SI23510A - Improved synthesis of biosynthetic product by ordered assembly of biosynthetic enzymes guided by the nucleotide sequence motif template - Google Patents

Abstract

Invention refers to a method producing a compound by a biosynthetic pathway by culturing a genetically modified host cell, wherein the cell is modified with one or more nucleic acids comprising nucleotide sequence encoding chimeric proteins composed of biosynthetic pathway enzyme or other functional polypeptide and nucleic acid binding factor and program nucleic acid sequence that contains target nucleic acid elements that are recognized by said nucleic acid binding factor from the chimeric protein and where the said program nucleic acid sequence provides arrangement of biosynthetic pathway enzymes or other functional polypeptides along the program nucleic acid sequence and said culturing providing for synthesis of said chimeric proteins in the genetically modified host cells resulting in a production of said compound. Invention refers to host cells with program nucleic acid sequence, nucleic acid sequence expressing chimeric proteins. Invention provides methods improving product synthesis.

Description

Improved synthesis of biosynthesis product by targeted assembly of biosynthetic enzymes based on nucleotide sequence motif

FIELD OF THE INVENTION

FIELD OF THE INVENTION Improved product synthesis is achieved by directed assembly of chymic proteins, which are composed of biosynthetic enzymes or other functional polypeptides and nucleic acid binding domains. Directed assembly of chimeric proteins is based on binding to recognizable sites on program nucleic acid. The invention is a biotechnological invention.

The state of the art

For industrial applications, biosynthetic pathways from a large number of enzymes have been modified in such a way as to achieve a higher yield of the desired biosynthetic products. Improving the efficiency of biosynthetic pathways in order to achieve higher yields of the final product has been extremely interesting in recent years. To date, various optimization strategies have been developed. The yields of the biosynthetic pathway end products were improved by: i) increasing the amount of substrate available and / or overexpressing the enzymes of the limit steps; ii) adding heterologous enzymes with better kinetic properties; iii) disabling the branching of the biosynthetic pathway; iv) compartmentalization of biosynthetic pathways by targeting enzymes of a particular biosynthetic pathway into specific cellular compartments or artificial compartments (eg metabolosomes), or; v) approximation of biosynthetic enzymes with targeted assembly on a protein framework (WO 2009/108774). While there are many advantages to a protein framework, such an approach is limited by the number of combinations of peptide linkers available, and such an approach does not control the spatial distribution and orientation of biosynthetic enzymes.

None of the above solutions enables the optimal arrangement of biosynthetic enzymes to give the desired order of biosynthetic reactions. In living cells, biosynthetic pathway enzymes and other functional polypeptides are often restricted via microlocation by multi-enzyme complexes or anchoring mechanisms. This type of organization increases the local concentration of enzymes and the efficiency of biosynthetic pathways. In order to achieve optimal concentration of individual enzymes and accelerate the formation of multi-enzyme complexes, two different approaches have been adopted: i) targeting the enzymes to microlocations, or. targeting organelles; and ii) formation of multi-enzyme complexes on the polypeptide framework (WO 2009/108774).

However, the protein framework has many disadvantages and limitations.

Although the number and distribution of enzymes in a multi-enzyme complex can be programmed with the sequence of a polypeptide framework, the three-dimensional distribution of enzymes is unpredictable due to the flexibility of the dimerization domains (Figures 1 and 2). In addition, the design of a polypeptide framework with bound protein domains can be difficult due to the limited number of dimerization domains, and in addition there are specific conditions for each dimerization domain under which functional interactions are twisted and formed.

Summary of the Invention

The problem of arranging biosynthetic pathways into an ordered sequence of biosynthetic enzymes cannot be solved by binding to a protein framework. The solution is a nucleic acid molecule with sequence motifs, also called program nucleic acid, which determines the regulation of chemistry proteins, biosynthetic enzymes, or other functional polypeptides associated with binding domains for nucleic acids.

The inventors have discovered that it is possible to determine, if desired, the three-dimensional arrangement of biosynthetic enzymes or other functional polypeptides with programmatic nucleic acid. The inventors designed chimeric polypeptides composed of nucleic acid binding domains and a biosynthetic enzyme or other functional polypeptide. Said heme proteins bind to the target nucleic acid motif via the nucleic acid binding domain. Programmatic nucleic acid determines the order of the target motifs and thus the sequence and spatial arrangement of biosynthetic enzymes or other functional polypeptides. Such an ordered biosynthetic pathway of chemical biosynthetic enzymes or other functional polypeptides, guided by a program nucleic acid, increases the yield of the biosynthetic product of this pathway compared to a protein framework that simply forms clusters of enzymes in no specific order.

The use of program nucleic acid to immobilize and determine the order of chemical biosynthetic enzymes or other functional polypeptides provides one or more of the following options: i) increase the flux or flow efficiency of the biosynthetic pathway, ii)

optimizes metabolic flux through the pathway, iii) reduces host cell metabolic burden, iv) allows for alternative branching of metabolic pathways, thus creating new products, v) permitting reprogramming of the order of the enzyme enzymes within the organism if a different program nucleic acid is added.

The nucleic acid sequence carries more than two target elements. These elements represent specific ligands or targets of nucleic acid binding domains. When the program nucleic acid is present in the solution with the chemical proteins, the chemical proteins bind to the target elements within the program sequence and form a protein-nucleic acid complex, allowing the formation of a multiprotein complex. The arrangement of chimeric proteins within such a complex is predictable because of: i) the classification of the target motifs is defined by the nucleic acid sequence due to the known three-dimensional nucleic acid structure, ii) because the order of the motifs is predictable, allows orderly sorting of the chemistry proteins, iii) such orderly classification of the chemistry proteins determines the order of biosynthetic reactions, which is defined by the nucleotide sequence of programmed nucleic acid and thus reduces the time required for diffusion of reaction intermediates between different enzymes of the biosynthetic pathway. The inventors conclude that for such assembly of biosynthetic pathways it is advantageous to have a large number of nucleic acid binding domains. For example, for 9 nucleotide motifs, there are as many as 262144 combinations that can be recognized by different binding domains, which allows for extremely high variability in biosynthetic pathway assembly.

The present invention has many advantages over the protein framework. Programmatic nucleic acid has no ripening problems and the ordered nucleotide sequence can be selected as desired. The binding of nucleic acid binding domains to the target nucleotide sequence is well characterized. Due to the proximity of the programmatic nucleic acid-bound proteins, other polypeptides that could reverse the synthesis are spatially excluded from the multi-enzyme complex. The binding of all components of the biosynthetic pathway takes place under the same conditions, since all the binding domains used in the assembly of the protein chemistry can be based on the same type of protein and interact with the target nucleotides by the same interaction mode. Conversely, different types of domain dimerization must be used in the protein framework, which twist and interact correctly under different conditions. The invention also has the advantage that it can direct the formation of different reaction products depending on the sequence of program nucleic acid added to the production cells or reaction medium, where different sequence of program DNA causes different order of binding of the biosynthetic enzymes.

The present invention provides a method for the production of compounds of biosynthetic pathways, which includes the cultivation of genetically modified host cells modified to express chimeric proteins composed of enzymes of the biosynthetic pathway or other functional polypeptides linked to nucleic acid binding domains, and sequences of program nucleic acids that calibrates the arrangement of chemical proteins into multi-enzyme biosynthetic complexes.

The present invention relates to processes involving at least three to about 100 steps of biosynthetic reactions. The three steps are the minimum size where the order of the reaction processes may vary. For example, three steps can arrange reaction steps 1,2,3 in the order of 1,3,2 or 3,1,2.

Another use of the invention involves the use of chimeric proteins composed of enzymes of the biosynthetic pathway or other functional polypeptides linked to the binding domains for nucleic acids produced in and isolated from host cells, and a programmed nucleic acid sequence that calibrates the regulation of chimeric proteins into multi-enzyme biosynthetic complexes. In this mode, the biosynthetic pathway is assembled in vitro, either in solution or by immobilizing program DNA to another molecule or to a solid phase. In order to carry out a biosynthetic reaction, an in vitro compound complex must be incubated under conditions that support biosynthesis, and the necessary substrate and cofactors required for the biosynthetic reaction or the enzyme complex for the generation of these cofactors must be added.

The present invention provides a description of programming nucleic acid for in vivo and in vitro use. Genetically engineered host cells are presented that combine a program nucleic acid sequence and a sequence that encodes chimeric proteins composed of biosynthetic pathway enzymes or other functional polypeptides linked to nucleic acid binding domains that bind to specific target elements within the program nucleic acid .

The present invention provides a nucleic acid comprising a program nucleic acid sequence for use in a method for improving the yield of biosynthetic products.

The present invention allows for the design of bioprocesses for the production of specific end products by excluding other polypeptides that could shift synthesis toward unwanted products due to the proximity of the functional domains of the chemical nucleic acid-bound proteins.

The invention relates to genetically engineered host cells expressing chimeric proteins and / or a duplicate nucleic acid sequence.

The invention also relates to a method used to produce a compound of any biosynthetic pathway that requires proximity to biosynthetic enzymes for the synthesis of that compound.

The invention also relates to processes such as information processing, chemical degradation, signaling and the like

Short description of the pictures:

Figure 1: Schematic illustration of the benefits of using program nucleic acid.

Figure 2: Effect of spacer sequence between target elements / nucleic acid motifs.

Figure 3: Chemistry proteins, biosynthetic enzymes linked to nucleic acid binding domains, bind to the target nucleic acid element. All tested binding domains of Znf_Zif268, Znf_Blues, ZnfPBSII, ZnfHivC and NicTAL (Table 1) bind specifically to the target nucleic acid element and inhibit the expression of the β-galactosidase reporter protein. [Α] Znf_Zif268, Znf_Blues, Znf_PBSII, ZnfHivC, and NicTAL were tested (Table 1). [Β] Binding domains specifically recognize target nucleic acid elements. The specific target element was replaced by an element specific to another binding domain. Expression of the β-galactosidase reporter protein was not inhibited when another target nucleic acid element was used.

Figure 4: In vitro binding of nucleic acid binding domains to the target nucleic acid. [Α] Program nucleic acid hybridization was performed by injecting 0.5-2 μΜ for up to 300 s to achieve a final response of 300 RU. Binding of heme proteins was observed by injecting different concentrations of heme proteins for 1 min, followed by a 5 s dissociation step. Regeneration was achieved by two 30 s injections of 50 mM NaOH and 24 s injections of 0.5% SDS. [Β] Top protein chemistry: ZnfGlil, Znf_Zif268, ZnfBlues, • ·

ZnfHivC, ZnfPBSII, ZnfJazz. [C] DNA was bound at the beginning of the cycle and the chyme protein was injected 1 min one after the other in two different sequences: linked line: Znf Jazz, ZnfBlues, Znf_Zif268, Znf PBSII, Znf HivC and ZnfGlil; dashed line: ZnfGlil, Znf PBSII, Znf HivC, Znf Jazz, Znf Blues and Znf_Zif268. The results show that the binding of chimeric proteins is weaker in the sequences shown if ZnfGliniquitinated first.

Figure 5. In vitro binding of nucleic acid binding domains to nucleic acid sequence - programmatic DNA. The binding of individual binding domains to the nucleic acids Znf Jazz, Znf Blues, Znf_Zif268, Znf PBSII, Znf HivC, and Znf Glil was determined by agarose gel electrophoresis mobility shift assay. The arrows indicate the position of the proteinDNA complex.

Figure 6. In vivo reconstitution of two non-functional halves of a green fluorescent protein (named split_GFP) linked to nucleic acid binding domains in the presence of programmatic DNA. [Α] schematic of reconstitution split_GFPjev. [Β] 75 ng of plasmid containing his ZnF badge Glil-binding peptide-nCFP and 75 ng of plasmid containing cCFP-binding peptide-ZnfHIVC-tag were co-transfected with 350ng of nucleic acid program (SPR Program) , split, FRET; Table 1). CFPja emission (475 nm) was detected after excitation at 433 nm. [C] 75 nm of each plasmid with a split YFP chimeric protein (His-Znf_PBSII_-binding peptide-nYFP and cYFP-binding peptide-Znf_Zif268-binding peptide-His) was co-transfected with 350 ng of plasmid, FRET Nucleic Acid Program (FRET program) Table 1). The YFP emission signal at 529 nm was measured after excitation at 513 nm under a confocal microscope.

Figure 7. In vitro reconstitution of two non-functional split-GFPs linked to nucleic acid binding domains in the presence of programmatic DNA. Lysates of HEK293 cells transfected with plasmids encoding his_Znf_Glil-binding peptide-nCFP and cCFP-binding peptide-Znf HIVC-his tag or his-tag-Znf_PBSII_binding peptide-nYFP binding-peptide-nYFP badge were mixed with 50μg of program DNA (SPR program, split, FRET; Table 1). After 18 hours of incubation at 4 ° C, the fluorescence spectrum for CFP and YFP was observed. The emission peaks are indicated by arrows.

Figure 8. In vivo reconstitution of two pairs of split fluorescent proteins (split CFP and split YFP) was demonstrated by the FRET method. HEK293 cells were co-transfected with plasmids that encoded hissign-ZnfGlil-binding peptide-nCFP, cCFP-binding peptideZnfHIVC-hissign, hissign-ZnfPBSIIj-binding peptide-nYFP, cYFP-peptide-bindingZYZP2 , which contained programming DNA (SPR program, split, FRET, Table 1). [A. top left] Displays CFP reconstitution from split CFPs. Excitation at 453 nm, emission from 470 dO 510 nm. [A. top right] View YFP reconstitution from split YFPjev. Excitation at 415 nm, emission from 525 to 560 nm. [A. bottom left] Simultaneous representation of CFP and YFP from above. [A. bottom right] The same cells stained with Hoechst 34580 dye. Arrows point to cells showing the FRET signal, demonstrating the colocalization of all four split GFP fusions within the cell nucleus. [B. left top] Donor view reconstituted CFP before photobleaching fluorophore deactivation. [A. top right] View recipient of reconstituted mCitrinin after fluorophore deactivation. The bleached area is indicated by arrows. [B. left bottom] View of donor reconstituted CFP after fluorophore deactivation. Increased donor fluorescence after recipient fluorophore deactivation is indicated by arrows.

Figure 9. Biosynthesis of violacein in the presence of programmatic DNA. Cross cultures of E. coli with plasmids encoding the protein chymes ZnfBlues-binding peptide-vioA, Znf_Zif268 binding peptide-vioB, Znf-PBSII-binding peptide-vioE, Znf-HivC-binding peptidvioD, ZnfGlil-binding peptide-vioC with or without plasmid containing program DNA (Table 1, biosynthesis program (123456) or biosynthesis program (341256)). After 17.5 hours, extraction, TLC analysis was performed and the amount of violacein was determined. The amount of violacein produced in the presence of program DNA (biosynthesis program 123456) was much higher than in the case of program DNA (biosynthesis program 341256) or. when no program was present in E. coli.

Detailed description of the invention:

Definitions:

The term "program / program nucleic acid / program nucleotide sequence / program DNA sequence" refers to a nucleic acid containing a target sequence of nucleotides of any length and number recognized by the nucleic acid binding domains. These target sequences are separated by nucleotide sequences of arbitrary lengths called spacers. The software can be inserted into the host cell as such, or inserted into any vector and multiplied in the host cell.

The term "target / target nucleotide sequence / target nucleotide sequence motif" used throughout the text refers to a nucleotide sequence of any length that is a substrate for nucleic acid binding domains.

The term "nucleic acid binding factor / binding domain" refers to any molecule with the ability to bind to nucleic acids. The binding factors can be a natural or artificially engineered whole protein or just a segment of a protein with the ability to bind to a specific nucleotide sequence of a nucleic acid. In the present invention, the nucleic acid binding element is used as a carrier for enzymes involved in the biosynthetic pathway or any other functional polypeptide. The nucleic acid binding domain is linked to an enzyme molecule by a chemical bond. This is achieved by the binding peptide. The nucleic acid binding domain binds to one or more specific sites on the nucleic acid sequence program and provides a high local concentration and correct spatial position of the enzymes involved in the biosynthetic pathway.

The term spacer / spacer sequence refers to a sequence of nucleotides of any length that separates the target nucleotide sequences.

The term "nucleic acid" refers to a polymeric form of nucleotides of any length, ribonucleotides or deoxyribonucleotides and is not limited to one, two or more strands of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers with phosphothiolated backbone pyrimidine bases or other natural, chemical or biochemical modified, unnatural or derivatized nucleotide bases.

The term "polypeptide", "protein", "peptide" refers to a polymeric form of amino acids of any length that may include ordinary and unusual amino acids, chemically or biochemically modified or derivatized amino acids and polypeptides with a modified peptide backbone. The term "functional polypeptide" as used herein refers to an polypeptide of any length that expresses any function such as structure formation, site-specific targeting, organelle targeting, facilitation and initiation of chemical reactions, binding to other functional polypeptides. The term "biosynthetic pathway enzyme" as used herein refers to an polypeptide of any length capable of forming a new chemical bond.

The term "heme protein" as used herein has a general meaning and refers to a polymeric form of amino acids of any length composed of more than one protein / domain / segment, possibly linked to one another via a linker of any length preferentially composed of 1 to 40 amino acids, and that at least one protein / domain / segment is a nucleotide binding factor and the whole or binding domain is the other enzyme in the biosynthesis pathway. any other functional polypeptide, whole or active domain.

The term "heterologous" refers to the context of genetically modified host cells and refers to a polypeptide to which at least one of the following is true: (a) the polypeptide is foreign ("exogenous") to the host cell (not found in nature) ; (b) the polypeptide is naturally occurring (“endogenous”) in a given host micro-organism or host cell, but is produced in unnatural (greater than expected or greater amounts than found in nature) quantities in the cell or varies in nucleotide sequences from the endogenous nucleotide sequence such that the same protein (having the same or substantially similar amino acid sequence) as the endogen is produced in unnatural (more than expected or more than is found in nature) amounts in the cell.

The term "homologous" as used herein refers to proteins or nucleic acids with a conserved amino acid or nucleotide sequence, preferably with at least 50% affinity, with a minimum of 20% conservation determined by protein or nucleic acid comparative techniques known to those skilled in the art. Homologous proteins are characterized by performing the same functions in the cell. Homologous nucleic acids are coding for homologous proteins.

The term "recombinant" as used herein means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction and / or ligation leading to a construct having structurally coding or non-coding sequences different from endogenous nucleic acids in natural systems. Generally, a DNA sequence encoding a structural coding sequence can be assembled from cDNA fragments or from short oligonucleotide linkers or from synthetic oligonucleotides from which synthetic nucleic acid is obtained that can be expressed from recombinant transcription units into cells or into non-cellular cells

transcription and translation systems. Such a sequence can be used in the form of an open reading frame and does not interfere with internal non-translated sequences or introns that are usually present in eukaryotic genes. Genomic DNA containing important sequences can also be used to form a recombinant gene or transcriptional unit. Non-translated DNA sequences may be present on the 5 'or 3' side of the open reading frame, where such sequences do not affect the manipulation or expression of the coding regions and may act as modulators of the production of the desired products through various mechanisms (see DNA regulatory sequences, below).

The term "host cell" as used herein refers to an in vivo or in vitro eukaryotic or prokaryotic cell, to a cell or multicellular organism (cell line) cultured as a unicellular entity, or used as a nucleic acid recipient (expression vector that contains a nucleotide sequence encoding one or more gene products of the biosynthetic pathway such as the mevalonate pathway gene products) and includes the offspring of an original cell that has been genetically modified by a nucleic acid. Of course, the offspring of a single cell are not necessarily completely identical to the parent in morphological form and in the entire DNA complement, due to the consequences of natural, accidental or planned mutations. A "genetically modified host cell" (also a "recombinant host cell") is a host cell into which a heterologous nucleic acid, an expression vector, has been introduced. For example, a prokaryotic host cell is a genetically engineered host cell (bacterium) that is formed by the introduction of a heterologous nucleic acid, meaning that it is an exogenous nucleic acid that is a foreign (not normally present in nature) prokaryotic host cell, or recombinant a nucleic acid not normally present in a prokaryotic host cell or a eukaryotic host cell that is genetically modified. A eukaryotic host cell produces a genetically modified host cell by introducing a heterologous nucleic acid into a primordial eukaryotic host cell, that is, an exogenous nucleic acid, which is a foreign eukaryotic host cell or a recombinant nucleic acid that is not normally present in the eukaryotic host.

The term "biosynthesis pathway" as used herein refers to a sequence of enzymatic and other reactions through which a compound is converted to another by forming new covalent bonds in the body or in vitro.

The term "in vitro" as used herein refers to a process that takes place not in living organisms or cells but in a controlled environment.

The term "binding peptide" refers to a short amino acid sequence whose role can only be the separation of individual fusion protein domains. The role of the binding peptide in the fusion protein may also be the presentation of the cleavage site or site for post-translational modifications, including the presentation of sites for improved antigen processing. The binding peptide is not limited in length but is normally up to 20 amino acids long.

Normally, a heterologous nucleic acid is introduced into an expression vector. Suitable vectors are: plasmids, viral vectors and others. Host cell-compatible expression vectors are well known to those skilled in the art and contain the necessary control elements for transcription and translation of nucleic acids. Typically, the expression vector comprises a cassette with antibiotic resistance, a sequence for the heme protein under the promoter suitable for guided expression into the ski cell host, a polyadenylation signal, and a transcription ski terminator.

Method for the synthesis of a biosynthetic product / precursor

The present invention provides a method for the synthesis of a product or precursor of a biosynthetic pathway in a genetically modified host cell or in vitro. The method involves the cultivation of genetically modified host cells under suitable conditions, wherein the genetically modified host includes: a) nucleic acids with a nucleotide sequence encoding for three or more binding factors associated with biosynthetic pathway enzymes or other functional polypeptides, and b) nucleic acids acid with one or more program nucleotide sequences.

In another embodiment of the invention, the method involves the formation of the product in vitro. The in vitro method includes: a) at least three or more binding factors that are associated with the biosynthetic pathway chemical enzymes or other functional polypeptides, b) a nucleic acid with one or more program nucleotide sequences, and c) a substrate of the first enzyme and cofactors present in the mixture .

The binding factor binds the target nucleic acid element within the program nucleotide sequence with sufficient affinity to provide a link between the chemical

biosynthetic pathway enzyme and program nucleotide sequence. The affinity between the biosynthetic pathway chemical enzyme and the program nucleotide sequence is high enough that the chemical enzymes of the biosynthetic pathway or other functional polypeptides are immobilized on the program nucleotide sequence. The sequence of a program nucleic acid determines the sequence of adjacent binding factors and thus the sequence of adjacent chemical enzymes or other functional polypeptides. The sequence of the nucleic acid target elements with the bound functional polypeptides can be arranged in such sequence as in a particular biosynthetic reaction. Likewise, the sequence of target nucleic acid elements with the corresponding functional polypeptides can be altered by changing the order of adjacent target sequences in the program nucleotide sequence, providing a different sequence of adjacent biosynthetic enzymes or other functional polypeptides. The host cell is grown in such a way that the substrate of the first enzyme is either: a) present in the cell or b) added to the cells from the outside. The enzymes of the biosynthetic pathway are synthesized in the cell and either a) convert the substrate into a product in the cell, or b) excrete from the cell and convert the substrate into the product outside the cell.

The use of a software nucleotide sequence to immobilize biosynthetic pathway enzymes provides at least one of the following effects: increased biosynthetic pathway efficiency and optimized metabolic flux, reduced metabolic loads and the concentration of potentially toxic free intermediates in the cytosol, the possibility of alternative branching of the metabolic pathways leading to new products of the biosynthesis process . The program nucleotide sequence provides balanced enzyme activity. For example, a program nucleotide sequence may be modified to include more repetitions of target nucleic acid elements for himme polypeptides having lower substrate conversion activity, resulting in a greater number of repetitions of himme polypeptides having lower substrate conversion activity than those with higher activity. This is advantageous in cases where a lower activity enzyme catalyzes the rate-limiting rate of the biosynthetic pathway. The use of a program nucleotide sequence increases efficiency and optimizes metabolic flux by reducing the distance between enzymes involved in the biosynthetic pathway; owing to increased efficiency and optimized metabolic flux, the product yield may be equal or higher with less enzyme content. Synthesis of a smaller amount of enzyme in a host cell is an advantage as it provides a smaller metabolic load for the host cell. As the conversion of biosynthetic pathway intermediates is more efficient using the program nucleotide sequence, the amount / concentration of biosynthetic pathway intermediates in the cytosol or cytoplasm is reduced. The reduced amount of free intermediates of the biosynthetic pathway is an advantage when these intermediates are toxic to the host cell.

In some embodiments, at least three chemically biosynthetic enzymes or other functional polypeptides are immobilized on a program nucleotide sequence. The first chimeric functional enzyme or polypeptide produces the first product that is a substrate of the next chimeric enzyme or other functional polypeptide. Chimeric proteins, biosynthetic pathway enzymes, or other functional polypeptides are positioned in proximity to each other. In this way, the effective concentration of the first product is high and the second chemistry enzyme of the biosynthetic pathway can effectively act on the first product.

Three or more (i.e., three, four, five, six, seven, or more) functional polypeptides may be mobilized to the nucleotide program sequence. In some embodiments, the nucleic acid program sequence includes (in the 5 'vs. 3' direction) a) one repetition of the target nucleotide sequence element for the first chemistry polypeptide or enzyme of the biosynthetic pathway, b) one repetition of the target nucleotide sequence element for the second chemistry polypeptide or enzyme biosynthetic and c) one repetition of the target nucleotide sequence element for the third chimeric polypeptide or biosynthetic pathway enzyme. In other embodiments, the nucleic acid program sequence includes (in the 5 'vs. 3' direction) a) one repetition of the target nucleotide sequence element for the first chimeric polypeptide or biosynthetic pathway enzyme, and b) two or more (i.e. two, three, four or more) ) repeating an element of the target nucleotide sequence for another chemistry biosynthetic pathway enzyme or other functional polypeptide. In this way, the ratio of any chemically functional polypeptide in the biosynthetic pathway can be altered. For example, the ratio of the first to second chimeric polypeptide of the biosynthetic pathway may range from about 0.1: 10 to about 10: 0.1, and the ratio between the second and third polypeptides from about 0.1: 10 to about 10: 0.1 and so on.

In some embodiments, at least three or more (i.e., three, four, five, six, seven or more) chimeric enzymes of the biosynthetic pathway are immobilized on the program nucleic acid. The first biosynthetic pathway enzyme produces the first product, which is the substrate for the second product, which is the substrate for the third biosynthetic pathway product: In these embodiments, the sequence of target nucleotide sequences in the program nucleic acid determines the order and number of copies of the biosynthetic pathway enzymes.

Programming nucleotide sequence programming

The program nucleotide sequence is designed to arrange enzymes of the biosynthetic pathway into a functional complex. A program nucleotide sequence consists of three or more target nucleotide elements for binding binding factors. Binding of the nucleic acid binding factors that are linked to the biochemical enzymes of the biosynthetic pathway to the target nucleotide element ensures immobilization of the enzyme to the program nucleotide sequence. Each target element has a corresponding binding partner in the biosynthetic pathway chemical enzyme or functional polypeptide. A given target nucleotide element may be directly adjacent to or separated from another target nucleotide element by a nucleotide interface.

The program nucleotide sequence can be involved in vivo in different types of host cells in different forms. Examples of various forms of program nucleotide sequence are, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagmids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (for example, but not limited to vaccine virus vectors, polioviruses adenoviruses, AA, SV40, herpes virus and the like), Pl-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes and other vectors. The program nucleotide sequence can be incorporated into various host cells, even as a linear nucleic acid molecule. All these forms of program nucleotide sequence can also be used in vitro. If the program nucleotide sequence is at least partially composed of RNA, it can be used per se, or DNA encoding the program nucleotide sequence can be used. DNA encoding a nucleic acid program sequence that can be used in vitro or in vivo exists in various forms as listed but not limited to the examples above.

In a production system, a program nucleotide sequence may be present in one or more copies. Multiple copies of one program nucleotide sequence can provide a greater number of functional biosynthetic complexes composed of immobilized chemistry enzymes of the biosynthetic pathway. Individual copies of program nucleotide sequences may be present on one or more nucleic acid molecules. Therefore, increasing the number of individual nucleic acid molecules also provides a greater number of copies of the program nucleotide sequence in the production system.

For example, but not limited to, a bacterial host cell may be transformed with self-duplicating plasmids or any other type of nucleic acid containing a program nucleotide sequence in one or more copies, thereby providing a higher overall program number nucleotide sequences in the production system. A plasmid containing one or more copies of a program nucleotide sequence may be present in the system in one or more copies (for example, but not limited to a large copy number plasmid that transforms a bacterial cell). Higher numbers of plasmids in a production system with one or more copies of the program nucleotide sequence also provide a higher total number of copies of the program nucleotide sequence in the production system.

For example, but not limitingly, a bacterial host cell may be transformed by a nucleotide sequence encoding for a single stranded, double stranded, partly double stranded multilayered or partly multilayered RNA molecule. A program nucleotide sequence may be present in one or more copies on a single RNA molecule. Multiple copies of a program nucleotide sequence on a single RNA molecule provide a higher total number of copies of a program nucleotide sequence in a production system. Also, the coding sequence for an RNA molecule may be present in the production system in one or more copies. A higher copy number of the coding sequence for an RNA molecule provides a higher number of RNA molecules in the production system and therefore a higher number of program nucleotide sequences in the production system. The total number of individual RNA molecules in a production system can be regulated by promoters used to regulate the transcription of coding nucleotide sequences into RNA molecules consisting of one or more copies of a program nucleotide sequence. Stronger promoters provide a higher copy number of RNA molecules in the production system and therefore provide a higher total number of individual program nucleotide sequences in the production system. A higher total number of program nucleotide sequences combines a higher number of functional biosynthetic complexes, which are composed of immobilized chemistry enzymes of the biosynthetic pathway.

The program nucleotide sequence consists of at least three target nucleotide sequences, that is, at least three corresponding binding elements in the three chemical enzymes of the biosynthetic pathway bind to the program nucleotide sequence. The program nucleotide sequence has one, two or more copies of each target nucleotide element. Each target nucleotide element may therefore be present in one or more copies. These copies may be directly next to each other or separated by a nucleotide spacer. Replicates of target elements may also be separated by one or more other target elements, which may be present in one or more copies. If only three target nucleotide elements are present in the program nucleotide sequence, they may be different, which ensures the correct spatial distribution or high local concentration of two different chimeric enzymes of the biosynthetic pathway or other functional polypeptides. If only three target nucleotide elements are present on a program nucleotide sequence, they can be of the same species, providing the correct spatial organization or high local concentration for three copies of the same chimeric biosynthetic enzyme.

The specific target nucleotide element determines the nucleotide sequence from the 5 'to 3' end of the nucleic acid molecule. On a two-stranded program nucleotide sequence or a two-stranded program nucleotide sequence section, the target nucleotide sequence may also be a reverse complement to the sequence of the original target nucleotide sequence. The same binding factor can bind to the target nucleotide element and its reverse complement, but in a different orientation and on the other side of the program nucleotide sequence. Therefore, the reverse complement of the target nucleotide sequence can be used instead of the target nucleotide sequence if two biosynthetic pathway enzyme chymes or two other functional polypeptides are too large to bind to the program nucleotide sequence directly next to each other, or to achieve a different spatial arrangement of the two copies of the same chimeric enzyme of the biosynthetic pathway or other functional polypeptide on a program nucleotide sequence.

A nucleotide spacer may be positioned between two target nucleotide elements to provide sufficient space for large chimeric enzymes of the biosynthetic pathway or other functional polypeptides acting sequentially in the biosynthetic pathway. In a two-stranded program nucleotide sequence or a two-stranded program nucleotide sequence section, the nucleotide spacer may also provide the correct spatial arrangement of two adjacent or nearby enzymes of the biosynthetic pathway or other functional polypeptides. For example, a program nucleotide sequence may be a double stranded DNA sequence that forms a helix. Therefore, varying the spatial length can be achieved by changing the length of the nucleotide interface

distribution of immobilized biosynthetic pathway enzyme enzymes, or greater distance between two adjacent biosynthetic pathway enzyme enzymes (Figure 2).

Each program nucleotide sequence has the general formula [((X or X ') n Sp)] m, where Xn is a target nucleotide sequence, Xn' is a reverse complement of the target nucleotide sequence, S is an optional nucleotide interface of any length, m is an integer greater or equal to 3 and represents the number of target nucleotide sequences with an optional nucleotide interface in the program nucleotide sequence, n indicates the type of target nucleotide element (for example XI, X2 and so on) and represents the various individual target nucleotide elements, p indicates the type of nucleotide interface (e.g. Fig. S2 and so on) and represents different individual nucleotide interfaces. Individual target nucleotide sequences in the program nucleotide sequence may be of any length and depend on the corresponding binding elements. For example, zinc fingers with three fingers can be used. In these cases, the target nucleotide sequence length is usually, but not always, 9 or 10 nucleotides.

In some embodiments of the invention, the program nucleotide sequence has the general formula (X1) (S1) (X2) (S2) (X3) (S3) (X4), wherein XI is the first target nucleotide sequence for the binding of the first biosynthetic pathway enzyme; wherein X2 is a second target nucleotide sequence for binding another chimeric enzyme of the biosynthetic pathway; wherein X3 is a third target nucleotide sequence for binding the third heme chemical enzyme of the biosynthetic pathway; wherein X4 is a fourth target nucleotide sequence for binding the fourth chimeric enzyme of the biosynthetic pathway; wherein Sl, if present, is the first nucleotide interface providing the correct spatial orientation of adjacent biosynthetic pathway enzyme enzymes; where S2, if present, is a second nucleotide interface providing the correct spatial orientation of adjacent biosynthetic pathway enzyme enzymes; where S3, if present, is the third nucleotide interface that provides the correct spatial orientation of adjacent biosynthetic pathway enzyme enzymes.

In some other embodiments, the program nucleotide sequence has the formula (X1) (S1) (X2) (S1) (X2) (S1) (X3), wherein XI is the first target nucleotide sequence for binding the first chimeric enzyme of the biosynthetic pathway; wherein X2 is a second target nucleotide sequence for the binding of a second chimeric enzyme of the biosynthetic pathway; wherein X3 is a third target nucleotide sequence for binding the third heme chemical enzyme of the biosynthetic pathway; where Sl, if present, is a nucleotide interface of the same type between each pair of target nucleotide sequences.

A sequentially repeated target nucleotide sequence (in this case, X2) may provide a higher local concentration of the chemical enzyme that catalyzes the rate of the rate-limiting biosynthetic pathway. This results in increased efficiency of metabolic pathway flow. Sequentially repeating the target nucleotide sequence (in this case X2) may also provide an increased degree of dimerization (or multimerization if the specific target nucleotide sequence is repeated more than twice in a row) of the dimeric (or multimemic) chimeric proteins of the biosynthetic pathway. A sequentially repeated target nucleotide sequence (in this example X2) may also provide increased metabolic flux efficiency in biosynthetic pathways where one enzyme acts on the substrate more than once in a row.

In some other embodiments, the nucleic acid program sequence has the formula (X1) (S1) (X2) (S2) (X1) (S3) (X3), wherein XI is the first target nucleotide sequence for binding the first biosynthetic pathway enzyme and is repeated in the program nucleotide sequence but not directly behind the first iteration; wherein X2 is a second target nucleotide sequence for the binding of a second chimeric enzyme of the biosynthetic pathway; wherein X3 is a third target nucleotide sequence for binding the third heme chemical enzyme of the biosynthetic pathway; wherein Sl, if present, is the first nucleotide interface providing the correct spatial orientation of adjacent biosynthetic pathway enzyme enzymes; where S2, if present, is a second nucleotide interface providing the correct spatial orientation of adjacent biosynthetic pathway enzyme enzymes; where S3, if present, is the third nucleotide interface that provides the correct spatial orientation of adjacent biosynthetic pathway enzyme enzymes. This program nucleotide sequence can be used to improve the efficiency of biosynthetic pathways, where one chemistry enzyme of the biosynthetic pathway (hereinafter, the chemistry enzyme of the biosynthetic pathway that binds to the target nucleotide sequence XI) occurs more than once in the biosynthetic pathway, but not in direct succession .

Design of binding factors

The invention relates to chimeric polypeptides consisting of a nucleic acid binding factor (hereinafter NABF) and an enzyme of the biosynthetic pathway or other functional polypeptide, which are linked to each other by an amino acid linker. NABFs can be either of natural origin or artificially designed and can be isolated from any organism. NABF can be any polypeptide, domain, protein or protein segment that contains at least one nucleic acid recognition motif. NABF interacts with nucleic acids by specifically recognizing the nucleotide sequence. Examples of NABF polypeptides include, but are not limited to: helix-loop-helix motif, zinc finger, leucine zipper, winged helix, wing-helix-loop-helix, helix-loop-helix motif, and HMG sequence.

Polypeptides with the basic helix-loop-helix motif are divided into leucine zipper factors, helix-loop helix motif factors, helix-helix and leucine zipper motif factors, NF-1, F-X and bHSH. They are determined by the structure of the two loopholes connected by a loop. Transcriptional factors that involve this domain are typically bound in the dimethyl form to the consensus palindromic sequence (CACGTG), also called the E-sequence. The bHLH transcription factors bind to non-palindromic sequences, which are often similar to the E-sequence.

In some other embodiments, NABFs are selected from a superclass of DNA-binding domains that coordinate the zinc ion. This subclass is divided into subclasses: Cys4 core receptor type zinc finger, various Cys4 zinc fingers, Cys2His2 zinc fingers, Cys6-Zn zinc fingers, and other composition zinc fingers. Individual zinc finger domains usually occur in tandem repeats with two, three, or more fingers, which is the DNA binding domain of the protein. These tandem zinc finger arrangements can bind into a large groove of a DNA molecule, usually every three base pairs. the α-helix of each domain can form nucleotide sequence-specific interactions with nucleotides in the nucleic acid molecule; the amino acid residues of the aforementioned α-helix can interact with 4 or more nucleotides, thereby forming an overlapping pattern of interactions with adjacent zinc fingers.

In some other embodiments, NABFs are selected from the parent-loop loop-helix motif, which is divided into six classes: homeodomain, fork head / winged helix, heat shock factors, tryptophan cluster, and TEA domains. The helix-loop is the main structural motif capable of binding to DNA. Both α-helices recognize and bind a DNA molecule, one helix is at the N- and the other at the C-end of the motif.

The fourth NABF class is represented by beta-framework factors that interact with the small groove. It is further divided into the following classes: RHR, STAT, p53, MADS, beta-barrel and α-helical transcription factors, TATA-binding proteins, heteromemic CCAAT factors, grainyhead, cooling shock factors and Runt.

Other transcription factors that bind to specific nucleotide sequences are also important, such as mutated restriction enzymes without restriction activity but with the ability to recognize the sequence.

The present invention relates, but is not limited to, polypeptides with a zinc finger domain that forms a smaller, independent coiled domain that contains a zinc ion and recognizes a specific nucleotide sequence. Zinc finger domains usually exist as tandem repeats with at least two, three or more zinc fingers that together make up the DNA-binding domain of the protein. Each finger recognizes and binds to places consisting of three base pairs. Amino acid residues at sites 1, 2, 3, and 6 in the α-helix within the smaller zinc finger domain are responsible for specific binding. A modular approach or combinatorics can multiply repeated smaller domains and achieve characteristic binding by variations in some key amino acid residues. Thus, soil with specificity for different triplets is combined to achieve specific recognition of longer nucleotide sequences. By combining at least two or more zinc finger domains, we can minimize the non-specific binding to nucleic acids present in the host organism and increase the specificity of binding of zinc finger containing polypeptides to program nucleic acid.

Zinc finger domains are stable structures whose conformation rarely changes when bound to a target. In the present invention, functional polypeptides are linked to at least one binding sequence that is covalently linked to a single enzyme of the biosynthetic pathway. The linking sequence determines the position of the enzyme biosynthetic pathway or other functional polypeptide relative to the zinc finger domain and relative to the adjacent enzyme biosynthetic pathway or other functional polypeptide. The binding sequences can vary in length and amino acid sequence and ensure that the covalently bound enzyme can form the correct tertiary structure and thus maintain its biological function.

The number of chemical enzymes of the biosynthetic pathway or other functional polypeptides bound to NABF may be, but is not limited to, three, four, five or more, and may be different from the number of individual target nucleotide elements on a program nucleotide sequence. The number of chemical enzymes of the biosynthetic pathway or other functional polypeptides associated with NABF depends on the number of steps in the biosynthetic pathway. If the biosynthetic pathway requires three, four, five different enzymes or other functional polypeptides to convert a substrate into a product or precursor, at least three, four, five different binding elements in a specific order will be included in the program nucleotide sequence.

In some embodiments of our invention, RNA molecules may be used as program nucleotide sequences. In this case, NABFs that recognize a specific RNA sequence are used. These may, but are not limited to: RNA recognition motif (RRM, RBD or RNP motif), heterogeneous core RNP K-homology domain (KH-domain), zinc finger domain (best characterized zinc finger domains that bind an RNA molecule, are those of the CCHH and CCCH type, but other types) and Pumilio (Puf) domains may be used.

Although RNA-binding domains that recognize specific secondary structures on RNA molecules can also be used as NABF (for example, but not limited to Sl domains), RNA-binding domains that recognize a specific sequence (e.g., but not restricted to the zinc finger RNA binding domains). The inventors propose the use of RNA-binding domains that recognize specific sequences of double-stranded RNAs as they allow proper spatial arrangement. Methods for the design of zinc fingers that bind to a specific double-stranded RNA sequence are described in the literature.

Design of biosynthetic notes.

The present invention relates to a method for increasing the yield of biosynthesis products. This method combines: (i) a program nucleotide sequence; and (ii) chimeric proteins with a biosynthetic pathway enzyme or other functional polypeptide and NABF. The two necessary components (i) and (ii) can be combined (i) in vitro, in solution in the presence of substrates and cofactors for enzymes of the biosynthetic pathway, or (ii) can be inserted and expressed in host cells in vivo.

In the embodiment, the biosynthetic pathway means any enzyme cascade that forms a new chemical bond on the substrate in a particular order, i.e. as it occurs in nature or artificially designed. An artificially designed biosynthetic pathway refers to the synthesis of a product unknown in nature or the enzymes of the biosynthetic pathway are not from the same organism or have been obtained through genetic manipulation.

The invention relates to a biosynthetic pathway, to any coupled enzyme reaction which must be carried out concurrently with at least three or more enzymes. The biosynthetic pathway (flow, efficiency) can be measured by measuring the concentration of precursors and end products by suitable methods known to those skilled in the art.

The invention relates to any biosynthetic pathway, which includes, but is not limited to:

• catabolic or anabolic pathways, • primary metabolic pathways, such as but not limited to the synthesis of amino acids, fatty acids, carbohydrates, pyrimidines, and purines, • secondary metabolic pathways, such as, but not limited to, the synthesis of secondary metabolites by biological activity and pharmacological properties (polyketides), unusual peptides, hormones, terpenoids (carotenoids, ...), pigments (violacein, melanin, ...) i. t. d.

Carotenoid biosynthetic pathway enzymes

Carotenoids fall into the category of tetraterpenoids. Astaxanthin, zeaxanthin incantaxanthin are β-carotene derivatives and their biosynthesis is catalyzed by the carotenoid biosynthetic pathway enzymes: crtE, crtB, lines, crtY, crtO, crtZ, where the order and participation of enzymes lead to the synthesis of different products. For example, a biosynthetic pathway involving crtE, crtB, lines, crtY, crtO, crtZ leads to astaxanthin synthesis, crtE, crtB, lines, crtY, crtZ lead to zeaxanthin synthesis, crtE, crtB, lines, crtY, crtO synthesis of canthaxanthin.

Violacein biosynthetic pathway

Viocein pigment biosynthesis genes are in an operon consisting of the genes vioE, vioD, vioC, vioB, and vioA. VioA is a FAD-dependent 1-tryptophan oxidase that synthesizes IPA imine. It is a substrate of the enzyme VioB, a hemoprotein oxidase, which converts it to a compound of unknown chemical formula (compound X). VioE is a recently discovered protein without characterized homologs and is a key enzyme in the synthesis of violacein. It works by converting compound X into intermediates that can be used as a substrate by the enzymes VioD and VioC, both of which are FAD-dependent monooxygenases that hydroxylate these compounds, leading to the formation of violacein.

Resveratrol biosynthetic pathway

Resveratrol (3, 5, 4′-transhydroxystilbene) is a plant polyphenol. The resveratrol biosynthetic pathway consists of four enzymes: phenylalanine / ammonia-lyase (PAL) and cinnamic acid 4-hydroxylase, which can be replaced by the enzyme tyrosine / ammonia-lyase (TAL), 4-coumarate / CoAligase (4CL) and stilben synthase (STS). ). The first two enzymes, PAL and C4H, convert phenylalanine into p-cumic acid (4-cumic acid). The third enzyme, 4CL, binds p-cumanoic acid to the pantothenic group coenzyme A (CoA), yielding the product 4-coumaroyl-CoA. The last enzyme in the pathway, STS, catalyzes the condensation of 4-coumaroyl-CoA and three malonyl-CoA molecules into resveratrol.

In the present invention, enzymes from an individual biosynthetic pathway, such as biosynthetic pathways for the synthesis of violacein, resveratrol or carotenoids, are coupled to NABF. By sequential binding of the enzymes to the program nucleotide sequence, the sequence of enzymatic reactions can be determined. This allows for faster and more efficient reactions and also represents the possibility of synthesizing new products by rearranging the order of enzymatic reactions (eg, if the order of enzymes is 13-2, the end product is different than if the order is 1-2-3). An advantage of our invention is the ability to synthesize artificial compounds with the desired properties.

Recombinant DNA

The invention employed standard molecular biological methods generally known to those skilled in the art.

The invented protein can be synthesized by expressing the DNA encoding for these proteins in a suitable host organism. The protein coding DNA is inserted into a suitable expression vector. Suitable vectors include, but are not limited to, plasmids, viral vectors, and more. Expression vectors that are compatible with the host organisms are known to those skilled in the art and include suitable control elements for transcribing and translating the nucleotide sequence.

Expression vectors can be prepared for expression in prokaryotic and eukaryotic cells. For example, prokaryotic cells are bacteria, most commonly Escherichia coli. In the invention, prokaryotic cells have been used to produce a sufficient amount of nucleic acids. Expression vectors generally include control elements operatively linked to the DNA of the protein-coding invention. Control elements are selected to induce efficient and tissue-specific expression. The promoter may be constitutive or inducible, depending on the pattern of expression desired. The promoter may be native or of foreign origin (does not exist in the cells in which it is used. The promoter must be selected to function in the target cells of the host organism. In addition, initiation signals are included to efficiently translate the chemical proteins, meaning ATG and When the vector used in the invention includes two or more reading frames, the reading frames must be independently operatively linked to the control elements and the control elements must be the same or different depending on the desired protein production.

Examples:

Cloning and purification of polypeptides

The DNA sequences of the nucleic acid binding domains and biosynthetic enzymes or other functional polypeptides described above were designed based on the amino acid sequences of the selected protein domains using the Designer tool in DNA 2.0 Inc. The Designer tool allows the user to design DNA fragments and optimize expression in desired host organisms (eg E.coli) with organism-specific codon optimization. Genes were ordered from GENEART AG (Im Gewerbepark B32, D-93059 Regensburg), excised by restriction enzymes, and cloned into a suitable vector containing appropriate sequence regulators using a standard procedure used by those skilled in the art. The DNA sequences of the half-fluorescent proteins were amplified by PCR, as the template DNA sequences of the fluorescent proteins were used. The vectors used include commercial vectors pET, pBluescript, pCDNA, pSBlA2 (pSBlA2 http://partsregistry.Org/Part:pSB 1A2) pSBlC3 (http://partsregistry.org/wiki/index.php?title ^: = Part: pSB 1C3 ), pSB 1AK3 (http://partsregistry.org/wiki/index.php?title=Part:pSB 1AK3), and plasmid vectors in low copies of pSB4K5 (http://partsregistry.Org/Part:pSB4K5) and pSB4C5 ( http://partsregistry.Org/Part:pSB4C5) that carry all the necessary properties such as antibiotic resistance, duplicate start site, and polyclonal site.

Molecular biology methods (DNA fragmentation by restriction enzymes, DNA amplification by polymerase chain reaction with polymerase - PCR, PCR ligation, determination of DNA concentration, agarose gel electrophoresis, purification of DNA fragments from agarose gels, ligation of DNA fragments into vector, transformation of chemically competent cells E .coli DH5a, plasmid DNA isolation with commercially available putty, screening and selection) were used to prepare DNA constructs. All procedures were performed under sterile conditions (aseptic technique). DNA sections were analyzed by restriction analysis and sequencing.

Molecular cloning procedures are well known to those skilled in the art and are described in detail in any manual of molecular biology.

The DNA constructs and the corresponding heme proteins are described in Table 1. All DNA constructs have a start codon (ATG) in front of the histidine marker or coding region. The constructs for the chimeric proteins were cloned into the pET19b vector for high expression, or into pSB4K5 and pSB4C5 to monitor the production of carotenoids and violacein in vivo. The 5 'to 3' expression cassette includes a T7 promoter, a polyclonal fusion protein site, and a T7 terminator. These regulatory elements allow expression of a protein in the E. coli prokaryotic cell line that contains the genetic record for T7 RNA polymerase.

DNA constructs were prepared using molecular biology methods described in any manual of molecular biology and known to those skilled in the art. Plasmids and final and intermediate constructs were transformed into E. coli DH5a or BL21 (DE3) pLysS cells.

Several constructs were prepared to demonstrate the feasibility of producing the chimeric proteins listed in Table 1.

Plasmids encoding the open reading frames of fusion proteins in Table 1 (SEQ ID No. 52 to SEQ ID No. 57) were transformed into competent E. coli BL21 (DE3) pLysS cells by chemical transformation. Selected bacterial colonies grown on LB plates with the appropriate antibiotic (ampicillin) were inoculated into 10 ml of LB medium with the appropriate antibiotic added. After an overnight incubation at 37 ° C, ΙΟ-ΙΟΟμΙΟΟ was inoculated into 100 ml of the selected medium and shaken overnight at 37 ° C. Cross culture was diluted 20-50 times to OD600 between 0.1 and 0.2. 500 ml diluted culture flasks were shaken at 25 ° C to OD600 between 0.6 and 0.8 when protein expression was induced by the addition of lmM IPTG Four hours after induction, the culture was centrifuged and the bacterial cells were resuspended in lysis buffer (lOmM Tris pH 8.0 , IM NaCl, 0.1% deoxycholate, 0.1 mM zinc sulphate and protease inhibitor cocktail addition) and frozen at -80 ° C overnight. Thawed cell suspensions were then lysed by sonication and centrifuged. The precipitate (cell membranes, body inclusion) and the supernatant were tested for expression of the constructs by SDS-PAGE and West transfer, and anti-His tag antibodies were used as primary antibodies when needed. Fusion proteins were mostly present in the insoluble part (inclusion bodies), which contained> 80% of the selected protein. Inclusion bodies were washed twice with lysis buffer, twice with IM urea and 10 mM Tris pH 8.0 and twice with 2M urea and 10 mM Tris pH 8.0. Typically, the result of this process was> 95% protein purity. Purified inclusion bodies were dissolved in 8M urea and 50 mM Tris pH 8.0 and purified using a Ni-NTA column. Cleaning under denaturing conditions was performed according to the manufacturer's instructions. Proteins were washed with 8 M urea, 50 mM Tris pH 8.0, and 250 mM imidazole. Protein retraining was performed by dialysis buffer with 50 mM Tris pH 8.0, 500mM NaF, 500 μΜ ZnSC> 4, 5 mM DTT, 0.005% Tvveen and 10% glycerol. Dialysis was performed at least twice after 4 hours at 4 ° C without shaking. Upon completion of the process, the dialysate was carefully transferred from the dialysis tubes to the centrifuges. Non-recurrent proteins were precipitated by centrifugation at 10,000 rpm to separate from the recurrent proteins in the soluble phase. Protein concentration was measured by spectrophotometry and Bradford method, and protein size was determined by SDS-PAGE / Commasie staining and Western blotting with anti-His tag antibodies.

Table 1: Composition of plasmids prepared for demonstration of the invention

Seq IE No. »Seq ID No. The composition of the construct Framework plasmid DNA protein Binding domains 1 2 Znf Glil pSBAK3 3 4 Znf HivC pSBAK3 5 6 Znf_Zif268 pSBAK3 7 8 Znf Jazz pSBAK3 9 10 ZnfPSBII pSBAK3 11 12 ZnfBlues pSBAK3 13 14 Znf Tyr pSBAK3 15 16 NicTAL pSBAK3 Biosynthetic pathway enzymes or other functional polypeptides

18 drawing pSBlA2

19 20 crtB pSBlA2 21 22 lines pSBlA2 23 24 crtY pSBlA2 25 26 crtZ pSBlA2 27 28 crtO _P SB1A2 29 30 TAL pSBlA2 31 32 4CL pSBlA2 33 34 STS pSBlA2 35 36 OMT pSBlA2 37 38 vioA pSBlA2 39 40 vioB pSBlA2 59 60 vioC pSBlA2 61 62 vioD pSBlA2 63 64 vioE pSBlA2 65 66 nCFP (N-terminal mCerulean) pSBAK3 67 68 nYFP (N-terminal mCitrine) pSBAK3 69 70 cCFP (C-terminal mCerulean) pSBAK3 71 72 cYFP (C-terminal mCitrine) pSBAK3 Auxiliary polypeptides 73 74 binding peptide pSBAK3 75 histidine marker pSBAK3 Nucleic acid program sequences 76 SPR program, split, FRET pBluescript 77 the NICTAL program pBluescript 78 biosynthesis program (123456) pBluescript 79 biosynthesis program (12346) pBluescript 80 biosynthesis program (341256) pBluescript

Chimeric proteins

81 82 Znf_Glil-binding peptide-OMT pSBlA2 83 84 Znf_HivC-binding peptide-OMT pSBlA2 85 86 Znf Zif268-binding peptide-4CL pSBlA2 87 88 Znf_PBSII-binding peptide-STS pSBlA2 89 90 Znf_Blues-binding peptide-TAL pSBlA2 91 92 TAL-binding peptide-ZnfBlues pSBlA2 93 94 Znf_Jazz-binding peptide-crtE pSBlA2 95 96 Znf_Blues-binding peptide-crtB pSBlA2 97 98 Znf_Zif268-binding peptide-crtl pSBlA2 99 100 Znf_PBSII-binding peptide-crtY pSBlA2 101 102 ZnfGlil-binding peptide-crtZ pSBlA2 103 104 ZnfHivC-binding peptide-crtO _P SB1A2 105 106 Znf_Blues-binding peptide-vioA pSBlA2 107 108 Znf_Zif268-binding peptide-vioB _P SB1A2 109 110 Znf_PBSII-binding peptide-vioE pSBlA2 111 112 Znf_HivC-binding peptide-vioD pSBlA2 113 114 Znf Glil-binding peptide-vioC pSBlA2 115 116 Histag-ZnfGli 1 -binding peptide-nCFP pSBAK8 117 118 Histag-ZnfBlues-binding peptide-nCFP pSBAK8 119 120 His tag-Znf PBSII-binding peptide-nYFP pSBAK8

121 122 cCFP-binding peptide-ZnfHIVC-Histag pSBAK8 123 124 cCFP-binding peptide-ZnfJazz-binding peptide-Histag pSBAK8 125 126 cYFP-binding peptide-Znf_Zif268-binding peptide His tag pSBAK8

Binding of binding domains to the target nucleic acid element

Method for measuring β-galactosidase enzyme activity

Bacterial cultures with plasmid constructs listed in Table 1 were inoculated into 10 ml LB medium with 5 μΐ IM IPTG (isopropyl-PD-thio-galactoside) added and increasing concentrations (0%, 0.0025%, 0.1% and 1%) L-arabinose and incubated in a shaker at 180 rpm and 37 ° C for 18 hours. Bacterial density was determined by measuring optical density (OD600). Measurements of β-galactosidase activity were performed in a microtiter plate reader preheated to 28 ° C. 5 μΐ of each culture was transferred to the wells of 96-well transparent bottom microtiter plates and then 100 μΐ Z-buffer with chloroform was added (Z-buffer: 0.06 M Na2HPO4 χ 7H20, 0.04 M NaH2PO4 χ H20, 0 , 1 M KC1, 0.001 M MgSO4x7H2O, pH 7; Z-chloroform buffer: Z-buffer, 1% β-mercaptoethanol, 10% chloroform). Bacterial cells were lysed by the addition of 50 μΐ Z-buffer with SDS (Z-buffer, 1.6 SDS), followed by incubation for 10 min at 28 ° C. 50 μΐ 0.4% of the ONPG solution in Z-buffer was added to each well and OD405 differences were measured at 30 sec intervals over a 20-min period.

Results

To determine whether transcription factors (NABFs) (zinc fingers Zif 268, Blues, PBSII, HivC) and His-Nictal bind to the corresponding DNA targets in vivo, we designed a reporter system. The reporter comprises a plasmid constructed from a) lacZ under a synthetic promoter containing a DNA binding site for a particular DNA binding protein and b) a corresponding DNA binding protein under the arabinose promoter. Successful binding of NABF to the synthetic promoter inhibits lacZ transcription, as evidenced by lower β-galactosidase activity. E. coli cultures with plasmids with different NABFs were grown overnight in LB medium with increasing concentrations of arabinose. βgalactosidase activity was measured as described in the text. For all constructs tested, β-galactosidase activity was shown to decrease in proportion to increasing arabinose concentration. It follows that NABF (Table 1) binds to the target DNA element within the synthetic promoter in vivo (Fig. 3A). Furthermore, β-galactosidase activity remained unchanged in the presence of inappropriate NABF and DNA target (e.g., Blues_O target element was replaced with PBSII target element PBSIIO, and HivCO target element with Glil_O target element), thus demonstrating the specificity of our system for NABF testing (Fig. 3B).

Surface plasmon resonance (SPR)

SPR analysis was performed on a Biacore T100 (Ge Healthcare). The Series S sensor chip SA (Ge Healthcare) was immobilized with biotinylated single stranded DNA probe at a lOOnM concentration, yielding a final concentration of approx. 300 RU. Complementary DNA hybridization (programmatic DNA sequencing with HivC zinc finger binding sites, Zif268, Jazz, Blues, PBSII, Glil) was performed by injecting 0.5-2 μΜ into the deposition buffer (lOmM HEPES, 150 mM NaCI, 0, lmM EDTA, 0.005% P20, pH 7.4) for up to 300s, resulting in a final response of approx. 300 RU. Analyte binding was monitored by injecting different concentrations of the analyte for 1 min, followed by dissociation after 5 min. Regeneration of the chip surface was achieved with two 30 s injections of 50 mM NaOH and one 24 s injection of 0.5% SDS. Subsequently, new DNA was injected to give a fresh binding base.

The Series S sensor chip CM5 (Ge Healthcare) was immobilized with 3000 RU of avidin via binding to an amino acid residue according to the manufacturer's protocol. The carboxymethylated surface of the CM5 chip was activated by a 7 min injection of 1: 1 NHS: EDC. Avidin in Na-acetate at pH 5.5 was then injected in several short pulses until a final response of 3000 RU. Avidin was bound only in the second flow cell, as the first served as the reference basis for subtracting the non-specific binding of the analytes to the dextran matrix of the sensor chip. Unreacted sites on the sensor surface were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5).

Results:

In vitro binding. chimeric proteins, biosynthetic enzymes bound to NABF on programmatic DNA (Figure 4). Figure 4A shows the binding of program DNA, followed by a chemically derived protein containing Glil NABF and substrate-chip regeneration. DNA hybridization was performed with up to 300s injection of 0.5–2 μΜ until a final response of 300 RU. Analyte binding was monitored by injecting different concentrations of the analyte for 1 min, followed by 5 min regeneration. Regeneration was achieved with two 30 s injections of 50 mM NaOH and 24 s injections of 0.5% SDS.

The binding of the analyte to program DNA and the binding of fresh DNA to each protein are presented in Figure 4B. Top analytes: Glil, Zif268, Blues, ZNF_HivC, PBSII, Jazz.

The sequential binding of NABF to programmatic DNA is presented in Figure 4C. DNA was captured at the beginning of the cycle and proteins were injected 1 min one after the other in two different sequences. Full line: ZnfJazz, Znf Blues, Znf_Zif268, ZnfPBSII, ZnfHivC, and Znf Glil; dashed line: Znf Glil, Znf PBSII, Znf HivC, Znf Jazz, ZnfBlues, and Znf_Zif268. Binding of all analytes in the sequence indicated that the binding of the analytes was weaker if Glil was injected than the former.

EMSA - electrophoresis mobility test

The specific DNA binding of synthetic domains from zinc fingers to program nucleic acid was tested by electrophoresis mobility assay. 1 pg of purified protein was incubated with different amounts of programmed nucleic acid - 500ng, 750ng, lOOOng - for 3 hours. Samples diluted with ultra-pure laboratory water to 20 μΐ were applied to a 2.0% agarose gel previously stained with ethidium bromide. The electrophoresis of the samples was run for 40 minutes at a constant voltage of 70V. Nucleic acid-protein complexes were visible under UV light.

Results:

Travel with ethidium bromide of stained program nucleic acid was dependent on the presence of NABF (i.e., the zinc finger DNA binding domain), which implied a slower travel compared to programmatic nucleic acid alone (Figure 5). These data represent NABF binding to specific target sequences in the DNA program (Fig. 5).

Confocal microscopy

A Leica TCS SP5 laser confocal microscope was used to detect half-fluorescence (i.e., NABF bound to nCFP, NABF bound to cCFP) proteins and reconstitution of FRET on program nucleic acid in vivo. Transfected HEK293 cells cultured overnight in a 37 ° C microscope dish were placed on top of the lens,

where we applied a drop of immersion oil. Cells were excited with 433 nm for CFP and 515 nm for YFP. FRET was determined using the microscope manufacturer's methodology (FRET AB and FRET SE wizard in the Leica LAS AF software package).

Split, FRET experiments

HEK293 cells were transfected with a mammalian expression vector with genes for half-fluorescent fusion proteins under the CMV promoter. Transfection was performed with jetPEI ™ transfection reagent and cells were finally fixed with 4% paraformaldehyde. Reconstitution of half-fluorescent proteins as well as the FRET effect was observed under a confocal microscope (described under Confocal microscopy ”). CFP (Znf Glil-binding peptide-nCFP and cCFP-binding peptideZnf HivC) and YFP (ZnfPBSII-binding peptide-nYFP and cYFP-binding peptideZnf_Zif268) were reconstituted only in the presence of specific, but not random, acid.

Results:

Semi-fluorescent proteins bind to adjacent target nucleotide sequences when plasmids with genes for half-fluorescent proteins are transfected simultaneously with a plasmid carrying the target program nucleic acid. Because half-fluorescent proteins are unable to form a functional chromophore in vivo by chance, the fluorescent signal is due to the binding of the heme protein to the DNA program nucleotide sequence.

Reconstitution of half-GFP Figure 6 shows in vivo reconstitution of two non-functional fluorophores (split-GFPs) bound to NABF only in the authenticity of the program nucleotide sequence (Figure 6a).

Plasmids, 75ng each, with genes for Znf Glil-binding peptide-nCFP and cCFP-binding peptide-Znf_HIVC were cotransfected with 350ng of program DNA into HEK293 cells. The emission signal of turquoise fluorescent protein was observed by exciting cells with 433nm laser light (Fig. 6b).

Plasmids, 75ng each, with Znf genes PBSII-binding peptide-nYFP and cYFP-binding peptide Znf_Zif268) were cotransfected with 350ng program DNA in HEK293

cells. YFP emission with a peak at 529nm was observed under a confocal microscope (Fig. 6C).

Reconstitution of half-GFPs in vitro .. HEK293 cells were cotransfected with ZnfGlil-binding peptide-nCFP and cCFP-binding peptide-ZnfHIVC and Znf PBSII-binding peptide-nYFP and cYFP-binding peptide Znf_Zif2. Cells were lysed in 150 μΐ Promega passive cell lysis buffer after 72 hours of cultivation and then 50 μg of program nucleic acid was added to the lysates. Functional fluorophore was observed in the presence of DNA program after 18 h growth at 4 ° C. This was detected using a PerkinElmer LS55 luminescent spectrometer.

The effectiveness of FRET. The FRET efficacy for the four NABF-associated GFP half-protein GFPs in the presence of program DNA is presented in Table 2. FRET was measured using the FRET AB wizard in a LAS AF program that allows microscopy on a Leica TCS SP5 laser confocal microscope . Emission signals in the CFP and YFP channels were set. The acceptor was destroyed by a 515 nm laser, and the FRET efficiency in the destroyed area was then calculated. HEK293 cells were transfected with software DNA and Znf Glil-binding peptide-nCFP, cCFP-binding peptide-Znf HIVC, Znf PBSII-binding peptide-nYFP, Znf_cYFP-binding peptide-Znf_Zif268. For negative control, cells were transfected with cytosolic CFP and YFP without DNA binding factors, and for positive control with CFP associated with YFP with the binding peptide.

Table 2: FRET efficiency of four half-GFPs, each linked to a different DNA binding factor.

FRET positive control FRET negative control FRET on a DNA program ± 2 1 ± 1 4 ± 2

Synthesis of violacein

Cross-culture E. coli cultures with plasmids encoding the chimeric proteins (see Table 1, construct combinations of ZnfBlues-binding peptide-vioA, Znf_Zif268-binding peptide-vioB, ZnfPBSII-binding peptide-vioE, ZnfHivC-binding peptide-vioD, Znf Glil binding peptide-vioC) with or without a plasmid encoding a DNA program (see Table 1, biosynthesis program (123456) or biosynthesis program (341256)) were diluted in fresh Luria Bertani medium to OD600 0.05 and grown in the presence of appropriate antibiotics. at 37 ° C. At different times during growth, fermentation broths (3 ml in total) were sampled. A representative point (17.5 h) is visible in Figure 9. Bacteria were lysed by addition of the same volume (1.5 ml) of 10% SDS, and violacein was extracted with ethyl acetate (1: 1 vol, vol). After short vortexing, the organic phase was collected and the absorbance spectrum at 575 nm was measured. Samples were analyzed by TLC, and quantitative analysis was performed by densiometry at 575 nm after direct spectral analysis by HPTLC. Violacein was also identified by mass spectrometry. We compared the amount of violacein production with or without programming DNA or in the presence of mixed DNA (Figure 9).

While the present invention is described with reference to the specific embodiments of the invention, it should be understood by those skilled in the art that minor changes can be justified and the equivalents can be replaced without departing excessively from the true spirit and purpose of the invention. Many changes can also be made to adapt a particular situation, material, material composition, process, process steps or steps to the object, spirit and scope of this invention

Claims (16)

1. A method for synthesizing a biosynthesis product by culturing host cells, wherein the cells are modified with one or more nucleic acids containing: a) a nucleotide sequence encoding at least three to about 100 chimeric proteins, each chimeric protein comprising at least one enzyme of the biosynthetic pathway or functional polypeptide and at least one factor that binds to the nucleic acid, wherein said enzyme is the biosynthetic pathway, or a functional polypeptide and a nucleic acid-binding factor associated with the binding polypeptide; b) a program nucleotide sequence comprising at least three to about 100 target nucleic acid elements, each of which recognizes a specific factor of the chimeric polypeptide that binds to the nucleic acid motif and said program nucleotide sequence regulates biosynthetic pathway enzymes or other functional polypeptides along the program sequence ; c) the substrate for the biosynthetic pathway present in the cell or extracellularly added to the host cell and said cultivation serves to synthesize said chimeric proteins in the genetically engineered host cells, as reflected in the production of the compound. 2. A method for the synthesis of a biosynthesis product by the addition of one or more ingredients, comprising: a) three to about 100 chimeric proteins, proteins wherein each chimeric protein consists of at least one biosynthetic pathway enzyme or functional polypeptide and at least one factor that binds to a nucleic acid motif, wherein said biosynthetic pathway enzyme or functional polypeptide and factor that binds to a nucleic acid linked to a binding polypeptide; b) a program nucleotide sequence comprising at least three to about 100 target nucleic acid elements, each of which recognizes a specific factor of the chemical polypeptide that binds to the nucleic acid sequence and said program nucleotide sequence regulates biosynthetic pathway enzymes or other functional polypeptides along the program sequence ; c) the biosynthetic pathway substrate and enzyme cofactors are added to the mixture or formed in the mixture, where said nucleic acid sequence is in the mixture or immobilized to another compound or solid phase. A method according to any one of claims 1 to 2, characterized in that the nucleic acid program sequence consists of target nucleic acid elements and a spacer sequence separating nucleic acid elements and said nucleic acid elements are defined nucleotide sequences determined by distinctive motifs nucleic acid binding factors; and is a sequence of spacers of any length, preferably 1 to 50 nucleotides. Method according to any one of claims 1 to 3, characterized in that said nucleotide sequence of each target nucleic acid element is selected according to a specific motif recognized by a factor that binds to the nucleic acid motif used in the method as an enzyme carrier biosynthetic pathways or other functional polypeptide; and each nucleic acid target nucleic acid target element is used one or more times; typically from 1 to 16; and three or more different nucleic acid motif elements are used on a nucleic acid program sequence, typically 3 to 16, preferably 3 to 7, depending on the state of oligomerization or the number of catalytic steps in the biosynthetic pathway. A method according to any one of claims 1 to 4, characterized in that the target nucleic acid sequence motif is any size greater than 4 nucleotides, optionally consisting of 4 to about 30 nucleotides, and the nucleic acid binding factor recognizes the target element nucleic acids at each site of the target element one nucleotide or any combination of two, three or four nucleotides, and each target element of the nucleic acid lies on the nucleic acid program sequence in one or more copies and in a sequence that determines the spatial arrangement of the protein chemistry in relation to one another . A method according to any one of claims 1 to 5, characterized in that the biosynthetic pathway enzyme or any other functional polypeptide is covalently linked via a binding peptide of 1 to about 100 amino acids in length to a nucleic acid-binding factor and is a natural source factor or artificially synthesized and having specific binding properties to nucleic acid elements; and a factor that binds to the nucleic acid motif is selected from, but not limited to: the helix-loop-helix nucleic binding domain, the zinc finger nucleic binding domain, the leucine zipper as the nucleic binding domain, the winged helix-loop-helical nucleic binding domain, helix-helical nucleus binding domain, HMG-box nucleic binding domain, inactive restriction endonucleases and transcription factors. A method according to any one of claims 1 to 6, characterized in that the nucleic acid program sequence is used as such or cloned into a vector suitable for the host cell and the vector is amplified extrachromosomally. A nucleic acid encoding a polypeptide composed of an enzyme biosynthetic pathway or functional polypeptide and a binding factor used in the method of any one of claims 1 to 6, and said nucleic sequence is inserted into a suitable vector under the control of a regulatory sequence that modulates the expression of the polypeptides and is vector suitable for expression in a ski cell host. Genetically engineered host cells containing one or more nucleic acid programming sequences according to any one of claims 3 to 5 and the host cell is selected from eukaryotic or prokaryotic cells. Genetically modified host cells containing one or more enzymes of the biosynthetic pathway or other functional peptides according to claim 6, and the host cell is selected from eukaryotic or prokaryotic cells. Genetically modified host cells comprising one or more nucleic acid programming sequences according to any of claims 3 to 5 and one or more functional polypeptides according to claim 6, and the host cell is selected from eukaryotic or prokaryotic cells. A method according to any one of claims 1 to 2, characterized in that the biosynthetic pathway is either catabolic or anabolic, the synthesis of primary metabolites, for example, but not limited to: amino acids, fatty acids, carbohydrates, pyrimidines, purines, citric acids , itaconic acid, ethanol, glycerol, methanol, butanol, propanol, isoprene and higher alcohols. Method according to any one of claims 1 to 2, characterized in that the biosynthetic pathway is either a synthesis of secondary metabolites, for example, but without limitation: polyketides, non-ribosomal peptides, hormones, terpenes, antioxidants, pigments. A method according to any one of claims 1 to 2, characterized in that the biosynthetic pathway is a synthesis of carotenoids. A method according to any one of claims 1 to 2, characterized in that the biosynthetic pathway is a synthesis of violacein. A method according to any one of claims 1 to 2, characterized in that the biosynthetic pathway is the synthesis of resveratrol or methylated resveratrol.