimi treoninovyi operon E. coli.

The RNA polymerase binding with two hybrid plasmids carrying threonine operon genes was studied. RNA polymerase binding sites were localized using nitrocellulose binding assay and electron microscopy visualization of the RNA polymerase--DNA complexes. To confirm that observed RNA polymerase binding sites are real promoters we analyzed RNA transcripts synthesized in vitro by hybridization with DNA fragments. The promoters of following genes were localized on the plasmid maps: ampicilline, threonine and RNA-primer of DNA replication. Two latter promoters bound RNA polymerase only being in the supercoiled DNA molecule. Several additional binding sites were found. The positions of some of these sites corresponded with known sites of rho-independent transcription termination.

[Distribution of DNA sequences recognized by specific endonucleases]. / Raspredelenie posledovatel'nostei DNK, uznavaemykh spetsificheskimi éndonukleazami.

The molecular weight distributions of fragments obtained after endonuclease treatment of DNA were studied. DNA's from pigeon and E. coli and restriction enzymes EcoRI and BamHI were used. The samples of 14C- and 3H-labelled DNA were treated by endonucleases, separated electrophoretically in 1% agatose gels, and radioactivity distributions along gels were measured. From these data weight and number distributions and the average molecular weights of DNA fragments were determined. EcoRI-fragments of phage DNA were used as standards for the molecular weight calibration. The experimental results are compared with the expected data calculated from the DNA GC content. The molecular weight distribution of fragments and the average molecular weights of BamHI-fragments of pigeon DNA and EcoRI- and BamHI-fragments of E. coli DNA differed from random ones. It is suggested that certain genomes contain regions in which the probability of endonuclease cleavage strongly differs from the average probability of such a cleavage for the entire genome.

[Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli cells. I. Construction of vectors for the cloning of transcription regulatory elements]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. I. Konstruirovanie vektorov dlia klonirovaniia reguliatornykh élementov transkriptsii.

New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions. In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor. Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype. As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present. The plasmids were sequenced and their restriction maps were established. Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments.

[Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli]. / Udalenie intronov metodom amplifikatsii DNK iz genomnogo gena interleikina 1 beta i ékspressiia gena interleikina 1 beta v Escherichia coli]

The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.

[Design of a species-specific DNA probe based on the pFra plasmid of the plague pathogen]. / Konstruirovanie vidospetsificheskogo DNK-zonda na osnove plazmidy pFra chumnogo mikroba.

The recombinant plasmid pBS1 carrying a 2 kb SalGI fragment of Yersinia pestis pFra plasmid was constructed by insertion of the fragment into a vector plasmid pBR327. SalGI-BspRI 400 bp subfragment was recloned into a pBR322 vector plasmid. Open reading frame was found in the fragment by DNA sequencing technique. The subfragment designated F1-probe permits one to identify specifically the Yersinia pestis strains harbouring pFra plasmid, thus, differing them from closely related Yersiniea and other representatives of Enterobacteriaceae family.

[Analysis of the nucleotide sequence of a small colicinogenic plasmid Cold gene coding for lysis]. / Analiz nukleotidnoi posledovatel'nosti gena lizisa maloi kolitsinogennoi plazmidy Cold.

The nucleotide sequence of a DNA fragment of a small colicigonenic plasmid Cold-CA23 containing the lysis gene cdl has been defined. An open reading frame has been found within the fragment for 48 amino acid polypeptide with the molecular mass 4920 Da. The polypeptide is homologous to the lysis proteins encoded by the small colicinogenic plasmids ColE1 and CloDF13. The homology was also found for the structural and functional arrangement of the cdl gene and the plasmid CloDF13 lysis gene. The analysis of the possible secondary structures of the lysis protein encoded by cdl gene has revealed the amino acid sequences able to form the alternative structures. The described feature is supposed to be required for lysis protein translocation from cytoplasmatic to outer membrane of Escherichia coli cells.

[Expression of the chloramphenicol acetyltransferase gene is under control of various promoters of E. coli and phage lambda]. / Ekspressiia gena khloramfenikolatsetiltransferazy pod kontrolem nekotorykh promotorov E. coli i bakteriofaga lambda.

Plasmids have been constructed with the structural region of the cat gene being under the control of the lactose (lacUV5), tryptophane (trpOP), operons of Escherichia coli, the hybrid trp-lac (tac) promoter and early bacteriophage lambda promoters (PL, PR and PLIT). The expression of chloramphenicolacetyltransferase gene in Escherichia coli cells harbouring such recombinant plasmids and pBR325 as well has been examined by determining the chloramphenicol resistance and studying the enzyme activity of Cm-acetylase. A high level of enzyme synthesis is connected with transcription from PL, PR and tac promoters.