BR102014017026A2 - method of obtaining the expression vector, expression cassette, expression vector and expression system for constitutive production and secretion of heterologous proteins - Google Patents

Abstract

method of obtaining the expression vector, expression cassette, expression vector and expression system for constitutive production and secretion of heterologous proteins. The present invention relates to a method of obtaining the pt7bsxa expression vector by steps (a) including specific restriction sites; (b) directed evolutionary construction of the randomly mutated variant library in the promoter region by error-prone pcr; (c) constructing, by directed evolution, the library of randomly mutated variants in the co1e1 (ori) replication origin region by error-prone pcr; and (d) inserting the protein gene of interest, in particular, oxidoreductase enzymes, transferases. hydrolases, lyases, isomerases and / or ligases. In addition, the present application relates to an expression cassette composed of (a) variants of the base promoter region (seq id no. 12a, seq id no. 27a, seq id no. 30a, seq id no. 32a, seq id 33a, seq id no 34a, seq id no 38a, seq id no 39a and / or combinations thereof obtained by site-directed mutagenesis, such as seq id no 40a (rpa), seq id no 41a (rpb), seq id no. 42a (rpab)), (b) variants of the origin of replication region (ori co1e1) (seq id no. 50, seq id no. 60, seq id no. 70, seq id no. 80) and (c) a gene of a protein of interest, a pt7bsxa expression vector, obtained by said procurement method, containing the expression cassette and an expression system for the constitutive production and secretion of heterologous proteins in in particular, oxidoreductase enzymes, transferases, hydrolases, lyases, isomerases and / or ligases, preferably the wild-type (xy1a) xylanase enzyme gene in escherichia coli.

Description

"METHOD OF OBTAINING EXPRESSION VECTOR, EXPRESSION CASSETTE, EXPRESSION VECTOR AND EXPRESSION SYSTEM FOR CONSTITUTIVE PRODUCTION AND SECRETION OF HETEROLOGICAL PROTEINS" Field of the Invention [001] The present invention relates to a method for obtaining the expression vector pTBxA through steps (a) inclusion of specific restriction sites; (b) constructing, by directed evolution, the randomly mutated variant library in the promoter region by error-prone PCR; (c) constructing, by directed evolution, the library of randomly mutated variants in the ColEl replication origin region (orz) by error-prone PCR and (d) insertion of the protein gene of interest, in particular, oxidoreductase enzymes, transferases hydrolases, lyases, isomerases and / or ligases. Additionally, the present application relates to an expression cassette composed of (a) BsXA promoter region variants (SEQ ID NO: 12a, SEQ ID NO: 27a, SEQ ID NO: 30a, SEQ ID NO: 32a, SEQ ID 33a, SEQ ID NO 34a, SEQ ID NO 38a, SEQ ID NO 39a and / or combinations thereof obtained by site-directed mutagenesis such as SEQ ID NO 40a (RPA), SEQ ID NO 41a (RPB), SEQ ID NO: 42a (RPAB)), (b) variants of the origin of replication region (ori ColEl) (SEQ ID NO. 50, SEQ ID NO. 60, SEQ ID NO. 70, SEQ ID NO. 80) and (c) a gene of a protein of interest, an expression vector pT7BsXA, obtained by said method of obtaining, containing the expression cassette and an expression system for the constitutive production and secretion of heterologous proteins in in particular, oxidoreductase enzymes, transferases, hydrolases, lyases, isomerases and / or ligases, preferably the wild-type xylanase A (XylA) enzyme gene in Escherichia coli.

Background of the Invention Recombinant DNA technology can produce genetic changes in microorganisms in order to improve biochemical and physiological characteristics that are of value to the industry and can be commercially exploited, as in the case of enzyme production. In addition, enzyme properties can be improved by streamlined design or directed evolution, and the combination of the two strategies will present a more successful route to improve the properties and function of an enzyme, or even create a new enzyme function (PAIVA, CLA and SA). -PEREIRA, P. Enzymes in Biotechnology: Production, Applications and Market The application of molecular biology in enzyme production p.30 2010).

Nature has produced enzymes by an evolutionary process without the use of "rational design" or any "knowledge" about the structure and function of these complex and intriguing biomolecules. Recent advances in recombinant DNA technology, coupled with high-throughpuf selection techniques and genetic and proteomic methodology have increased the development of new biocatalysts and biocatalysis processes. There are three different routes to obtaining high stability enzymes: (1) isolating enzymes from organisms that live in extreme conditions; (2) by a rational method of mutagenesis (classical protein engineering) or (3) by directed evolution (ED). In particular, Directed Evolution technology has emerged as a powerful tool in engineering new enzymes of biotechnological interest (ARNOLD, FH; VOLKOV, AA (1999) Curr Opin Chem Biol. 3: 54-59; ROHLIN, L .; OH. J. K .; LI AO, JC (2001) Curr. Opin. Microbiol. 4 (3): 330-335).

[004] Plasmid vectors are widely used to modify living cell properties and the understanding of replication and copy number regulation of the plasmid has been a focus of research for over four decades (CAMPS, M. Modulation of ColEl-like plasmid replication for recombinant expression gene Recent PatDNA Gene Seq 4 (2010) 58-73; DEL SOLAR, G., GIRALDO, R., RUIZ-ECHEVARRIA, MJ, ESPINOSA, M., DIAZ-OREJAS, R. Replication and control of circular bacterial plasmids Microbiol Mol Biol Rev 62 (1998) 434-464).

[005] Xylanases (1,4 β-D xylanhydrolases EC 3.2.1.8) are endo-xylanases that randomly hydrolyze xylose β-1,4 glycosidic bonds of the xylan structure (plant cell wall hemicellulose polysaccharide) releasing xyloligosaccharides. For the industrial use of this enzyme it is important that it is thermostable, and in this sense, technologies have focused a great effort to increase the catalytic activity of this enzyme at elevated temperatures.

ColE1-like plasmids are the most popular vectors for recombinant protein expression. The replication of plasmid ColEl is tightly controlled by a highly dynamic antisense RN A mechanism, optimizing the metabolic load of the plasmid with the physiological state of the host. Plasmid homeostasis is unbalanced in inducing recombinant protein expression due to non-physiological levels of expression and because of the variable amino acid composition of recombinant proteins. Deregulation of plasmid replication is a major cause of the collapse of plasmid-based expression systems because of a simultaneous increase in metabolic load (due to increased mean copy number) and the likelihood of plasmid free cell generation (due to increase in copy number variation). Interference between regulatory elements of co-resident plasmids has comparable effects on plasmid stability due to incompatibility between plasmids with different origin of replications. A high copy number of the plasmid aims to obtain a high dosage of genes, while preserving the stability of the expression system (CAMPS, M. Modulation of ColEl-like plasmid replication for recombinant gene expression. ; 4 (1): 58-73).

ColEl replication is under the control of a 550 bp sequence known as the origin of replication of the plasmid or ori. Selzer and his collaborators observed remarkable conservation between the ColEl, pMBl, p15A, RSF1030 and CloDF13 ori sequences and proposed that such sequences could have comparable replication mechanisms (SELZER, G., SOM, T., ITOH, T., TOMIZAWA, J. The origin of replication of plasmid p5A and comparative studies on nucleotide sequences around the origin of related plasmids (Cell 1983; 32: 119-129). This was later confirmed, and these plasmids are known as ColEl family plasmids (DEL SOLAR, G., GIRALDO, R., RUIZ-ECHEVARRIA, MJ, ESPINOSA, M., DIAZ-OREJAS, R. Replication and control of circular bacterial plasmids Microbiol Mol Biol Rev 1998; 62: 434-464).

In the ColEl plasmid family, ori encodes a pre-primer RNA that forms a stable hybrid with a size of 30 nucleotides with the template DNA strand. This stable hybrid, known as the R-loop, is processed by RNAseH to create a 3 '-OH end. Correct placement of the 3'-OH end of the pre-primer is critical for RNA processing (MASUKATA, H., TOMIZAWA, J. Effects of point mutations on formation and structure of the RNA primer for ColEl DNA replication. Cell 1984; 36 : 513-522), and to the extension Pol I (YANG, YL, POLISKY, B. Suppression of ColEl high-copy-number mutants by mutations in the polA gene of Escherichia coli. J Bacteriol 1993; 175: 428-437) .

Replication is regulated by the transcription of a 108 nt long antisense RNA encoded by the plasmid (known as RNA I), which is transcribed from the PI antisense promoter (CESARENI, G. , HELMER-CITTERICH, M., CASTAGNOLI, L. Control of ColEl plasmid replication by antisense RNA Trends Genet 1991; 7: 230-235; POLISKY, B. ColEl replication control circuitry: sense from antisense Cell 1988; 55: 929 -932). The pre-primer (known as RNA II) is transcribed from a sense promoter, P2. The "antisense" promoter is much stronger, which results in a 100-fold excess of the inhibitor over the pre-initiator. This constitutes a negative feedback loop as inhibitor levels are proportional to the number of plasmids. The result is a specific number of copies per cell for a given set of conditions. Plasmid copy number regulation, however, is very dynamic and responds to metabolic fluctuations and the presence of additional plasmids in the host. The short half-life of RNA I (only 2 minutes during exponential growth) facilitates rapid response to environmental conditions. An antisense regulatory system is unnecessary for plasmid replication, but the resulting sustained increase in plasmid copy number (known as runaway plasmid replication) may be lethal to the host. The 5 'end of ori ( to position -386) can be deleted, which makes the plasmid copy number proportional to P2 transcription (PANAYOTATS, N. DNA replication regulated by priming promoter. Nucleic Acids Res 1984; 12: 2641-2648) .

[010] ColEl plasmids are the most commonly used plasmids with a broad spectrum of applications (GRABHERR, R., BAYER, K. Impact of targeted vector design on Co / El plasmid replication. Trends Biotechnol 20 (2002) 257-260 ; WANG, Z., YUAN, Z., HENGGE, UR Processing of plasmid DNA with ColEl-like replication (Plasmid 51 (2004) 149-161), including all pBR322 derivatives used for DNA cloning, such as , pUC plasmids (YANISCH-PERRON, C. VIEIRA, J., MESSING, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33 (1985) 103-119), pBluescript ( ALTING-MEES, MA, SHORT, JM pBluescript II: gene mapping vectors (Nucleic Acids Res 17 (1989) 9494) and most vectors used for heterologous protein expression (such as the pET series). All ColE1 plasmids include a 590 bp region that controls plasmid copy numbers known as the origin of replication (on). On ColEl encodes two antisense RNAs, called RNAI and RNAII, respectively, which are transcribed from complementary strands of a partial overlapping region within the origin of replication (CESARENI, G., HELMER-CITTERICH, M. , CASTAGNOLI, L. Control of ColEl plasmid replication by antisense RNA Trends Genet 7 (1991) 230-235; EGUCHI,,, ΙΤΟΗ, T., TOMIZAWA, J. Antisense RNA Annu Rev Biochem 60 (1991) 631- 652). The formation of the complex between RNAI and RNAII leads to a reduction in RNA primer and plasmid replication. The interaction between RNAI and RNAII is stabilized by a small helical protein, Rom (EGUCHI, Y., TOMIZAWA, J. Complex formed by complementary RNA stem-loops and its stabilization by a protein: function of ColEl Rom protein. Cell 60 ( 1990) 199-209; HELMER-CITTERICH, M., ANCESCHI, MM, BANNER, DW, CESARENI, G. Control of ColEl replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII EMBO J 7 (1988) 557-566) and in pUC plasmids, the deleted Rom / Rop gene leads to reduced stability of the RNAI / RNAII complex resulting in plasmids that have a high copy number compared to the plasmid pBR322 family (TOMIZAWA, J., SOM Control of ColEl plasmid replication: enhancement of binding of RNA I to the transdermal polymer by the Rom protein Cell 38 (1984) 871-878).

An important element in the process of gene expression is the interaction of the enzyme RNA polymerase with the promoter region of the gene. Analyzes show that the gene promoter has certain regions that aid in its recognition by RNA polymerase. The RNA polymerase in prokaryotes has five subunits (α, β, β ’, σ, ω) and the subunit σ is mainly responsible for the recognition phase of the promoter. The interaction of the RNA polymerase (R) holoenzyme with the promoter (P) initiates a series of structural transitions from the closed promoter complex (RPc) to the open complex (RPo). RPc and RPo are in equilibrium, and this equilibrium depends on many factors, including temperature, Mg2 + concentration, and nucleotide sequence of the promoter region. Transcription begins when the RNA polymerase, associated with factor o, binds between 40 to 60 base pairs of the promoter to form the closed promoter complex. RNA polymerase binds tightly to the promoter to produce an intermediate RNA-promoter (I) complex and open the double strand for approximately 14 nucleotides before the first nucleotide of the gene, giving access to the genetic information contained in the template strand to generate finally, the open RNA-promoter complex. Transcription termination is signaled by a nucleotide sequence found at the end of the gene, called the terminator (MURAKAMI, KS, DARST, S.A, 2003, Bacterial RNA polymerases: the whole story, Current Opinion in Structural Biology 13 (1), 31-39).

In the promoter are found two specific regions: a sequence of approximately six nucleotides centered at -35 from the initial transcription site and a hexamer, centered at -10. The sequence at -35 acts as a signal for recognition of the σ portion of RNA polymerase, while the sequence at -10 allows the complex to be closed to open. Moreover, the distance between them is relevant, despite the variable size and low conservation in the nucleotide bases that make up DNA, as it may be critical in the interaction of hexamers with RNA polymerase (ΟΡΡΟΝ, EC) geometry. prediction algorithms: a choice for small training dataset? ”, Dissertation Department of Computer Science, University of the Western Cape, 2000). In addition, a greater number of contacts (in addition to the -10 and -35 regions) between RNA polymerase and the promoter DNA may increase the affinity between both (AIYAR, SE, GOURSE, R. L, ROSS, W. Upstream A- tracts increase bacterial promoter activity through interactions with the RNA subunit RNA polymerase Proc Natl Acad Sci (1998) USA 95: 14652-14657; ESTREM, ST, ROSS, W., GAAL, T., CHEN, ZW, NIU, W. , EBRIGHT, RH, GOURSE, RL Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit. Genes Dev. 1999 13: 2134-2147). Prolonged RNA polymerase - DNA interaction may influence closed to open complex isomerization (DAVIS, CA, CAPP, MW, RECORD, T.. T., JR & SAECKER, RM) Effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase, Proc. Natl Acad. Sci. USA, (2005) 102, 285-290). In this sense, the involvement between the DNA in the closed complex is strongly dependent on the promoter region nucleotide sequence (CELLAI, S., M. ANGIAROTTI, L., VANNINI, N., NARYSHKIN, N., KORTKHONJIA, K., EBRIGHT. , R., RIVETTI, C. Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-open promoter complex (EMBO reports (2007) 8, 271-278).

Through the pioneering work of Arnold (ARNOLD, FH (1996) Chemical Engineering Science. 51 (23): 5091-5102) and Stemmer (STEMMER, WPC (1994) Nature. 370 (6488): 389-391) who introduced powerful tools that significantly improved the applied methods of mutation and screening in protein engineering. This new approach encouraged broad acceptance of the enzyme-driven evolutionary technique for biotechnological application.

[014] Directed evolution (DE) can be defined as; A process in which a class of a particular agent (drugs, enzymes, etc.) is generated and selected with the best performance for a given characteristic. ED usually begins with the creation of a mutant gene library. The gene product that shows the best performance for a given property of interest is identified by selection methods and the isolated gene, which encodes this enzyme, is submitted to new cycles of mutagenesis and selection, aiming at an accumulation of mutations favorable to the "evolution" of. certain feature. This process of directed evolution mimics the process of Darwinian evolution (relating mutations, recombinations to selection pressures). Gene diversity is typically introduced by two main techniques: random PCR (error-prone PCR) or in vitro gene recombination (DNA shuffling) methods (PATTEN, P.; HOWARD, RJ; STEMMER, WPC (1997 ) Curr Opin Biotechnol 15: 523-530; REETZ, T., JAEGER, KE (2000) Chemistry 6: 407-412).

[015] Taq DNA polymerase, a standard that is used in conventional PCR reactions, has a mutation error ratio that usually decreases when in directed mutagenesis experiments. For example, enzymes with corrective subunits such as TaqP / id have a mutation ratio ranging from 10 6 to 107, while enzymes lacking the corrective subunits (such as TaqDNApol) have an error ratio of 10 4 to 10 5. This ratio, however, can be significantly increased by the conditions of polymerase use, such as variation in magnesium cofactor concentrations, partial replacement of Mg + 2 (magnesium) by Mn + 2 (manganese), optimization of dNTP concentration in proportions. unbalanced and variations in the number of PCR cycles. This method called error-prone PCR (epPCR) is simple, efficient and restricted to the gene region of interest.

Interest in enzyme engineering by ED has grown significantly in recent years (CHIRUMAMILLA, RR; MURALIDHAR, R.; MARCHANT, R.; NIGAM, P. (2001) Mol Cell Biochem. 224 (1-2): 159-68). Many successful examples are known of the directed evolution of enzymes for industrial use. Properties such as substrate specificity, enzymatic activity, enantioselectivity and thermal stability are some of the main enzymatic characteristics that have been altered by the application of native enzyme directed evolution techniques (CHIRUMAMILLA, RR; MURALIDHAR, R .; MARCHANT, R .; NIGAM , P. (2001) Mol Cell Biochem. 224 (1-2): 159-68).

International publication WO 97/25410 deals with a method for in vivo production of a cell library comprising a multiplicity of mutant genetic elements, wherein an error-prone PCR is used on each ancestor cell to replicate all or one part of a genetic element regardless of the host chromosomal replication mechanism. The genetic element comprising i) an origin of replication from which replication begins, ii) optionally a genetic marker, for example, a gene conferring resistance to an antibiotic, iii) a gene encoding the polypeptide of interest. Further, the international publication discloses methods for generating a DNA sequence encoding a desired variant of a polypeptide of interest and for determining such a DNA sequence.

[018] International publication WO 2007/039461 relates to a method for the in vivo generation of DNA, RNA, peptides and protein libraries by means of a genetic element harboring a viral or fanatic origin of replication that is independently reproduced by a viral or phantasmal error-prone polymerase not physically linked to the genetic element within a host cell further containing viral or phage helper nucleotide sequences and proteins that are required for replication of the viral or phage genetic element. The nucleotide or nucleotides of interest to be diversified are introduced into the genetic element and physically linked to the viral or phage origin of replication.

US Patent Application 2012083012 deals with an expression system for the production of a multienzyme complex, the system includes a nucleic acid molecule containing a promoter operably linked to a nucleotide sequence including multiple genes encoding enzymes which are components of the multienzyme complex.

[020] International publication WO 01/18218 provides for increased production of a protein of interest from a fungal host. The invention discloses nucleotide sequences comprising a regulatory region, in operative association with a xylanase secretion sequence and a gene of interest. The gene of interest encodes a protein selected from a nutraceutical, pharmaceutical, industrial, feed, food additive and enzyme product. Preferably, the gene of interest encodes a cellulase, hemicellulase, a lignin, pectinase, protease or peroxidase degradation enzyme. The present invention also relates to vectors and hosts comprising these nucleic acid sequences, as well as methods for producing a protein of interest.

Brazilian patent PI 9503454 refers to a new xylanase, the processes for preparing this xylanase, its uses and compositions comprising it. Further, the invention describes a novel strain of microorganism producing this xylanase and a DNA molecule comprising the nucleotide sequence encoding this xylanase, as well as vectors containing this DNA molecule and the strains transformed by these vectors.

US 2003166053 discloses that in an expression vector having a ColEl replication system, RNAI and RNAII homology of the ColEl origin of replication to uncharged tRNAs are modified mutations in the RNAI gene coding region and corresponding mutations in the RNAII gene. The mutation results in one or more loop 1 and / or loop 2 and / or loop 3 base exchanges of RNAI and RNAII. Also, the application shows that in methods using this vector for recombinant protein production, the copy number of the plasmid is stably maintained and in methods for plasmid production a high copy number of the plasmid is obtained.

US 6,136,567 discloses plasmids comprising: (1) an origin of replication, (2) an additional sequence required for plasmid replication or, preferably, a gene that generates a selective advantage, and (3) two expression cassettes each, which are located between (1) and (2), but are separated by (1) and (2) from each other, and which are free of inverted repeated sequences (except in ( 3)) which are highly persistent despite successive generations of microorganisms containing them.

[024] International publication WO 2011/007005 provides a host vector system and plasmid DNA methods and recombinant protein production. The system allows control of the copy number of a CoIE1 plasmid in B. coli by an RNA molecule that is transcribed from the host genome, and which interacts with the RNAI or RNAII of the transcribed plasmid. The system can be extended to combine PCN control with antibiotic free selection.

[025] Brazilian patent application PI 0515436 is for a vector-host system that uses the RNA-based copy number control mechanism of CoIE1-type plasmids to regulate expression of a marker gene that allows selection. antibiotic-free plasmid and is useful for the production of plasmid DNA and recombinant proteins.

Chinese patent CN 102154361 claims an expression vector for efficiently expressing xylanase and a method of constructing the expression vector, wherein the expression vector comprises a CoIE1 replication in Escherichia coli, a Bacillus subtilis replication (B. sub rep), selection marker and promoter, wherein a signal peptide sequence and an XynA xylanase gene sequence are arranged downstream of said promoter; Escherichia coli / Bacillus subtilis are taken as a transport vector, the signal peptide with strong xylanase expression is classified to efficiently express xylanase.

US 6,472,177 describes an expression vector adapted for inducible amplification and transcription of a heterologous polynucleotide operatively linked to a transcription promoter provided in the vector. The invention further provides a method for using the vector for producing large amounts of a heterologous polypeptide encoded by the heterologous polynucleotide. The pBAC of the invention is derived from a BAC cloning vector by modifying the BAC cloning vector to include a conditional ori for amplifying pBAC and an inducible promoter for transcriptional regulation of an inserted gene. Both conditionally or inducible promoter can be activated together or separately by suitable signals in a host cell. Once a polynucleotide of interest is inserted into pBAC and pBAC is inserted into a host cell, the level of the polynucleotide-encoded product can be controlled by regulating the copy number of the expression vector or the activity of the promoter to which the transcription is transcribed. of the inserted polynucleotide is controlled or both.

[028] International publication WO 2006/083240 deals with a codon optimized xylanase coding sequence and the expression of xylanases in microorganisms and yeast. The invention further relates to the use of multiple copies of the xylanase expression construct for high levels of protein expression. The invention also provides the use of xylanases as food additives or foods. Additionally, the international publication is directed to methods for the expression of enzymes to increase thermotolerance by expressing them in organisms than glycosylated proteins compared to expression of the same enzyme without glycosylation.

In the light of the foregoing, the Depositor has developed an expression system for the constitutive production and heterologous secretion of the xylanase enzyme gene from Bacillus subtilis in Escherichia coli and a vector / plasmid (pT7BsXA) containing an expression cassette including a promoter region, a replication origin region, and a xylanase enzyme gene.

Description of the Figures [030] Figure 1 shows a banded agarose gel electrophoresis that characterizes pT7BsXA vector digestions following PCR site-directed mutagenesis to introduce Apcâ and Xhol restriction sites into the pT7BsXA vector, where, ( A) 2% agarose gel showing restriction digestions of pT7BsXA vector with Apal (lane A), Xhol (lane X), both showing the same pattern as negative control 0. EcoRI digestion (lane E) cleaves a single restriction site to produce the linear shape of the vector and (B) the enhanced contrast image of a 1.5% agarose gel showing the pT7BsXA (L10) vector (lane V) and the same vector after ApaI digestion (lane) A) and Apal + Xhol (lane A / X). Double digestion releases a 650 bp fragment containing the ColE 1 M origin of replication being the molecular size marker and the open and closed arrows indicating the 750 and 3,000 bp markers, respectively.

[031] Figure 2 shows the activities of xylanases extracted from broth, which were performed using each of the selected mutant vectors. In the xylanase catalytic activity measurement assay, xylose release occurs, and its dosage is performed through oxidation by ADNS, so that the xylose concentration is directly proportional to the absorbance at 540 nm. Figure 2 also shows the xylanase activities produced by the different mutant vectors and the L10 vector containing the unchanged ori (positive control). EDI, ED2 and ED3 samples are variants selected from the initial solid medium screening that grew after 16 hours. Values show the means and standard deviations of five measurements, with the mutant with a significant increase (p> = 0.05 in a paired t-test) indicated with an asterisk and selected for nucleotide sequencing.

Figure 3 shows the quantification of plasmid copy number by real time PCR. Plasmids pT7BsXA (IOL) and pT7BsXA (ED2 (SEQ ID NO. 70)) were used to transform E. coli DH5a and the cultures were grown to 0.6 µM OD in LB medium containing 100 mg / ml ampicillin. Copy numbers were calculated from the rtPCR experiments, assuming a cell density of 2.2 x 10 7 at an OD 100 of 0.6.

Figure 4 shows the location of the regulatory and structurally relevant regions at the ColEl origin of replication. The total 590 nt ori ColEl contains the regions of the promoter PI and P2 for transcription of 108 nt RNAI and 555 nt RNAII, respectively (-10 and -35 sites shown). Both RNAI and nascent RNAII (<200 nt) include three hairpin loops (denoted I, II and III) in regions -452 / -481, -484 / -512 and -519 / -540, respectively. The RNAI / RNAII complex forms through interactions between the unpaired complementary bases at the tips of loops I-III and, after stabilization by Rom protein (also known as ROP), RNAII can be further transcribed for inclusion in the coding region of the I-III loop. rod b (b, -359 / -380) and rod g (g, -303 / -324) which form a loop that further stabilizes the RNAI / RNAII complex. In the absence of RNAI binding, bases included in stem b of longer (> 200 nt) RNAII transcripts may form paired base interactions with stem a (a, -460 / -481). The interaction of the a / b stems in the longer RNAII transcripts results from the redistribution of the paired base loop I, leading to an alternative secondary RNAII structure in which loop I and II are absent and the RNAI / RNAII complex not present. can be formed. RNAII / DNA interactions are mediated by guanine-rich regions (G) and hairpin Η1 and H2 loops play a role in RNANH processing by RNaseH and correct primer formation for DNA polymerase I. The numbering used is in relation to the first base. of DNA extended from the RNAII primer (t, +1).

[034] Figure 5 shows halos resulting from enzymatic activity in selective solid culture: (A) being the variant library colonies obtained after epPCR reaction and transformation into E. coli. Black arrows indicate the colonies that formed halos due to xylan degradation by bacteria-secreted xylanase, and red arrows indicate non-xylanase-secreting colonies, while (B) in 1 (top panel) highlights an unformed colony. halo and in 2 (bottom panel) a halo colony.

[035] Figure 6 shows the tests of xylanhydrolytic activity in liquid medium. In the xylanase catalytic activity measurement assay, xylose release occurs which is measured by oxidation with ADNS, the xylose concentration is directly proportional to the absorbance at 540 nm. The different variants selected from the activity-to-optical density ratio (Ativs4o / D06oo) demonstrated activity relatively superior to the activity of the recombinant wild-type xylanase.

Figure 7 shows the nucleotide sequences of the promoter region. The DNA sequence corresponds to the promoter region of the pT7BsXA vector between bases 2619 and 2751. All variant DNA sequences were analyzed using the Bioedit Sequence Alignment Editor program. In the boxes: (A) - corresponds to the -55 region from the beginning of the xylanase A gene translation and (B) - corresponds to the region - 95. The epPCR-altered nucleotide bases are highlighted within each box.

[037] Figure 8 shows the result of amplification reactions for insertion of promoter region point mutations, where (A) shows PCR amplification reactions using the RPA and PSS3 (channel 1) or RPB and PSS5 oligonucleotide pairs (channel 2) and (B) representing the extension and amplification reaction using the amplified fragments in figure A.

Figure 9 shows the xylanolytic activity curves of E. coli expressed and secreted wild-type xylanase A transformed with the pT7BsXA vectors containing the RPA, RPB and RPAB promoter regions. Duplicate experiments of pT7BsXA vector expression with the native promoter region (pT7BsXAl (- -) and pT7BsXA2 (- · -) curves compared to vectors with the RPA promoter regions (Al (-À- ) and A2 (- ▼ -)), RPB (curves BI (-A-) and B2 (- ▼ -)) and RPAB (curves AB1 (-A-) and AB2 (-Y-)).

Description of the Invention The present invention relates to a method of obtaining the pT7BsXA expression vector by steps (a) including specific restriction sites; (b) constructing, by directed evolution, the randomly mutated variant library in the promoter region by error-prone PCR; (c) directed evolutionary construction of the randomly mutated variant library in the ColEl replication origin region (on) by error-prone PCR and (d) insertion of the protein gene of interest, in particular, oxidoreductase enzymes, transferases hydrolases, lyases, isomerases and / or ligases. Additionally, the present application relates to an expression cassette composed of (a) BsXA promoter region variants (SEQ ID NO: 12a, SEQ ID NO: 27a, SEQ ID NO: 30a, SEQ ID NO: 32a, SEQ ID 33a, SEQ ID NO 34a, SEQ ID NO 38a, SEQ ID NO 39a and / or combinations thereof obtained by site-directed mutagenesis such as SEQ ID NO 40a (RPA), SEQ ID NO 41a (RPB), SEQ ID NO: 42a (RPAB)), (b) variants of the origin of replication region (ori ColEl) (SEQ ID NO. 50, SEQ ID NO. 60, SEQ ID NO. 70, SEQ ID NO. 80) and (c) a gene of a protein of interest, an expression vector pT7BsXA, obtained by said method of obtaining, containing the expression cassette and an expression system for the constitutive production and secretion of heterologous proteins in in particular, oxidoreductase enzymes, transferases, hydrolases, lyases, isomerases and / or ligases, preferably the wild-type xylanase A (XylA) enzyme gene in Bscherichia coli.

In a first embodiment of the present invention, the method of obtaining the pT7BsXA expression vector utilizes the pT7T3318 primer plasmid containing a 590 bp (Ori) ColE1 origin of replication region.

[041] In step (a) of the pT7BsXA expression vector method, the inclusion of specific restriction sites by the enzymes Xhol and / or ApaI (endonucleases) is accomplished by PCR site-directed mutagenesis at the flanking positions. of the ColEl origin of replication at positions 1383 and 2062, respectively. In addition, the oligonucleotides (primers) specific APAP / Apar (APAP; 5 'TTCCGGGCCCGCAAAAGGCCAG 3', Apar, 5 'GGATGGGCCCTTTGCTCACATG 3') and XhoF / XhoR (XhoF; 5 'TGTTCTCGAGAACTCACGTTAAGGG 3', XhoR; 5 'CTGGCTCGAGCCACTGAGCGTCAG 3') are used in the method of the invention.

[042] In step (b) of the method of obtaining the pT7BsXA expression vector, the directed evolutionary construction of the random mutant promoter region library by error-prone PCR occurs through the steps of (a) performing PCR's with 40 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 1 minute and then gel purification of the generated fragments; (b) screening of random libraries to select variants that produce higher levels of activity; (c) sequencing of promoter mutants of selected variants, plasmid DNA being extracted from mutant cell pellets with a significant increase in hydrolytic xylan activity and the promoter region nucleotide sequence determined by automated DNA sequencing and compared to the original promoter sequence using ClustalW and (d) site-directed mutagenesis combination of the individual mutants identified in steps (b) and (c) above to produce other sequences, for example, RPA, RPB and RPAB.

[043] In step (c) of the pT7BsXA expression vector method, the directed evolutionary construction of the error-prone PCR random mutant origin of ColEl (orí) replication occurs through steps of (a) performing PCR's with 40 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 1 minute and subsequent gel purification of the generated fragments; (b) selection of the high variant ori variants of the antibiotic resistant vector; (c) sequencing the resistant ori variants, particularly EDI (SEQ ID NO. 60), ED2 (SEQ ID NO. 70) and ED3 (SEQ ID NO. 80) and the nucleotide sequences of the ColEl region determined by sequencing the Automated DNA and compared to original ColE1 sequence using ClustalW program; (d) Enzyme assay for variant selection, preferably ED2 (SEQ ID NO. 70), which demonstrates high enzyme production in the high copy number vector mutant source and (e) polymerase chain reaction (PCR) in Real time variant, preferably ED2 (SEQ ID NO. 70) used a pT7T318U template purified from cultured cell cultures to an OD 100 of 0.6. Also, serial dilutions of all samples are analyzed and quantitative analysis of the plasmid copy number is performed by comparing the measured signal with the standard curves.

[044] In step (d) of the pT7BsXA expression vector method, the protein of interest gene is inserted, in particular, oxidoreductase enzymes, transferases, hydrolases, liases, isomerases and / or ligases, preferably wild-type xylanase A enzyme from Bacillus subtilis (XylA) in Escherichia coli [045] In a second embodiment, the present application relates to an expression cassette composed of (a) BsXA promoter region variants (SEQ ID NO 12a, SEQ ID NO: 27a, SEQ ID NO: 30a, SEQ ID NO: 32a, SEQ ID NO: 33a, SEQ ID NO: 34a, SEQ ID NO: 38a, SEQ ID NO: 39a and / or combinations thereof obtained by site-directed mutagenesis such as SEQ ID NO 40a (RPA), SEQ ID NO 41a (RPB), SEQ ID NO 42a (RPAB), (b) variants of the origin of replication region (ori ColEl) (SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80) and (c) a gene of a protein of interest, in particular oxidoreductase enzymes, transferases, hydrolases, lyases , isomerases and / or ligases, preferably the wild-type xylanase A (XylA) enzyme gene.

[046] In a third embodiment, the present invention provides an expression vector pT7BsXA obtained by said obtaining method containing the expression cassette and restriction sites at the flanking positions 1383 and 2062 of the ColE 1 origin of replication.

In a fourth embodiment, the patent application provides an expression system for constitutive production and secretion of heterologous proteins comprising the use of the pT7BsXA vector in Escherichia coli, where the heterologous proteins may be the oxidoreductase enzymes, transferases, hydrolases, lyases, isomerases and / or ligases, preferably the wild-type xylanase A (XyLA) enzyme gene from Bacillus subtilis (XylA).

Example Method for Obtaining Expression Vector pT7BsXA Introducing Restriction Sites for ApaI and XhoI in pT7BsXA

Plasmid pT7T318U (GE Healthcare, Pittsburgh, PA; Genbank U13869.1) included the ori ColEl of 590 bp. This plasmid was previously used for construction of the 3653 bp pT7BsXA expression vector (RULLER, R., ROSA, JC, KNIFE, VM, GREENE, LJ, WARD, RJ Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5a under Biotechnol Appl Biochem 43 (2006) 9-15), which contains a 791 bp genomic fragment of Bacillus subtilis that includes a 639 bp open reading frame encoding xylanase A (XynA) 185 amino acids, plus an initial 28 amino acid sequence. The genomic fragment also included a recognition site for a MerR-type transcriptional regulator (-40 to -27) and a Shine-Dalgarno 5'-AGGAGGT-3'em -6 to -12 sequence (initiation codon numbering) dexylA), E. coli transformation with pT7BsXA resulted in constitutive expression of xylA and efficient secretion of mature protein from culture supernatants. Plasmid copy number was under the regulation of a ColEl origin of replication and the presence of the β-lactamase gene from plasmid pT7T318U conferred an ampicillin resistant phenotype.

Restriction sites for XhoI or ApaI were absent in pT7BsXA and PCR site-directed mutagenesis was used to introduce unique restriction sites for these enzymes at the flanking positions of the ColEl origin of replication at positions 1383 and 2062, respectively. The pT7BsXA vector was used as a template in standard PCR reactions with 10 pmol of the ApàF / ApaR oligonucleotide pair (.ApaF; 5'TTCCGGGCCCGCAAAAGGCCAG 3 ', ApaR; 5'GGATGGCCCTTTGCTCACATG 3', underlined restriction sites) PCR (94 ° C for 1 minute, 55 ° C for 2 minutes, 72 ° C for 4 minutes) using 2.5U TaqDNA high-fidelity polymerase (TrueStart TaqPol - Fermentas-Lithuania), resulting in the amplification of a 3680 bp which corresponded to the complete vector sequence with a ApoI restriction site at both 5'and 3 'ends. Following ApoI digestion, the amplified vector was circularized with T4 DNA ligase. Plasmid DNA was purified from E. coli DH5a which was transformed with this ApcH digested construct to confirm the presence of a unique restriction site. The mutated plasmid was used as a template in a second PCR reaction using the XhoF / XhoR oligonucleotide primers (XhoF; 5'TGTTCTCGAGAACTCACGTTAAGGG 3 ', XhoR; 5'CTGGCTCGAGCCACTGAGCGTCAG 3 ') to give a plasmid containing restriction sites for both Apcd and XhoI. This plasmid was named pT7BsXA (L10) and was used as a template for the creation of the randomized library.

Construction of ColEI Random Mutant Library by gpPCR

An ori ColEl random mutant library was constructed using the error-prone PCR (epPCR) technique (CADWELL, R. C., JOYCE, G.F. Mutagenic PCR. PCR Methods Appl 3 (1994) S136-140). Ten micro liters of buffer reagent (70 mM MgCh, 500 mM KCl, 100 mM Tris-HCl - pH 8.3 and 0.1% glycerol), 10 pL mutagenic DNTPmix (5 mM dGTP, 2 mM ATP, 10 mM dCTP, 10 mM dTTP and 0.5 mM MgCk), 10 pM ApaF and XhoR primers, 10 ng pT7BsXA (L10) were used as DNA template, 2.5 units Taq DNA polymerase (Promega, Madeson, WI) and 50 pL MnCh (700mm). PCR was performed with 40 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute and 72 ° C for 1 minute. After gel purification, the 692 bp reaction product was digested with the ApoI and XhoI restriction endonucleases and ligated to pT7BsXA (L10), which was previously digested with the same two enzymes to eliminate the native ori ColEl sequence.

Selection of High Ampicillin-Resistant Resistant Mutants [051] In order to screen the random mutant library for antibiotic-resistant variants, the ampicillin concentration that prevented the growth of pT7BsXA (L10) -transformed bacteria was determined. Escherichia coli DH5a was transformed with 10 ng of pT7BsXA (L10), and 100 pLe plated on a LB solid medium series containing ampicillin concentrations between 800 and 2,400 pg / mL. No colonies above 1800 pg / mL of ampicillin were observed, and therefore this antibiotic concentration was chosen for random mutant library screening. Following transformation of E. coli DH5a with the random mutant ColEl library into pT7BsXA (L10), cells were plated on solid LB plates containing 1,800 pg / mL ampicillin and the three colonies appearing after 16 hours of incubation. 37 ° C were selected and named as EDI (SEQ ID NO. 60), ED2 (SEQ ID NO. 70) and DI3 (SEQ ID NO. 80), respectively.

Enzyme Assay for Variant Selection [052] The EDI (SEQ ID NO. 60), ED2 (SEQ ID NO. 70) and ED3 (SEQ ID NO. 80) colonies grown on solid medium with high concentration of antibiotic were inoculated into LB liquid medium containing ampicillin and incubated at 37 ° C for 16 hours, and the hydrolytic xylan activity of the supernatant culture was estimated by reducing the sugar production measured by the DNS (3,5-dithrosalicylic) microassay (MILLER, GL Use of dinitrosalicylic acid). acid reagent for determination of reducing sugar Analytical Chemistry 31 (1959) 426-428). Ten microliters of supernatant culture were incubated with 40 µl buffer (200 mM NaAc - pH 6.0) and 50 µl 1% beechwood beech xylan at 55 ° C. After 10 minutes, 100 µl of DNS was added and then heated to 99 ° C for 5 minutes, the reaction was cooled with ice and 1 mL of water was added. After a brief centrifugation at 12,000 g, the absorbance at 540 nm was measured and enzyme activity determined.

Nucleotide Sequencing of Selected Mutants [053] Plasmid DNA was extracted from cellular pellets of such mutants that showed a significant increase in hydrolytic xylan activity and the ColEl region nucleotide sequence was determined by automated DNA sequencing. The obtained sequences were analyzed and compared with the original ColEl sequence using ClustalW (http: //www.ebi.ac.uk/Tools/clustalw2/index.html). Real Time PCR [054] Plasmid copy number per bacterial cell was estimated by real time PCR using as a purified pT7T318U template from cultured cell cultures to an OD100 of 0.6. Reactions were performed using the SYBR green qPCR Supermix-UDG ROX Platinum Kit (Invitrogen, Carlsbad, CA) in a total volume of 25 pL containing 1 pmol of each RT-F and RT-R oligonucleotide primer (RT-F, 5 'TTCCATGCGTTCACATGATT 3 '; RT-R, 5' GATGGCGATCGCACTACTTT 3 ') using an ABI 7500 thermal cycler (Applied Biosystems, Carlsbad, CA). The fluorescent signal was calibrated to known pT7BsXA (L10) copy number values using the softvuare supplied with the apparatus (7500 Real Time PCR System Sequence Detection Software v 1.4). Serial dilutions of all samples were analyzed and quantitative analysis of the plasmid copy number was performed by comparing the measured signal to standard curves. Plasmid copy number per cell was calculated assuming a cell density of 2.2 x 10 7 cells / ml at an OD 60 of 0.6.

Directed Evolution of the pT7BsXA Vector Promoter Region

[055] In order to obtain the mutant promoter vector library, called variants, it was constructed using the error-prone PCR (epPCR) technique (CADWELL, RC, JOYCE, GF Mutagenic PCR. PCR Methods Appl 3 (1994) SI36 -140). However, the pT7BsXA promoter region between nucleotides 2619 and 2834 was amplified using an epPCR reaction performed with mutagenic PCR buffer (10x) by mixing 70 mM MgCb, 500 mM KC1, 100 mM tris-HCl (pH 8 , 3) and 0.1% gelatin. A dNTP (mutagen) mixture was composed of 2 mM dGTP, 2 mM ATP, 10 mM dCTP and 10 mM dTTP, and a 0.5 mM MnCh solution. For the preparation of the mutagenesis reaction, 10pL of the mutagenic PCR buffer, 10 μΕ of dNTPmix (mutagen), 10pMol of each of the oligonucleotides (PSS3 5'-CGACTCACTATAGGGAATTC-3 and PSS5'5'-AGTAGTCTGTGCTAGC-3 ') were used. , 10pg template DNA (pT7BsXA), 5U Taq DNA polymerase with low correction rate (Fermentation) and 50 μΜ MnCU, yielding a final volume of 100 μΕ The reaction was thermocycled for 30 cycles of 94 ° C for 1 minute, 45 ° C per minute and 72 ° C for 1 minute, together with negative control without the mold. Subsequently, a reaction sample was separated and analyzed on 1.5% agarose gel.

SUBCLONING OF MUTANT FRAGMENTS

After the mutagenic polymerase chain reaction the mutant DNA fragments and the pT7BsXA expression vector were analyzed and purified from the 1.5% agarose gel and digested with NheI and EcoRI restriction endonucleases (10U each - both from Fermentas brand). ), together with compatible Fast Digest buffer for both enzymes, completing the reaction volume with sterile water to 30μ1. Reactions were incubated for 3 hours at 37 ° C, followed by inactivation of enzymes at 65 ° C for 20 minutes.

Expression Vector Digestion pT7BsXA

A culture containing 500 mL of selective liquid LB medium was inoculated with pT7BsXA transformed DH5a cells which were grown for 16 hours at 37 ° C at 180rpm. Plasmid DNA from this culture was extracted using the standard alkaline lysis technique (SAMBROOK, J. and RUSSEL, DW (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York) and DNA isolated. was quantified by spectrophotometry. In about 20pg of this vector, 20U of CIAP (sheep intestine alkaline phosphatase) was added to dephosphorylation of the vector and prevent re-ligation of cohesive ends. The reaction was incubated for 30 minutes at 37 ° C and subsequently purified on 1.5% agarose gel.

Fragment Binding and Vector to Create the Random Mutant Library [058] After quantification of the mutated fragments and the linearized and dephosphorylated vector (pT7BsXA), both were diluted to the ratio of 100pg vector and 6C ^ g of fragments in a final volume. of 20pL. 3pL 10x binding buffer and 1 μΐ T4 DNA ligase (as described in the T4 DNA ligase Kit -FERMS manual) were added and the reaction occurred for 14 hours at 16 ° C.

Transformation into Escherichia coli (DH5al [058]) After quantification of the epPCR fragments and the linearized and dephosphorylated vector (pT7BsXA), both were diluted to 100pg vector and 60pg fragments in a final volume of 20pL. 3μ adicionados were added 10x binding buffer and 1 μΐ of T4 DNA ligase (as described in the T4 DNA ligase Kit -FINEMENTS manual) the reaction occurring for 14 hours at 16 ° C.

Transformation of Random Mutant Library into E. coli DHSaCOMFETENT Cells

A Petri dish containing solid LB medium (1% Tryptone, 1% NaCl, 0.5% yeast extract, 1.5% agar) was striated with E. coli DH5a strain cells preserved at - 196 ° C in 10% glycerol suspension and incubated at 37 ° C overnight. Subsequently, some colonies were inoculated into tubes containing 5 ml of LB liquid medium for 12 hours while stirring at 180 rpm at 37 ° C. After bacterial growth, 3 ml of the culture in 500 ml of LB was inoculated into 2 L Erlenmeyer flasks, allowed to stir at 180 rpm for 2 to 3 hours until the optical density of the culture reached 0.360. The culture was transferred to sterile polypropylene tubes and centrifuged at 2000 # for 12 minutes at 4 ° C and the supernatant discarded. Cells were resuspended in 34 ml FSB buffer (100 mM KCl, 45 mM MnCl 2) 10 mM CaCl 2> 3 mM HACoCl 3> 3 mM AcK, 10% glycerol) pH 7.5, incubated for 15 minutes at 4 ° C and centrifuged again under the same conditions. Cells were resuspended in 16 ml FSB buffer, and 10 ml DMSO were added and the cells incubated without ice for 15 minutes. The cells were aliquoted in cryotubes and stored in liquid nitrogen (-196 ° C).

[060] The 20 μΐ volume of gene-vector binding was diluted in IX, 5X and 10X fractions in aqueous solution and used to transform competent cells, which after regeneration (growth in liquid LB medium for 1 hour). stirring at 200 rpm and 37 ° C) were diluted and plated until a satisfactory colony distribution was achieved throughout the 180 mm diameter plate area containing selective solid LB.

Selection of Solid Xylanase Producing Variants [061] Plates containing the newly transformed colonies were covered with a layer about 2 mm thick of a substrate-containing medium (100 mM Hepes, 170 mM NaCl pH 7, 0.2% agar and 1% xylan) and left until solidification. Subsequently, a 1 hour incubation at 55 ° C was performed in a preheated oven for the catalytic activity of secreted xylanase to degrade xylan in the medium. After the activity time the Congo red reaction was performed to identify the variants expressing xylanase based on the technique developed by Teather and Wood (TEATHER, RM, WOOD, PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacterium from the bovine rumen Appl Environ Microbiol 1982, 43 (4): 777-780). Congo red interacts with xylan to form a deep red color, so the regions around the xylanase producing colonies became clear due to xylan degradation. The petri dish was then flooded with 0.1 mg / ml Congo red (diluted with water) and incubated for 15 minutes at room temperature. After discarding the dye, it was added to the 1 M plate (NaCl) and incubated for 10 minutes at 25 ° C. The saline solution was discarded and then 0.5% acetic acid was added to the plate for color development and identification of the clear region around the colony.

[062] The variants with the largest diameter of the clear region formed due to xylan degradation, when compared to the control, were identified, isolated and stored in TE buffer (10 mM Tris HCl and 1 mM EDTA pH 7.4).

[063] The sample containing bacteria was heated for 10 minutes at 96 ° C for agar solubilization, disrupting the cells to release their plasmid DNA. The DNA-containing solution was centrifuged at 20,000g for 20 minutes and the supernatant separated and precipitated with sodium acetate and ethanol. Total DNA was resuspended in 10 µl DNAse free sterile water and a 5 µg aliquot used to transform competent DH5a cells.

Analysis of Enzyme Activity of Xylanases Produced by Selected Variants [064] Transformed cells were grown in selective solid LB medium for 16 hours and isolated colonies were individually used to inoculate 3 mL of selective liquid LB medium and grown for 16 hours at 37 ° C. 200 rpm. After growth, the cultures were subjected to optical density analysis at 600 nm in a spectrophotometer (UV-Visible Spectrophotomerer 50 Bio - Varian) in order to estimate the number of cells present in the samples. Subsequently, the cells were separated from their supernatant by centrifugation and the supernatant used for the determination of enzymatic activity. To this end, about 10 μ contendo ^ of the enzyme-containing culture supernatant was mixed with 50 mL of substrate (Sigma 1% oat spelt xylan) completing the reaction volume to 100 pL with a 100 mM buffer solution and pH 6.0. (0.2M Na2HPÜ4 and 0.1M acetic acid). The activity test was performed at a balanced temperature of 55 ° C for 10 minutes. After this time, the reaction was stopped by the addition of 0.1 ml ADNS solution (3-5 dinitrosalicylic acid), reacted for 5 minutes at 100 ° C, cooled in ice for 5 minutes and then readings taken. absorbance at 540 nm.

As the purpose of the promoter-directed evolution procedure was to improve xylanase production in E. coli, the final selection of variants was made to choose the cells in culture (with the estimated absorbance at 600 nm of the bacterial culture ) that produce the highest xylanase activity. The criterion used for final selection was to choose the highest values resulting from divisions between activity and Optical Density (Ativ / DOeoo) at a predefined 16-hour cultivation time.

Nucleotide Sequencing of Selected Variants [066] Plasmid DNA was extracted from cell pellets of such mutants that showed a significant increase in hydrolytic xylan activity and the promoter region nucleotide sequence was determined by automated DNA sequencing using the SeqRP oligonucleotide (5 '- CCCGCCAGACCCATTGACAG - 3'). The obtained sequences were analyzed and compared with the native promoter region sequence using the ClustalW program (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Nucleotide Sequence Analysis of Selected Variants and Combination of Mutations by Site-Directed Mutagenesis [067] In order to combine the observed mutations in nucleotide sequences in the promoter region, a site-directed mutagenesis strategy was improved. To this end, two oligonucleotides were designed, RPA (5'-ATACATTTAGGGCATCTTTAATTA - 3 ') and RPB (5'-ATTACGCTGAAGAGGACGATCA - 3'), which incorporate all mutations observed after selection of the best promoter region variants by enzymatic activity tests. as compared to native promoter (bases shown in bold, see SEQ ID NO. 12a, SEQ ID NO. 27a, SEQ ID NO. 30a, SEQ ID NO. 32a, SEQ ID NO. 34a, SEQ ID NO. 38a, SEQ ID NO 39a). The site-directed mutagenesis reaction was outlined in three reactions.

Reaction 1 [068] In this step, oligo PSS5 was incorporated and amplified in distinct reactions together with RPB mutagen oligo or PSS3 oligo with RPA mutagenic oligo producing 210 bp or 90 bp DNA fragments, respectively. For this purpose, 5 pL dNTP mix, 5U Taq DNA polymerase (Fermentas) and 3 μΐ MgCh (50 mM) were obtained for each reaction, obtaining a final volume of 50 pL with sterile water. The reaction was thermocycled through 35 cycles of 95 ° C for 1 minute, 50 ° C for 2 minutes and 72 ° C for 1 minute, along with negative control. Subsequently, each reaction was analyzed and purified on 2% agarose gel.

Reaction 2 [069] Called the extension reaction, it consisted in extending and completing the sequence of mutated DNA fragments generated in reaction 1. The 210 bp or 90 bp purified fragments were used in a 5 pL reaction of dNTP mix, 5U Taq DNA polymerase (Fermentas) and 3 µg MgCh (50 mM) in the presence of the pT7BsXA template and making the final volume to 50 pL with sterile water. The reaction was thermocycled for 10 cycles of 95 ° C for 1 minute, 45 ° C for 2 minutes and 72 ° C for 1 minute.

Reaction 3 This step consisted of amplifying the DNA strands obtained by the extension reaction by adding 5 pMol of the oligos PSS5 and PSS3. Subsequently, the PCR reaction was run in 30 cycles of 95 ° C for 1 minute, 50 ° C for 2 minutes and 72 ° C for 1 minute. The DNA fragments produced in reaction 3 encoded the promoter regions containing the mutations introduced by the RPA or RPB oligonucleotides, and were separated and purified on 1.5% agarose gel, with subsequent enzymatic digestion with Nhel and jBcoRI enzymes, and ligated. in the pT7BsXA expression vector. Binding between the mutant fragment and the vector, followed by transformation into Eschenchia coli (DH5a) was performed as described above.

To obtain a promoter region sequence that combines all mutations observed in the directed evolution process (called RPAB), the sequence of reactions 2 through 3 were repeated using a mixture of the amplified fragments using the RPA and RPB oligonucleotides obtained in the reaction. 1. The three RPA, RPB and RPAB constructs were subjected to the automatic DNA sequencing reaction (SEQ ID NO. 40a (RPA), SEQ ID NO. 41a (RPB) and SEQ ID NO. 42a (RPAB)).

Xylanase Expression Level Analysis Using pT7BsXA Vector With RPA, RPB, and RPAB Promoter Region Sequences

After growth of colonies in liquid culture medium LB, all clones were subjected to optical density analysis at 600 nm on a spectrophotometer (UV-Visible Spectrophotomerer 50 Bio-Varian) in order to estimate the number of cells present in the samples. . Subsequently, all samples were centrifuged and the enzymatic activity in the supernatant was determined by the amount of reducing sugars produced during the reaction using the 3,5-ADNS oxidation method (MILLER, GL Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31 (1959) 426-428). To this end, about 10 µl of the enzyme-containing culture supernatant was mixed with 50 µl of substrate (1% oat spelt xylan from Sigma) by completing the reaction volume to 100 µl with a 100 mM sodium acetate pH 6 buffering solution. 0 (0.2 M Na 2 HPPC> 4 and 0.1 M acetic acid). The activity test was performed at a balanced temperature of 55 ° C for 10 minutes. After this time the reaction was stopped by the addition of 0.1 ml ADNS solution (3-5 dinitrosalicylic acid), reacted for 5 minutes at 100 ° C, cooled in ice for 5 minutes and then readings of absorbance at 540 nm.

ColEI Replication Origin Results

Unique restriction sites for both ApaI and XhoI were introduced by site-directed mutagenesis in flanking positions at 5 'and 3' ends of the origin of replication resulting in the pT7BsXA vector (L10). PT7BsXA (L10) was linearized by ApaI or Xhol digestion and by double digestion with 3,000 bp and 650 bp ApaI and Xhol produced fragments, the smallest of which contains the ColEI origin of replication. The expression of XylA in E. coli DH5a transformed with pT7BsXA (L10) was compared with that of E. coli DH5a transformed with the original vector pT7BsXA, which showed that cells transformed with pT7BsXA (L10) showed an activity. xylanitic acid against xylan for bacteria transformed with the unmodified plasmid (results not shown), which demonstrates that insertion of the ApaI and Xhol enzyme sites did not result in loss of enzyme constitutive expression. Finally, pT7BsXA (L10) nucleotide sequencing confirmed that the ColEI origin of replication was unchanged and that the only changes were those introduced by site-directed mutagenesis (results not shown).

[074] The pT7BsXA (L10) vector included the gene encoding β-lactamase and, therefore, the selection strategy for high copy number mutants explored the phenotype of increased ampicillin antibiotic resistance, and a concentration of 1,800 pg / mL ampicillin was used for the final selection. The library of random onColEl mutants constructed using error-prone PCR (epPCR) was used to transform E. coli DH5a, and after selection in LB solid medium containing Petri dishes containing 1800 pg / mL ampicillin, three colonies from the ColEl random mutant library grew after 16 hours at 37 ° C. The xylanase activity of the supernatants of these three mutants (named as EDI (SEQ ID NO. 60), ED2 (SEQ ID NO. 70) and ED3 (SEQ ID NO. 80)) was measured (Figure 2), where it was found that The mutant ED2 (SEQ ID NO. 70) showed an 83% increase in enzyme activity measured in the culture supernatant when compared to bacteria transformed with pT7BsXA (L10).

[075] Mutant ED2 (SEQ ID NO. 70) was selected for further evaluation by real-time PCR to analyze the possible relationship between increased activity and higher vector copy number. Copy number of mutant ED2 (SEQ ID NO. 70) and native vector pT7BsXA (L10) was compared and showed that the plasmid copy number per cell increased by 36% from 1320 in pT7BsXA (L10) to 1800 in the mutant. DI2 (SEQ ID NO. 70), (Figure 3). The origin of replication (on) encodes the sense / antisense pair RNAI / RNAII transcribed from partial overlapping complementary strands. Multiple structural determinants influenced both RNAI and RNAII structure and RNAI / RNAII complex formation and the present results confirmed that mutations in the ori sequence resulted in variation of the plasmid cell copy number.

The complete nucleotide sequences of the ColEl origin of replication from the ED2 mutant (SEQ ID NO. 70) were compared with the unchanged ori ColEl, as revealed by the six nucleotide substitutions shown in Table I, in which the numbering Standard for the origin of replication was used to designate the positions of the individual substitutions (TOMIZAWA, JE, SOM, T. Control of ColEl plasmid replication: enhancement of binding of RNA I to the transcription protein by the Rom protein. Cell 38 (1984) 871-878). The positions of the mutations within the ori sequence are shown in figure 4.

Table I

Of the six mutations observed in ED2 (SEQ ID NO. 70), four are located in the RNAI / RNAII overlap region or in the RNAI promoter (Figure 4). Although detailed experimental analysis is beyond the scope of the present invention, comparison with prior art studies may provide information on the effects of some of the mutants that have been identified. RNAI transcription was triggered by a constitutive Pl promoter, which guarantees a high copy number over RNAII (ITOH, T., TOMIZAWA, J. Formation of an RNA primer for replication of ColEl DNA by ribonuclease H. Proc Natl Acad Sci USA 77 (1980) 2450-2454). The A -> G substitution at position -437 on the RNAI coding tape is immediately adjacent to the conserved P10 promoter region -10, and was therefore well positioned to influence RNAI transcription. A reduction in RNAI transcription and reduction in RNAI cytoplasmic levels would reduce the formation of the RNAI / RNAII complex and thus lead to a high copy number of the plasmid. Alternatively, the U437C mutation may alter the RNAII α / β structure, and it should be noted that mutants at positions -422, -423 and -426 were previously proposed to act in this manner (FITZWATER, T., ZHANG, XY, ELBLE, R., POLISKY, B. Conditional high copy number ColEl mutants: resistance to RNAI inhibition in vivo and in vitro (EMBO J 7 (1988) 3289-3297).

The mutation may also result in changes in the stability of the RNAI / RNAII complex (KIM, D., RHEE, Y., RHODES, D., SHARMA, V., SORENSON, O., GREENER, A., SMIDER. , V. Directed evolution and Identification of control regions of ColEl plasmid origins using only nucleotide deletions (J Mol Biol 351 (2005) 763-775), and it has been shown that most of the changes that increase the number of plasmid copies are loops I, II and II in the RNAI / RNAII overlap region (KIM, D., RHEE, Y., RHODES, D., SHARMA, V., SORENSON, O., GREENER, A., SMIDER, V. Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions J Mol Biol 351 (2005) 763-775). In the present invention, the mutation at the U - * A point at positions -490 and - 491 is located in the loop rod region II, and is predicted to result in the loss of two AU base pairing interactions. Both positions are immediately adjacent to a predicted bulge in the loop II stem structure, and loss of base pairing interaction in the double mutant may extend this bulge in mutant ED2 (SEQ ID NO. 70), and the Modified structure may result in reduced stability of the RNAI / RNAII complex enhanced RNAII preparation. Alternatively, the altered loop rod structure II may result in a decrease in RNAI half-life and secondary structure stability of native RNA and ED2 mutant (SEQ ID NO. 70) calculated using the free energy algorithm minimum folding, as implemented in the Mfold software (ZUKER, M., Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31 (2003) 3406-3415) were -42.3 and -39.7 kcal / mol respectively corroborating the result that the ED2 mutant (SEQ ID NO. 70) reduces RNAI stability.

In summary, the use of directed molecular evolution (ED) techniques, coupled with an appropriate selection strategy has proven to be useful for increasing the copy number of an expression vector by carrying the ColEl origin of replication, which results. in an increase in the expression levels of the heterologous protein. Although directed evolution is most commonly used in protein engineering studies, here it has been shown that this strategy can be successfully used for RNA engineering, and results have shown that random mutations in ori ColEl tend to accumulate. in the RNAI / RNAII overlapping region, which may reflect the importance of RNA sense / antisense interactions in regulating plasmid copy number.

Promoter Results A total of 24,396 variants of the promoter region random mutant library were analyzed, and after growth of colonies in Petri dish and xylanase expression, diluted substrate (1% xylan) was introduced into the plate. of activity and agar to be incubated at 55 ° C. The activity reaction was interrupted (removing the plate from the greenhouse) when the first halos of the positive controls were visually identified. To identify the best variants, a reaction with Congo red was performed, obtaining halos as shown in figure 5.

Following reaction with Congo red, a total of 95 colonies were isolated and plasmid DNA extracted from these variants was again used to transform competent E. coli cells. After transformation, the variants were grown in solid medium and one colony of each variant was inoculated into 3 ml LB medium with 0.1 g.L * 1 ampicillin. After 16 hours of expression, cell density was estimated by a 600nm optical density measurement (OD60), and the broth was centrifuged at 10,000 g and the supernatant used for enzymatic activity testing. The enzymatic production in liquid medium of each variant was evaluated as an average of three repetitions of the Activ54o / DOeoo ratio. Figure 6 shows the results obtained from Activated 540 / DO600, in reduced units, from native xylanase (nat) and 7 variants (variants 12a, 27a, 30a, 32a, 33a, 38a and 39a) showing the highest Activ54o / ratio. DOeoo compared to E. coli DH5a transformed with the native promoter region (Nat) plasmid.

Nucleotide sequencing of the promoter region between position 2633 (the EcoRI restriction site) and position 2751 (the Ndel restriction site) was done for the seven selected variants (SEQ ID NO. 12a, SEQ ID NO. 27a, SEQ ID NO: 30a, SEQ ID NO: 32a, SEQ ID NO: 34a, SEQ ID NO: 38a, SEQ ID NO: 39a). Sequence alignment is shown in Figure 7, where it shows that the distribution of mutations is localized in two promoter regions, delimited by boxes A and B.

[083] The seven identified mutations were combined using site-directed mutagenesis. Figure 8 shows a 1.5% agarose gel for PCR amplification reactions, where mutations encoded in the RPA and RPB oligonucleotides were incorporated into the amplified DNA. The 90 base pair band refers to the amplification reaction of the PSS3 and RPA oligos and the 210 bp band refers to the gene amplification using the PSS5 and RPB oligos. Both fragments were purified and used for extension reaction.

[084] The extension reaction is further shown in Figure 8 showing a 1.5% agarose gel displaying the 230 bp fragment, the result of the extension and gene amplification reaction on the DNA strand of reaction 2 and 3. After enzymatic digestion of the 230 bp fragment, together with the pT7BsXA expression vector and binding reaction between them, followed by transformation into E. coli (DH5a), several colonies were obtained on plates containing solid LB selective culture medium. Some of these colonies were then selected for plasmid DNA extraction and after purification, samples were sent for automatic sequencing. The sequences are shown in the alignment in figure 7.

The variants were identified and showed the insertion of mutations encoded by the RPA (SEQ ID NO. 40a) or RPB (SEQ ID NO. 41a) oligonucleotides and the two regions together in a construct called RPAB (SEQ ID NO. 42a). . These sequences confirm that the adopted technique of amplification and extension of two oligos containing point mutations simultaneously was efficient to obtain the mutated promoter regions in just two PCR steps.

Xylanase expression assays, comparing the native vector pT7BsXA and the vectors with the modified promoter regions, were performed by monitoring the enzymatic activity for a total period of 48 hours. Xylanase production was performed in liquid LB selective culture medium at 180 rpm and 37 ° C and the results are shown in Figure 9.

[087] All vectors showed in the first 15 hours a significant increase in enzymatic activity, and therefore in their expression, when compared with the native vector. However, the RPB vector in the first 18 hours doubled the rate of enzymatic activity. All enzymatic activity tests showed a relative decrease after 24 hours due to the decrease of nutrients present in the culture medium throughout the assay, as well as a possible enzyme instability expressed under temperature and pH conditions during the whole production period. .

Claims

Claims (20)

1. METHOD FOR OBTAINING THE pT7BsXA EXPRESSION VECTOR, characterized in that it comprises the steps (a) inclusion of specific restriction sites; (b) constructing, by directed evolution, the randomly mutated variant library in the promoter region by error-prone PCR; (c) constructing, by directed evolution, the library of randomly mutated variants in the ColEl origin of replication region (origin by error-prone PCR and (d) insertion of the protein gene of interest). Method according to claim 1, characterized in that it comprises the use of the pT7T3318 primer plasmid containing a 590 bp (on) ColE1 origin of replication region. Method according to Claims 1 and 2, characterized in that the inclusion of the specific restriction sites comprises the use of the XhoI and / or Apca endonucleases. Method according to Claims 1 to 3, characterized in that the inclusion of specific restriction sites further comprises the use of specific oligonucleotides such as ApaF / ApaR and XhoF / XhoR. Method according to Claims 1 to 4, characterized in that the inclusion of specific restriction sites is performed by PCR site-directed mutagenesis at the flanking positions of the ColEl origin of replication, respectively, at positions 1383 and 2062. Method according to Claim 1, characterized in that the directed evolutionary construction of the error-prone PCR random mutant variant library comprises the steps of (a) performing 40-cycle PCR's 94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 1 minute and then gel purification of the generated fragments; (b) tracking of random libraries for selection of variants; (c) sequencing promoter mutants of selected variants; and (d) site-directed mutagenesis combination of the individual mutants identified in steps (b) and (c) above. Method according to Claim 1, characterized in that the directed evolutionary construction of the library of variants with random mutations in the ColEl origin of replication region (ori) by error-prone PCR comprises the steps of (a) performing PCR's with 40 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 1 minute and subsequent gel purification of the generated fragments; (b) selection of the high variant ori variants of the antibiotic resistant vector; (c) sequencing the resistant ori variants and nucleotide sequences of the ColE1 region; (d) enzymatic assay for variant selection and (e) real-time polymerase chain reaction (PCR) of the variant. Method according to claim 7, characterized in that the sequencing of the resistant ori variants comprises in particular the variants EDI (SEQ ID NO. 60), ED2 (SEQ ID NO. 70) and DI3 (SEQ ID NO. 80). Method according to Claim 7, characterized in that the enzymatic assay for variant selection preferably comprises variant ED2 (SEQ ID NO. 70). Method according to claim 7, characterized in that the real-time polymerase chain reaction (PCR) of the variant preferably comprises variant ED2 (SEQ ID NO. 70). Method according to claim 1, characterized in that the insertion of the protein gene of interest comprises the insertion of heterologous proteins, such as enzymes, particularly oxidoreductases, transferases, hydrolases, liases, isomerases and / or ligases. Method according to Claim 11, characterized in that the enzymes preferably comprise the wild-type xylanase A enzyme from Bacillus subtilis (XylA) in Bscherichia coli. EXPRESSION CASSETTE, as obtained by the production method described in claims 1 to 12, characterized in that it comprises (a) variants of the BsXA promoter region; (b) variants of the origin of replication region (oh ColEl) and (c) a gene of a protein of interest. EXPRESSION CASSETTE according to Claim 13, characterized in that the BsXA promoter region variants comprise SEQ ID NO. 12a, SEQ ID NO. 27a, SEQ ID NO. 30a, SEQ ID NO. 32a, SEQ NO. 33a, SEQ ID NO. 34a, SEQ ID NO. 38a, SEQ ID NO. 39a and / or combinations thereof obtained by site-directed mutagenesis, such as SEQ ID NO. 40a (RPA), SEQ ID NO. 41a (RPB), SEQ ID NO. 42a (RPAB). EXPRESSION CASSETTE according to Claim 13, characterized in that the replication region (oh ColEl) variants comprise SEQ ID NO. 60, SEQ ID NO. 70, SEQ ID NO. 80 EXPRESSION CASSETTE according to Claim 13, characterized in that the protein gene of interest comprises the oxidoreductases, transferases, hydrolases, lyases, isomerases and / or ligases enzymes, preferably the wild-type xylanase A enzyme gene. (XylA) EXPRESSION VECTOR, obtained by the procurement method described in claims 1 to 12, characterized in that it comprises plasmid pT7BsXA. EXPRESSION VECTOR according to claim 17, characterized in that it comprises the expression cassette as described in claims 13 to 16 and restriction sites at the flanking positions 1383 and 2062 of the ColEl origin of replication. Expression system for the constitutive production and secretion of heterologous proteins, characterized in that it comprises the vector pT7BsXA as described in claims 17 and 18 in Escheríchia coli. EXPRESSION SYSTEM according to claim 19, characterized in that the heterologous proteins comprise, in particular, the oxidoreductases, transferases, hydrolases, lyases, isomerases and / or ligases enzymes, preferably the xylanase A-gene. (XylA) from Bacillus subtilis (XylA).