EP2609204A1 - Biocatalyseur de cellule entière - Google Patents

Biocatalyseur de cellule entière

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Publication number
EP2609204A1
EP2609204A1 EP11749822.0A EP11749822A EP2609204A1 EP 2609204 A1 EP2609204 A1 EP 2609204A1 EP 11749822 A EP11749822 A EP 11749822A EP 2609204 A1 EP2609204 A1 EP 2609204A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
cell
redox factor
redox
regenerating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11749822.0A
Other languages
German (de)
English (en)
Inventor
Joachim Jose
Ruth Maas
Eva Kranen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zyrus Beteiligungs GmbH and Co Patente I KG
Original Assignee
Zyrus Beteiligungs GmbH and Co Patente I KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zyrus Beteiligungs GmbH and Co Patente I KG filed Critical Zyrus Beteiligungs GmbH and Co Patente I KG
Publication of EP2609204A1 publication Critical patent/EP2609204A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/03—Oxidoreductases acting on NADH or NADPH (1.6) with oxygen as acceptor (1.6.3)
    • C12Y106/03001—NAD(P)H oxidase (1.6.3.1), i.e. NOX1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the invention relates to a nucleic acid molecule comprising (1) a portion encoding a signal peptide, (2) a portion which omits a heterologous redox factor-regenerating polypeptide or a variant thereof, (3) optionally, a portion encoding a protease recognition site (4) a portion encoding a transmembrane linker; and (5) a portion encoding a transporter domain of an autotransporter or a variant thereof.
  • Oxidoreductases are characterized by comprising not only at least one polypeptide chain but also one or more redox-reactive groups, which may be one or more redox-reactive amino acid side chains or prosthetic groups.
  • the substrates of the oxidoreductases include metabolic products and redox factors.
  • these substrate redox factors do not act as catalysts, but participate in the reaction in stoichiometric amounts and undergo chemical reactions with other substrates.
  • redox factors are generally much more expensive than other organic substrates, the feasibility of industrial syntheses using oxidoreductases is limited because of the need to provide suitable reduced or oxidized factors. To solve this problem, scientists have proposed the use of redox factor regenerating enzymes.
  • FDH formate dehydroxygenase
  • GDH glucose dehydrogenase
  • Such enzymes should be immobilized on suitable supports for several cycles of catalysis to avoid having to repeatedly purify large quantities of the polypeptides.
  • redox factor regenerating enzymes are often unstable, especially when present as immobilized enzymes.
  • the object of the present invention is to provide an agent with a redox factor-regenerating activity, wherein the source of the activity is not only readily accessible to substrate molecules, but can be easily amplified, recycled and regenerated to allow multiple catalytic steps, without it being necessary to repeatedly prepare new agent.
  • Another object of the present invention is to provide an agent which by itself or in the presence of potentially inactivating chemicals, such as inhibiting metal ions, or at acidic or basic pHs, has a redox factor regenerating activity with better properties than the original form of the corresponding Enzyme in terms of kinetics, thermal stability, shelf life and stability.
  • an object of the present invention It is to provide an agent which, in the presence of oxygen or oxygen-releasing agents or under oxidizing conditions, exhibits a stable redox factor-regenerating activity, the stability of which is better than that of the original form of the corresponding enzyme.
  • redox-factor regenerating enzymes can be expressed on the surface of an entire cell using the autotransporter system and are intrinsically surprisingly stable with respect to the presence of oxygen and various temperatures and conditions.
  • the subject invention has surprisingly found that redox factor regenerating enzymes can be incubated and remain stable for a long time in various solutions and buffers under various conditions.
  • Autodisplay is an elegant tool for presenting recombinant proteins to the bacterial surface.
  • This expression system is based on the secretory mechanism of the protein family of car transporters belonging to the type V secretion system.
  • Autotransporter proteins are synthesized as precursor proteins that fulfill all structural requirements for transport to the cell surface (Jose, 2006). They are synthesized with an N-terminal signal peptide, which is typical of the See pathway, which allows traversing the inner membrane. Once in the perip! Asma, the C-terminal part of the precursor folds into the outer membrane after truncation of the signal peptide as a porin-like structure, a so-called ⁇ -barrel.
  • the N-terminal bound passenger domain is transiently transcribed to the surface (Jose et al., 2002). There, it can be cleaved off - either autoproteolytically or by an additional protease - or can remain anchored to the cell membrane via the transporter domain. Replacement of the natural passenger with a recombinant protein results in its surface translocation. To do this, a non-natural precursor must be constructed using genetic engineering techniques a signal peptide, the recombinant passier, the ⁇ -barrel, and a linker region therebetween, which is required for unrestricted access to the surface.
  • the AIDA-I car transporter has been used successfully for efficient surface presentation of various passenger domains (Henderson et al., 2004).
  • self-association of subunits to an active enzyme was observed, for example, in the dimeric enzyme sorbitol dehydrogenase (Jose, 2002, Jose and von Schwichow, 2004).
  • auto-display technology is an expression method for certain proteins on the outer membrane surface of E. coli and other Gram-negative bacteria, the auto-display system being based on the natural secretory mechanism of auto transporter proteins (A. Banerjee et al. (2002)) ,
  • the transport of the recombinant passenger protein can be carried out simply by introducing, in reading frame, its coding sequence between the signal peptide and the translocating domain of the autodisplay vector using standard genetic engineering techniques.
  • the signal peptide may be derived from a subunit of the cholera toxin, and it may be combined with a non-natural promoter.
  • the passenger protein that is to undergo translocation through the outer membrane is expressed as a recombinant fusion protein with another protein, called an auto transporter, on the outer membrane of E. coli (AIDA-i) (Jose, 2006).
  • the C-terminal part of the car transporter protein forms a porin-like structure ( ⁇ -barrel) within the outer membrane of E. coli. This porin-like structure allows translocation of the recombinant pass protein to the outer membrane surface of E. coli (Jose, 1995, 2006, 2007).
  • a nucleic acid molecule comprising (1) a portion encoding a signal peptide, (2) a portion containing a heterologous redox factor regenerating polypeptide or a variant thereof, (3) optionally a portion encoding a protease recognition portion, (4) a portion encoding a transmembrane linker, and (5) a portion encoding a transporter domain of an autotransporter or a variant thereof ,
  • the nucleic acid molecule is operably linked to a sequence for expression regulation.
  • expression repression sequence refers to a nucleic acid sequence which can regulate the level of expression of a nucleic acid molecule, preferably downstream of the expression regulation sequence
  • the sequence for expression regulation may be, for example, a promoter.
  • the person skilled in the art is familiar with sequences suitable for expression regulation and methods for the functional connection of these sequences with a nucleic acid molecule.
  • the nucleic acid molecule is part of a recombinant plasmid.
  • the nucleic acid molecule comprises SEQ ID NO: 1 or variants thereof.
  • heterologous refers to a nucleic acid constructed using genetic engineering techniques, for example, by assembling an expression regulation sequence and a sequence to be expressed that does not normally have that sequence for expression regulation subordinate, or by Use a sequence that has a point mutation with respect to the original sequence. Consequently, even if only a section of a construct is called heteroiog, it implies that the entire construct is heteroiogical.
  • the person skilled in the art is familiar with genetic engineering methods.
  • heteroiog means that the portion of the nucleic acid encoding the redox factor-regenerating polypeptide is from at least one other organism another section, such as the transporter domain or the expression regulation sequence or the transmembrane linker, has been obtained or taken.
  • the redox factor-regenerating polypeptide is heterologous when recovered from Lactobacillus brevis but all other sequences were obtained from E. coli.
  • heteroiog means that the nucleic acid sequence termed heteroiogue is from an organism other than the host or intended host used to express or amplify that nucleic acid sequence.
  • the term "signal peptide” refers to a sequence of amino acids, preferably at the N-terminus of a polypeptide, which causes the polypeptide, when expressed in the cytosome of a host cell, to become one
  • the host cell is a Gram-negative bacterial cell and the signal peptide causes the resulting or final polypeptide to translocate into the peripiasma or outer membrane of the Gram-negative bacterial cell.
  • protease recognition site refers to a particular amino acid sequence motif in a polypeptide, this sequence motif of said protease is specifically recognized such that it binds and cleaves the polypeptide.
  • transmembrane linker refers to a flexible polypeptide portion useful for linking the auto-transporter domain to the redox factor-regenerating polypeptide but flexible enough to independently fold and / or transport the protein redox factor-regenerating polypeptide.
  • the term "transporter domain of an auto-transporter” refers to a domain that can be used to obtain the expression product of the nucleic acid molecule when synthesized ribosomally in the interior of the cell, preferably in bacterial cytopiasma, and to the outside Membrane of the cell, preferably translocated to the side of the outer membrane exposed to the cell environment
  • the transporter domain causes said expression product to be on the outer surface of the outer membrane
  • the transporter domain of an auto-transporter is a protein located on the outer membrane of the cell, and the NADH oxidase-encoding portion is part of or fused to a domain, loop, or other portion of the transporter domain, so that the di e NADH oxidase is presented on the surface of the cell.
  • the transporter domain of an auto-transporter is a protein of a system that can be used to present polypeptides on the surface of a cell.
  • the transporter domain is a transporter domain of the autodisplay system, also referred to as the autotransporter pathway, of the AIDA-I type Gram-negative bacterial tern.
  • variants of amino acids or nucleic acid sequences which are explicitly referred to in the present application, for example by the name or the accession number or even by the term “variant of”, or implicitly, for example by a description of the function, are included of the present invention
  • the term “variant” as used herein includes amino acid or nucleic acid sequences corresponding to 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% with the reference
  • the term "variant" with respect to an amino acid sequence includes those amino acid sequences that have a conservative amino acid exchange or multiple conservative amino acid substitutions with respect to the reference sequence.
  • the terms "variants" of an amino acid sequence or nucleic acid sequence include active portions and / or fragments of the amino acid sequence or nucleic acid sequence
  • the term “active portion” as used herein refers to an amino acid sequence or nucleic acid sequence shorter than the full length amino acid or nucleic acid sequence, but at least some of its essential biological activity, e.g. As an NADH oxidase.
  • the term "variant" of a nucleic acid includes the nucleic acids of the complementary strand that hybridize to the reference nucleic acid, preferably under stringent conditions
  • the stringency of hybridization reactions is readily determinable by one of ordinary skill in the art and is usually empirical as a function of In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures
  • Hybridization generally depends on the ability of the denatured DNA to renaturate when complementary strands are present in an environment whose temperature is lower than the melting temperature of the strands, the higher the degree of homology sought between the probe and a hybridizable sequence, the higher the relative Temperature that can be applied.
  • the term "variant" of a nucleic acid or amino acid refers to a nucleic acid or amino acid having, at least to some extent, the same biological activity and / or function as the reference nucleic acid or amino acid
  • the term “variant” refers to a Nucleic acid sequence, as used herein, to another nucleic acid sequence encoding an amino acid sequence that is similar to the reference amino acid sequence.
  • the term "redox factor” refers to an organic compound that is redox-reactive, ie, that can be oxidized or reduced under physiological conditions.
  • the redox factor does not comprise any redox-reactive amino acid side chains
  • the redox factor is a free redox factor, that is, it is not covalently bound to a peptide backbone or peptide.
  • the redox factor has a standard reduction potential of not more than 0.85 (ie, for high 0.6V), 0.5, 0.4, 0.2, 0, -0.1, -0.15, -0.2, -0.25, -0.3, or -0.4V up.
  • the redox factor has a standard reduction potential of -0.325 to -0.2V.
  • the redox factor has a standard reduction potential of -0.35 to -0.3.
  • the term "redox factor-regenerating polypeptide” as used herein refers to a polypeptide that catalyzes the regeneration of a redox factor.
  • the term "regenerating a redox factor” as used herein refers to the ability to to reduce an oxidized redox factor that acts as a reducing agent in its reduced form in a chemical synthesis, or the ability to oxidize a reduced redox factor, which in its oxidized form in a chemical synthesis as Oxidationsmitte! acts.
  • the term "regeneration of a redox factor” as used herein refers to the restoration of the redox state of a redox factor, preferably the state in which it could be used for a synthesis of interest, most preferably a synthesis by For example, NAD + consumed in a reaction can be regenerated by oxidizing NADH to NAD + .
  • the redox factor which is regenerated by the redox factor-regenerating polypeptide is selected from the group comprising NADH, NADPH, FADH 2l heme, metal ions, Glutathione, pyrroloquinoline quinone (PQQ), pyridoxal phosphate, thiamine pyrophosphate and ascorbate.
  • Metal ions include, but are not limited to, Fe, Cu, Zn, Ni, Co, Mn, Cr, and Mg ions.
  • each of the aforementioned redox factors has both the oxidized and reduced forms or forms of the corresponding conjugated redox couple, for example, both NADH and NAD 1 " in the case of the redox factor NADH, although only one form is expiicitly mentioned.
  • the redox factor-regenerating polypeptide comprises one or more flavin cofactors.
  • flavin cofactor refers to a redox-reactive cofactor based on the isoafoxazine ring system
  • flavin cofactor is selected from the group comprising riboflavin, flavin mononucleotide (FMN) and fiavin adenine dinucleotide (FAD), both in their oxidized and reduced forms.
  • the redox factor-regenerating polypeptide is selected from the group comprising NADH oxidase, formate dehydrogenase and glucose dehydrogenase.
  • the redox factor-regenerating polypeptide is selected from the group comprising the NADH oxidases from the genera Lactobacillus, T ermus, Brevibacterium and Streptococcus, preferably the NADH oxidase from Lactobacillus brevis, and variants of it.
  • the transporter domain of an autotransporter is selected from the group comprising Ssp, Ssp-h1, Ssp-h2, PspA, PspB, Ssa1, SphB1 , AspA / NalP, VacA, AIDA-I, IcsA, MisL, TibA, Ag43, ShdA, AutA, Tsh, SepA, EspC, EspP, Pet, Pic, SigA, Sat, Vat, EpeA, EatA, Espi, EaaA, EaaC , Pertactin, BrkA, Tef, Vag8, PmpD, Pmp20, Pmp21, AgA1 protease, App, Hap, rOmpA, rmpmp, ApeE, EstA, Lip-1, McaP, BabA, SabA, AlpA, Aae, NanB and variants thereof.
  • the term "transporter domain of an auto-transporter” as used herein includes a domain presenting a polypeptide other than the transporter domain, particularly a redox factor-regenerating polypeptide, on the surface of a cell when said protein has been inserted into or fused to the amino acid sequence of the transporter domain.
  • the redox factor-regenerating polypeptide is fused to a transporter domain of an auto-transporter.
  • the transporter domain of the autotransporter according to the invention may be any transporter domain of an auto-transporter and is preferably capable of forming a ⁇ -barrel structure.
  • a detailed description of the ⁇ -barrel structure and preferred examples of ⁇ -barrel auto-transporters are disclosed in WO 97/35 022 and incorporated herein by reference.
  • Henderson et al. describe auto-transporter proteins with suitable auto-transporter domains (a summary can be found in Table 1 of Henderson et al., 2004). The disclosure of Henderson et al. (2004) is incorporated herein by reference.
  • the transporter domain of the autotransporter can be selected from: Ssp (P09489, S. marcescens), Ssp-h1 (BAA33455, S. marcescens), Ssp-h2 (BAA 11383, S. marcescens), PspA (BAA 36466, P. fluorescens), PspB (BAA36467, P. fluorescens), Ssa1 (AAA80490, P. haemolytica), SphB1 (CAC44081, pertussis), AspA / NalP (AAN71715, N. meningitidis), VacA (Q48247, H.
  • aeruginosa Lip-1 (P40601, X. luminescens), McaP (AAP97134,. catarrhalis), BabA (AAC38081, py / o / 7), SabA (AAD06240, H. py / ⁇ ), AlpA (CAB05386, H. py / on), Aae (AAP21063, ⁇ . actinomycetemcomitans), NanB (AAG35309, P haemolytica) and variants of these car transporters.
  • Genbank accession numbers and species from which the autotransporter can be obtained are given in the form of a killer.
  • the transporter domain of the autotransporter is the AIDA-i protein of E. coli or a variant thereof, such as those described by Niewert et al. (2001).
  • the AIDA autotransporter system has been associated with F18 and Stx2e in pig / o-isolates from pigs diagnosed with edema and so-called post-weaning.
  • Variants of the above-described auto transporter sequences can be obtained, for example, by altering the amino acid sequence in the loop structures of the ⁇ -barrir which do not belong to the transmembrane sections.
  • the nucleic acids encoding the surface shingles can be completely deleted.
  • conservative amino acid substitutions ie, the replacement of one hydrophilic with another hydrophilic amino acid and / or the replacement of one hydrophobic with another hydrophobic amino acid, can be made.
  • a variant at amino acid level has a sequence identity of at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% with the corresponding naturally occurring sequence of the autotransporter domain, especially in the area of beta-sheet structures.
  • the redox factor-regenerating polypeptide is oxygen sensitive.
  • oxygen sensitive means that the corresponding polypeptide undergoes rapid deactivation in the presence of oxygen, preferably at normal, ie, atmospheric, concentrations.
  • the redox factor-regenerating polypeptide is a soluble polypeptide.
  • the term "soluble polypeptide" as used herein refers to a polypeptide that is not bound to a membrane in its natural environment
  • the Lactobacillus brevis NADH oxidase, a cytosolic protein is a soluble polypeptide
  • the redox factor-regenerating polypeptide is a membrane-bound polypeptide, ie, a polypeptide that is covalently or non-covalently bound or attached to a cell membrane or an integral membrane protein
  • the complex IV of the respiratory chain is a membrane-bound polypeptide.
  • the object of the present invention is achieved by a polypeptide encoded by a nucleic acid molecule according to an embodiment of the first aspect of the present invention.
  • the polypeptide comprises SEQ ID NO: 2 or variants thereof.
  • the object of the present invention is achieved by a cell which has on its surface a polypeptide according to the second aspect expressed or transformed using a nucleic acid molecule according to an embodiment of the first aspect of the present invention.
  • the cell or host cell is a prokaryotic cell, preferably a Gram-negative bacterial cell, most preferably an E. coli cell
  • the cell is a eukaryotic cell
  • the cell or host cell is a spore of a prokaryote.
  • the object of the present invention is achieved by a membrane fraction which can be obtained from the cell according to the third aspect of the present invention.
  • a bacterial cell comprises a series of compartments separated by hydrophobic membranes.
  • a Gram-positive bacterial cell has a plasma membrane that confines the cytosol, the interior of the cell. The plasma membrane is surrounded by a peptidoglycan layer.
  • Gram-negative bacteria have, in addition to the plasma membrane, another membrane called the outer membrane.
  • a polypeptide according to the present invention is expressed on the outside of the outer membrane of a Gram-negative bacterial cell
  • a polypeptide according to the present invention on the inside of the outer membrane of a Gram-negative Bacterial cell expressed.
  • the polypeptide of the invention is expressed on the outside of a spheroplast, which is a Gram-negative bacterial cell in which the outer membrane has been removed.
  • a spheroplast which is a Gram-negative bacterial cell in which the outer membrane has been removed.
  • the polypeptide according to the invention is expressed on the outside of a gram-positive bacterial cell.
  • the membrane fraction or the membrane preparation comprises a redox factor-regenerating polypeptide according to the first aspect of the invention, preferably in a catalytically active state.
  • the terms "membrane fraction” and “membrane preparation” as used herein preferably refer to a product that accumulates in membrane constituents, preferably constituents of the outer membrane of a Gram-negative bacterium.
  • membrane preparations One skilled in the art will be familiar with methodology and procedures that can be used to prepare membrane preparations.
  • bacterial cells can be harvested from a culture and lysed, for example by freeze-thaw cycles, sonication, resuspension in lysis buffer or the like, followed by differential centrifugation to isolate membrane fractions of the cells.
  • the membrane preparation is an outer membrane preparation, ie a preparation in which the outer membrane constituents are enriched compared to the constituents of other membranes and compartments, such as the cytosol, inner membrane and periplasm are.
  • an outer membrane preparation ie a preparation in which the outer membrane constituents are enriched compared to the constituents of other membranes and compartments, such as the cytosol, inner membrane and periplasm are.
  • One of ordinary skill in the art will be familiar with methodology and procedures that may be used to isolate or enrich the outer membrane or components thereof, such as lysozyme treatment of bacterial cells and subsequent centrifugation steps.
  • the membrane fraction may be a treated membrane fraction, ie, the content or properties of the membrane fraction have been altered, for example by purifying a protein component of the membrane fraction or by solubilizing the membrane fractions and / or incorporation of components the membrane fraction in vesicles.
  • the membrane fraction may be immobilized - for example on the surface of a vessel or column.
  • the object of the present invention is achieved by a method for regenerating a redox factor, comprising the following steps: a) providing the polypeptide according to the second aspect, the membrane preparation according to the fourth aspect or the cell according to the third aspect of the present invention and b) contacting the polypeptide according to the second aspect, the membrane preparation according to the fourth aspect or the cell according to the third aspect with at least one substrate of the redox factor-regenerating polypeptide.
  • step a) and / or step b) take place in the absence of a reducing agent and / or in an oxidative environment.
  • the object of the present invention is achieved by using the cell according to the third aspect or the membrane fraction according to the fourth aspect or the polypeptide according to the second aspect for regenerating a redox factor.
  • the object of the present invention is achieved by the use of the cell according to the third aspect, the membrane preparation according to the fourth aspect and the polypeptide according to the second aspect of the present invention for adjusting the redox environment of an aqueous solution, preferably by deoxidizing the aqueous Solution.
  • the term "adjusting the redox environment of an aqueous solution” as used herein refers to any action directly related to the redox conditions in an aqueous solution, ie, the ability of the solution to oxidize or bind a compound
  • the term "deoxygenating an aqueous solution” as used herein refers to any action that can be taken to reduce the concentration of oxygen in that solution.
  • the person skilled in the art is familiar with methods for measuring the oxygen concentration in an aqueous solution can be used, such as the use of oxygen electrodes.
  • the object of the present invention is achieved by a method of producing a cell presenting on its surface a redox factor-regenerating polypeptide, comprising: (a) introducing the nucleic acid into a cell according to an embodiment of the first aspect of the present invention and b) optionally contacting the cell with one or more redox reactive prosthetic groups.
  • the object of the present invention is achieved by a method for producing the product of a redox factor-dependent polypeptide, comprising the following steps: a) providing a redox factor-dependent enzyme and (b) contacting the redox-factor-dependent polypeptide with one or more of its substrates in the presence of the line according to the third aspect, the membrane preparation according to the fourth aspect and the polypeptide according to the second aspect of the present invention, whereby the polypeptide according to the second aspect regenerates the redox factor on which the redox factor-dependent polypeptide depends .
  • the term "redox factor-dependent polypeptide" as used herein refers to a polypeptide having a biological activity, preferably an enzyme activity, which requires a redox factor as cofactor, prosthetic group or, preferably, substrate.
  • the object of the present invention is achieved by a composition comprising the cell according to the third aspect, the membrane preparation according to the fourth aspect and the polypeptide according to the second aspect of the present invention, and a redox factor-dependent polypeptide and the redox factor.
  • the redox factor-dependent polypeptide is presented using the autotransporter system, ie, its expression is effected by a nucleic acid comprising: (1) a portion encoding a signal peptide, (2) a portion that is a preferably heterologous one redox factor-regenerating polypeptide or a variant thereof, (3) optionally one Section encoding a protease recognition site; (4) a section encoding a transmembrane linker; and (5) a section encoding a transporter domain of an autotransporter or a variant thereof.
  • Fig. 1 shows the restriction map of the plasmid pAT-NOx.
  • Figure 2 shows an SDS gel showing NADH oxidase in the outer membrane of E. coli as well as orientation by whole cell digestion.
  • M molecular weight standard; 1) E. coli UT 5600 ⁇ DE3); 2) E. coli UT5600 (DE3) pAT-NOx, not induced; 3) E. coli UT5600 (DE3) pAT-NOx induced; 4) E. coli UT5600 (DE3) pAT-NOx induced, digested with proteinase K; 5) E. coli UT5600 (DE3) pAT-NOx induced, digested with trypsin.
  • Fig. 3 shows absorbance spectra of the supernatant following an NADH oxidase assay using A) E. coli UT5600 (DE3) pAT-NOx and ⁇ ) E. coli BL21 (DE3) paT-NOx, each after 1 h of induction ,
  • Fig. 4 shows the absorption spectra of the supernatant following an NADH oxidase assay using A) E. coli UT5600 (DE3) pAT-NOx and B) E. coli BL21 (DE3) paT-NOx, each after 4 hours Induction.
  • Figure 5 shows the absorption spectra of the supernatant following an NADH oxidase assay using A) E. coli UT5600 (DE3) pAT-NOx and B) E, coli BL21 (DE3) paT-NOx, each after 20 h Induction.
  • Fig. 6 shows the results of the NADH oxidase assay performed on microtiter plates using E. Coli BL21 (DE3) pAT-NOx after growth in M9 medium. The figure shows the decrease in absorbance at 340 nm over time.
  • Figure 7 shows the results for the NOX whole cell biocatalyst stored at + 8 ° C for more than 7 weeks.
  • the activity test was performed with cells pre-stored [A] and cells given one week [B], two weeks [C], three weeks [D], four weeks [E], five weeks [F], six weeks [G] and seven weeks [H] had been stored.
  • Figure 8 shows the results for the NOX whole-cell biocatalyst stored at -18 ° C for more than 7 weeks.
  • the activity test was carried out with cells before storage [A] and lines lasting one week [B], two weeks [C], three weeks [D], four weeks [E], five weeks [F], six weeks [G ] and seven weeks [H] had been stored.
  • Figure 9 shows the results for the NOX whole cell biocatalyst stored at -70 ° C for more than 7 weeks.
  • the activity test was carried out with cells before storage [A] and cells that lasted one week [B], two weeks [C], three weeks [D], four weeks [E], five weeks [F], six weeks [G ] and seven weeks [H] had been stored.
  • Fig. 10 shows the determination of the number of cells in samples stored for stability tests for more than seven weeks. It is the decrease in the number of cells in samples stored at -18 ° C ( ⁇ ), as well as the decrease in the number of cells in samples stored at + 8 ° C ( ⁇ ) and in samples stored at -70 ° C (A).
  • 50 ⁇ were taken from a stored sample and used to prepare a series of dilutions. 50 ml of each of the 10 ⁇ 9 - and 10 "0 dilutions was plated on 30 mg / i kanamycin LB agar plates The plates were incubated at 37 ° C overnight, and the next day, the colonies were counted the number.. of cells / ml was calculated based on the number of co-ionizing units.
  • Figure 13 shows the regeneration of NAD + , more specifically the concentration of NADH as a function of absorbance at 340 nm over time, especially the NADH concentration during the AIDH reaction and during the AIDH reactions using E. coli BL21 (DE3) pAT-NOx.
  • the NADH concentration was normalized by calculating the quotient E 3 ( , 0 / E59o relative to the number of cells: AIDH 30 mU, E. coli BL21 (DE3) pAT-NOx OD 5, E. coli BL21 (DE3) OD 1, reaction started by 400 ⁇ NAD + .
  • NADH oxidase The activity of NADH oxidase was detected by a photometric assay.
  • NADH shows two extinction maxima, namely at the wavelengths 260 nm and 340 nm, while NAD + shows only one maximum, at 260 nm. This difference can be exploited to follow the oxidation of NADH to NAD * photometrically.
  • FAD was added to the nutrient media or buffers used to allow proper folding of the enzyme as well as uptake of FAD.
  • the cells were first stored on ice for 15 minutes and then pelleted by sedimentation at 5000 rpm for 10 minutes. The sediment was washed twice in 20 ml of 0.1 M potassium phosphate buffer, pH 7.5, 10 ⁇ FAD. The OD of the cells was then adjusted to 50 with the same buffer.
  • the test for detecting NADH oxidase activity was carried out with 1 m! Samples containing 0.1 M potassium sulfate buffer, pH 7.5, 1 mM DTT and 100 ⁇ M NADH as substrate. Cells were assayed at a final OD 578 of 1 in the test After one hour of incubation at room temperature, the cells were removed from the samples by centrifugation. The supernatant spectra were recorded at wavelengths between 200 and 400 nm.
  • coli BL21 (DE) pAT-NOx uninduced cells convert about half of the NADH offered into NAD + , while induced cells transduce all NADH present thus explain that the T7 system used for the expression of NADH oxidase is not "dense", ie that the repression of the T7 promoter is incomplete, which leads to low expression of the gene by T7 polymerase even in the uninduced state .
  • E. coli UT5600 (DE3) shows no activity in the uninduced state, however, induced cells convert only about half of the NADH.
  • the optical assay was transferred to microtiter plates to allow parallel observation of as many samples as possible.
  • NADH oxidase assay on microtiter plates The NADH oxidase assay for 96-well microtiter plates was performed in samples of 240 ⁇ per well in 0.1 M potassium phosphate buffer. The cells were used in amounts such that an OD578 of 1 was achieved in the assay. 200 ⁇ NADH was added to start the reactions. After a 0, 3, 7, 15, 30, 45, or 60-minute incubation at room temperature, the assay was monitored in a microtiter plate at 340 nm (Mithras, Berthold Technologies). In order to avoid sedimentation of the cells, the plate was shaken before the absorbance measurement. As a reference, a cell suspension of the corresponding OD in buffer was used.
  • the product tint of the NADH oxidase whole cell catalyst could be optimized to have i) higher activities, ii) increase the difference between induced and uninduced cells, and iii) only one in the nonsense controls low activity occurred.
  • the culture used to measure NADH oxidase must be in an optimal state.
  • Figure 6 shows the results of an NADH oxidase assay performed under standard conditions as described in the section "Methods".
  • the NADH oxidase assay was then performed directly in the cell suspension as described above in the microtiter plate and observed. This makes handling much easier and the data is easier to reproduce. A determination of absolute values of the enzyme activity is not possible.
  • the activity of enzymes determined by a photometric method is calculated according to Lambert-Beer's law. In order to apply this equation, the layer thickness of the cuvette or the 96-weli plate must be known. In the present experimental approach, this parameter can not be determined. Therefore, the activity of NADH oxidase was determined with a photometer, using cuvettes with a defined layer thickness of 1 cm. Consequently, the Lambert-Beer law can be applied.
  • the activity in the LB medium was determined to be 12.23 mU / ml.
  • the activity at 30 ° C in the M9 medium was slightly higher, namely 16.38 ml / ml.
  • Hummel et al. (2003) reported the activity of a readily purified enzyme at 10-15 U / mg.
  • Step Example! 3 Stability and storage stability test of the NADH oxidase whole cell catalyst
  • induced cells were stored at 8 ° C in the refrigerator or at -18 ° C or at -70 ° C in the freezer. These cells were checked weekly by triplicate with the NADH oxidase assay.
  • 20% glycerol was added to the freeze buffer.
  • the cells were diluted to OD 573 -1 in 200 ⁇ l samples with the above-mentioned buffer and then 0.2mM NADH was added to start the reaction.
  • the host strain E. coli BL21 (DE3) was similarly stored and subjected to the same type of activity assay as the whole cell biocatalyst BL21 (DE3) pAT-NOX.
  • the panels in Figure 7 show the decrease in NADH concentration in cell suspensions of different ages stored at 8 ° C.
  • the host strain E. coli BL21 (DE3) used as a control does not show any NADH activity before storage.
  • a marked decrease in the NADH concentration in the test can be detected in these host cells, the decrease being more pronounced the longer the storage takes.
  • the number of living cells has been determined parallel to the activity of the biocatalyst.
  • the results are shown in FIG.
  • the number of live cells decreases by three within the first four weeks Orders of magnitude.
  • the number of living cells remains relatively constant. This curve can be observed even at storage of the biocatalyst at -18 ° C and -70 ° C, respectively.
  • the decrease in the number of live lines during the first four weeks is only two orders of magnitude.
  • the decrease in the number of live cells does not correlate with the activity of the biocatalyst over a period of seven weeks.
  • the tents were spun down and resuspended in 160 ⁇ M potassium phosphate buffer. Again, 0.2 mM NADH was added to start the reaction and the oxidation was followed photometrically. This procedure was repeated for a total of 5 cycles.
  • the host strain E. coli BL21 (DE3) was subjected to the same test as the whole-cell biocatalyst BL21 (DE3) as a negative contrast.
  • Example 5 Regeneration of NADH to NAD + using the whole-cell biocatalyst E. coli BL21 (DE3) pAT-NOx
  • AIDH aldehyde dehydrogenase
  • Figure 13 shows in the upper half of the panel the AIDH mediated NADH increase.
  • the host strain E. coli BL21 (DE3) pAT-NOx had already been added to this reaction to increase the NADH concentration ⁇ as absorbance at 340 nm) with respect to the number of cells.
  • the biocatalyst E. coli BL21 (DE3) pAT-NOx was added to the AIDH reaction to increase the NADH concentration ⁇ as absorbance at 340 nm) with respect to the number of cells.
  • no increase in NADH could be detected over time, because of AIDH formed NADH was continuously reoxidized to NAD + . Consequently, this system is in equilibrium.

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Abstract

Une molécule d'acide nucléique comprend un segment codant un peptide signal, un segment comprenant un polypeptide hétérologue régénérateur de facteur d'oxydo-réduction, un segment optimal codant un site de reconnaissance de protéase, un segment codant un lieur transmembranaire et un segment codant un domaine de transporteur d'un autotransporteur ou d'une variante correspondante. La molécule d'acide nucléique permet l'expression d'enzymes régénératrices de facteur d'oxydo-réduction.
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