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  • 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)
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
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  • C12N1/14—Fungi; Culture media therefor
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • 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)
  • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
  • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
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  • 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
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
  • C12P7/20—Glycerol
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
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  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
  • C12R2001/865—Saccharomyces cerevisiae
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• TITLE Metabolically engineered microbial cell comprising a modified redox activity.
• the invention is in the area of microbial biotechnology and relates to a metabolically engineered microbial cell comprising an expressible enzyme activity which, when expressed, is capable of providing an alteration in the redox level of said cell.
• An alteration of said redox level comprises any increase or decrease in e.g. the rate of synthesis and/or amount produced of one or more components of a redox system such as e.g. a nicotinamide adenine dinucleotide coenzyme in its oxidised form (NAD) or reduced form (NADH), or a nicotinamide adenine dinucleotide phosphate coenzyme in its oxidised form (NADP) or reduced form (NADPH).
• a nicotinamide adenine dinucleotide coenzyme in its oxidised form (NAD) or reduced form (NADH) e.g. a nicotinamide adenine dinucleotide coenzyme in its oxidised form (NADP) or reduced form (NADPH).
• the increase or decrease of a rate of synthesis and/or an amount of coenzyme produced results in a redirection of a metabolic flux in said cell.
• the redirected metabolic flux is exploited in the generation of an increased or decreased production of a cellular metabolite.
• a complex metabolic network of more than a thousand different and simultaneously occurring reactions in a cell is regulated strictly and sensitively in order to avoid an unde- sirable accumulation or deficiency of metabolic intermediates and/or metabolic end products produced by said cell.
• a strict and sensitive regulation ensures that reactions of great mechanistic complexity and stereochemical selectivity may proceed smoothly under normal physiological conditions.
• the complex metabolic network comprises numerous interrelated metabolic pathways, the totality of which is resulting in an overall cellular flux of metabolites. Any alteration of said metabolic flux may in principle be perceived as a form of metabolic engineering. Accordingly, metabolic engineering achieved e.g. by means of in vitro and/or in vivo genetic engineering techniques represents a purposeful designing of metabolic networks and generates a change in and/or a redirection of the flow of metabolites in a microbial cell under e.g. aerobic or anaerobic conditions. Product formation of e.g. anaerobically growing microbial cells is well established in the art and has been reported in the aca- demic literature (Oura, 1977; van Dijken & Scheffers, 1986; Nissen et al, 1997).
• EP 0 733 712 Al discloses a method of production of preferably an amino acid by culturing a metabolically engineered microbial cell preferably an Escherichia coli cell, with a supposedly increased expression or productivity of NADPH and isolating said amino acid.
• WO 96/41888 discloses yeast having a modified alcohol sugar fermentation due to an altered expression of a gene encoding an NADH dependent glycerol-3 -phosphate dehydrogenase activity.
• EP 0 785 275 A2 discloses a yeast comprising constitutive expression of a gene encoding an enzyme activity involved in hexose transport.
• EP 0 645 094 Al discloses the use of a yeast comprising a glycolytic pathway comprising a futile cycle generated by means of a constitutive expression of genes encoding fructose- 1,6-biphosphatase and phosphoenolpyruvate carboxykinase.
• US 5,545,556 discloses a yeast strain having a reduced or increased production of glycerol mediated by mutations in various gene-encoded products.
• microorganisms are capable of growing in both the presence and absence of oxygen.
• some microbial cells are strictly aerobic and depend absolutely upon an oxidative form of metabolism known as respiration, i.e. the coupling of energy generation to an oxidation of nutrients by oxygen.
• respiration i.e. the coupling of energy generation to an oxidation of nutrients by oxygen.
• NADH is reoxidised through the mitochondrial electron transport chain in a process that generates additional energy and results in an ultimate transfer of electrons to oxygen.
• microorganisms either can or must grow in anaerobic environments while deriving their metabolic energy from processes that do not involve oxygen.
• Most of such anaerobically growing microbial organisms derive their energy from fermentations characterised by energy-yielding catabolic pathways such as glycolysis. Pyruvate formed in this pathway may be further reduced to a variety of metabolic end products such as e.g. ethanol, lactic acid and acetic acid.
• Glycolysis is a major catabolic pathway for degradation of carbohydrates in both aerobi- cally and anaerobically growing microbial cells.
• the major input to glycolysis is glucose and the pathway, comprising a total of 10 different reactions, leads to the conversion of one molecule of glucose to two molecules of pyruvate, with the concomitant generation of ATP as well as a reduced form of a coenzyme termed nicotinamide adenine dinucleotide.
• the coenzyme may exist in either a reduced form, NADH. or an oxidised form, NAD + .
• NADH For the glycolytic pathway to operate anaerobically, NADH must be reoxidised to NAD + by means of a transfer of electrons to a suitable electron acceptor so that a steady metabolic flux can be maintained. Microbial cells growing in the absence of oxygen have different ways of transferring such electrons.
• a simple route used by lactic acid bacteria consists of simply using the synthesised NADH to reduce py vate to lactate, via the enzyme lactate dehydrogenase. NADH is reoxidised in the process:
• the lactic acid fermentation i.e. conversion of glucose to lactic acid
• Another important fermentation involves a conversion of pyruvate to acetaldehyde and CO 2 and a reduction of acetaldehyde to ethanol mediated by alcohol dehydrogenase:
• Glycerol is another metabolic end product produced by numerous microbial cells. Glycerol is formed from dihydroxyacetone phosphate (DHAP) by a two-step reaction mediated by glycerol-3 -phosphate dehydrogenase and glycerol 3-phosphate phosphatase. respectively, as indicated below:
• DHAP dihydroxyacetone phosphate
• NADH must be reoxidised to NAD + by means of a transfer of electrons to a suitable electron for the glycolytic pathway to operate anaerobically.
• the reoxidised coenzyme in the form of NAD + contains a nicotinamide ring structure that is readily reducible and thus serves as an oxidising agent.
• the reduced form of the coenzyme, NADH is dissociated from the enzyme and is reoxidised by any suitably available electron-acceptor system in the cell.
• the NAD + so formed is capable of repeating another cycle of coupled reduction and oxidation. NAD + and NADH thus differ from most substrates in that they are continually recycled.
• NADPH nicotinamide adenine dinucleotide phosphate
• NADP + and NADPH are identical to NAD" and NADH. respectively, except that the form has an additional phosphate esterified at C-2' on the adenyl- ate moiety.
• NAD + and NADP + are equivalent in their thermodynamic tendency to accept electrons and they have similar standard reduction potentials.
• nicotinamide nucleotide-linked enzymes that act in catabolic metabolism usually use the NAD NADH coenzyme pair, whereas those acting in anabolic pathways tend to use NADP' NADPH.
• NADPH can be synthesised either from NADP + in the pentose phosphate pathway or from NADH through the action of an enzyme exhibiting a transhydro- genase activity.
• ethanol production reached an estimated 31.3 billion litres in 1996. Approximately 80% were produced by anaerobic fermentation of various sugar sources by Saccharomyces cerevisiae. Accordingly, ethanol is one of the most important biotechnologi- cal products with respect to both value and amount. Two thirds of the production is located in Brazil and in the United States with the primary objective of using ethanol as a renewable source of fuel. The demand and growth of this market is expected to give rise to a substantial growth in the ethanol production industry in the future. Hence, there are strong economic incentives to further improve the ethanol production process.
• the price of the sugar source is a very important process parameter in determining the overall economy of ethanol production. Hence, it is of great interest to optimise the ethanol yield in order to ensure an efficient utilisation of the carbon source.
• glycerol is both undesirable and a limiting factor in ethanol production
• an increased production of glycerol may also be desirable, as glycerol is known to provide desirable organoleptic qualities in many wines. Accordingly, it is also desirable to reduce and/or eliminate formation of ethanol and to redirect the metabolic flux towards an increased production of glycerol.
• the invention concerns a metabolic engineering of the capability of a microbial cell to produce one or more metabolic products such as e.g. ethanol. glycerol and lactic acid.
• Metabolic end products may either be produced concomitantly or the production of at least one metabolic end product may be increased while the production of additional end products is decreased accordingly.
• Increases and decreases in metabolic end product formation are guided by the metabolic potential of a cell and the flux of metabolites under certain metabolic conditions.
• Ethanol is a particularly preferred metabolic end product and is produced by a yeast cell under anaerobic conditions.
• a microbial cell comprising a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity.
• a microbial cell comprising a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said expression of said first expressible enzyme activity in said mi- crobial cell being either novel to said cell or altered as compared to the expression of said first enzyme activity in a comparable wild-type microbial cell or a comparable isolated microbial cell
• a microbial cell comprising a first expressible en- zyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity, said expression of said first expressible enzyme activity in said microbial cell being either novel to said cell or altered as compared to the expression of said first enzyme directed by an expression signal natively associated with said first expressible enzyme activity.
• a microbial cell comprising a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity, said expression of said first expressible enzyme activity being operably linked to an increased production of a first metabolite.
• a microbial cell comprising a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity, said expression of said first expressible enzyme activity being operably linked to an increased production of a first metabolite and a decreased production of a second metabolite.
• the microbial cell according to the invention as described herein above also comprises a further expressible enzyme activity, said further expressible enzyme activity, when expressed, mediates a first biosynthetic reaction resulting in a production of a first metabolite, said further expressible enzyme activity, when expressed at an increased level, results in an increased production of said first metabolite, said increased expression of said further expressible enzyme activity and/or said increased production of said first metabolite is operably linked to an increased expression of a first expressible enzyme activity controlling an intracellular redox system of said cell, said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity.
• a microbial cell comprising a further expressible enzyme activity, said further expressible enzyme activity, when expressed, mediates a first biosynthetic reaction resulting in a production of a first metabolite, said further expressible enzyme activity, when expressed at an increased level, results in an increased production of said first metabolite, said increased expression of said further expressible enzyme activity and/or said increased production of said first metabolite is operably linked to an increased expression of a first expressible enzyme activity resulting in maintenance and/or alteration of an intracellular redox system of said cell, said expression of said first enzyme activity in said microbial cell being either novel or altered as compared to the expression of said first enzyme activity in a comparable wild-type microbial cell or a comparable isolated microbial cell.
• compositions comprising a microbial cell and a physiologically acceptable carrier, a nucleotide sequence encoding a transhydrogenase enzyme activity, a recombinant replicon in the form of a vector harbouring said nucleotide sequence, a microbial cell transformed with said nucleotide sequence or said vector, and an amino acid sequence being encoded by said nucleotide sequence.
• a microbial cell or a composition for use in a production of a first metabolite or in a preparation of a drinkable or an edible product In a further aspect there is provided a microbial cell or a composition for use in a method of generating an alternative intracellular NADH or NADPH reoxidation.
• a microbial cell or a composition in a production of a first metabolite or in a preparation of a drinkable or an edible product.
• a microbial cell or a composition in a method of generating an alternative intracellular NADH or NADPH reoxidation.
• a method of constructing a microbial cell comprising introducing into said cell a capability and/or increasing a capability of said cell to express a first expressible enzyme activity which, when expressed in said microbial cell, is controlling a redox system of said cell, said first expressible enzyme activity being operably linked to an expression signal not natively associated with said activity.
• a method of constructing a microbial cell comprising the steps of
• the invention concerns a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell.
• the expressible enzyme activity is a transhydrogenase activity the expression of which in a microbial cell is resulting in a modified i.e. redirected carbon flux or a redirected metabolic flux resulting at least in an increased production of a first metabolite and/or in a decreased production of a second metabolite.
• glucose is metabolised and biomass and metabolites are generated under i) an oxidation of NADPH to NADP and ii) a reduction of NAD to NADH.
• NADPH is regenerated e,g. when NADP is reduced in the pentose phosphate pathway. Conversion of pyruvate via acetaldehyde to acetate also contributes to a reduction of NADP and NADPH regeneration.
• Oxidation of NADH and regeneration of a pool of NAD is occurring under anaerobic conditions by conversion of dihydroxy acetone phosphate (DHAP) via glycerol-3-phos- phate to glycerol in a two-step reaction being mediated by glycerol-3 -phosphat dehydro- genase and glycerol-3 -phosphat phosphatase.
• DHAP dihydroxy acetone phosphate
• a microbial cell preferably a yeast cell or a lactic acid bacterial cell, capable of performing NADH reoxidation without a concomitant generation of glycerol.
• a microbial cell preferably a yeast cell or a lactic acid bacterial cell, capable of performing NADH reoxidation without a concomitant generation of glycerol.
• Such an alternative reoxidation of NADH would in principle make it possible to direct a majority of an available carbon source into the production of ethanol without any significant production of - in this context - an undesirable "waste product" such as glycerol.
• NADH reoxidation is essential to the microbial cell, as the synthesis, under anaerobic conditions, of biomass and an extensive and diverse range of reducible metabolites such as organic acids such as e.g. succinate. acetate, pyruvate and the like, results in a net formation of intracellular NADH which evidently cannot be reoxidised by means of the respiratory chain, as the respiratory chain is not functioning under anaerobic conditions.
• Intracellular NADH formation must therefore be balanced by a mechanism whereby NADH is reoxidised to NAD + in order to avoid depletion of the NADH pool under anaerobic conditions.
• NADH may be reoxidised to NAD + via the conversion of organic acids and/or aldehydes to organic alcohols such as e.g. glycerol, ethanol, pro- panol and/or lactic acid, since synthesis of these reduced metabolites leads to reoxidation of NADH and alleviates NADH depletion.
• organic acids and/or aldehydes such as e.g. glycerol, ethanol, pro- panol and/or lactic acid
• a hypothesis will have to be revised or even partially redrafted several times before it adequately and/or sufficiently accurately may describe e.g. i) a cellular metabolism or part thereof .
• ii) a metabolic flux or the flux of a carbon source in a cell iii) an intricate allosteric regulation mechanism and its effect on e.g. enzyme activity and/or the rate of metabolite synthesis and or product yield, or iv) a set of interconnected anabolic and/or catabolic metabolic pathways and changes therein caused e.g. by a change in the metabolic flux, which may in turn be caused by subjecting said cell to metabolical engineer- ing.
• a microbial cell comprising a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity.
• the expression of said first expressible enzyme activity is either novel to said cell or altered as compared to the expression of said first enzyme activity in a comparable wild- type microbial cell or a comparable isolated microbial cell. Said expression of said first expressible enzyme activity is preferably operably linked to an increased production of a first metabolite.
• the cell comprises a further expressible enzyme activity, said further expressible enzyme activity, when expressed, mediating a first biosynthetic reaction resulting in production of a first metabolite, said further expressible enzyme activity, when expressed at an increased level, resulting in an increased production of said first metabolite, said increased expression of said further expressible enzyme activity and/or said increased production of said first metabolite being operably linked to an increased expression of said first expressible enzyme activity.
• a microbial cell wherein said expression of said first expressible enzyme activity is operably linked to an increased production of a first metabolite and a decreased production of a second metabolite.
• a comparable wild-type microbial cell or a comparable isolated microbial cell is a cell of the same species, preferably the same subspecies, as that of the microbial cell according to the invention.
• the expressible enzyme activity is preferably operably linked to an expression signal not natively associated with said enzyme activity.
• an altered expression will be understood to comprise any expression which is altered as compared to the expression when being directed by an expression signal natively associated with said activity in a natural host organism.
• the microbial cell in question may thus be a microbial eukaryote or a microbial prokary- ote.
• microbial eukaryotes are many yeast and fungal cells preferred, such as yeast cells of the species Saccharomyces. Schizosaccharomyces and Pichia, such as e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe. Pichia pastor is and the like, as well as algae such as e.g. Chlamydomonas reinhardi, slime moulds such as e.g. Dictyos- telium discoideum and filamentous fungi.
• Preferred filamentous fungi are species of Neu- rospora such as e.g. Neurospora crassa. and species of Aspergillus such as Aspergillus nidulans. Aspergillus niger. Aspergillus oryzae and Penicillium species such as e.g. Penicillium chrysogenum. Penicillium roqueforti and Penicillium camemberti. Particularly preferred are industrially relevant yeast cells, slime moulds and filamentous fungi providing production of products such as e.g. antibiotics, steroids, pigments, enzymes, organic alcohols and acids, amino acids, polysaccharides and the like.
• bacterial cells such as Gram-positive species such as Bacillus species like e.g. Bacillus subtilis. Bacillus thuringensis. Bacillus li- cheniformis. Bacillus amyloliquefaciens. Bacillus cereus, Bacillus lentus and Bacilus stearothermophilus, species of Corynebacterium like e.g. Corynebacterium glutamicum, and species of Propion ⁇ bacterium as well as Gram-negative species such as Escherichia coli. Particularly preferred are also lactic acid bacterial species such as e.g. Lactococcus lactis. Lactococcus lactis subsp.
• Gram-positive species such as Bacillus species like e.g. Bacillus subtilis. Bacillus thuringensis. Bacillus li- cheniformis. Bacillus amyloliquefaciens. Bacillus cereus, Bacillus lentus and Bacilus stear
• lactis Lactococcus lactis subsp. cremoris. Lactococcus lactis subsp. diacety lactis, Leuconostoc species such as e.g. Leuconostoc oenos. Lacto- bacillus species, such as Lactobacillus acidophilus, Lactobacillus plantarum. Lactoba- cillus delbr ⁇ ckii subsp. bulgaricus, and Lactobacillus helveticus, Pediococcus species, Brevibacterium species, Propionibacterium species and similar industrially relevant species like e.g. Bifidobacterium.
• a particularly preferred microbial cell according to the invention is deposited with the DSM under Accession Numbers 12267 as Saccharomyces cerevisiae strain TN4.
• An altered expression of said first expressible enzyme activity in a microbial cell according to the invention shall be understood to comprise any expression that differs with respect to the rate of product formation or with respect to the amount of product formed as compared to a comparable microbial cell. Accordingly, if a wild-type microbial cell is subjected to the metabolic engineering manipulations according to the invention, the skilled person will compare the expression of said first expressible enzyme activities provided in the metabolically engineered cell with the expression of the same activities in the wild-type microbial cell.
• the person skilled in the art will preferably analyse - and compare with one another - similar or near identical microbial cells such as identical cells with and without a first expressible enzyme activity according to the invention.
• This is standard laboratory practise and the person skilled in the art will know how to conduct such an analysis so that it may form a basis for a direct comparison of e.g. an expressed enzyme activity or an expressed coenzyme or an expressed redox system within the meaning of those terms as set out herein below.
• the person skilled in the art will want to compare microbial cells to cells of at least the same species and more preferably to compare said cells to cells of at least the same subspecies.
• an isolated microbial cell such as e.g. an industrial strain or a strain in a culture collection
• the skilled person will compare the expression of said first expressible en- zyme activity provided in the metabolically engineered cell with the expression of the same activity in the industrial strain or the microbial cell from the culture collection.
• the skilled artisan will know how to culture comparable strains such as strains of the same species or subspecies under identical or substantially similar conditions so as to provide a basis for performing the comparison between the relevant enzyme activities.
• the person skilled in the art will also know how to perform an enzymatic assay for use in said comparison and being indicative of the formation of e.g. a biosynthetic product resulting directly or indirectly from the action of said first expressible activity, when ex- pressed, and he will be aware of the potential of transcriptional and/or translational fusions in monitoring expression of said expressible enzyme activities under comparable conditions.
• the skilled person will also be able to perform immunoassays including quantitative immunoprecipitations.
• An analysis of gene expression is available in e.g. Old and Primrose (1985): Principles of Gene Manipulation - An introduction to genetic engineering (Third edition), Blackwell Scientific Publications, Oxford, England.
• the altered expression of said first expressible enzyme activity in a microbial cell shall preferably be understood to com- prise an increased expression.
• the expression of said first expressible enzyme activity is increased by a factor of at least 1.02. such as a factor of at least 1.04, for example 1.06. such as 1.08. for example 1.10. such as at least 1.12. for example 1.14, such as 1.16, for example 1.18. such as 1.2, for example 1.25, such as 1.3, for example 1.4, such as 1.5. for example 1.6.
• an increased expression of said first expressible enzyme activity in the microbial cell according to the invention is operably linked to an increased expression of a further expressible enzyme activity, said increased expression of said further activity is increased by a factor of at least 1.02, such as a factor of at least 1.04, for example 1.06, such as 1.08.
• 1.3, for example 1.4, such as 1.5, for example 1.6, such as 1.7, for example 1.8. such as 1.9, for example 2.0, such as 2.25, for example 2.5, such as 3, for example 3.5.
• an increased expression of said first expressible enzyme activity in the microbial cell according to the invention is operably linked to an increased expression of a further expressible enzyme activity, said increased expression of said further activity results in an increased production of a first metabolite, said increased production of said first metabolite is increased by a factor of at least 1.02, such as a factor of at least 1.04.
• the first metabolite is preferably a primary or secondary metabolite and more preferably an amino acid, an alcohol or an acid, such as e.g. glutamat, lysin, threonin. aspartate, ethanol, glycerol, acetic acid, propionic acid. Ethanol and glycerol are particularly preferred.
• said secondary metabolite is preferably selected from the group of secondary metabolites consisting of a ⁇ -lactam, a polyketide. a terpene, a steroid, a quinone, a coumarin, a flavonoid, an alkaloid, a piperi- dine. a pyridine, and the like.
• an altered expression of said first expressible activity shall not be limited to an increased expression.
• a reduced expression of said first expressible activities shall also be understood to be comprised by the term altered expression.
• said first expressible enzyme activity is one encoded by a nucleotide sequence designated SEQ ID NO:l, as illustrated herein below, or a functional derivative of said nucleotide sequence.
• redox system and intracellular redox system shall be understood to comprise any redox system comprising a coenzyme that is present in corresponding oxidised and reduced forms.
• Pre- ferred intracellular redox systems are coenzymes in corresponding oxidised/reduced forms such as e.g. NAD/NADH, NADP/NADPH and FAD/FADH 2 .
• Any redox system can generally be perceived to contribute to the provision of a certain redox level in a cell.
• the totality of all such redox systems in a cell determines the redox level of said cell.
• the redox level of a cell thus comprises the presence and or amount of the totality of reducing equivalents and oxidising equivalents present in said cell.
• An alteration of an intracellular redox system can be measured either by monitoring the increase or decrease of a specific redox system, i.e. an increase or decrease in both the oxi- dised form as well as in the reduced form of a coenzyme constituting said redox system, or alternatively, said alteration can be monitored by measuring an overall cellular redox level.
• the first expressible enzymatic activity is expressed and results in maintaining said desirable level. Accordingly, when it is desirable to increase a cellular redox level from an undesirable low level to a desirable higher level, said first expressible enzymatic activity is expressed and results in such an increase. Also, when it is desirable to decrease a cellular redox level from an undesirable high level to a desir- able lower level said first expressible enzymatic activity is expressed and results in such a decrease. It will be understood that redox level means both the cellular koncentration of an entire redox system, i.e.
• the first expressible enzymatic activity either maintains a redox level or promotes changes in said level.
• said first expressible enzymatic activity it is possible to regulate the redox level of a cell and promote the synthesis of desirable metabolites by providing a suitable redox level facilitating said synthesis or by e.g. increasing the intracellular yield of a coenzyme essential for the formation of said desirable metabolites.
• maintenance of an intracellular redox system shall be understood to comprise the action exerted by a first expressible enzymatic activity which, when expressed, is acting e.g. in a pathway leading to the synthesis of one or more components of said sys- tern or by acting in a recycling reaction or indeed in any cyclical reaction involving such components, preferably a reaction involving an oxidisation of a reduced coenzyme and or a reduction of an oxidised coenzyme. It will be understood that maintenance and/or alteration involves controlling the redox level of a cell by increasing or decreasing the formation of an oxidised or reduced coenzyme such as e.g.
• control will be understood to comprise e.g. maintaining a redox level in the cell, when said level would have been either reduced or increased in the absence of expression of said first expressible enzymatic activity.
• Controlling the redox level will also be understood to comprise the provision of a desirable alteration i.e. a decrease or an increase of redox level in the cell, said alteration would have occurred in the absence of expression of said first expressible enzymatic activity.
• a desirable alteration i.e. a decrease or an increase of redox level in the cell
• the first expressible enzyme activity is facilitating an alteration of said intracellular redox system.
• the terms increase and decrease relate to a level of expression or synthesis or to a concentration of a coenzyme and/or a redox system.
• the term level is used interchangeably in the art with terms such a synthesis rate and concentration. The person skilled in the art will be familiar with such terms and attach the cor- rect meaning to their use in different contexts.
• said first expressible enzyme activity is controlling the maintenance of a redox system.
• Maintenance also comprises maintaining a redox level under conditions wherein said level would have been substantially decreased or increased had the cell not harboured said first expressible enzyme activity.
• the above-described maintenance of said redox system may well lead to an increased rate of synthesis of any one or more components of said system. Maintenance may also lead to an increase or decrease in the pool of any one component of said redox system, such as an increase or decrease of a reduced or oxidised form of a coenzyme.
• the person skilled in the art will know how to assess an increase or decrease of any form of a coenzyme or of any redox system and he will know that he must compare the levels of that same coenzyme or redox system in a comparable wild-type microbial cell or an isolated microbial cell grown under identical or substantially similar conditions that allow for a direct comparison of said levels by exploiting state of the art monitoring techniques such as those described by Weuster and de-Graff (1996) in Adv. Biochem. Eng. Biotechnol, vol. 54, pages 75 - 108 and by Wiechert and de-Graff (1996) in Adv. Bio- chem. Eng. Biotechnol. vol. 54. pages 109 - 154.
• the person skilled in the art will preferably analyse similar or substantially identical microbial cells with and without said first expressible enzyme activity. Similar or substantially identical microbial cells are e.g. cells of the same species or the same subspecies.
• the expression results in an increased or decreased level, preferably an increased level, of at least one intracellular coenzyme in its oxidised or reduced form.
• Said coenzyme in its oxidised reduced form is preferably selected from the group consisting of FAD/F ADH>. NAD/NADH and NADP/NADPH.
• the level of at least one intracellular coenzyme in its oxidised or reduced form is either increased or decreased, preferably increased, such as increased by a factor of at least 1.005, for example 1.010, such as 1.015, for example 1.020. such as a factor of at least 1.025. for example a factor of at least 1.030, such as 1.035, for example 1.040, such as 1.045, for example 1.050, such as 1.055. for example 1.060. such as at least
• alteration is by no means limited to an increase in the level of at least one intracellular coenzyme in its oxidised or reduced form. Said alteration shall also comprise any decrease in the level of at least one intracellular coenzyme in its oxidised or reduced form.
• the expression of said first expressible en- zyme activity results in an increase or a decrease, preferably an increase, in the level i.e. concentration of an intracellular redox system, i.e. an increase or decrease, preferably an increase, of both of two corresponding oxidised and reduced forms of a coenzyme, such as an increase by a factor of at least 1.005, for example 1.010, such as 1.015, for example 1.020, such as a factor of at least 1.025, for example a factor of at least 1.030, such as 1.035. for example 1.040, such as 1.045, for example 1.050. such as 1.055, for example 1.060, such as at least 1.065.
• a factor of at least 1.090 such as 1.095, for example 1.100, such as 1.105, for example 1.110, such as 1.115, for example 1.120, such as at least 1,125, for example a factor of at least 1.130, such as 1.135, for example 1.140, for example 1.145, such as 1.150, for example 1.155, such as at least 1.160, for example a factor of at least 1.165, such as a factor of at least 1.170. for example a factor of at least 1.175, such as 1.180, for example 1.185, such as 1.190, for example 1.195, such as a factor of at least 1.200.
• Said first expressible enzyme activity may well result in an increase or a decrease, prefer- ably an increase, of more than one intracellular redox system. It shall be understood that in one embodiment of the invention, said first expressible enzyme activity, when expressed, is resulting in an increased level of at least one intracellular redox system.
• said first expressible enzyme activity is an intracellular transhydrogenase activity, preferably a pyridine nucleotide transhydrogenase activity, and even more preferably the pyridine nucleotide transhydrogenase activity of CTH of Azotobacter vinelandii as harboured by Saccharomyces cerevisiae TN4 as deposited under DSM Accession Number 12267, or a functionally equivalent activity.
• a functionally equivalent activity is any activity capable of carrying out a substantially similar reaction with the provision of a similar outcome as that resulting from the reaction being carried out by the above-mentioned CTH encoded polypeptide when harboured by Saccharomyces cerevisiae.
• the pyridine nucleotide transhydrogenase activity is either endogenous or heterologous to said microbial cell, and said activity is either exhibited by a polypeptide which is membrane-bound in a natural host organism or located i.e. present in the cytoplasm of a natural host organism, said natural host organism preferably being selected from the group of mammalian cells, eukaryotic cells including microbial eukaryotic cells, and prokaryotic microbial cells, preferably a Gram-positive microbial prokaryote or a Gram- negative microbial prokaryote.
• pyridine nucleotide transhydrogenase activity in said microbial cell in one preferred embodiment of the invention results in an increased conversion of NADPH and NAD to NADH and NADP.
• said expression of said pyridine nucleotide transhydrogenase activity results in an increased consumption of NADPH and an increased formation of NADH.
• the expression has the effect of increasing the consumption of NADH and increasing the formation of NADPH.
• the expression results in an increased formation of NADH and/or NADP.
• said expression may also result in a decreased formation of NADPH and or a decreased NADPH/NADP retio.
• the pyridine nucleotide transhydrogenase activity is preferably expressed in said microbial cell under anaerobic growth conditions and preferably results in the ratio of NADPH/NADP being lower than the ratio of NADH/NAD.
• the transhydrogenase activity preferably an activity that is membrane-bound in a natural host organism, is inserted into the plasma membrane of a microbial cell, preferably a yeast cell.
• the transhydrogenase activity mediates a re- action consuming NADP and generating NADPH.
• an E. coli transhydrogenase is expressed in a yeast cell and leads to an increased level of NADPH. The expression of the yeast plasma membrane located E. coli transhydrogenase is coupled to a proton gradient across said membrane similar to the coupling observed in a natural host organism i.e. E. coli.
• the microbial cell according to invention is preferably one suitable for storage in the form of a frozen or freeze-dried preparation such as a lyophilisate from which the microbial cell is partly or wholly reconstitutable.
• the microbial cell has been metabolically engineered as described above and is capable of alternative NADH re-oxidation.
• Said alternative NADH re-oxidation is mediated at least by expression of said first expressible enzyme activity and optionally also by expression of a further expressible enzyme activity.
• Alternative NADH re-oxidation shall be understood to comprise the introduction of a novel major pathway for NADH re-oxidation or a generation of a substantially altered pathway for NADH-reoxidation in a microbial cell.
• Alteration in respect of a pathway for alternative NADH oxidation shall be understood in the context of the rate of a reaction mediating a conversion of one metabolite to another, said reaction also resulting in NADH re-oxidation.
• the rate of said re-oxidation reaction in a microbial cell capable of alternative NADH re-oxidation is substantially increased as compared to the rate of said reaction in a comparable microbial cell.
• the definition of the term comparable in respect of microbial cells is introduced herein above.
• Alternative NADH re-oxidation in one example is a microbial cell wherein the expression of a transhydrogenase activity, in combination with several additionally preferred expressible enzyme activities, is capable of generating a purposeful redesigning of a complex network of metabolic reactions.
• the redesigned microbial cell is invented by replacing or supplementing a normally dominant first metabolic reaction with a second reaction that is normally insignificant in relation to reaction rate and/or product formation as compared to said first dominant reaction.
• a microbial cell capable of alternative NADPH re-oxidation or alternative NADP reduction.
• composition comprising the microbial cell and a carrier, preferably a physiologically acceptable carrier and more preferably a water-based liquid such as a broth suitable for culturing said microbial cell.
• a carrier preferably a physiologically acceptable carrier and more preferably a water-based liquid such as a broth suitable for culturing said microbial cell.
• the composition in a preferred embodiment is a fermentation starter culture.
• nucleotide sequence encoding a novel and industrially relevant transhydrogenase enzyme activity, said sequence comprising SEQ ID NO:l as illustrated herein below, or part thereof, including functionally equivalent derivatives thereof encoding a transhydrogenase enzyme activity, preferably but not limited to conservative nucleotide substitutions and/or nucleotide deletions and/or nucleotide insertions.
• Said functionally equivalent derivatives may thus be at least 99.9 percent identical to SEQ ID NO: l, such as at least 99.8 percent identical to SEQ ID NO:l, for example at least 99.7 percent identical, such as at least 99.6 percent identical, , for example at least 99.5 percent identical, such as at least 99.4 percent identical, for example at least 99.3 percent identical, such as at least 99.2 percent identical, for example at least 99.1 percent identical, such as at least 99 percent identical to SEQ ID NO:l, for example at least 98.5 percent identical to SEQ ID NO:l, such as at least 98.0 percent identical, for example 97.5 percent identical, such as at least 97.0 percent identical to SEQ ID NO:l, for exam- pie at least 96.5 percent identical, such as at least 96.0 percent identical, for example at least 95.5 percent identical, such as at least 95.0 percent identical, for example at least 94.5 percent identical, such as at least 94.0 percent identical, for example at least 93.5 percent identical, such as at least 93.0 percent identical to S
• Functionally equivalent derivatives may have an altered nucleotide sequence and may encode a polypeptide having an altered amino acid sequence as compared to that encoded by SEQ ID NO:l.
• said polypeptides are characterised by a similar or substantially identical enzymatic activity and are thus functionally equivalent.
• the functionally equivalent nucleotide sequence is provided by means of homologous or semi-homologous recombination of identical or substantially identical nucleotide sequences being part of different genes and located e.g. on the same or different extrachromosomal replicons.
• the transhydrogenase gene according to the invention is composed of nucleotide sequences originating from different genes such as e.g. different genes encoding a cytoplasmatically located transhydrogenase and/or a membrane-bound transhydrogenase. This technique is also termed gene-shuffling in the art.
• the nucleotide sequence when being expressed in a microbial cell, is preferably operably linked to an expression signal not natively associated with said nucleotide sequence.
• the expression signal is preferably one generating at least a substantially constitutive expression, such as the PGK promoter, an inducible expression signal such as a growth phase regulated promoter or a promoter induced by e.g. pH or temperature and changes therein.
• the expression signal is preferably a regulable expression signal such as a regulable tran- scription initiation signal and/or a regulable translational initiation signal, such as an expression signal regulatable in response to an alteration in a value, level and/or concentration of a factor such as a physiological growth parameter, preferably a parameter selected from the group consisting of pH. temperature, salt content including osmolarity, anaero- bicity, aerobicity including oxygen level, energy level including a membrane potential and a proton motive force.
• a regulable expression signal such as a regulable tran- scription initiation signal and/or a regulable translational initiation signal, such as an expression signal regulatable in response to an alteration in a value, level and/or concentration of a factor such as a physiological growth parameter, preferably a parameter selected from the group consisting of pH. temperature, salt content including osmolarity, anaero- bicity, aerobicity including oxygen level, energy level including a membrane potential and a proton motive force.
• the promoter is preferably a promoter being either growth phase regulated, inducible and/or repressible and/or. in a natural host organism, directing expression of a gene encoding a gene product involved in mediating a reaction of a biosynthetic pathway and/or a major metabolic pathway, preferably a pathway selected from the group of pathways consisting of glycolysis. gluconeogenesis. citric acid cycle, and pentose phosphate pathway.
• the expression signal may be further regulated by an upstream activating sequence (UAS), by an enhancer element or by a silencer element.
• UAS upstream activating sequence
• the person skilled in the art will be aware of general molecular biology techniques for use in the construction in vitro of a recombinant DNA molecule. Such techniques are described e.g. in Sambrook et al. (1989) and in Old and Primrose (ibid.). Said skilled artisan will further be aware of the academic literature including general textbooks on molecular biology and genetic engi- neering and he will be able to combine various expression signals such as putative or recognised promoter regions with a range of regulatory nucleotide sequences generally known to exert an effect on the level of gene expression.
• An ex- pression signal can be a cloned expression signal or an in vitro synthesised expression signal.
• Expression signals in prokaryotic microbial cells are known to comprise so-called -35 and -10 regions and numerous examples of such regions are available from various databases.
• Expression signals may be optimised by increasing the promoter strength, by adjusting translational initiation sequences, by optimising the choice of codons by using so-called highly expressed codons, by adjusting the secondary structure of the mRNA. by increasing the efficiency of transcriptional termination, by increasing or decreasing a copy num- ber of a vector, or by increasing or decreasing the stability of said vector.
• a recombinant DNA-replicon in the form of a vector comprising the nucleotide sequence designated SEQ ID NO:l as illustrated herein above including functionally equivalent derivatives.
• the nucleotide se- quence designated SEQ ID NO: 1 is operably linked to an expression signal comprised in said replicon, said expression signal directing expression of said nucleotide sequence.
• a microbial cell microbial cell preferably a yeast cell, harbouring the nucleotide sequence designated SEQ ID NO: 1 or a recombinant replicon in the form of a vector harbouring said nucleotide sequence.
• the recombinant DNA-replicon is preferably one capable of replicating in a yeast cell and/or in a prokaryotic microbial cell such as a lactic acid bacterial cell.
• Preferred yeast vectors comprise a selectable marker and one or more sites in a nucleotide sequence specific for a restriction endonuclease.
• An autonomously replicating sequence (ARS) medi- ates replication of said replicon when harboured in a yeast cell.
• the vector is preferably based on a plasmid selected from the group consisting of a yeast episomal plasmid (Yep), such as the 2 ⁇ m plasmid, a yeast replicating plasmid (Yrp), a yeast centromeric plasmid (Yep) and a yeast integrating plasmid (Yip).
• a yeast episomal plasmid such as the 2 ⁇ m plasmid
• Yrp yeast replicating plasmid
• Yep yeast centromeric plasmid
• Yip yeast integrating plasmid
• Vectors capable of being maintained in a prokaryotic microbial cell such as a lactic acid bacterial cell are well described in the literature and preferably contain a replicon directing e.g. rolling circle replication or ⁇ -replication, a selectable marker such as a nonsense mutation preventing selection and/or replication unless suppressed by a suppresser comprised by a cell comprising said vector, and one or more sites cleavable by a restriction endonuclease.
• Functionally equivalent derivatives may have an altered amino acid sequence as compared to that encoded by SEQ ID NO:2.
• said polypeptides are characterised by a similar or substantially identical enzymatic activity and are thus functionally equivalent.
• Said functionally equivalent derivative of said amino acid sequence designated SEQ ID NO:2 may thus be at least 99 percent identical to SEQ ID NO:2. such as at least 98 percent identical to SEQ ID NO:2. for example at least 97 percent identical, such as at least 96 percent identical for example at least 95 percent identical such as at least 94 percent identical, for example at least 93 percent identical, such as at least 92 percent identical, for example at least 91 percent identical, such as at least 90 percent identical to SEQ ID NO:2, for example at least 89 percent identical to SEQ ID NO:2, such as at least 88 percent identical, for example 87 percent identical, such as at least 86 percent identical to SEQ ID NO:2, for example at least 85 percent identical to SEQ ID NO:2.
• SEQ ID NO:2 is a sequence that is synthesised partly or wholly in vitro.
• the microbial cell according to the invention in one embodiment further comprises a second and a third expressible enzyme activity which, when independently expressed in said microbial cell, are facilitating or controlling assimilation in said cell of a nutrient source, preferably ammonia, said expression of said second and third expressible enzyme activities in said microbial cell being either novel or altered as compared to the expression of said second enzyme activity in a comparable wild-type microbial cell or a comparable isolated microbial cell, said second expressible enzyme activity being non-identical to said first expressible enzyme activity.
• Said second and/or third expressible enzyme activities are preferably operably linked to an expression signal such as a promoter not operably linked to said activities in a natural host organism.
• Said altered expression is an expression being different from that directed by an expression signal natively associated with said expressible activities in a natural host organism.
• the nutrient source is capable of sustaining microbial growth by e.g. being assimilated into a biosynthetic product that can be utilised by a microbial cell or be converted i.e. metabolised into a further biosynthetic product, said utilisation and/or metabolism involving one or more energy yielding metabolic reactions.
• a nutrient source is any source that is potentially assimilable and metabolisable by a microbial cell.
• the step of assimilation shall be understood to comprise both uptake of said source into said call as well as conversion of said source into a biosynthetic product - an intermediate metabolite - within said cell.
• assimilation is the conversion that takes place within the cell without necessarily being limited to this step of the assimilation process.
• Preferred assimilable nutrient sources comprise ammonia, ammonium ions, nitrite ions, and nitrate ions.
• the assimilable nitrogen source according to the invention is more preferably ammonia and ammonium ions and most preferably ammonia. It will be understood that the invention pertains to all nitrogen containing nutrient sources capable of being converted into ammonia by oxidation including biological oxidation or by reduction including biological reduction.
• a biosynthetic reaction mediated by said second expressible enzyme activity, when expressed is preferably a reaction capable of being carried out by action of a metabolite synthase enzyme, more preferably by an allosteric metabolite synthase enzyme, and even more preferably is said reaction carried out by an expressible enzyme activity which, when expressed, is exhibited by a glutamate synthase.
• said glutamate synthase activity is that of GLTl of Saccharomyces cerevisiae TN17 as deposited under DSM Accession Number 12275, or an activity functionally equivalent therewith.
• a functionally equivalent activity is any activity capable of carrying out the same reaction with the provision of a similar outcome as that resulting from the reaction being carried out by the above- mentioned GLTl encoded polypeptide of Saccharomyces cerevisiae.
• a biosynthetic reaction mediated by said third expressible enzyme activity, when expressed, is preferably a reaction capable of being carried out by action of a metabolite synthetase enzyme, and more preferably is said reaction carried out by an expressible enzyme activity which, when expressed, is exhibited by a glutamine synthetase.
• said glutamine synthetase activity is that encoded by GLNl of Saccharomyces cerevisiae TNI 5 as deposited under DSM Accession Number 12274. or an activity functionally equivalent therewith.
• a functionally equivalent activity is any activity capable of carrying out the same reaction with the provision of a similar outcome as that resulting from the reaction being carried out by the above-mentioned GLNl encoded activity of Saccharomyces cerevisiae.
• a microbial cell preferably a yeast cell
• said second expressible enzyme activity is a metabolite synthase activity, more preferably an allosteric metabolite synthase activity, and even more preferably a glutamate synthase activity
• said third expressible enzyme activity is a metabolite synthetase activity, preferably a glutamine synthetase activity.
• said second or third expressible enzyme activity a is li- gase activity such as e.g. or a NADH-dependent glutamate dehydrogenase activity or a NADPH-dependent glutamate dehydrogenase activity.
• a microbial cell comprising a reduced expression or no expression of a fourth expressible enzyme activity which, when being present in said cell, is operably linked to an expression signal not natively associated with said fourth activity, said fourth expressible enzyme activity facilitates or controls as- similation in said cell of a nutrient source, said expressible fourth enzyme activity may optionally be operably linked to an expression signal not natively associated with said activity, said fourth expressible enzyme activity being non-identical to each and both of said second and third expressible enzyme activities.
• the activity may also be deleted from any other eukaryotic microbial cell or from a prokaryotic microbial cell.
• the fourth ex- pressible enzyme activity is preferably a metabolite dehydrogenase activity, and more preferably a glutamate dehydrogenase activity, and most preferably a NADPH-dependent glutamate dehydrogenase activity, which is either present in said microbial cell in a reduced amount, more preferably in a substantially reduced amount, or eliminated from said cell by means of e.g. deletion of a nucleotide sequence encoding said activity or by effectively repressing the expression of said expressible fourth enzyme activity.
• an altered expression in said microbial cell according to the invention of said fourth expressible enzyme activity shall be understood to comprise an expression reduced by at least 1 percent, such as decreased by at least 2 percent, for example 4 percent, such as 6 percent, such as at least 8 percent, for example at least 10 percent, such as 12 percent, for example 14 percent, such as 16 percent, such as at least 18 percent, for example at least 20 percent, such as 22 percent, for example 24 percent, such as 26 percent, such as at least 28 percent, for example at least 30 percent, such as 32 percent, for example 34 percent, such as 36 percent, for example 38 percent, such as at least 40 percent, for example 42 percent, such as 44 percent, for example 46 percent, such as 48 percent, such as at least 50 percent, for example 52 percent, such as 54 percent, for example 56 percent, such as 58 percent, such as at least 60 percent, for example 62 percent,
• an altered expression shall not be limited to a decreased expression.
• An in- creased expression of said fourth expressible enzyme activity shall also be understood to be comprised by the term-altered expression.
• said glutamate dehydrogenase activity is that of a GDH1 encoded polypeptide of Saccharomyces cerevisiae TN9 de- posited with the DSM under Accession Number 12268, or an activity functionally equivalent therewith.
• a microbial cell wherein the expression of said second and third expressible enzyme activities is in- creased, preferably substantially increased, whereas the expression of said fourth expressible enzyme activity is decreased, preferably substantially decreased, or eliminated, such as the level of expression of said activities in e.g. Saccharomyces cerevisiae strain TN 19 deposited with DSM under Accession Number 12276.
• a microbial cell according to the invention is preferably a yeast cell wherein the expression of said second and third expressible enzyme activities is increased, preferably substantially increased, whereas the expression of said fourth expressible enzyme activity is decreased, preferably substantially decreased, and said cell may in one embodiment produce a first metabolite, such as e.g.
• said production of said first me- tabolite is increased as compared to an expression of said metabolite in a comparable wild-type or isolated cell, said increase is increased by a factor of at least 1.005, for example 1.010, such as 1.015, for example 1.020, such as a factor of at least 1.025, for example a factor of at least 1.030, such as 1.035, for example 1.040, such as 1.045, for example 1.050, such as 1.055, for example 1.060, such as at least 1,065, for example a factor of at least 1.070, such as 1.075, for example 1.080, such as 1.085, for example a factor of at least 1.090, such as 1.095, for example 1.100, such as 1.105, for example 1.110, such as 1.115, for example 1.120, such as at least 1,125, for example a factor of at least 1.130, such as 1.135, for example 1.140, for example 1.145, such as 1.150
• Said microbial cell, most preferably yeast, having an increased production of a first metabolite has, in another preferred embodiment, a decreased production of a second me- tabolite, preferably glycerol or ethanol.
• Said decreased production of said second metabolite is decreased by at least 0.5 percent, for example at least 1 percent, such as at least 2 percent, for example 4 percent, such as 6 percent, such as at least 8 percent, for example at least 10 percent, such as 12 percent, for example 14 percent, such as 16 percent, such as at least 18 percent, for example at least 20 percent, such as 22 percent, for exam- pie 24 percent, such as 26 percent, such as at least 28 percent, for example at least 30 percent, such as 32 percent, for example 34 percent, such as 36 percent, for example 38 percent, such as at least 40 percent, for example 42 percent, such as 44 percent, for example 46 percent, such as 48 percent, such as at least 50 percent, for example 52 percent, such as 54 percent, for example 56 percent, such as 58 percent, such as at
• the maximum specific growth rate of said cell according to the invention is substantially unaltered as compared to a comparable wild-type microbial cell or to a comparable isolated microbial cell
• a microbial cell characterised by a decrease in the maximum specific growth rate is also preferred according to the invention, such as a microbial cell, preferably a yeast cell, having a maximum specific growth rate that is decreased by less than 1 percent, such as 1.5 percent, for example 2.0 percent, such as by less than 2.5 percent, for example 3.0 percent, such as 3.5 percent, for example by less than 4.0 percent, such as 4.5 percent, for example 5.0 percent, such as by less than 5.5 percent, for example 6.0 percent, such as 6.5 percent, for example by less than 7.0 percent, such as 7.5 percent, for example 8.0 per- cent, such as by less than 8.5 percent, for example 9.0 percent, such as 9.5 percent, for example by less than 10.0 percent, such as 12 percent, for example 14 percent, such as by less than 16 percent, for example 18 percent,
• a microbial cell preferably a yeast cell or a bacterial cell, or a composition comprising said cell, for use in a production of a first metabolite such as a primary or secondary metabolite, preferably a primary metabolite and more preferably an amino acid, an alcohol or an acid, such as e.g. ethanol, glycerol, acetic acid and propionic acid, ethanol and/or glycerol being particularly preferred.
• a first metabolite such as a primary or secondary metabolite, preferably a primary metabolite and more preferably an amino acid, an alcohol or an acid, such as e.g. ethanol, glycerol, acetic acid and propionic acid, ethanol and/or glycerol being particularly preferred.
• said secondary metabolite is preferably selected from the group of secondary metabolites consisting of a ⁇ -lactam, a polyketide, a terpene, a steroid, a quinone, a coumarin, a flavonoid. an alkaloid, a piperi- dine, a pyridine, and the like.
• any state of the art down-stream processing technique said first metabolite in an organism such as e.g. a fungal cell, a yeast cell or a bacterial cell.
• an organism such as e.g. a fungal cell, a yeast cell or a bacterial cell.
• Any of said eukaryotic or prokaryotic cells for use in said production preferably qualify for GRAS status ("Generally Regarded As Safe") with the Federal Drug Administration of the United States of America.
• a microbial cell preferably a yeast cell, for use in said production of said first metabolite, said cell further producing a second metabolite, preferably glycerol or ethanol, said production of said second metabolite being substantially decreased as compared to the production of said second metabolite in a comparable wild-type cell or a comparable isolated microbial cell.
• the microbial cell is a prokaryotic cell such as e.g. a lactic acid bacterial cell for use in the production of a first metabolite
• said first metabolite is selected from the group consisting of lactic acid and an aroma component such as acetoin.
• the microbial cell is a cell such as e.g. a Bacillus cell for use in the production of a first metabolite
• said cell is capable of further producing e.g. a protease, an amylase, a cellulase, a ⁇ -glucanase. an endoglucanase, a phosphatase, a xylanase, a lipase, a ⁇ -lac- tamase, or a xylosidase.
• the microbial cell or the composition according to the invention is preferably used in a production of a first metabolite or used in a method of generating alternative intracellular NADH re-oxidation. Accordingly, the microbial cell is providing a novel or, in terms of efficiency and/or overall rate of reaction, a much improved path- way for alternative NADH re-oxidation for the purpose of providing, supplementing and/or increasing a pool of intracellular NAD, said provision, supplementation and/or increase being used in a process of altering, directing and/or redirecting the flux of primary and or secondary metabolites in said cell.
• a microbial cell for use in a production of a first metabolite, said cell harbouring a novel or, in terms of efficiency and/or overall rate of reaction, a much improved pathway for alternative NAD reduction for the purpose of providing, supplementing and or increasing a pool of intracellular NADH, said provision, supplementation and/or increase being used in a process of altering, directing and/or redirecting the flux of primary and/or secondary metabolites in said cell.
• a microbial cell for use in the production of a first metabolite, said cell harbouring a novel or, in terms of efficiency and/or overall rate of reaction, a much improved pathway for alternative NADPH re-oxidation for the purpose of providing, supplementing and/or increasing a pool of intracellular NADP.
• said provision, supplementation and/or increase being used in a process of altering, directing and/or redirecting the flux of primary and/or secondary metabolites in said cell.
• a microbial cell for use in the production of a first metabolite, said cell harbouring a novel or, in terms of efficiency and or overall rate of reaction, a much improved pathway for alternative NADP reduction for the purpose of providing, supplementing and/or increasing a pool of intracellular NADPH. said provision, supplementation and/or increase being used in a process of altering, directing and/or redirecting the flux of primary and/or secondary metabolites in said cell.
• a microbial cell according to invention for use in a preparation of a drinkable or an edible product.
• a microbial cell for use in a production of a first metabolite for use in a drinkable or an edible product preferably a product having desirable organoleptic qualities.
• said first metabolite has and/or provides a desirable organoleptic quality to said product.
• said first metabolite is ethanol.
• the microbial cell for use in a production of a first metabolite according to the invention in one embodiment, further produces a second metabolite, the production of said second metabolite being substantially decreased as compared to the production of said second metabolite in a comparable wild-type cell or a comparable isolated microbial cell, said decreased production resulting in a provision of a desirable organoleptic quality to said product.
• said product is a functional food.
• a microbial cell or a composition comprising said cell in a production, preferably an increased production, of a first metabolite, said metabolite being a primary metabolite or a secondary metabolite.
• the microbial cell is a yeast cell or a prokaryotic microbial cell and that said first metabolite is an alcohol or an acid, preferably ethanol. glycerol, acetic acid, lactic acid or propionic acid.
• Further preferred metabolites are selected from the group consisting of a ⁇ -lactam, a polyketide, a terpene, a steroid, a quinone. a coumarin. a flavonoid, an alkaloid, a piperidine. a pyridine. and the like.
• a microbial cell preferably a yeast cell, further pro- ducing a second metabolite, said production of said second metabolite being substantially decreased as compared to the production of said second metabolite in a comparable wild- type cell or a comparable isolated microbial cell.
• a second metabolite is ethanol or glycerol or an undesirable aroma component naturally produced by a lactic acid bacterial cell.
• Another preferred use of said microbial cell is in preparation of a drinkable or edible product or in a production of a first metabolite for use in said drinkable or edible product, said first metabolite having and/or providing a desirable organoleptic quality to said product.
• the first metabolite is ethanol or, when the microbial cell is a lactic acid bacterial cell, an aroma component produced by said lactic acid bacterial cell, preferably acetoin and/or diacety lactis.
• a much preferred use of said microbial cell in said preparation of said drinkable or edible product is that of a microbial cell according to the invention, preferably a yeast cell or a lactic acid bacterial cell, further producing a second metabolite, said production of said second metabolite being substantially decreased as compared to the production of said second metabolite in a comparable wild-type cell or a comparable isolated microbial cell, said decreased production resulting in a provision of a desirable organoleptic quality to said product.
• a microbial cell preferably a yeast cell or a lactic acid bacterial cell
• the method comprises cultivation of any microbial cell including a microbial eukaryotic cell or a microbial prokaryotic cell.
• microbial eukaryotes are many yeast and fungal cells preferred, such as yeast cells of the species Saccharomyces. Schizosaccharomyces and Pichia. such as e.g. Saccharomyces cerevisiae. Schizosaccharomyces pombe, Pichia pastor is and the like, as well as algae such as e.g. Chlamydomonas rein- hardi, slime moulds such as e.g. Dictyostelium discoideum and filamentous fungi.
• Preferred filamentous fungi are species of Neurospora such as e.g.
• Neurospora crassa and species of Aspergillus such as Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Penicillium species such as e.g. Penicillium chrysogenum, Penicillium ro- queforti and Penicillium camemberti.
• Penicillium chrysogenum e.g. Penicillium chrysogenum
• Penicillium ro- queforti e.g. Penicillium chrysogenum
• Penicillium ro- queforti Penicillium camemberti
• Particularly preferred are industrially relevant yeast cells, slime moulds and filamentous fungi providing production of products such as e.g. antibiotics, steroids, pigments, enzymes, organic alcohols and acids, amino acids, poly- saccharides and the like.
• bacterial cells such as Gram-positive species such as Bacillus species like e.g. Bacillus subtilis, Bacillus thuringensis, Bacillus li- cheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus lentus and Bacilus stearothermophilus, species of Corynebacterium like e.g. Corynebacterium glutamicum, and species of Propionibacterium as well as Gram-negative species such as Escherichia coli.
• Particularly preferred are also lactic acid bacterial species such as e.g. Lactococcus lactis. Lactococcus lactis subsp.
• lactis Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. diacetylactis, Leuconostoc species such as e.g. Leuconostoc oenos, Lactobacillus species, such as Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus delbruckii subsp. bulgaricus, and Lactobacillus helveticus, Pediococcus species, Brevibacterium species, Propionibacterium species and similar industrially relevant spe- cies like e.g. Bifidobacterium.
• Embodiments of this aspect of the invention comprise a method wherein said first metabolite is either a primary metabolite or a secondary metabolite.
• Particularly preferred metabolites are selected from the group consisting of an alcohol, an acid, ethanol glyce- rol acetic acid, lactic acid, propionic acid, a ⁇ -lactam, a polyketide, a terpene, a steroid, a quinone. a coumarin, a flavonoid, an alkaloid, a piperidine, a pyridine, and the like.
• said metabolite is preferably diacetyl, acetaldehyde, 2,3-butylene glycol acetoin, or lactic acid.
• Said metabolite may be produced by a cell further capable of producing a gene product heterologous to said cell, preferably a product selected from the group consisting of an a protease, an amylase, a cellulase, a ⁇ -glucanase, an endoglucanase, a phosphatase, a xylanase, a lipase, a ⁇ -lactamase, a ⁇ -galactosidase, a ⁇ -glucoronidase, and a xylosidase.
• a product selected from the group consisting of an a protease, an amylase, a cellulase, a ⁇ -glucanase, an endoglucanase, a phosphatase, a xylanase, a lipase, a ⁇ -lactamase, a ⁇ -galactosidase, a
• the method according to the invention pertains in one embodiment to an increased production of said first metabolite, such as a substantially increased production, as compared to the production of said first metabolite in a comparable wild-type cell or a comparable isolated microbial cell. Accordingly, there is provided a method by which said production of said first metabolite is increased at least by a factor of 1.01, such as 1.02, for ex- ample 1.03, such as a factor of at least 1.04, for example 1.05, such as 1.06, for example 1.07, such as 1.08, for example 1.09, such as 1.10, for example 1.11, such as at least 1.12.
• said metabolite When being isolated or when being isolated and purified, said metabolite is isolated or isolated and purified according to any available state of the art techniques for isolating or isolating and purifying a metabolite.
• the method pertains to the production in a yeast cell or in a lactic acid bacterial cell of a first metabolite such as a primary or secondary metabolite, preferably a primary metabolite and more preferably an alcohol or an acid, such as e.g. ethanol, glycerol acetic acid and propionic acid, with ethanol being particularly preferred.
• a first metabolite such as a primary or secondary metabolite, preferably a primary metabolite and more preferably an alcohol or an acid, such as e.g. ethanol, glycerol acetic acid and propionic acid, with ethanol being particularly preferred.
• a first metabolite such as a primary or secondary metabolite
• a primary metabolite preferably a primary metabolite and more preferably an alcohol or an acid, such as e.g. ethanol, glycerol acetic acid and propionic acid
• ethanol is particularly preferred.
• a method wherein said cell
• a microbial cell preferably a yeast cell or a lactic acid bacterial cell, said cell further produc- ing a second metabolite, the production of said second metabolite being substantially decreased as compared to the production of said second metabolite in a comparable wild- type cell or a comparable isolated microbial cell.
• said decrease of said production of said second metabolite is at least 2 percent, such as 4 percent, for example at least 6 percent, such as 8 percent, for example at least 10 percent, such as 12 percent, for example 14 percent, such as 16 percent, for example 18 percent, such as at least 20 percent, for example 24 percent, such as at least 30 percent, for example 35 percent, such as at least 40 percent, for example 50 percent, such as 60 percent, for example at least 70 percent, such as 80 percent, for example at least 90 percent, such as decreased by at least 92 percent, for example 94 percent, such as 96 percent, for example 98 percent, such as decreased by 99 percent or decreased to such an extent that said second metabolite is virtually unassayable using state of the art assays for identifying and/or quantifying said second metabolite.
• the second metabolite is glycerol.
• the second metabolite when the cell is a lactic acid bacterial cell the second metabolite is an undesir- • able aroma component naturally produced by a lactic acid bacterial cell.
• a method for generating an alternative re-oxidation of a reduced coenzyme consisting essentially of providing in a microbial cell a novel or, in terms of efficiency and/or overall rate of reaction, a much improved pathway for alternative NADH and/or NADPH re-oxidation for use in providing, supplementing and/or increasing a pool of intracellular NAD and/or NADP.
• said provi- sion, supplementation and/or increase being used in a process of altering, directing and/or redirecting the flux of primary and/or secondary metabolites in said cell.
• a method for generating an alternative reduction of an oxidised coenzyme consisting essentially of providing in a microbial cell a novel or, in terms of efficiency and/or overall rate of reaction, a much improved pathway for alternative NAD and/or NADP reduction for the purpose of providing, supplementing and/or increasing a pool of intracellular NADH and/or NADPH, said provision, supplementation and or increase being used in a process of altering, directing and/or redirecting the flux of primary and/or secondary metabolites in said cell.
• a method of constructing a microbial cell comprising introducing into said cell a capability and/or increasing a capability of said cell to express a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said expression of said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity.
• the first expressible enzyme activity is that of SEQ ID NO:2, or a functional derivative thereof, as encoded by the nucleotide sequence designated SEQ ID NO:l, or a functional derivative thereof.
• a method of constructing a microbial cell comprising introducing into said cell a capability and/or increasing a capability of said cell to express a first expressible enzyme activity which, when expressed in said microbial cell, is controlling an intracellular redox system of said cell, said expression of said first expressible enzyme activity in said microbial cell being operably linked to an expression signal not natively associated with said first expressible enzyme activity.
• the term introducing into said cell a capability to express an expressible enzyme activity shall be understood to comprise any means by which said cell subsequently becomes capable of increasing an expression of said expressible enzyme activity.
• said capability is a first nucleotide sequence comprising an expression signal, said expression signal being operably linked to a second nucleotide sequence, said second nucleotide sequence comprising said expressible enzyme activity.
• Said expression signal may direct a substantially constitutive expression, a constitutive expression during growth of said cell in a particular growth phase, an inducible expression in response to the presence and/or level of an inducer or the absence and/or level of a repressor.
• the expression signal is preferably a regulable expression signal such as a regulable transcription initiation signal and/or a regulable translational initiation signal, such as an ex- pression signal regulable in response to an alteration in a value, level and/or concentration of a factor such as a physiological growth parameter, preferably a parameter selected from the group consisting of pH, temperature, salt content including osmolarity, anaero- bicity, aerobicity including oxygen level, energy level including a membrane potential and a proton motive force.
• a regulable expression signal such as a regulable transcription initiation signal and/or a regulable translational initiation signal, such as an ex- pression signal regulable in response to an alteration in a value, level and/or concentration of a factor such as a physiological growth parameter, preferably a parameter selected from the group consisting of pH, temperature, salt content including osmolarity, anaero- bicity, aerobicity including oxygen level, energy level including a membrane potential and a proton motive force.
• the promoter is preferably a promoter being either growth phase regulated, inducible and/or repressible and/or, in a natural host organism, directing expression of a gene encoding a gene product involved in mediating a reaction of a biosynthetic pathway and/or a major metabolic pathway, preferably a pathway selected from the group of pathways consisting of glycolysis, gluconeogenesis, citric acid cycle, and pentose phosphate pathway.
• the expression signal may be further regulated by an upstream activating sequence (UAS), by an enhancer element or by a silencer element.
• UAS upstream activating sequence
• the person skilled in the art will be aware of general molecular biology techniques for use in the construction in vitro of a recombinant DNA molecule. Such techniques are described e.g. in Sambrook et al. (1989) and in Old and Primrose (ibid.). Said skilled artisan will further be aware of the academic literature including general textbooks on molecular biology and genetic engi- neering and he will be able to combine various expression signals such as putative or recognised promoter regions with a range of regulatory nucleotide sequences generally known to exert an effect on the level of gene expression.
• An expression signal can be a cloned expression signal or an in vitro synthesised expression signal.
• Expression signals in prokaryotic microbial cells are known to comprise so-called -35 and -10 regions and numerous examples of such regions are available from various databases.
• Expression signals may be optimised by increasing the promoter strength, by adjusting translational initiation sequences, by optimising the choice of codons by using so-called highly expressed codons, by adjusting the secondary structure of the mRNA. by increasing the efficiency of transcriptional termination, by increasing or decreasing a copy num- ber of a vector, or by increasing or decreasing the stability of said vector.
• the microbial cell is preferably a fungal cell, a yeast cell, or a bacterial cell. Particularly preferred is a microbial eukaryotic cell or a microbial prokaryotic cell.
• microbial eukaryotes are many yeast and fungal cells preferred, such as yeast cells of the species Saccharomyces, Schizosaccharomyces and Pichia. such as e.g. Saccharomyces cerevisiae. Schizosaccharomyces pombe.
• Pichia pastor is and the like, as well as algae such as e.g. Chlamydomonas reinhardi, slime moulds such as e.g. Dictyostelium discoideum and filamentous fungi.
• Preferred filamentous fungi are species of Neurospora such as e.g. Neurospora crassa, and species of Aspergillus such as Aspergillus nidulans. Aspergillus niger. Aspergillus oryzae and Penicillium species such as e.g. Penicillium chrysogenum, Penicillium roqueforti and Penicillium camemberti. Particularly preferred are industrially relevant yeast cells, slime moulds and filamentous fungi providing production of products such as e.g. antibiotics, steroids, pigments, enzymes, organic alcohols and acids, amino acids, polysaccharides and the like.
• bacterial cells such as Gram-positive species such as Bacillus species like e.g. Bacillus subtilis, Bacillus thuringensis, Bacillus li- cheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus lentus and Bacilus stearothermophilus, species of Corynebacterium like e.g. Corynebacterium glutamicum, and species of Propionibacterium as well as Gram-negative species such as Escherichia coli.
• Particularly preferred are also lactic acid bacterial species such as e.g. Lactococcus lactis, Lactococcus lactis subsp.
• Lactis Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. diacetylactis.
• Leuconostoc species such as e.g. Leuconostoc oenos, Lactobacillus species, such as Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus delbr ⁇ ckii subsp. bulgaricus, and Lactobacillus helveticus.
• Pediococcus species Brevibacterium species. Propionibacterium species and similar industrially relevant species like e.g. Bifidobacterium.
• the first expressible enzyme activity is preferably any transhydrogenase activity and more preferably any pyridine nucleotide transhydrogenase activity, or any activity which, when expressed, results in an increased level of at least one intracellular coenzyme in its oxidised or re- prised form, preferably a coenzyme in its oxidised/reduced form selected from the group consisting of FAD/FADH 2 . NAD/NADH and NADP NADPH.
• Said first expressible enzyme activity when expressed, may also provide an increased level of at least one intracellular redox system consisting of a coenzyme in its corresponding oxidised and reduced forms, said redox system being preferably selected from the group consisting of FAD/FADH 2 , NAD NADH and NADP/NADPH.
• said first expressible enzyme activity is that of the transhydrogenase activity encoded by CTH of Azotobacter vinelandii as harboured by Saccharomyces cerevisiae TN4 deposited under DSM Accession Number 12267, or a function- ally equivalent activity within the meaning of that term as defined herein above.
• Said transhydrogenase activity in a preferred embodiment is operably linked to an expression signal not natively associated with said activity.
• the method of the invention further comprises, in a particularly preferred embodiment, a further step of freezing or freeze-drying the microbial cell in the preparation of a recon- stitutable lyophilisate.
• the microbial cell is a lactic acid bacteria as exemplified herein, and said first expressible enzyme activity is, at least in its native host organism, a cytoplasmic transhydrogenase. said expression of said cytoplasmic transhydrogenase resulting in an altered and/or novel product formation and/or metabolite pro- duction of said lactic acid bacteria.
• Lactic acid bacteria metabolise pyruvate through a number of different pathways.
• the metabolite is converted into lactate by lactate dehydrogenase. into acetyl-CoA and CO by pyruvate dehydrogenase, into formate by pyruvate formate lyase and into acetolactate and CO 2 by acetolactate decarboxylase.
• the carbon flux distribution through these pathways is dependent on the external growth conditions. This control is exerted through changes in the intracellular NADH/NAD " ratio (C. Garrigues, P. Loubiere. N. D. Lindley and M. Cocaign-bousquet (1997). J.
• Lactic acid bacteria with an increased formation of the secondary metabolite diacetyl are relevant for a number of industrial applications. It has been shown that overexpression of NADH-oxidase from Streptococcus mutans in L. lactis results in a shift from homolactic (production of lactic acid) to mixed acid fermentation (production of lactic acid, acetic acid, acetoin and diacetyl) under aerobic growth conditions. This effect is ascribed to a decrease in the intracellular NADH/N AD + ratio of the recombinant strain (F. Lopez de Felipe, M. Kleerebezem, W. M. de Vos and J. Hugenholtz (1998). J. Bacteriology 180, 3804-3808). Expression of the cytoplasmic transhydrogenase in lactic acid bacteria is expected to have a similar effect on the NADH/NAD + ratio if the reaction occurs in the direction from NADH to NADPH.
• lactate dehydrogenase is activated by a high NADH/NAD T ratio it is expected that the flux towards lactate can be increased by expressing transhydrogenase in lactic acid bacteria under conditions where the transhydrogenase reaction occurs in the direction from NADPH to NADH.
• Figure 1 shows a comparison of the amino acid sequences of CTH of Azotobacter vinelandii as described herein and a pyridine nucleotide transhydrogenase from P. fluore- scens (French et al., 1997).
• Figure 2 shows the consumption of glucose and formation of ethanol, glycerol and carbon dioxide in strain TN4.
• Figure 3 shows the consumption of glucose and formation of ethanol, glycerol and carbon dioxide in strain TN3.
• Figure 4 shows the formation of succinate, pyruvate, acetate and 2-oxoglutarate in strain TN4.
• Figure 5 shows the formation of succinate, pyruvate, acetate and 2-oxoglutarate in strain
• This example contains a description of a cloning of a gene encoding a cytoplasmic transhydrogenase from A. vinelandii and the expression of said gene in a S. cerevisiae strain derived from the industrial model strain S. cerevisiae CBS8066 ( issen et al. 1997; Verduyn et al. 1990).
• the constructed strain has been cultivated anaerobically in batch culture in a high performance bioreactor in order to perform a quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH.
• NAD + . NADPH and NADP T are examples of a quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH.
• Saccharomyces cerevisiae strains were generated from Saccharomyces cerevisiae T23D which was kindly provided by Jack Pronk from the Department of Microbiology and Enzymology, Kluyver Laboratory of Biotechnology, Delft University of Technology, The Netherlands. The strain was maintained at 4°C on YPG agar plates, monthly prepared from a lyophilized stock kept at -80°C.
• Escherichia coli DH5 ⁇ (F F80d/ ⁇ cZ DM15 O(lacZYA- argF) U169 deoR recAl endAl hsdRllfa m k * ) supE44 1 " thi-1 gyra96 relAl) (GIBCO BRL, Gaithersburg, MD. USA) was used for subcloning.
• Azotobacter vinelandii ATCC 478 was purchased from Centraalbureau voor Schimmelcultures (Baam & Delft, The Netherlands). The Strain was maintained at 4°C on agar plates consisting of Winegradskis media monthly prepared from a lyophilized stock kept at -80°C.
• Plasmid DNA from E. coli was prepared with Qiagen colums (Qiagen GmbH, D ⁇ sseldorf, Germany) following the manufacturer's instructions.
• Qiagen colums Qiagen GmbH, D ⁇ sseldorf, Germany
• the desired fragments were separated on 0.8% agarose gels, excised and recovered from agarose using the Qiagen DNA isolation kit (Qiagen GmbH. D ⁇ sseldorf. Germany).
• Plasmid constructions A plasmid, pRY253, containing the DNA sequence from 1100 bp upstream of HO to 1500 bp downstream of the open reading frame was digested with the restriction enzyme Sspl and two fragments of 594 bp (-119 bp to 475 bp) and 180 bp (+50 bp to 230 bp) was isolated. The fragments were ligated into the Smal and EcoRV sites of plasmid pFA6A-kanMX3 (Wach et al, 1994), respectively. The correct direction of the inserts was verified by PCR. The resulting plasmid, pHOdel, was digested with BamHI and Spel prior to transformation and a 3350 bp fragment consisting of G4 f flanked by the two inserts was isolated.
• a plasmid, pRB58, containing the chromosomal region around SUC2 was kindly donated by Danisco Biotechnology (Copenhagen, Denmark).
• a 3.9-kb fragment (-906 bp to +1383) containing the open reading frame and its promoter and terminator was isolated by digestion with EcoRI and Clal.
• the EcoRI site was made blunt by treatment with Klenow fragment and the fragment was inserted into the StuI/BstBI sites of the URA3 gene in the yeast integration plasmid YIp5.
• the resulting construct, pSUC2 was linearised with Pstl prior to transformation.
• E. coli DH5 ⁇ was transformed by electro- transformation using the Bio-Rad electroporation equipment (Biorad Laboratories. Richmond. USA). Transformants were selected on L broth plates containing 100 mg/ml ampicillin. S. cerevisiae cells were made competent for plasmid uptake by treatment with lithium acetate and polyethyleneglycol (Schiestl & Gietz. 1989). 5 ⁇ g of DNA was used for each transformation. Transformants were plated directly on selective media except for the G418 resistant transformants. These were suspended in YPD for 24 hours prior to plating on selective media in order to obtain expression of the G418 resistance gene. Correct integration of the fragments from pHOdel and pSUC2into the chromosome was verified by PCR analysis using extracted DNA from the transformants.
• the diploid Saccharomyces cerevisiae T23D (Wenzel et al. 1992) is a meiotic progeny of the tetraploid.
• industrial model strain CBS8066 The strain was chosen as the parent strain in this study in order to facilitate the introduction of genetic changes in the organism while maintaining the genetic background of CBS8066.
• haploid strain which was isogenic to T23D.
• HO was deleted in one of the alleles in T23D.
• the gene encodes a homothallic switching endonuclease that enables meiotic progeny of a diploid to switch mating type.
• T23D was transformed with a 3350 bp fragment from pHOdel and transformants with integration of the fragment in the HO locus were selected on YPD plates containing 300 mg/1 G418.
• Loop out of G41tY by homologue recombination of the two direct repeats in the insert was obtained with a frequency of 1/10000 colonies after cultivation of one of the transformants in non-selective YPD media for 30 generations. It was verified by PCR that the loop out transformants contained both the wildtype HO and the expected deletion in the gene (results not shown).
• One of the loop out transformants was sporulated and the resulting spores were tested for mating types.
• TNI haploid-like organism
• a 3.9-kb fragment containing SUC2, encoding invertase was inserted into the URA3 locus of TNI. This was done by transformation with pSUC2 after linearisation with Pstl. Transformants were selected on minimal media containing sucrose and uracil, since T23D and its haploid progeny did not grow on this carbon source while the transformants did. Single colonies of the transformants were isolated on minimal media containing sucrose and 5-fluoroorotic acid. The latter is lethal to cells with an intact URA3 locus. The transformants did not grow in the absence of uracil in the media confirming their auxotrophy towards this compound.
• One of the uracil auxotrophs was designated TN2 and used for expression of the cytoplasmic transhydrogenase as described below.
• CTH CTH in S. cerevisiae.
• CTH was ligated into YEp24 behind the strong constitutive promoter of PGK resulting in plasmid Yep24-pPGK-CTH. This plasmid was transferred into strain TN2 resulting in strain TN4.
• Yep24-pPGK was transferred into strain TN2 resulting in strain TN3. This strain served as a negative control in the physiological studies.
• the yeast was cultivated in a mineral medium prepared according to Verduyn et ⁇ /.(1990). Vitamins were added by sterile filtration following heat sterilization of the medium. The concentrations of glucose and (NH ⁇ ) 2 SO 4 was 25 g l "1 and 7.5 g 1 " . respectively. Growth of S. cerevisiae under anaerobic conditions requires the supplementary addition to the medium of ergosterol and unsaturated fatty acids, typically in the form of Tween 80 (Andreasen & Stier, 1953; Libudzisz et al, 1986).
• Ergosterol and Tween 80 were dissolved in 96 %(v/v) ethanol and the solution was autoclaved at 121°C for 5 min.
• the final concentrations of ergosterol and Tween 80 in the medium were 4.2 mg g DW “1 and 175 mg g DW "1 , respectively.
• 75 ⁇ l l "1 antifoam (Sigma A- 5551) was added to the medium.
• Experimental setup for the batch cultivations Anaerobic batch cultivations were performed at 30°C and at a stirring speed of 800 rpm in in-house manufactured bioreactors. The working volume of the reactors were 4.5 liters. pH was kept constant at 5.00 by addition of 2 M KOH.
• the bioreactors were equipped with off-gas condensers cooled to 2°C.
• the bioreactors were continuously sparged with N 2 containing less than 5 ppm O 2 , obtained by passing N 2 of a technical quality (AGA 3.8), containing less than 100 ppm O 2 , through a column (250x30 mm) filled with copper flakes and heated to 400°C.
• the column was regenerated daily by sparging it with H 2 (AGA 3.6).
• a mass flow controller (Bronkhorst HiTec F201C) was used to keep the gas flow into the bioreactors constant at 0.50 1 nitrogen min "1 liter "1 Norprene tubing (Cole-Parmer Instruments) was used throughout in order to minimize diffusion of oxygen into the bioreactors.
• the bioreactors were inoculated to an initial biomass concentration of 1 mg l "1 with precultures grown in unbaffled shake flasks at 30°C and 100 rpm for 24 hours.
• the anaerobic batch cultivations of strains TNI, TN3 and TN4 were each carried out three times with identical results.
• RSD relative standard deviation
• the loss of ethanol through the reflux condenser of the bioreactor was determined to be between 4% and 9% of the ethanol formed by the bioreaction depending on the dilution rate (Schulze, 1995). In the carbon balances the measured ethanol fluxes were corrected for this loss through evaporation.
• 2-oxoglutarate was identified in the extracellular samples by an enzymatic assay. 100 ⁇ l ammonium (50mM), 40 ⁇ l NADPH (40 mM), 100 units of glutamate dehydrogenase (Boehringer Mannheim) and 1 ml KPO -buffer (50 mM, pH 7.0) was pipetted into a quartz cuvette. At time zero 100 ⁇ l of a sample was added and the absorbency at 340 nm was monitored.
• the intracellular nucleotides were extracted from cells of S. cerevisiae, growing anaerobically in batch cultivations. 5.0 ml of culture liquid was withdrawn from the bioreactor and sprayed into 20 ml of 60% methanol (-40°C) within 1 second. Except for the following changes, the further proceedings were carried out as described earlier for the cold methanol extraction method (de Koning & van Dam, 1992). Instead of storing the samples in a freezer after quenching the cells in cold methanol, the nucleotides were extracted from the cells and quantified in one step to avoid degradation.
• A.vinelandii ATCC 478 was cultured in 14 litres of Winegradskis nitrogen-free medium at 37°C for 5 days and harvested by centrifugation (3000xg, 20 minutes) and resuspended in buffer A (50 mM Tris-Cl (pH 7.5) with 5 mM dithiothreitol). Phenylmethylsulfonyl fluoride was added to buffer A at a concentration of 1 mM and intracellular protein was extracted from 30 g cells (wet weight) by treating the cells with lysozyme followed by passage through a French press at approximately 14 MPa. These procedures were carried out at 2°C.
• Cell-free extract was isolated by ultracentrifugation (60000xg, 30 min, 2°C).
• the extract contained 710 units of transhydroge- nase activity at a specific activity of 0.39 U/mg protein.
• the extract was loaded onto a 2',5'-ADP-Sepharose-4B affinity column with a 10-ml bed volume (1.2 x 9 cm, Pharmacia) at a flow rate of 10 ml/hour.
• the ligands of the column bind specifically to NADP + - dependent dehydrogenases.
• transhydrogenase The fractions containing transhydrogenase were identified by measuring the enzyme activity. Active fractions were pooled and concentrated by ultrafiltration with an Amicon ultrafiltration cell fitted with a membrane with a nominal M r cut-off of 30000. The concentrate contained 287 units of transhydrogenase activity at a specific activity of 240 U/mg protein, corresponding to a 615-fold purification at a yield of 40%.
• SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis
• transhydrogenase from A. vinelandii has an apparent M r of 54000 (Voordouw et al, 1980). Since the isolated protein described above with this size was blocked in the N-terminus, it was digested with trypsin and the resulting polypeptides were separated by hydrofobicity on HPLC (reference). 5 polypeptides were sequenced by automated Edman degradation (underlined in Figure 1).
• a DNA fragment of 1300 bp was obtained. This fragment was subcloned into the Smal site of pUC18 and partly sequenced. The remaining 100 bp of the gene was identified by inverse PCR (Triglia et al, 1988). Chromosomal DNA from A. vinelandii was digested with PvuII (Promega). No site for this restriction enzyme was present in the 1300 bp fragment while two sites on each side of the fragment turned out to be conveniently located for the further proceedings. Selfligation of the linearised chromosomal DNA fragments was obtained by treatment with T4 DNA ligase (Promega).
• the deduced amino acid sequence of CTHp is listed in SEQ ID NO:2.
• the protein has a M t of 51345, as determined by the Compute pI/Mw program on the world wide web (Bjellqvist et al, 1993). This is slightly lower than the M r of 54000 expected from the migration position in SDS-PAGE. Furthermore, the amino acid composition fitted well with earlier determinations (Voordouw et al, (1980); Middleditch et al, 1972).
• FAD flavoproteins which contain an active site with the consensus pattern G-G-X-C- (L/I/V/A)-X-G-C-(L/I/V/M)-P where the two cysteines are involved in the transfer of reducing equivalents from the FAD cofactor to the substrate (Kuriyan et al, 1991).
• a similar pattern is observed in the two soluble transhydrogenases from amino acid 41 to 51 in Figure 1, but one of the cysteine residues is missing.
• the characteristic active site, that is observed in other flavoproteins. is absent in the soluble transhydrogenases.
• a high similarity of 75% was observed to an unknown dehydrogenase from E. coli.
• the protein is encoded by udhA, which is located next to oxyR in the chromosome of the bacterium. Since oxyR is a part of the positive regulation of genes involved in the defence against oxidative damage (Tao et al, 1989), udhA could have a role in this process. Defense against oxidative stress is dependent on consumption of NADPH through glutathione reductase in most organisms and the high similarity between the two soluble transhydrogenases and udhA could indicate a physiological role of the three enzymes in synthesis of NADPH when the cell is subjected to oxidative stress.
• One site binds both NADP(H) and NAD(H) and constitutes the active site where transfer of reducing equivalents between the two cofactor systems occurs through a simple ping-pong mechanism (Widmer & Kaplan, 1977).
• the second site binds only to NADP(H) and is involved in allosteric regulation of the enzyme activity by NADP(H) and 2'-nucleotides.
• NAD(H) binding site of these enzymes in addition to the Rossman fold motif consists of an acidic residue (aspartate or glutamate) 18- 19 residues upstream of the last glycine residue in the Rossman fold and a glycine residue 13 residues further upstream of the acidic residue (Olausson et al, 1995). This is observed for the Rossman motif located from amino acid 12 to 17 which indicates that NAD(H) binds to this site and thus, that it is the active site of the enzyme.
• Anaerobic batch cultivations Anaerobic, glucose-limited batch cultivations of strains TNI . TN3 and TN4 were carried out in order to analyse the effect of CTH expression on the maximum specific growth rate, product formation, enzyme activities and the intracellular levels of the four nucleotides NAD, NADH, NADP + and NADPH.
• Figures 2 and 3 show the consumption of glucose and formation of ethanol. glycerol and carbon dioxide in strains TN4 and TN3, respectively.
• strain TN4 In order to verify correct expression of CTH in strain TN4, cells of strains TNI, TN3 and TN4 were sampled from the batch cultivations at different time points in the exponential growth phase and disrupted as described above. The specific enzyme activities of transhydrogenase, glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase/glucokinase (HXK+GLK) in the extracts were measured and did not vary throughout the exponential growth phase (Table 1).
• G6PDH glucose-6-phosphate dehydrogenase
• HXK+GLK hexokinase/glucokinase
• Table 1 Specific enzyme activities measured in vitro in cell free extracts from samples from the exponential growth phase of strains TNI, TN3 and TN4 grown in anaerobic, glucose-limited batch cultivations. No transhydrogenase activity could be detected in extracts from strains TNI and TN3 while a specific transhydrogenase activity of 4.53 U/mg protein was measured in the extract from strain TN4 containing plasmid Yep24-pPGK-CTH. Hence, it was concluded that expression of CTH in S. cerevisiae was successful. Based on the specific activity of 575 U/mg for the purified transhydrogenase from A.
• CTHp formed approximately 0.8% of the protein pool in strain TN4. This level is approximately 10 times higher than in the protein extracts from A. vinelandii from this study.
• PPP pentose phosphate pathway
• transhydrogenase probably converts NADPH to NADH in the cytoplasm of strain TN4.
• pyruvate decarboxylase In the compartment pyruvate decarboxylase and the NADP + -dependent cytoplasmic aldehyde dehydrogenase, respectively (Tamaki & Hama, 1982; Meaden et al, 1997; Nissen et al, 1997).
• NADPH 1 mole of NADPH is synthesised in the cytoplasm per mole of pyruvate consumed.
• the maximum specific growth rates, ⁇ mas , of TNI and TN3 were 0.39 h "1 and 0.35 h "1 , respectively.
• the reduction in the maximum specific growth rate of TN3 must be due to the presence of the control plasmid in multiple copies.
• Introduction of plasmid Yep24 pPGK-CTH into strain TN4 resulted in a further decrease in ⁇ mas to 0.18 h "1 .
• a minimal medium was used whereby the intracellular precursors for biomass synthesis had to be synthesised by the cells from glucose and ammonia. In this process glutamate is involved in several reactions as a donor of ammonium in the formation of proteins.
• RNA and DNA Jones & Fink.
• the intracellular concentrations of NAD(H) and NADP(H) were measured in cells of TNI, TN3 and TN4 sampled in the exponential growth phase. This was done to analyse the direction of the flux through the reaction catalysed by the soluble transhydrogenase in more detail.
• the direction of the reaction in equation 1 is determined by the size of Gibb's free energy of the reaction as described in equation 2.
• ⁇ G -RTln((aNAD+.cyt * SNADPH, cyt)/(aNADH. cyt * aNADP+.cyt )) (eq. 2)
• Table 3 The intracellular concentrations ofNAD(H) and NADP '(H) in ⁇ mol per gram biomass (dry weight) in cells of strains TNI, TN3 and TN4 sampled during exponential growth in anaerobic glucose- limited batch cultivations.
• the cytoplasmic enzyme, encoded by IDP2 is glucose-repressed and thus, not active during the growth conditions used in this work.
• the two mitochondrial enzymes, encoded by IDH and IDP1. are dependent on NADH and NADPH, respectively, and are active during growth on glucose. If the reactions encoded by isocitrate dehydrogenases function as a redox shunt in animal cells, this could also be the case in yeast. Hence, the system consisting of the coupled reactions catalysed by the two isocitrate dehydrogenases could have a role in conversion of surplus NADH, formed in synthesis of biomass and organic acids, into NADPH.
• cth encoding the cytoplasmic transhydrogenase from Azotobacter vinelandii, was cloned by PCR using primers Bglll-cth (5'-tacgaagatctGCTGTATATAAC- TACGATGTGGTGG-3') (SEQ ID NO:9) and CTH-XhoI (5'-tagcactcgagt- taAAAAAGCCGATTGAGACC-3') (SEQ ID NO:10) and pfu polymerase. The resulting DNA fragment was digested with the restriction enzymes Bglll and Xhol and inserted into the multi cloning site of the E. coli/L.
• lactis shuttle vector pTRKH2-pl 70 behind a strong constitutive derivate of the promoter pl70 (S. M. Madsen, J. Arnau. A. Vrang, M. Givskov amd H. Israelsen (1999). Molecular Microbiology 32, 75-87).
• the resulting plasmid was denoted pTRKH2-pl70-cth.
• the promoter region of the vector and the inserted cth were sequenced, whereby it was verified that the gene had been inserted correctly into the shuttle vector.
• E. coli DH5 ⁇ and L. lactis subsp. cremoris were both transformed with pTRKH2-pl70- cth and transformants were selected on plates containing complex medium (LB and

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