Genetically engineered bacterium as a platform for reductive whole-cell biocatalysis

Abstract

The present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene; a deletion of one or more nuo genes; a deletion of ldhA gene; and preferably a deletion of mqo gene, wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuoI, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a chinone species endogenously, preferably ubiquinone; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (H+-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously. The genetically engineered bacterium is only able to grow if an external electron acceptor (substrate) and a corresponding NADH-consuming reaction are present. In this way, growth is directly coupled to a biotechnologically usable whole-cell catalysis. Thus, the growth of the genetically engineered bacterium of the present invention is absolutely dependent on the conversion of a substrate into an industrially interesting product.

Description

[0001] Genetically engineered bacterium as a platform for reductive whole-cell biocatalysis

[0002] The present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene; a deletion of one or more nuo genes; a deletion of IdhA gene; and preferably a deletion of mqo gene, wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a chinone species endogenously, preferably ubiquinone; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously. The genetically engineered bacterium is only able to grow if an external electron acceptor (substrate) and a corresponding NADH-consuming reaction are present. In this way, growth is directly coupled to a biotechnologically usable whole-cell catalysis. Thus, the growth of the genetically engineered bacterium of the present invention is absolutely dependent on the conversion of a substrate into an industrially interesting product.

[0003] Background of the invention

[0004] Enzymatic reductions are mainly carried out industrially with purified enzymes. Wholecell catalysis has some advantages over the use of cell-free (in vitro) approaches, e.g. no external addition of costly components such as cofactors, cofactor regeneration systems and enzymes. However, such systems are sensitive to oxygen, which serves as primary electron acceptor in cellular metabolism, and thus sinks away reducing power needed for catalysis. Thus, there is a need for engineered bacteria that can be used as a platform for reductive whole-cell biocatalysis even in presence of oxygen.

[0005] Under aerobic conditions, thus in the presence of oxygen, the NADH generated during glycolysis is re-oxidized in the respiratory chain which involves ubiquinone (Q) mediated electron transfer from dehydrogenases to cytochrome oxidases. NADH dehydrogenase I (NDH-1 ) and NADH dehydrogenase II (NDH-2) are two NADH dehydrogenases in E. coli that oxidize NADH to NAD+by donating electrons via quinone species to the cytochrome oxidases which in turn reduce O2 to H2O. The three cytochrome oxidases in E. coli are cytochrome bo oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC). The present invention comprises engineered bacteria that allow reductive whole-cell biocatalysis in presence or absence of oxygen.

[0006] The objective of the present invention is to provide engineered bacteria that can be used as a platform for reductive whole-cell biocatalysis even in presence of oxygen.

[0007] The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.

[0008] Brief description of the invention

[0009] The present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously.

[0010] Thus, the present invention preferably relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (H+-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously.

[0011] Thus, the present invention preferably also relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and a deletion of mdh gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously.

[0012] A strain where GIpD is intact allows the quinone-dependent use of glycerol while a strain with intact GpsA allows NAD(P)H-dependent use of glycerol. Depending on the application, either can be desired. However to avoid the formation of a minicycle, it is sufficient that either glpD gene or gpsA gene is deleted.

[0013] Thus, in some preferred embodiments, the genetically engineered bacterium further comprises a deletion of one or more genes selected from the group comprising: did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0014] Moreover, in some preferred embodiments, the genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising: did gene, , poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0015] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is an E. coli.

[0016] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, and preferably mqo gene or mdh gene, more preferably a deletion of mqo gene. Thus, the present invention further relates to a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, and mqo gene. Thus, the present invention further also relates to a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, and mdh gene.

[0017] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0018] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0019] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0020] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0021] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0022] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogeneously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expresses one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogeneously.

[0023] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expresses cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0024] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of an ubi gene.

[0025] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

[0026] The present invention further relates to a method for producing sugar acids comprising the following steps: a) providing a genetically engineered bacterium as decribed herein, wherein the genetically engineered bacterium expresses a quinone-dependent dehydrogenase, b) providing a culture medium comprising a carbon source and a substrate of the quinone-dependent dehydrogenase, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the carbon source is selected from the group comprising glucose, xylose, arabinose, ribose, lyxose, allose, fucose, mannose, galactose, and meliobiose, or a combination thereof.

[0027] Preferably the carbon source is selected from the group comprising D-glucose, D-xylose, L-arabinose, D-ribose, L-lyxose, D-allose, D-fucose, D-mannose, D-galactose, and meliobiose, or a combination thereof.

[0028] Preferably, the quinone-dependent dehydrogenase is Quinoprotein glucose dehydrogenase (gcd), and wherein the substrate is pyrroloquinoline quinone (PQQ). The present invention further relates to a method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium of any one of the claims 1 - 12, wherein the genetically engineered bacterium expresses a NADH consuming enzyme preferably selected from NADH oxidase, phosphoketolase, lactate dehydrogenase, sarcosine oxidase, methanol dehydrogenase; b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is absolutely dependent on the conversion of the substrate.

[0029] Description of the invention

[0030] The present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene; a deletion of one or more nuo genes; a deletion of IdhA gene, preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (H+- transporting) (EC 7.1.1.3) and / or quinol oxidases (electrogenic, proton-motive force generating) (EC 7.1.1.7).

[0031] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene; a deletion of one or more nuo genes; a deletion of IdhA gene, preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species; and wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC), preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC).

[0032] With other words, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene; a deletion of one or more nuo genes; a deletion of IdhA gene, preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species; and wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC), preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC).

[0033] With other words, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene; a deletion of one or more nuo genes; a deletion of IdhA gene, preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species; and wherein the genetically engineered bacterium is capable of expressing one or more of of CydAB, CyoABCD, and CbdAB, preferably wherein the genetically engineered bacterium is capable of expressing CydAB, CyoABCD, and AppBC.

[0034] Thereby, wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species means that the genetically engineered bacterium does not comprise a deletion of a gene involved in the biosynthesis of said quinone species. Thus, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone means that the genetically engineered bacterium does not comprise a deletion of a gene involved in the biosynthesis of ubiquinone (ubi genes), e.g. does not comprise a deletion of ubiA gene, ubiC gene, or ubiCA gene etc. Thus, the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferably, the genetically engineered bacterium biosynthesizes ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. According to the invention, the genetically engineered bacterium produces the quinone species without genetic modification. Thus, the quinone species is an endogenous quinone species already produced by the wild-type strain of the genetically engineered bacterium.

[0035] Moreover, wherein the genetically engineered bacterium is capable of expressing ubiquinol oxidases (IT-transporting) (EC 7.1.1.3) and / or quinol oxidases (electrogenic, proton-motive force generating) (EC 7.1.1.7), preferably capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC), more preferably preferably capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) means that the genetically engineered bacterium does not comprise a deletion of a gene involved in the biosynthesis of ubiquinone ubiquinol oxidases (IT-transporting) (EC 7.1.1.3) and / or quinol oxidases (electrogenic, protonmotive force generating) (EC 7.1.1.7) etc. Preferabl, the genetically engineered bacterium does not comprise a deletion of the cydAB genes, cyoABCD genes and / or appBC genes. Thus, the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferably, the genetically engineered bacterium expresses cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Thus, the genetically engineered bacterium expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously. With other word, the genetically engineered bacterium naturally expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating). Therefore, the one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) are endogenous enzymes alreadpy expressed by the wild-type strain of the bacterium, i.e. are expressed without genetic modifications.

[0036] In order to provide a genetically engineered bacterium as a platform for reductive wholecell biocatalysis, the inventors of the present invention envisaged to create a thermodynamically favorable NADH / NAD+ratio; a ratio that already exists under anaerobic conditions, but since oxygen-dependent enzymes were to be used in this invention, another solution had to be found to deprive the cell of the possibilities of NADH oxidation.

[0037] An important advantage of the genetically engineered bacterium of the present invention is that due to the possibility of fermentation in the presence of oxygen completely new fermentative pathways are possible. For example, quinone-dependent dehydrogenases can convert sugars into sugar acids. The quinol formed is then used to form ATP via the respiratory chain. Examples of this new type of fermentation are the quinone-dependent oxidation of glucose, xylose or galactose. The high yields that can be achieved by selecting for ATP provision open up new possibilities for fermentative product formation.

[0038] Moreover, the genetically engineered bacterium of the present invention can be used for the selection of NADH-consuming enzyme reactions. The genetically engineered bacterium of the present invention lacks any possibility of transferring electrons from NADH to the respiratory chain and thus generating ATP via the resulting proton gradient. The genetically engineered bacterium of the present invention has been further modified by deleting lactate dehydrogenase, which is essential for fermentative reactions. Since the regeneration of NADH to NAD+is no longer possible, the genetically engineered bacterium of the present invention can no longer grow on minimal medium with e.g. glucose. The genetically engineered bacterium of the present invention can only grow if an external electron acceptor (substrate) and a corresponding NADH-consuming reaction are present. In this way, growth is directly coupled to a biotechnologically usable whole-cell catalysis. It should be clarifiled that the advantage of the genetically engineered bacterium of the present invention does not lie in the use of a strain for reductive whole-cell biocatalysis, but in the fact that the growth of the strain used is absolutely dependent on the conversion of a substrate into an industrially interesting product.

[0039] The genetically engineered bacterium of the present invention is useful for screening and adaptive evolution approaches, which can be used as follows:

[0040] - into a process where the growth of the strain depends on the formation of the product.

[0041] - in adaptive evolution experiments for selection on faster rates of known activities.

[0042] - adaptive evolution experiments for changes in substrate specificity (selection for growth yes or no), e.g. on minimal medium agar plates.

[0043] - screening experiments of libraries, e.g. generated by MAGE or CRISPR, or screening of rationally produced enzymes, e.g. on minimal medium agar plates. Optimally, in this whole-cell catalysis, the entire carbon source (e.g. sugar or glycerol) is oxidized and the electrons are bound in NADH and then transferred to the desired catalytic reactant. A maximum of 1 mol product / mol substrate can be formed (e.g. in a simple reduction).

[0044] The inventors of the present invention have established a proof of concept for the usability of the strain for the selection of NADH-consuming or quinol-forming reactions.

[0045] Since the described E. coli strain cannot transfer electrons from NADH into the electron transport chain, oxygen limitations do not play a role, which is particularly important in oxigenase or peroxidase reactions. In addition, the electrons originating from the substrates are to a very large extent available for the reduction. Many biocatalytic reductions are carried out as processes in vitro, using as electron donors substances that are either relatively expensive (formate), lead to toxic intermediates (methanol), or contribute only a small fraction of their electrons to the process (glucose when using glucose dehydrogenase). The above points summarize the following advantages over currently used whole cell biocatalysis systems: - Oxygen tolerance (important for oxigenases), no competition with high-affinity cytochrome oxidases - High NADH availability - Selection of product formation - Evolution of optimized conversion in the process. Essentially, the genetically engineered bacterium of the present invention is a strain where the main NADH dehydrogenases (Ndh & Nuo) are deleted together with lactate dehydrogenase (LdhA). This results in a strain that cannot grow on glycolytic substrates anymore, as it cannot ferment (Figure 1).

[0046] The genetically engineered bacterium carries preferably deletions in ndh, nuoEFG and IdhA and was called “MiniKat”. The strain cannot use substrate level phosphorylation or the electron transport chain for ATP generation. Although this minimal setup of deletions results in the desired phenotype additionally deleting further genes ensure that the strain can be used for long-term cultivations without the possibility to mutate or activate bypasses. The additional deletions can be roughly grouped into

[0047] Group A: NADH-dehydrogenase(s): ndh, nuoEFG;

[0048] Group B: NADPH dehydrogenase activity described: kefF, wrbA, yieF, mdaB, ygiN;

[0049] Group C: quinone reducing reactions from central metabolism: poxB, sdhABCD;

[0050] Group D: reactions of NADH dehydrogenase bypassing activities: mqo, mdh, did, putA, glcDEF, dadA, fadE, IldD, glpABC, IhgO, gpsA (for glycerol processes) or alternatively glpD.

[0051] The resulting strain could be classified as an auxotrophic strain for redox factors NAD+and NADP+as well as ATP. Note that the strain in principle still has the ability to synthesize NAD+, NADP+ or ATP and therefore exhibits a special form of auxotrophy as it normally relies on the recycling of these molecules from NADH, NADPH or ADP / AMP, respectively.

[0052] Due to its phenotype, the genetically engineered bacterium of the present invention does not grow (i) in minimal media (M9), casamino acid supplemented minimal media (M9 + casein hydrolysate), or complex medium such as LB. Since E. coli possesses an additional quinone dependent dehydrogenase, GCD, a Pyrrolochinolinchinon- dependent (PQQ) sugar dehydrogenase, that oxidizes sugars into their corresponding sugar acids while reducing PQQ, which transfers the electrons to the electron transport chain. Hence, upon supplementation of PQQ (a cofactor E. coli cannot synthesize itself), the strains were able to grow on glucose or xylose media again.

[0053] For applications, the genetically engineered bacterium of the present invention represents an immense playground as it allows for the growth-coupled selection for ATP as well as NAD+ / NADP+ producing reactions. Definitions

[0054] The term “NADH dehydrogenase”, as used herein, relates to an enzyme of EC class 1 .6.5.11 .that catalyzes the chemical reaction

[0055] NADH + H++ a quinone NAD++ a quinol

[0056] The term “nuo gene”, as used herein, relates to a gene that encodes one subunit of the protein complex NADH:ubiquinone oxidoreductase I (NuoABCDEFGHIJKLMN, NDH-1 ). The nuo genes in E. coli include 14 genes: nuoABCDEFGHIJKLMN genes, encoding 13 - 14 subunits, nuoC and nuoD are thought to form one subunit. Thus, NADH:ubiquinone oxidoreductase I of E. coli is made up of 13 - 14 different subunits, the NuoA to NuoN subunits. This organization in 13-14 different subunits is conserved in several other bacteria, including Salmonella typhimurium, Paracoccus denitrificans, Rhodobacter capsulatus, and Thermus thermophilus. The nuoA gene encodes the subunit NuoA of NDH-1. The nuoB gene encodes the subunit NuoB of NDH-1. The nuoC gene encodes the subunit NuoC of NDH-1 and the nuoD gene encodes the subunit NuoD of NDH-1 , or nuoC and nuoD genes encode the nuoCD subunit. The nuoE gene encodes the subunit NuoE of NDH-1. The nuoF gene encodes the subunit NuoF of NDH-1. The nuoG gene encodes the subunit NuoG of NDH-1. The nuoH gene encodes the subunit NuoH of NDH-1. The nuol gene encodes the subunit Nuol of NDH-1. The nuoJ gene encodes the subunit NuoJ of NDH-1. The nuoK gene encodes the subunit NuoK of NDH-1. The nuoL gene encodes the subunit NuoL of NDH-1. The nuoM gene encodes the subunit NuoM of NDH-1. The nuoN gene encodes the subunit NuoN of NDH- 1 ,NADH:ubiquinone oxidoreductase I (NDH-1 ) of E. co / / consists of three components: a soluble fragment composed of the NuoE, F and G subunits, an amphipathic connecting fragment composed of the NuoB, CD and I subunits, and a hydrophobic membrane fragment composed of the NuoA, H, J, K, L, M and N subunits. Thereby, the soluble NADH:ubiquinone oxidoreductase I fragment consisting of the NuoE, NuoF and NuoG subunits represents the electron input part of NADH:ubiquinone oxidoreductase I.

[0057] The term “NADH:ubiquinone oxidoreductase I” (NDH-1), as used herein, relates to a protein complex of the respiratory chains of many organisms from bacteria to humans. NADH:ubiquinone oxidoreductase I belongs to EC class 7.1.1.2. In E. coli, NADH:ubiquinone oxidoreductase I (NDH-1 ) is one of two distinct NADH dehydrogenases that catalyze the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane and is able to generate a proton electrochemical gradient.

[0058] The term “ndh gene”, as used herein, relates to a gene that encodes the enzyme NADH:quinone oxidoreductase II. The term “NADH:quinone oxidoreductase II” (NDH-2), as used herein, relates to an alternative, non-proton pumping NADH:quinone oxidoreductase that delivers electrons to the respiratory chain by oxidation of NADH and reduction of quinones. Thus, NADH:quinone oxidoreductase II is one of two distinct NADH dehydrogenases that catalyze the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane but does not generate an electrochemical gradient as NDH-1 does. NADH:quinone oxidoreductase II. NADH:ubiquinone oxidoreductase II belongs to EC class 1 .6.5.9.

[0059] The term “KefF”, (synonym: yabF), as used herein, relates to the Glutathione-regulated potassium-efflux system ancillary protein. KefF is an activator of potassium transport mediated by the KefC antiporter. KefF also has enzymatic activity as a quinone oxidoreductase, thereby reducing the redox toxicity of electrophilic quinones.

[0060] The term “wrbA gene”, as used herein, relates to a gene that encodes a NAD(P)H dehydrogenase (quinone).

[0061] The term “WrbA”, as used herein, relates to a protein that has NAD(P)H:quinone oxidoreductase activity. WrbA is related to the flavodoxin family of proteins. Unlike the flavodoxins, WrbA does not have a stabilized semiquinone state. It rapidly takes up two electrons, generating the fully reduced form.

[0062] The term “yieF gene” (synonym: chrR), as used herein, relates to a gene that encodes a quinone reductase.

[0063] The term “YieF”, as used herein, relates to a flavoprotein containing the FMN cofactor that belongs to the flavodoxin superfamily of enzymes. YieF was shown to possess quinone reductase activity which may guard against oxidative stress by preventing redox cycling of quinones which would otherwise generate ROS, and by maintaining a pool of reduced quinone in the cell that is able to quench ROS directly. The quinone reductase activity of YieF is considered as the primary biological role of this enzyme.

[0064] The term “ygiN gene”, as used herein, relates to a gene in E. coli that encodes the probable quinol monooxygenase YgiN. The ygiN gene may be transcribed in an operon together with mdaB, indicated as mdaB-ygiN.

[0065] The term “YgiN”, as used herein, relates to a protein “probable quinol monooxygenase” that is able to re-oxidize menadiol that has been reduced by “MdaB quinone reductase” in vitro. The two enzymes “probable quinol monooxygenase” and “MdaB quinone reductase” may form a quinone redox cycle. The biological role of a quinone redox cycle is considered to maintain an intracellular pool of menadione and ubiquinone using a catalytic mechanism that avoids the formation of a semiquinone intermediate, and to act as a quinone buffer.

[0066] The term mdaB gene”, as used herein, relates to a gene that encodes the NADPH:quinone oxidoreductase MdaB.

[0067] The term “MdaB”, as used herein, relates to the protein “MdaB quinone reductase” that is specific for NADPH and is most active with quinone derivatives and ferricyanide as electron acceptors. In vitro, YgiN is able to reoxidize menadiol that has been reduced by MdaB quinone reductase; the two enzymes may form a quinone redox cycle.

[0068] The term mdaB-ygiN genes” or mdaB-ygiN operon”, as used herein, relates to the genes or operon encoding a NADPH:quinone oxidoreductase and a “probable quinol monooxygenase” presumably forming a quinone redox cycle.

[0069] The term “putA gene” (synonym: poaA), as used herein, relates to a gene in that encodes the bifunctional protein PutA.

[0070] The term “PutA”, as used herein, relates to the bifunctional protein PutA that is involved in step 1 and 2 of the sub-pathway that synthesizes L-glutamate from L-proline. The bifunctional protein PutA includes the domains proline dehydrogenase and pyrroline-5- carboxylate dehydrogenase. PutA is a flavoprotein with mutually exclusive functions as a transcriptional repressor and membrane-associated enzyme. The switch between the two activities is due to conformational changes triggered by the redox state of FAD. In the presence of L-proline, PutA is associated with the cytoplasmic membrane and acts a bifunctional enzyme catalyzing both reactions of the proline degradation pathway: the oxidation of proline by proline dehydrogenase and subsequent oxidation to glutamate by pyrroline-5-carboxylate (P5C) dehydrogenase. The kinetics of the coupled reaction is best described by substrate channeling. In the absence of proline, PutA is cytoplasmic and functions as a transcriptional repressor of the put regulon. Proline dehydrogenase activity requires the presence of an electron acceptor. The reaction is split into a reductive half reaction, the reduction of the FAD cofactor by oxidation of proline, and an oxidative half reaction, the re-oxidation of reduced FADH2 by transfer of electrons to the quinone pool in the cytoplasmic membrane.

[0071] The term “glcDEF genes” (synonyms: gox, yghM), as used herein, relates to the genes glcD, glE, and glcF that encode the subunits of the enzyme glycolate oxidase. More in particular, the term “GlcDEF”, as used herein, relates to the three subunits of the enzyme glycolate oxidase GlcD, GlcE and GlcF. GlcDEF is a component of a complex that catalyzes the oxidation of glycolate to glyoxylate. GlcDEF is required by bacteria to grow on glycolate as a sole carbon source. The ability to oxidize D-lactate has been described in connection with GlcDEF. GlcDEF does not link directly to O2.

[0072] The term “did gene”, as used herein, relates to a gene that encodes the enzyme quinone-dependent D-lactate dehydrogenase.

[0073] The term “Did”, as used herein, relates to the enzyme quinone-dependent D-lactate dehydrogenase that is a FAD-dependent peripheral membrane dehydrogenase catalyzing the oxidation of D-lactate to pyruvate. D-lactate dehydrogenase (Did) is a respiratory enzyme; electrons derived from D-lactate oxidation are transferred to the membrane soluble quinone pool.

[0074] The term mqo gene” (synonym: yojH), as used herein, relates to a gene that encodes the enzyme malate:quinone oxidoreductase (Mqo).

[0075] The term “Mqo”, as used herein, relates to the enzyme the malate:quinone oxidoreductase which is a membrane-associated enzyme that catalyzes the oxidation of malate to oxaloacetate. Electrons are likely donated to the electron transfer chain at the quinone level.

[0076] The term “mdh gene”, as used herein, relates to a gene that encodes the enzyme malate dehydrogenase (Mdh).

[0077] The term “Mdh”, as used herein, relates to the enzyme the malate dehydrogenase which catalyzes the chemical reaction of L-malate to oxaloacetate.

[0078] The term dadA gene” (synonym: dadR), as used herein, relates to a gene that encodes the enzyme D-amino acid dehydrogenase (DadA).

[0079] The term “DadA”, as used herein, relates to the enzyme D-amino acid dehydrogenase. E L- and D-alanine can be used by bacteria as the sole source of carbon, nitrogen and energy. D-amino acid dehydrogenase is the second enzyme of the L-alanine degradation I pathway. The enzyme has broad substrate specificity; it catalyzes the oxidative deamination of many D-amino acids, although D-alanine is the best substrate. The enzyme is membrane-associated and linked to the respiratory chain. The term “fadE gene” (synonym: yafH), as used herein, relates to a gene that encodes the enzyme Acyl-coenzyme A dehydrogenase (FadE). fadE mutants are unable to utilize oleate and other fatty acids as the sole source of carbon.

[0080] The term “FadE”, as used herein, relates to the enzyme Acyl-coenzyme A dehydrogenase that catalyzes the first step in the degradation of fatty acids via the [3- oxidation cycle.

[0081] The term glpD gene” (synonym: yafH), as used herein, relates to a gene that encodes aerobic glycerol-3-phosphate dehydrogenase (GlpD).

[0082] The term “GlpD”, as used herein, relates to the enzyme aerobic glycerol-3-phosphate dehydrogenase that catalyzes the oxidation of sn-glycerol 3-phosphate to dihydroxyacetone phosphate. GlpD is a respiratory enzyme and shuttles electrons via a non-covalently bound FAD cofactor to reduce ubiquinone. Glycerol 3-phosphate is an obligatory intermediate in phospholipid biosynthesis and thus glpD expression is regulated to ensure that phospholipid biosynthesis is maintained while the energy needs of the cell are met. GlpD is required for aerobic growth with glycerol or glycerol 3- phosphate.

[0083] A glpD deletion is not essential to metabolize a particular carbon source, such as a carbon source selected from the group comprising glucose, fructose, xylose, arabinose, galactose, mannose, sucrose, cellobiose, and hemicellulose or a combination thereof. However, a glpD deletion seems to induce higher stability in bacteria when growing on a carbon source selected from the group comprising glucose, fructose, xylose, arabinose, galactose, mannose, sucrose, cellobiose, and hemicellulose. In contrast, a glpD deletion seems to be necessary in E. coli strain growing on a carbon source being glycerol.

[0084] The term IldD gene” (synonym: IctD), as used herein, relates to a gene that encodes L- lactate dehydrogenase.

[0085] The term “LldD”, as used herein, relates to the enzyme L-lactate dehydrogenase that is an FMN-dependent membrane-associated dehydrogenase. It functions in aerobic respiration and also has a role in anaerobic nitrate respiration. L-lactate dehydrogenase is associated with the inner membrane. LldD is one of three lactate dehydrogenase enzymes which interconvert pyruvate and lactate. The other two enzymes are specific for D-lactate: the soluble LdhA, an NAD-linked fermentative enzyme, and Did, a membrane-associated respiratory enzyme. L-lactate dehydrogenase is induced by aerobic growth on L-lactate and L-fucose and in the presence of lactate under nitrate, fumarate and TMAO respiration conditions. The term “glpABC genes”, as used herein, relates to the genes that encode anaerobic glycerol-3-phosphate dehydrogenase (GlpABC).

[0086] The term “GlpABC”, as used herein, relates to the enzyme anaerobic glycerol-3- phosphate dehydrogenase that catalyzes the oxidation of glycerol-3-phosphate to dihyroxyacetone phosphate. GlpABC is a respiratory enzyme; anaerobic growth of bacteria on glycerol and fumarate induces expression of an anaerobic glycerol-3- phosphate dehydrogenase and fumarate reductase and is associated with proton translocation and the generation of a proton motive force. The GlpABC enzyme is loosely associated with the cell membrane. Bacteria, such as E. coli K-12, contain two glycerol-3-phosphate dehydrogenases encoded by the glpABC and glpD genes. GlpABC is required for anaerobic growth with glycerol or glycerol-3-phosphate and fumarate as the terminal electron acceptor while GlpD is required for aerobic growth with glycerol (or glycerol-3-phosphate).

[0087] The term “IhgO gene” (synonym: IhgD), as used herein, relates to a gene that encodes- 2-hydroxyglutarate dehydrogenase (LhgO, synonym LhgD).

[0088] The term “LhgO”, as used herein, relates to the enzyme L-2-hydroxyglutarate dehydrogenase which is an electron transport chain-coupled dehydrogenase that feeds electrons from the reaction into the membrane quinone pool. LhgD contains an FAD cofactor which is not covalently attached, and whose reduction potential is relatively high at -25 mV. LhgD is associated with the cytoplasmic membrane, and its activity is only found in the membrane fraction.

[0089] The term “poxB gene”, as used herein, relates to a gene that encodes pyruvate dehydrogenase [ubiquinone] (PoxB).

[0090] The term “PoxB”, as used herein, relates to the enzyme pyruvate dehydrogenase [ubiquinone] which is a peripheral membrane enzyme that catalyzes the oxidative decarboxylation of pyruvate to form acetate and CO2. The reaction is coupled to the electron transport chain via ubiquinone. Metabolism of pyruvate by pyruvate oxidase is less efficient than the route via pyruvate dehydrogenase (PDH); however, the pyruvate oxidase route is important for wild-type growth efficiency and responsible for a significant amount of pyruvate metabolism under aerobic conditions.

[0091] The term “sdhABCD genes”, as used herein, relates to the genes encoding succinate dehydrogenase or succinate:quinone oxidoreductase (SdhABCD). The term “SdhABCD”, as used herein, relates to the enzyme succinate:quinone oxidoreductase that catalyzes the oxidation of succinate to fumarate concomitant with the reduction of ubiquinone to ubiquinol. SdhABCD plays an important role in cellular metabolism and directly connects the TCA cycle with the respiratory electron transport chain. As part of the TCA cycle succinate is oxidized to fumarate by SdhABCD and electrons are transferred to the membrane quinone pool for entry into the electron transport chain. SdhABCD does not contribute to the proton motive force; the sites of quinol reduction and succinate oxidation are both located on the cytoplasmic side of the membrane and there is no separation of charge across the membrane during catalysis. SdhABCD is a membrane bound heterotetramer. Subunits SdhA and SdhB are hydrophilic and attached to the cytoplasmic surface of the plasma membrane via interactions with the two hydrophobic integral membrane subunits, SdhC and SdhD. SdhA contains the FAD cofactor and the dicarboxylic acid binding site. Electrons from the oxidation of succinate are transferred through the iron-sulphur protein, SdhB, to a quinone binding site located at the interface of the SdhB, SdhC and SdhD subunits. The SdhC and SdhD subunits each contain three transmembrane helices and anchor the complex to the membrane. A single heme b556 cofactor bridges the SdhC and SdhD subunits.

[0092] The term gpsA gene”, as used herein, relates to a gene that encodes the enzyme glycerol-3-phosphate dehydrogenase (GpsA).

[0093] The term “GpsA”, as used herein, relates to the enzyme glycerol-3-phosphate dehydrogenase catalyzes the NAD(P)H-dependent reduction of the glycolytic intermediate dihydroxyacetone-phosphate to produce glycerol-3-phosphate, a precursor for the biosynthesis of phospholipids.

[0094] The term “IdhA gene”, as used herein, relates to a gene that encodes the enzyme D- lactate dehydrogenase (LdhA).

[0095] The term “LdhA”, as used herein, relates to the enzyme D-lactate dehydrogenase. LdhA is a soluble NAD-linked lactate dehydrogenase (LDH) that is specific for the production of D-lactate.

[0096] The term ubiC gene” as used herein, relates to a gene that encodes the enzyme chorismate pyruvate-lyase (UbiC).

[0097] The term “UbiC”, as used herein, relates to the enzyme chorismate pyruvate-lyase that catalyzes the first reaction step in the biosynthesis of ubiquinone which involves the formation of 4-hydroxybenzoate from chorismate. ubiC mutants are deficient in the formation of ubiquinone and are characterized by the inability to grow aerobically on oxidizable substrates such as succinate.

[0098] The term ubiA gene” as used herein, relates to a gene that encodes the enzyme 4-hydroxybenzoate octaprenyltransferase (UbiA).

[0099] The term “UbiA”, as used herein, relates to the enzyme 4-hydroxybenzoate octaprenyltransferase that catalyzes the second reaction step in the biosynthesis of ubiquinone which involves the prenylation of 4-hydroxybenzoate with an all-trans polyprenyl group.

[0100] The term ubiCA genes” or "ubiCA operon" as used herein, relates to the operon encoding the enzymes chorismate lyase and 4-hydroxybenzoate transferase for the first two committed steps of ubiquinone (also named Coenzyme Q, or Coenzyme Q10, UQ) biosynthesis.

[0101] The term “chinone species”, as used herein, relates to chinone compounds produced by bacteria. Quinones function as membrane bound electron carriers between dehydrogenases and oxidases in the aerobic and anaerobic respiratory chains. For example, the electron transport chain of E. coli contains three different quinone species, ubiquinone (UQ), menaquinone (MK) and demethylmenaquinone (DMK). Quinones are conjectured to occur in all respiring organisms. Some serve as electron acceptors in electron transport chains such as those in aerobic respiration (ubiquinone).

[0102] The term “Escherichia coli’ (E. coli), as used herein, relates to the well-known Gramnegative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia. The term “Escherichia coli strain”, (E. coli strain) as used herein, relates to a subgroup within the species that has unique characteristics that distinguish it from other strains. The E. coli K-12 and B strains are well-known and used routinely in molecular biology as both a tool and a model organism. The E. coli strains K-12, B, C, and W are thought of as model organism strains. These are classified in Risk Group 1 in biosafety guidelines. The term “Escherichia coli K-12 strain”, (E. coli K-12 strain) as used herein, relates to an E. coli strain isolated from a stool sample of a patient convalescent from diphtheria and was labelled K-12 in 1922 at Stanford University. The “Escherichia coli K-12 strain MG1655”, Genotype: F- lambda- ilvG- rfb-50 rph-1 Serotype: OR:H48:K-was sequenced by the Blattner laboratory because it approximates wild-type E. coli and "has been maintained as a laboratory strain with minimal genetic manipulation, having only been cured of the temperate bacteriophage lambda and F plasmid by means of ultraviolet light and acridine orange, respectively." The mutations listed in the genotype are present in most E. coli K-12 strains and were probably acquired early in the history of the laboratory strain. An extensive list of Escherichia coli K-12 strain derivatives and their individual construction, genotypes, phenotypes, plasmids and phage information can be viewed at Ecoliwiki. E. coli MG1655 has been engineered to express the genes encoding an arabinose inducible lambda Red recombineering system and a rhamnose inducible flippase recombinase to allow fast turnover for multiple deletions. However, the present invention is not restricted to a particular E.coli strain and different E. coli strains are suitable for carry out the invention.

[0103] Preferred E. coli strains within the scope of the present invention are selected from the group comprising: E. coli MG1655, BW25113 strain, W3110 strain, JM109 strain, B21 strain, BL21 (D3) strain, HMS174 strain, RV308(DE3) strain, and AD494 strain.

[0104] Thus, the present invention relates to a genetically engineered Escherichia coli as disclosed above, wherein the Escherichia coli is an E. coli MG1655 or an E. coli MG1655 further comprising one or more recombination systems. Preferred recombination systems for the present invention are lambda Red recombinases, Rec recombinases, ET recombination, flippase recombinase.

[0105] The term "evolved strain" refers to a bacterial strain that has been cultivated for longer time in culture medium under standard culture conditions selecting for an improvement of growth characteritics. Said process is referred to as "adaptative laboratory evolution". Usually "adaptative laboratory evolution" requires a prolonged bacterial cultivation.

[0106] The recombinase system of lambda Red recombinases comprises three phage-derived lambda Red proteins: Gam, Exo and Beta, which are necessary to complete dsDNA recombination (Murphy, K.C. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180, 2063-2071 (1998) , Gam prevents degradation of foreign linear double stranded DNA by the E.coli nucleases, Exo degrades dsDNA to form a single stranded DNA (ssDNA) and Beta binding facilitates recombination. The exact mechanism on how a desired construct recombines with the chromosome in the presence of the three lambda Red proteins has been highly debated.

[0107] However, the genetically engineered E. coli of the present invention can be obtained also by other gene editing methods, which are known to the skilled person of the art, such as classical homologous recombination using restriction enzymes and DNA ligase, CRISPR / Cas (Robb, G B 2019. Genome editing with CRISPR-Cas: an overview. Current Protocols Essential Laboratory Techniques, 19, e36). In alternatively preferred embodiments, the present invention related to a genetically engineered E. coli as described above, wherein the E. coli is an E. coli MG1655. In more preferred embodiments, the present invention related to a genetically engineered E. coli as described above, wherein the E. coli is an E. coli MG1655 further comprising one or more recombination systems selected from the group comprising lambda Red recombinases, Rec recombinases, and flippase recombinase.

[0108] The inventors of the present invention suggested that the controlled regulation of oxygen uptake without completely preventing it will allow the genetically engineered bacteria to produce ATP from O2 if reduced ubiquinone (Q) is produced. Thus, the deletion of the nuo genes and ndh gene were initially targeted in the bacteria, as the NADH:ubiquinone oxidoreductase I (NDH-1 , NuoABCDEFGHIJKLMN) and NADH:quinone oxidoreductase II (NDH-2) are the two distinct NADH dehydrogenases that catalyze the oxidation of NADH to NAD and mainly catalyze the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane.

[0109] Thus, a genetically engineered bacterium having a deletion of ndh gene and of one or more nuo genes was obtained in the first step. In particular, a deletion of ndh gene and of nuoEFG genes was performed in an E. coli K-12 strain MG1655. The so obtained genetically engineered Anuo, AnuoEFG E. coli strain did not show a reduction in biomass production compared to the wild type. Thus, further manipulations were required.

[0110] It has been described in the prior art that the deletion of the three cytochrome oxidases, i.e. a deletion of the cyoABCD genes, cydAB genes and cbdAD (appBC) genes and ygiN gene results in utilization of anaerobic respiration pathways even under aerobic growth conditions and in a shift in the quinone pool from ubiquinones to menaquinones. The disruption of the biosynthesis of ubiquinone in E. coli by deletion of one or more ubi genes leads to a shift in the quinone pool from ubiquinones to menaquinones, as the ubiquinones are not available anymore. However, the inventors have found that in presence of oxygen the E. coli strains having a deletion of one or more ubi genes try to compensate for the lack of ubiquinone in other ways, e.g. evolutionary adaptions and mutations.

[0111] Thus, for provision of the desired genetically engineered bacterium a different strategy was necessary. The inventors of the present invention have first identified further suitable enzymes that catalyze quinone-dependent reactions for further gene deletions in Anuo, AnuoEFG E. coli strain. The following genes have been selected for deletions in the Anuo, A nuoEFG bacteria: kefF gene, wrbA gene, yieF gene, mdaB-ygiN genes, genes putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, IldD gene, glpABC genes, glpD gene, IhgO gene, poxB gene, sdhABCD genes and gpsA gene.

[0112] The genetically engineered bacterium according to the present invention particularly does not have the disadvantages of instability of the genetically engineered bacteria AubiCA and Anuo, AnuoEFG, AubiCA.

[0113] Thus, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, and a deletion of IdhA gene, preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene, wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0114] Preferably, the present invention relates to a genetically engineered bacterium (“MiniKat”) comprising a of ndh gene, a deletion of one or more nuo genes, and a deletion of IdhA gene, wherein the one or more nuo genes are selected from nuoE gene, nuoF gene, and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0115] With other words, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, and a deletion of IdhA gene, and a deletion of nuoEFG genes (i.e. nuoE gene, nuoF gene, and nuoG gene).

[0116] In a further embodiment the genetically engineered bacterium according to the present invention may further comprise a deletion of nuoEFG together with deletion of the nuo genes nuoA gene, nuoB gene, nuoCD genes, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene. Thus, in further embodiments of the present invention the genetically engineered bacterium may comprises a deletion of ndh gene, a deletion of nuoEFG genes, a deletion of IdhA gene, preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene, and further comprising a deletion of one or more nuo genes selected from the group nuoA gene, nuoB gene, nuoCD gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0117] Thus, the present invention further relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and a deletion of glpD gene, wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0118] In preferred embodiments, the present invention further relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and a deletion of gpsA gene, wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0119] In a preferred embodiment, the genetically engineered bacterium may further comprise a deletion of did gene, and mqo gene. Preferably, the present invention preferably relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, a deletion of mqo gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0120] In a preferred embodiment, the genetically engineered bacterium may further comprise a deletion of did gene, and mdh gene. Preferably, the present invention preferably relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, a deletion of mdh gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0121] In a preferred embodiment, the genetically engineered bacterium may further comprise a deletion of poxB, and sdh genes enconding for enzymes whose activities are directly coupled to central metabolism. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0122] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, a deletion of mqo gene; a deletion of poxB gene and / or of sdhABCD gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0123] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, a deletion of mdh gene; a deletion of poxB gene and / or of sdhABCD gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0124] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of glcDEF genes, IhgO gene and / or putA gene. Preferably, the present invention preferably relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of glcDEF genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0125] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of glcDEF genes, IhgO gene and / or putA gene. Preferably, the present invention preferably relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, mqo gene, glcDEF genes, IhgO gene and / or putA gene. Preferably, the present invention preferably also relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, mdh gene, glcDEF genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferably, the present invention preferably relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene and / or putA gene. Preferably, the present invention preferably also relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0126] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of IldD gene and / or dadA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0127] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene; and further comprising a deletion of IldD gene and / or dadA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0128] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mqo gene; poxB gene and / or of sdhABCD gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0129] Preferably, the present invention also relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mdh gene; poxB gene and / or of sdhABCD gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0130] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0131] Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0132] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of kefF gene and / or mdaB-ygiN genes. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0133] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of kefF gene and / or mdaB-ygiN genes. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0134] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of glpABC genes and / or of gpsA gene. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, glpABC genes, gpsA gene, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0135] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of glpABC genes and / or of gpsA gene. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, glpABC genes, gpsA gene, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0136] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of wrbA gene and / or of yieF gene. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, glpABC genes, gpsA gene, wrbA gene yieF gene, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0137] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of wrbA gene and / or of yieF gene. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, glpABC genes, gpsA gene, wrbA gene yieF gene, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0138] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of fadE gene. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, glpABC genes, gpsA gene, wrbA gene yieF gene, fadE gene, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0139] In preferred embodiments, the genetically engineered bacterium may further comprise a deletion of fadE gene. Preferably, the present invention relates to a genetically engineered bacterium comprising a deletion of ndh gene, a deletion of one or more nuo genes, a deletion of IdhA gene, and further comprising a deletion of IldD gene, dadA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, kefF gene, mdaB-ygiN genes, glpABC genes, gpsA gene, wrbA gene yieF gene, fadE gene, IhgO gene and / or putA gene. Preferably, the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene and nuoN gene, more preferably nuoE gene, nuoF gene and nuoG gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0140] A stable genetically engineered bacterium was obtained by deletion of the nuo and ndh gene IdhA gene along with one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF gene, did gene, mqo gene, dadA gene, fadE gene, IldD gene, glpABC gene, IhgO gene, poxB gene, sdhABCD gene, and gpsA gene, instead of the ubiCA genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0141] A stable genetically engineered bacterium was obtained by deletion of the nuo and ndh gene IdhA gene along with one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF gene, did gene, mdh gene, dadA gene, fadE gene, IldD gene, glpABC gene, IhgO gene, poxB gene, sdhABCD gene, and gpsA gene, instead of the ubiCA genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0142] This genetically engineered bacterium is still able to synthesize ubiquinone via the common ubiquinone biosynthesis pathway. The genes kefF, wrbA, yieF, mdaB encode enzymes with NADPH dehydrogenase activity, the genes putA, glcDEF, did, mqo, dadA, fadE, gpsA, IldD, glpABC, IhgO encode enzymes with reactions of NADH dehydrogenase bypassing activities and the genes poxB, sdhABCD encode enzymes with quinone reducing reactions from central metabolism. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0143] The genetically engineered bacterium according to the present invention preferably comprises a deletion of at least one of the nuo genes selected from nuoE gene, nuoF gene, and nuoG gene, and the ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0144] The genetically engineered bacterium according to the present invention preferably comprises a deletion of at least one of the nuo genes selected from nuoE gene, nuoF gene, and nuoG gene, and the ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0145] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0146] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0147] In a further preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of at least one of the nuo genes selected from nuoE gene, nuoF gene, and nuoG gene, and a deletion of ndh gene, and IdhA gene, and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF gene, did gene, mqo gene, dadA gene, fadE gene, IldD gene, glpABC genes, IhgO gene, poxB gene, sdhABCD genes, gpsA gene, but comprises glpD gene. Thus, in such preferred embodiments the genetically engineered bacterium does not comprise a deletion of glpD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0148] In a further preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of at least one of the nuo genes selected from nuoE gene, nuoF gene, and nuoG gene, and a deletion of ndh gene, and IdhA gene, and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF gene, did gene, mdh gene, dadA gene, fadE gene, IldD gene, glpABC genes, IhgO gene, poxB gene, sdhABCD genes, gpsA gene, but comprises glpD gene. Thus, in such preferred embodiments the genetically engineered bacterium does not comprise a deletion of glpD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0149] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, IldD gene, glpABC genes, IhgO gene, poxB gene, sdhABCD genes, gpsA gene, but comprises glpD gene. Thus, in such preferred embodiments the genetically engineered bacterium does not comprise a deletion of glpD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0150] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, IldD gene, glpABC genes, IhgO gene, poxB gene, sdhABCD genes, gpsA gene, but comprises glpD gene. Thus, in such preferred embodiments the genetically engineered bacterium does not comprise a deletion of glpD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0151] An alternative genetically engineered bacterium according to the present invention preferably comprises a deletion of at least one of the nuo genes selected from nuoE gene, nuoF gene, and nuoG gene, and ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0152] An alternative genetically engineered bacterium according to the present invention preferably comprises a deletion of at least one of the nuo genes selected from nuoE gene, nuoF gene, and nuoG gene, and ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0153] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0154] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of one or more genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0155] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of kefF gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0156] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of wrbA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0157] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of yieF gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0158] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of mdaB gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0159] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of putA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0160] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of dadA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0161] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of fadE gene. In preferred embodiments, the genetically engineered bacterium is an E. coli. In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of IldD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0162] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of glpABC genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0163] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of IhgO gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0164] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and further comprises a deletion of poxB gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0165] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0166] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of did gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0167] In a particularly preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of mqo gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0168] In a particularly preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of mdh gene. In preferred embodiments, the genetically engineered bacterium is an E. coli. In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the kefF gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0169] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the wrbA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0170] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the yieF gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0171] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the mdaB gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0172] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the putA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0173] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the dadA gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0174] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the fadE gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0175] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the IldD gene. In preferred embodiments, the genetically engineered bacterium is an E. coli. In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the glpABC genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0176] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the IhgO gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0177] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the poxB gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0178] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0179] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the did gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0180] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the mqo gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0181] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, IdhA gene, and gpsA gene and further comprises a deletion of the mdh gene. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0182] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene and IdhA gene and further comprises a deletion of at least two, three or four genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF gene, did gene, mqo gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0183] In a most preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene and IdhA gene further comprises a deletion of kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0184] In a preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene and IdhA gene and further comprises a deletion of at least two, three or four genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF gene, did gene, mdh gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0185] In a further preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene and IdhA gene further comprises a deletion of kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, gpsA gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0186] In another preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of at least two, three or four genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0187] In another preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene and further comprises a deletion of at least two, three or four genes selected from kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli. In another preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene further comprises a deletion of kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mqo gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0188] In another preferred embodiment, the genetically engineered bacterium according to the present invention preferably comprises a deletion of nuoEFG genes, ndh gene, and IdhA gene further comprises a deletion of kefF gene, wrbA gene, yieF gene, mdaB gene, putA gene, glcDEF genes, did gene, mdh gene, dadA gene, fadE gene, glpD gene, IldD gene, glpABC genes, IhgO gene, poxB gene, and sdhABCD genes. In preferred embodiments, the genetically engineered bacterium is an E. coli.

[0189] In some embodiment, instead of deleting the mqo gene, the mdh gene may be deleted as alternative as Mqo and Mdh are both involved in the same minicycle.

[0190] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously. In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0191] Thus, the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferably, the genetically engineered bacterium biosynthesizes ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. According to the invention, the genetically engineered bacterium produces the quinone species without genetic modification. Thus, the quinone species is an endogenous quinone species already produced by the wild-type strain of the genetically engineered bacterium.

[0192] Thus, the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferably, the genetically engineered bacterium expresses cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Thus, the genetically engineered bacterium expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously. With other word, the genetically engineered bacterium naturally expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating). Therefore, the one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) are endogenous enzymes alreadpy expressed by the wild-type strain of the bacterium, i.e. are expressed without genetic modifications.

[0193] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; the genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and glpD gene or the genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and glpD gene.

[0194] In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK), more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0195] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene, wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; the genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and gpsA gene or did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0196] In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0197] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; wherein the nuo genes are selected from nuoEFG genes.

[0198] In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0199] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; the genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and glpD gene, or did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and glpD gene, wherein the nuo genes are selected from nuoEFG genes.

[0200] In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0201] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; the genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene, or did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene, wherein the nuo genes are selected from nuoEFG genes. In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0202] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; wherein the genetically engineered bacterium further comprises a deletion of did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene, or wherein the genetically engineered bacterium further comprises a deletion of did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene, wherein the nuo genes are selected from nuoEFG genes.

[0203] In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8.

[0204] Thus, the present invention relates to a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH: ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously, preferably ubiquinone-8; and wherein the genetically engineered bacterium is capable of expressing one ore more of ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously, more preferably wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and / or cytochrome bd-\\ oxidase (AppBC) endogenously; wherein the nuo genes are selected from nuoEFG genes wherein the genetically engineered bacterium further comprises a deletion of did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene or wherein the genetically engineered bacterium further comprises a deletion of did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0205] In preferred embodiments, the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Previously, the inventors have found an obligate aerobic fermentative strain called “NNmini”. This strain allowed the re-balancing of otherwise unbalanced fermentations. The inventors could show the growth-coupled production of D-lactate as well as isobutanol from glycolytic carbon sources as well as reduced carbon sources like glycerol.

[0206] Here, the inventors present a unique metabolism and further modifications and applications of the NNmini strain.

[0207] Essentially, the NNmini strain is a strain where the main NADH dehydrogenases (Ndh & Nuo) are deleted together with the quinone-dependent lactate dehydrogenase (Did). This forces the strain to produce lactate as a fermentation product even in the presence of oxygen.

[0208] Here the inventors present a different approach. Instead of deleting Did, the inventors have deleted its NADH-dependent counterpart, LdhA. This results in a strain that cannot grow on glycolytic substrates anymore, as it cannot ferment (Figure 1).

[0209] The resulting strain carries deletions in ndh gene, nuoEFG genes, and IdhA gene and was called “MiniKat”. The strain cannot use substrate level phosphorylation or the electron transport chain for ATP generation. Although this minimal setup of deletions results in the desired phenotype, it is recommended additionally deleting the genes as decribed herein to ensure that the strain can be used for long-term cultivations without the possibility to mutate or activate bypasses. The deletions as decribed herein can be roughly grouped into

[0210] Group A: NADH-dehydrogenase(s): ndh, nuoEFG;

[0211] Group B: NADPH dehydrogenase activity described: kefF, wrbA, yieF, mdaB, ygiN;

[0212] Group C: quinone reducing reactions from central metabolism: poxB, sdhABCD',

[0213] Group D: reactions of NADH dehydrogenase bypassing activities: mqo, mdh, did, putA, glcDEF, dadA, fadE, glpD, HdD, glpABC, IhgO, gpsA (important for glycerol processes)

[0214] The resulting strain could be classified as an auxotrophic strain for redox factors NAD+ and NADP+ as well as ATP. Note that the strain in principle still has the ability to synthesize NAD+, NADP+ or ATP and therefore exhibits a special form of auxotrophy as it normally relies on the recycling of these molecules from NADH, NADPH or ADP / AMP, respectively.

[0215] Due to its phenotype, both the MiniKat strain and the MaxKat strain do not grow (i) in minimal media (M9), casamino acid supplemented minimal media (M9 + casein hydrolysate), or complex medium such as LB. However, E. coli possesses an additional quinone dependent dehydrogenase, GCD, a Pyrrolochinolinchinon-dependent (PQQ) sugar dehydrogenase, that oxidizes sugars into their corresponding sugar acids while reducing PQQ, which transfers the electrons to the electron transport chain. Hence, upon supplementation of PQQ (a cofactor E. coli cannot synthesize itself), the strains were able to grow on glucose or xylose media again (Figure 2).

[0216] For applications, the MiniKat strain and the Maxkat strain represent an immense playground as both allow for the growth-coupled selection for ATP as well as NAD+ / NADP+ producing reactions.

[0217] Uses

[0218] When ndh gene, nuoEFG genes and IdhA gene were deleted, growth on glucose as well as glucose and acetate was abolished. However, when PQQ was added to the medium, growth could be restored. This is due to the activity of Quinoprotein glucose dehydrogenase (gcd). This enzyme is constitutively expressed by E. coli as an apoenzyme, which only becomes a holoenzyme when PQQ is supplied to the medium as E. coli lacks the biosynthetic pathway for the production of this cofactor. Once PQQ is provided together with a suitable substrate like glucose, xylose, arabinose, ribose, lyxose, allose, fucose, mannose, galactose or melibiose, these substrates are converted to their respective sugar acids and the electrons are transferred to the ubiquinone pool which reactivates the electron transport chain and allows the respiratory production of ATP. Preferably the substrate is selected from the group comprising D-glucose, D-xylose, L-arabinose, D-ribose, L-lyxose, D-allose, D-fucose, D-mannose, D-galactose, and meliobiose, or a combination thereof. When pyrroloquinoline quinone (PQQ) is supplied, Gcd oxidizes D-glucose to form D-gluconolactone, which spontaneously hydrolyses to form D-gluconate. Electrons are transferred via PQQ to the ubiquinone (UQ) pool and ultimately to O2 to form a proton gradient for ATP synthesis via oxidative phosphorylation. Both D-glucose and D-gluconate can serve for biomass formation (Figure 17).

[0219] For the production of the sugar acids, supplying the respective sugar and either PQQ or a PQQ biosynthesis pathway is required. In theses cases, the gcd reaction itself allows for ATP generation and thereby allows for growth with results in a selection for the production of sugar acids (Figures 5, 6, 8, 9, 18, 19, 20, 21 , 22). Structural analogues of PQQ are also possible as long as they can function as a cofactor for gcd.

[0220] Thus, the present invention further relates to a method for producing sugar acids comprising the following steps: a) providing a genetically engineered bacterium of the present invention as described herein, wherein the genetically engineered bacterium expresses a quinone-dependent dehydrogenase, b) providing a culture medium comprising a carbon source and a substrate of the quinone-dependent dehydrogenase, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the carbon source is selected from the group comprising glucose, xylose, arabinose, ribose, lyxose, allose, fucose, mannose, galactose, and meliobiose, or a combination thereof. Preferably the carbon source is selected from the group comprising D-glucose, D-xylose, L-arabinose, D-ribose, L-lyxose, D-allose, D-fucose, D-mannose, D- galactose, and meliobiose, or a combination thereof.

[0221] Furthermore, the present invention further relates to a method for producing sugar acids comprising the following steps: a) providing a genetically engineered bacterium of the present invention as described herein, wherein the genetically engineered bacterium overexpresses a quinone-dependent dehydrogenase, b) providing a culture medium comprising a carbon source and a substrate of the quinone-dependent dehydrogenase, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), d) measuring gluconate or xylonate in the culture medium of step c); wherein the carbon source is selected from the group comprising glucose, xylose, arabinose, ribose, lyxose, allose, fucose, mannose, galactose, and meliobiose, or a combination thereof. Preferably the carbon source is selected from the group comprising D-glucose, D-xylose, L-arabinose, D-ribose, L-lyxose, D-allose, D-fucose, D-mannose, D- galactose, and meliobiose, or a combination thereof.

[0222] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium expresses a quinone-dependent dehydrogenase.

[0223] Thus, the present invention further relates to a method for producing sugar acids comprising the following steps: a) providing a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene, wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, wherein the genetically engineered bacterium expresses a quinone-dependent dehydrogenase, b) providing a culture medium comprising a carbon source and a substrate of the quinone-dependent dehydrogenase, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the carbon source is selected from the group comprising glucose, xylose, arabinose, ribose, lyxose, mannose, galactose, and meliobiose, or a combination thereof. Thus, the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferably, the genetically engineered bacterium biosynthesizes ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. According to the invention, the genetically engineered bacterium produces the quinone species without genetic modification. Thus, the quinone species is an endogenous quinone species already produced by the wild-type strain of the genetically engineered bacterium.

[0224] Thus, the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferably, the genetically engineered bacterium expresses cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Thus, the genetically engineered bacterium expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously. With other word, the genetically engineered bacterium naturally expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating). Therefore, the one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) are endogenous enzymes alreadpy expressed by the wild-type strain of the bacterium, i.e. are expressed without genetic modifications.

[0225] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0226] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0227] Preferably the genetically engineered bacterium is an E. coli. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat). Preferably, the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, IdhA gene and mqo gene.

[0228] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat). Preferably, the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, IdhA gene and mdh gene.

[0229] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0230] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0231] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0232] Also preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0233] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK), more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferably, the genetically engineered bacterium biosynthesizes ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. According to the invention, the genetically engineered bacterium produces the quinone species without genetic modification. Thus, the quinone species is an endogenous quinone species already produced by the wild-type strain of the genetically engineered bacterium.

[0234] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expresses one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0235] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expresses cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0236] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene.

[0237] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

[0238] One way to allow growth of the MiniKat strain and / or MaxKat strain is to introduce a quinone dependent reaction as well as the corresponding substrate. As shown for PQQ dependent sugar dehydrogenase as an example, the addition of glucose (or xylose or other substrates) and PQQ allows growth of the MiniKat strain and / or MaxKat strain. Here, reduced PQQ donates its electrons to the electron transport chain, protons are pumped to generate a proton motive force that allows ATP generation via ATPase. However, it is still beneficial to supplement acetate as the cell exhibits a slight acetyl- CoA auxotrophy due to suboptimal activity of pyruvate dehydrogenase due to high intracellular NADH concentrations. To show that the strain indeed grows because of the reactivation of the electron transport chain and not gluconate production, the inventors demonstrate that addition of gluconate (together with PQQ and acetate) does not rescue growth of the strain.

[0239] Enzyme Selection Platform

[0240] The genetically engineered bacteria strains of the present invention (e.g. Minikat strain and Maxkat strain) can serve as platforms for growth-coupled selection of enzyme activities. Enzyme activities which can be selected in these strains are either consuming (oxidizing) NAD(P)H or reducing quinones. In both cases ATP limitation of the genetically engineered bacteria strains of the present invention (e.g. Minikat strain and Maxkat strain) is released. While NAD(P)H consumption allows carbon to pass through glycolysis to generate ATP via substrate level phosphorylation, quinone reduction allows electron flux from carbon metabolites (enzyme’s substrates) into the respiratory chain and hence ATP generation via oxidative phosphorylation is enabled.

[0241] Since strains are only able to grow if enzymes consuming (oxidizing) NAD(P)H or reducing quinones are active, the strains can be used to test enzyme reactions, select best variants from large enzyme libraries, and use adapted laboratory evolution (ALE) to optimize enzyme variants further.

[0242] Examples for NAD(P)H consuming reactions:

[0243] Oxidoreductases

[0244] Alcohol dehydrogenases

[0245] Lactate dehydrogenase ketol-acid reductoisomerase

[0246] Aldehyde reductases

[0247] Enoyl-CoA reductase

[0248] Reductive aminases (amino acid dehydrogenases)

[0249] Imine reductase

[0250] Oxygenases

[0251] Monooxygenases

[0252] P450

[0253] Dioxygenases

[0254] Examples for Quinone reducing reactions

[0255] PQQ-dependent enzymes

[0256] PQQ-dependent alcohol Dehydrogenase

[0257] PQQ-Ethylene glycol Dehydrogenase PQQ-Aldose Dehydrogenase

[0258] Ubiquinone dependent enzymes

[0259] Succinate dehydrogenase

[0260] Malate dehydrogenase

[0261] Pyruvate dehydrogenase

[0262] In the following, the inventors demonstrate the advantageous uses of the Minikat strain and Maxkat strain as a platform for the growth-coupled selection of NAD(P)H consuming enzymes as well as quinone reducing enzymes. Furthermore, the inventors demonstrate the advantageous uses of the Minikat strain and Maxkat strain for the novel growth-coupled bioprocesses that rely on Quinone dependent oxidations for net ATP generation.

[0263] For this purpose, the Minikat strain and Maxkat strain provide plasmid-based expression of an enzyme (NADH oxidase, monooxygenase, phosphoketolase, alanine dehydrogenase, lactate dehydrogenase, sarcosine oxidase + methanol dehydrogenase).

[0264] Thus, the present invention further relates to a method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium of the present invention as described herein, wherein the genetically engineered bacterium overexpresses an (NADH consuming) enzyme preferably selected from a NADH oxidase, a monooxygenase, phosphoketolase, lactate dehydrogenase, alanine dehydrogenase, and / or sarcosine oxidase and methanol dehydrogenase; b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is dependent on the conversion of the substrate.

[0265] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium is modified for plasmid-based overexpression of NADH oxidase, monooxygenase phosphoketolase, lactate dehydrogenase, alanine dehydrogenase and / or sarcosine oxidase and methanol dehydrogenase.

[0266] Thus, the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferably, the genetically engineered bacterium biosynthesizes ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. According to the invention, the genetically engineered bacterium produces the quinone species without genetic modification. Thus, the quinone species is an endogenous quinone species already produced by the wild-type strain of the genetically engineered bacterium.

[0267] Thus, the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferably, the genetically engineered bacterium expresses cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Thus, the genetically engineered bacterium expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously. With other word, the genetically engineered bacterium naturally expresses one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating). Therefore, the one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) are endogenous enzymes alreadpy expressed by the wild-type strain of the bacterium, i.e. are expressed without genetic modifications.

[0268] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0269] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0270] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0271] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0272] Preferably the genetically engineered bacterium is an E. coli.

[0273] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat).

[0274] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0275] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene. Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0276] Also preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0277] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone endogenously, more preferably ubiquinone-8.

[0278] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressies one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0279] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressescytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0280] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene.

[0281] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes. NADH dependent

[0282] NADH oxidase (Nc

[0283] The present invention further relates to a method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium as disclosed herein, wherein the genetically engineered bacterium overexpresses NADH oxidase (Nox); b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is dependent on the conversion of the substrate.

[0284] The genetically engineered bacterium of the present invention as described herein has been further modified for plasmid-based overexpression of NADH oxidase (Nox).

[0285] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (H+-transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium is modified for plasmid-based overexpression of NADH oxidase.

[0286] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0287] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0288] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0289] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0290] Preferably the genetically engineered bacterium is an E. coli.

[0291] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat).

[0292] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0293] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene. Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0294] Also preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0295] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone endogenously, more preferably ubiquinone-8.

[0296] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressies one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0297] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressescytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0298] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

[0299] As a proof of principle for the selection for NAD+ recycling reactions, the inventors could surprisingly demonstrate how two different NADH oxidases that either produces H2O or H2O2 restore growth of the MaxKat strain on glucose or glycerol. The inventors showed that expression of the FhO-forming NADH oxidase leads to better growth compared to H2O2-forming NADH oxidase likely due to the oxidative stress from H2O2 (Figure 3).

[0300] Interestingly, when growing on glucose stoichiometric amounts of acetate (1 :2 ratio) are produced, probably because this allows the Maxkat strain to yield an extra ATP from the activity of acetate kinase.

[0301] The conversion glucose to acetate is an example of unbalanced fermentations that the MaxKat strain can perform.

[0302] Experimental modification: overexpression of an NADH-consuming enzyme (from plasmid as demonstrated or also genomic integration). Transformation via electroporation or other transformation method. Selection via antibiotic resistance on plasmid.

[0303] Monooxygenase

[0304] The present invention further relates to a method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium as disclosed herein, wherein the genetically engineered bacterium overexpresses a monooxygenase; b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is dependent on the conversion of the substrate.

[0305] The genetically engineered bacterium of the present invention as described herein has been further modified for plasmid-based overexpression of NADH oxidase (Nox).

[0306] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium is modified for plasmid-based overexpression of a monooxygenase.

[0307] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0308] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0309] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0310] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene. Preferably the genetically engineered bacterium is an E. coli.

[0311] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat).

[0312] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0313] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0314] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0315] Also preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0316] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressies one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0317] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressescytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0318] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene.

[0319] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

[0320] Methanol dehydrogenase (MeDH)

[0321] The present invention further relates to a method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium of the present invention as described herein, wherein the genetically engineered bacterium overexpresses methanol dehydrogenase (MeDH) and sarcosine oxidase (soxA); b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is absolutely dependent on the conversion of the substrate.

[0322] Preferably, step b) includes providing pyrroloquinoline quinone (PQQ).

[0323] The genetically engineered bacterium of the present invention as described herein has been further modified for plasmid-based overexpression of methanol dehydrogenase (MeDH) and soxA.

[0324] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (I -transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium is modified for plasmid-based overexpression of methanol dehydrogenase (MeDH) and sarcosine oxidase (soxA).

[0325] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0326] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0327] Preferably the genetically engineered bacterium is an E. coli.

[0328] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat).

[0329] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0330] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0331] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone endogenously, more preferably ubiquinone-8.

[0332] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressies one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0333] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressescytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0334] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene.

[0335] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes. Next, the inventors could surprisingly demonstrate that the reverse activity of NAD+ dependent methanol dehydrogenase (reductive activity) rescues growth of the Maxkat strain as well. Methanol dehydrogenase (MeDH) is an important enzyme to engineer methylotrophy. In the Maxkat strain, engineering it for accelerated turnover is possible, which is important to enhance growth and bioproduction on methanol.

[0336] First, the inventors demonstrate that reduction of formaldehyde to methanol by (MeDH) restores growth if an ATP source (glucose + PQQ) is present. In the strain, formaldehyde is produced intracellularly by sarcosine oxidase (soxA) to reduce its toxicity on the cell. Taken together, the inventors could demonstrate a proof of principle for an orthogonal fermentation pathway (sarcosine — formaldehyde (+glycine) — methanol) to rescue growth where an NAD+ dependent dehydrogenase where formaldehyde serves as a substrate that is non-native to E. coli (Figure 4).

[0337] Interestingly, reverse activity of MeDH is not enough to allow growth on glucose and sarcosine alone. Instead, supplementation of PQQ for ATP generation from electron transport chain is required in addition.

[0338] Alanin dehydrogenase (AlaD)

[0339] Here, the inventors aimed to demonstrate growth rescue of the Maxkat strain by the overexpression of alanine dehydrogenase (alaD). This enzyme exhibits the same stoichiometry like lactate dehydrogenase (IdhA) but produces the industrially relevant amino acid D-alanine (Figure 10 and 11).

[0340] The present invention further relates to a method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium of the present invention as described herein, wherein the genetically engineered bacterium overexpresses alanine dehydrogenase (alaD); b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is absolutely dependent on the conversion of the substrate.

[0341] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium is modified for plasmid-based overexpression of alanine dehydrogenase (alaD).

[0342] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0343] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0344] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0345] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene. Preferably the genetically engineered bacterium is an E. coli.

[0346] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat).

[0347] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0348] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0349] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0350] Also preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0351] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressies one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0352] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressescytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0353] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene.

[0354] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

[0355] Selection of quinone-dependent reactions quinone-dependent formate dehydrogenase (Fdo)

[0356] In the MaxKat strain, quinone-dependent formate dehydrogenase (Fdo) is still intact but not active as within common growth conditions, no formate is present in the medium. Therefore, feeding formate is an option to feed electrons into the otherwise inactive electron transport chain (ETC) to restore ATP production and thereby reactivate growth. In this setup, the oxidation of formate to CO2 is decoupled from metabolism as neither substrate nor product enter central metabolism in substantial amounts. Formate only serves as an energy source as electrons are transferred from formate to the ubiqunone (UQ) pool to drive the formation of a proton gradient and thereby allow ATP generation via oxidative phosphorylation. As a first step, the inventors tested the effect of formate supplementation to LB medium and found that growth of the strain was restored when formate was supplied (Figure 12). To confirm whether Fdo was indeed responsible for this formate-dependent growth, the inventors deleted either the catalytic (fdoG) or the electron-transferring (fdoH) subunit of Fdo and found that formate-dependent growth in MaxKat AfdoG and MaxKat AfdoH was abolished. The inventors next investigated the functionality of the formate oxidation module on minimal medium. We found that formate dependency of biomass formation was linear in a range between 2 mM to 24 mM supplied formate (Figure 13)

[0357] PQQ dependent glucose and xylose dehydrogenase (gcd)

[0358] Here addition of PQQ rescued growth of the MiniKat and MaxKat strain in glucose or xylose minimal medium. The activity of gcd reduces PQQ to PQQH2 and subseguently PQQH2 donates its electrons to the ETC. The ETC then pumps protons across the inner membrane generating a proton motive force (PMF) over the membrane. This PMF is utilized by ATPase to generate ATP from ADP and Pi. Hence, PQQ reduction by gcd rescues growth of the MaxKat strain via reactivation of the electron transport chain (Figures 5, 6, 8, 9, 18, 19, 20, 21 , 22)

[0359] Bioprocesses

[0360] Acetate production from Glucose and Glycerol

[0361] Here, the inventors aimed to demonstrate acetate production from glucose and glycerol of the Maxkat strain by the overexpression of NADH oxidase (Nox) or phosphoketolase (PKT) (Figure 7).

[0362] The present invention further relates to a method for production of comprising the following steps: a) providing a genetically engineered bacterium of the present invention as described herein, wherein the genetically engineered bacterium overexpresses NADH oxidase (Nox) or phosphoketolase (PKT); b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the growth of the strain used is absolutely dependent on the conversion of the substrate.

[0363] The genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; and preferably a deletion of mqo gene or mdh gene, more preferably a deletion of mqo gene; wherein the one or more nuo genes are selected from nuoA gene, nuoB gene, nuoC gene, nuoD gene, nuoE gene, nuoF gene, nuoG gene, nuoH gene, nuol gene, nuoJ gene, nuoK gene, nuoL gene, nuoM gene, and nuoN gene; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, protonmotive force generating) endogenously, wherein the genetically engineered bacterium is modified for plasmid-based overexpression of NADH oxidase (Nox) and phosphoketolase (PKT).

[0364] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0365] Also preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0366] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0367] Preferred is a genetically engineered bacterium further comprising a deletion of one or more genes selected from the group comprising or consisting of: did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0368] Preferably the genetically engineered bacterium is an E. coli.

[0369] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises at least a deletion of ndh gene, nuoEFG genes, and IdhA gene (MiniKat). Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0370] Also preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

[0371] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mqo gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene (MaxKat).

[0372] Preferred is a genetically engineered bacterium, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, did gene, mdh gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

[0373] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone (UQ), menaquinone (MK) and / or demethylmenaquinone (DMK) endogenously, more preferably ubiquinone (UQ), more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, more preferably ubiquinone-8. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium biosynthesizes ubiquinone endogenously, more preferably ubiquinone-8.

[0374] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressies one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd- I oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously. Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium expressescytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

[0375] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a ubi gene.

[0376] Preferred is a genetically engineered bacterium, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

[0377] Preferred Bacteria

[0378] The genetically engineered bacterium preferably may be Enterobacteriaceae, Staphylococcaceae, Streptococcaceae, Lactobacillaceae, Vibrionaceae, Pasteurellaceae, Pseudomonidae, Bacilli, Corynebacteria, Gluconobacteria, Acetobacteria, Methylobacteria and the like.

[0379] Since several bacterial genomes have been sequenced and are online available in different specific databases, as listed at en.wikipedia.org / wiki / List_of_sequenced_bacterial_genomes, a person of ordinary skill in the art can easily confirm homologous gene sets by sequence alignment, and in particular using amino acid sequences, which diverge less than gene sequences in the prokaryotes.

[0380] Therefore, a preferred embodiment of the present invention discloses a genetically engineered bacteria comprising the gene deletions disclosed above, wherein the genetically engineered bacteria is a facultative anaerobic bacteria selected from the group comprising Enterobacteriaceae, Staphylococcaceae, Streptococcaceae, Lactobacillaceae, Vibrionaceae, and Pasteurellaceae.

[0381] In particularly preferred embodiments, the genetically engineered bacterium is an E. coli. In preferred embodiments, the E. coli is derived from the well-known E. coli K-12 MG1655 strain. In even more preferred embodiments, the E. coli is an E. coli K-12 MG1655 further comprising one or more recombination systems. The one or more recombination systems are preferably selected from an arabinose inducible lambda Red recombineering system and a rhamnose inducible flippase recombinase. Description of the Figures

[0382] Figure 1 Scheme depicting the simplified metabolic function of the Maxkat strain. A: Metabolic scheme highlighting metabolic fluxes and key deletions in the Maxkat strain. While ATP generation from the respiratory chain is prevented through the deletion of ubiquinone-dependent enzymes, it can be reactivated through the feeding of PQQ, which is required for the activity of ubiquinone-reducing glucose dehydrogenase. B: Simplified scheme depicting the inability of the Maxkat strain to operate catabolic metabolism due to missing NAD+regeneration routes. C: Through the reintroduction of a heterologous fermenting enzyme or pathway, aerobic fermentation can rescue growth. D: When both, a heterologous fermentation route and a ubiquinone-reducing enzyme are introduced, respiratory growth can be restored. Abbreviations: DHAP - dihydroxyacetone phosphate; GAP - Glyceraldehyde 3-phosphate; Sox - oxidized substrate; Pred - reduced product.

[0383] Figure 2 Effect of deleting ndh, nuoEFG and IdhA (“Minikat”) Growth of a Minikat strain on different carbon sources as well as PQQ.

[0384] Figure 3 Effect from the overexpression of different NADH oxidases on the Maxkat strain A: Growth of a Maxkat strain expressing FhO-forming NADH oxidase “nox” on different glucose concentrations as sole carbon source. B: Growth of a Maxkat + GIpD strain expressing H2O-forming NADH oxidase “nox” on different glycerol concentrations as sole carbon source. C: Comparison of H2O2- with H2O-forming NADH oxidase. D: measured acetate concentrations of a Maxkat strain expressing H2O2-forming NADH oxidase growing on different glucose concentrations.

[0385] Figure 4 Production of methanol from sarcosine rescues growth through an orthogonal fermentation pathway. A: Only the co-expression of sarcosine oxidase (soxA) and methanol dehydrogenase (MeDH) together with feeding sarcosine together with PQQ supplementation rescue growth of the Maxkat strain on glucose. B: scheme depicting the orthogonal fermentation pathway from sarcosine to methanol.

[0386] Figure 5 Investigating the effect of co-feeding PQQ with other carbon sources to the Maxkat strain. Scheme depicting PQQ-dependent glucose oxidation mechanism of Gcd and the electron transfer to the ubiquinone pool, which enables the use of molecular oxygen as a terminal electron acceptor and thereby proton gradient formation.

[0387] Figure 6 Investigating the effect of co-feeding PQQ with other carbon sources to the Maxkat strain. Growth experiment investigating the growth phenotype of the Maxkat strain with and without the addition of PQQ. In the case of PQQ addition, 1 pM PQQ was fed. Abbreviations: ace = acetate, Gcd = PQQ- dependent glucose dehydrogenase, UQ = ubiquinone.

[0388] Figure 7 Expression of phosphoketolase (PKT) in Maxkat. Comparison of Maxkat strain growing in minimal medium (M9) or minimal medium with 5g / L casamino acid supplementation (MX) on different carbon sources.

[0389] Figure 8 shows NNmini DldhA gluconate production.

[0390] Figure 9 shows NNmini DldhA xylonate production.

[0391] Figure 10 Growth experiment investigating a MaxKat_GlpD strain expressing alanine dehydrogenase from B. subtilis (alaD_Bs, PDM019) grown on different glycerol concentrations. All strains were grown on minimal medium (M9) with added 20mM NH4+ to account for NH4+ consumption by alanine dehydrogenase.

[0392] Figure 11 Alanine concentrations measured from a MaxKat_GlpD strain grown on different glycerol concentrations. Alanine concentrations were determined via LC / MS. All strains were grown on minimal medium (M9) with added 20mM NH4+ to account for NH4+ consumption by alanine dehydrogenase. Average conversion rates were 74.7 ± 1 .67% glycerol to alanine.

[0393] Figure 12 Growth curves of the MaxKat strain with or without deletions of different subunits of quinone-dependent formate dehydrogenase grown on LB medium with or without the supplementation of 10 mM formate.

[0394] Figure 13 Plot depicting the reached maximum optical density (ODeoo) of the MaxKat strain grown on minimal medium supplemented with 2.5 g / L casamino acids for biomass formation and varying formate concentrations. Figure 14 Minicycles were reintroduced to NNmini in a randomized manner relying on P1 transduction with a lysate from a nuo ndh strain and subsequent selection for growth on different carbon sources.

[0395] Figure 15 Growth of NNmini and two single colonies isolated from glucose (Glucl , Gluc2), Xylose (Xyl1 , 2) or Acetate (Ace1 , Ace2) on 10 mM glucose (left) or 20 mM pyruvate (right).

[0396] Figure 16 Schematic representation of the minicycles allowing quinone reduction. In the center three representative minicycles and the relevant genes are shown.

[0397] Figure 17 Scheme depicting the physiological orientation and interaction of membranebound glucose dehydrogenase (Gcd) with the ETC. When pyrroloquinoline quinone (PQQ) is supplied, Gcd oxidizes D-glucose to form D- gluconolactone, which spontaneously hydrolyses to form D-gluconate. Electrons are transferred via PQQ to the ubiquinone (UQ) pool and ultimately to O2 to form a proton gradient for ATP synthesis via oxidative phosphorylation. Both D-glucose and D-gluconate can serve for biomass formation.

[0398] Figure 18 Growth of the MaxKat strain on minimal medium with different carbon sources with and without the addition of PQQ.

[0399] Figure 19 Growth of the MaxKat strain on minimal medium with 10 mM acetate, 0.1 pM PQQ and a gradient of D-glucose concentrations.

[0400] Figure 20 Growth of the MaxKat strain on minimal medium with 10 mM acetate, 0.1 pM PQQ and a gradient of D-xylose concentrations.

[0401] Figure 21 Sugar acid production by the MaxKat strain grown with 10 mM acetate, 0.1 pM PQQ and a gradient of D-glucose or D-xylose concentrations.

[0402] Figure 22 Growth of the MaxKat strain on different sugars in LB or minimal medium supplemented with 10 mM acetate, 0.1 pM PQQ and 40 mM of the respective sugar. A condition without PQQ serves as a negative control to confirm that gcd-dependent sugar acid production is responsible for restored growth.

[0403] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0404] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

[0405] EXAMPLES

[0406] Methods

[0407] Strain and plasmids construction. All E. coli strains used in this study are listed in Table 1. An E. coli MG1655 further expressing a A-red recombinase system and a flippase recombinase, E. coli SIJ_488, was a gift from Alex Nielesen (Addgene palsmid #68246) and used as wildtype (E. coli WT). Gene deletions were performed by A-Red recombineering or P1 -transduction.

[0408] Table 1 : E. coli strains and plasmids used in this study

[0409] Information on genotypes of strains from Keio collection can be found at https: / / cgsc.biology.yale.edu / StrainQueryForm.php E. coli culture media. LB medium (1% NaCI, 0.5% yeast extract, 1 % tryptone) was used for cloning, generation of deletion strains, and strain maintenance. When appropriate, kanamycin (25 pg / mL), ampicillin (100 pg / mL), streptomycin (100 pg / mL), or chloramphenicol (30 pg / mL) were used. Growth experiments were carried out without antibiotics in standard M9 minimal medium free of any carbon source or amino acids, and containing 50 mM Na2HPO4, 20 mM KH2PO4, 1 mM NaCI, 20 mM NH4CI, 2 mM MgSO4and 100 pM CaCI2, 134 pM EDTA, 13 pM FeCI3'6H2O, 6.2 pM ZnCI2, 0.76 pM CuCl2'2H2O, 0.42 pM CoCI2-2H2O, 1.62 pM H3BO3, 0.081 pM MnCI2-4H2O. Carbon sources were added as indicated in the Examples and Figures.

[0410] Example 1 Gene deletion via P1 Phage transduction.

[0411] Gene deletions of putA, did, mqo, dadA, poxB, kefF, wrbA, fadE, yieF, glpD, gpsA, HdD, IhgO were generated by P1 phage transduction (Thomason et al., Curr Protoc Mol Biol. 2007, Ch. 1 :1.17.1-1.17.8). Donor strains were strains JWxxxx from the KEIO collection listed in Table 1 with a kanamycin-resistance gene (KmR). The strains of E. coli with the desired deletions were obtained by genetic recombination with the P1 phage lysate and selected for by plating on kanamycin containing plates. A successful gene deletion was verified by determining the size of the genomic locus by PCR with DreamTaq polymerase (Thermo Scientific, Dreieich, Germany) and the respective KO- Ver primers (Table 2). Furthermore, a PCR with DreamTaq polymerase (Thermo Scientific, Dreieich, Germany) and internal primers (“int”) binding inside of the gene coding sequence was performed to confirm that no copy of the gene to be deleted was present in the genome of the transduced strain. For removal of the selective marker, a fresh culture was grown to ODeoo ~ 0.2, followed by addition of 50 mM L-Rhamnose and cultivating for ~4h at 30°C for flippase expression induction. Colonies that only grew on LB medium in absence of the respective antibiotic were isolated and successful removal of the KmR gene from the respective locus was confirmed by PCR using the locus specific KO-Ver primers and with DreamTaq polymerase (Thermo Scientific, Dreieich, Germany).

[0412] Table 2: Oligonucleotide primers used (Sequences in Sequence Protocol)

[0413] ‘KO’ primers were used to amplify the Km knockout cassette from pKD4 with 50 bp gene-specific upstream and downstream sequences. To verify gene replacement by kanamycin resistance cassette and cassette removal by flippase, ‘KO-Ver’ -primers (knockout-verification) were used. Internal primers were used to verify successful removal of the gene from the genome.

[0414] Example 2: Gene deletion by recombineerinq.

[0415] Gene deletion by recombineering involved PCR with “KO” primers (Table 2 and Table 3) with 50bp homologous overhangs and pKD4 plasmid (Addgene # 45605; http: / / n2t.net / addgene:45605; RRID: Addgene_45605) as template and PrimeStar GXL polymerase (Takara Bio) was performed to generate kanamycin resistance cassettes. E. coli WT cells were prepared for gene deletion by inoculating fresh cultures in LB, followed by induction of the recombinase genes by addition of 15 mM L-arabinose at OD ~0.4-0.5, followed by incubation for 45 min at 37°C. The cells were harvested (11 ,000 rpm, 30 sec, 2°C) and washed three times with ice cold 10 % glycerol. For electroporation, ~300 ng of Km cassette PCR-product was transformed (1 mm cuvette, 1.8 kV, 25 pF, 200 Q). Gene deletions were confirmed by selection on kanamycin containing plates and via one PCR using ‘KO-Ver’ primers (Table 2) and one PCR using internal (int) primers (Table 2), both of these being done with DreamTaq polymerase (Thermo Scientific, Dreieich, Germany). The Km cassette was removed by adding 50 mM L-rhamnose to an exponentially growing 2 ml LB culture at OD 0.5 for induction of flippase gene expression. After induction, cells were incubated for > 3 h at 30°C. After screening colonies for kanamycin sensitivity, removal of antibiotic resistance cassette was confirmed by PCR using ‘KO-Ver’ primers and DreamTaq polymerase (Thermo Scientific, Dreieich, Germany).

[0416] E.coli was deleted of the genes related to two NAD(P)H:quinone oxidoreductases (ndh, nuoEFG, kefF, mdaB-ygiN, wrbA, yieF) and of the genes encoding quinone-dependent dehydrogenases sdhABCD, HdD, poxB, fadE, did, mqo, putA, glpD, glpABC, glcDEF, dadA, IhgO. The latter eight genes could potentially form a “mini-cycle” with an immediate NAD(P)H-dependent counterpart. The resulting genetically engineered bacterium strain was called “NNmini” where “NN” denotes the deletion of the 2 main NADH dehydrogenases and “mini” denotes the elimination of all other quinone-reducing reactions including the mini-cycles (Figure 3B).

[0417] To investigate whether the aerobic fermentative phenotype of the NNmini strain could be achieved with less genetic modifications, the ubiquinone biosynthesis (encoded by ubiCA) was deleted either alone (AubiCA strain) or in the AnuoEFG Andh background. The corresponding bacterial strains were genetically engineered. The AnuoEFG Andh AubiCA strain was called “NNQ” where “NN” denotes the deletion of the major NADH dehydrogenases and “Q” denotes the deletion of ubiquinone biosynthesis.

[0418] Table 3: List of gene deletions to obtains the genetically engineered bacteria

[0419] Group A: NDH activity, group B: described NDH activity, group C: quinone reducing reactions from central metabolism; group D: NDH bypassing activities. Example 4

[0420] The genetically engineered bacterium as disclosed herein has been further modified for plasmid-based overexpression of NADH oxidases (Nox). These oxidases oxidize NADH to NAD+and H2O or H2O2 and thereby support NAD+cofactor recycling that allows further glycolytic flux and ATP generation.

[0421] As a proof of principle for the selection for NAD+ recycling reactions, the inventors could demonstrate how two different NADH oxidases that either produces H2O or H2O2 restore growth of the MaxKat strain on glucose or glycerol. The inventors showed that expression of the FhO-forming NADH oxidase leads to better growth compared to H2O2- forming NADH oxidase likely due to the oxidative stress from H2O2 (Figure 3).

[0422] Interestingly, when growing on glucose stoichiometric amounts of acetate (1 :2 ratio) are produced, probably because this allows the Maxkat strain to yield an extra ATP from the activity of acetate kinase.

[0423] The conversion glucose to acetate is an example of unbalanced fermentations that the MaxKat strain can perform.

[0424] Experimental modification: overexpression of an NADH-consuming enzyme (from plasmid as demonstrated or also genomic integration). Transformation via electroporation or other transformation method. Selection via antibiotic resistance on plasmid.

[0425] Example 5

[0426] The genetically engineered bacterium as disclosed herein has been further modified for plasmid-based overexpression of NADH dependent methanol dehydrogenase (MeDH) together with sarcosine oxidase (SoxA). The combination of MeDH with soxA oxidizes NADH to NAD+by transferring electrons from NADH to formaldehyde, which was generated by soxA from sarcosine. Hence the enzymes support NAD+cofactor recycling that allows glycolytic flux and substrate-level phosphorylation.

[0427] In particular the minimal medium is composed of a carbon source, e.g. glucose and sarcosine. While glucose is converted to pyruvate generating ATP and NADH. In an absence of a metabolic electron sink (e.g. lactate dehydrogenase), ATP generation from glycolysisis stopped and the cells cannot grow. By providing sarcosine, soxA generates glycine and formaldehyde. The formaldehyde is then utilized by MeDH which uses electrons from NADH to generate methanoland thereby regenerates NAD+. The recycled NAD+can then be used in substrate level phosphorylation of glycolysis generating ATP, which restores growth of the strain. When growing on glucose and sarcosine stoichiometric amounts of methanol (1 :1 ratio for sarcosine, 1 :2 ratio for glucose) are produced.

[0428] Example 6

[0429] The genetically engineered bacterium as disclosed herein has been further modified for plasmid-based overexpression of NADH dependent alanine dehydrogenase (aldA). This enzyme transfers electrons from NADH to pyruvate. During the reaction ammonium is fixed to generate L-alanine. Hence aldA supports NAD+cofactor recycling that in turn restores glycolytic flux and therefore ATP generation and growth.

[0430] In particular the minimal medium is composed of a carbon source, e.g. glucose or glycerol. The carbon source is converted to pyruvate generating ATP and NADH. In the absence of a metabolic electron sink (e.g. lactate dehydrogenase), ATP generation from glucose is stopped and the cells cannot grow. By providing aldA NADH is consumed to make L-alanine from pyruvate, hence an electron sink using an internal metabolite is introduced, recovering NAD+. NAD+then is used in substrate level phosphorylation of glycolysis generating ATP. Strain’s growth is restored as a result of aldA activity. When growing on glucose stoichiometric amounts of L-alanine (1 :2 ratio) are produced. When growing on glycerol stoichiometric amounts of L-alanine (1 :1 ratio) are produced.

[0431] The conversion of glycerol to L-alanine is an example of unbalanced fermentations that the MaxKat strain can perform.

[0432] Example 7

[0433] The genetically engineered bacterium as disclosed herein has been further modified for plasmid-based overexpression of phosphoketolase (pkt) that provides an NAD+independent way of ATP generation via substrate level phosphorylation. This enzyme cleaves several sugar-phosphates. During these reactions inorganic phosphate is fixed to generate acetyl-phosphate. Subsequently, acetate kinase can utilize acetylphosphate to generate ATP and acetate. Hence the pkt supports NAD+independent ATP production and consequently growth.

[0434] The medium used in this experiment composed of minimal medium compounds with and without casamino acids as well as a carbon source.

[0435] Example 8

[0436] Identification of essential deletions via phage assisted reintroduction of potential minicycles

[0437] To investigate which of the quinone reducing reactions in the MaxKat strain are most essential (i.e. able to replace NADH dehydrogenase activity and hence allow respiration), individual minicycles were reintroduced in the NNmini strain (to facilitate growth) and the resulting strains characterized. Specifically, the inventors randomized the minicycle reintroduction by transducing the NNmini strain with a phage-lysate generated from the ndh nuo ancestor strain. To directly select for introduction of minicycles allowing cell respiration, the cells were directly plated on minimal medium plates containing glucose, xylose or acetate (Fig. 14). The inventors decided to use multiple carbon sources, since metabolite concentrations and hence levels of substrate for the different potential mini-cycle reactions might vary depending on the entry point for each carbon source into metabolism. Grown colonies were isolated, and the growth of two colonies from each carbon source was compared to that of the ancestral NNmini strain. Indeed, all tested strains transduced with the ndh nuo lysate grew faster than the NNmini strain, and to three times its biomass yield on glucose (Fig. 15). Furthermore, in contrast to the NNmini strain all tested clones were able to grow on 20 mM pyruvate, which would not allow growth in a purely fermentative manner due to resulting redox imbalance (Fig. 15). To identify minicyles responsible for the changed growth phenotype, the genotype of all obtained colonies was analyzed by PCR on all sixteen minicycle genes. Indeed, for most clones analyzed and the ones tested in growth experiments, either mqo or did were reintroduced (Table 1 ). Since both mqo and did dependent minicycles (Fig. 16) are part of central metabolism, we reasoned that these minicycles might be carrying most flux and thus have the highest quinone reducing turnover.

[0438] Figure 14. Minicycles were reintroduced to NNmini in a randomized manner relying on P1 transduction with a lysate from a nuo ndh strain and subsequent selection for growth on different carbon sources.

[0439] Figure 15. Growth of NNmini and two single colonies isolated from glucose (Glucl , Gluc2), Xylose (Xyl1 , 2) or Acetate (Ace1 , Ace2) on 10 mM glucose (left) or 20 mM pyruvate (right).

[0440] Table 4. Genes reintroduced in the colonies grown on glucose or pyruvate.

Claims

Claims1 . A genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NDH-II; a deletion of one or more nuo genes, wherein the nuo genes encode NDH-I; a deletion of IdhA gene; a deletion of mqo gene or mdh gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (IT-transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously.

2. The genetically engineered bacterium according to claim 1 or 2 further comprising a deletion of one or more genes selected from the group comprising: did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

3. The genetically engineered bacterium according to claim 1 or 2 further comprising a deletion of one or more genes selected from the group comprising: did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

4. The genetically engineered bacterium according to any one of claims 1 - 3, wherein the genetically engineered bacterium is an E. coli.

5. The genetically engineered bacterium according to any one of claims 1 - 4, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, and mqo gene.

6. The genetically engineered bacterium according to any one of claims 1 - 4, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, and mdh gene.

7. The genetically engineered bacterium according to any one of claims 1 - 5, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mqo gene, did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

8. The genetically engineered bacterium according to any one of claims 1 - 5, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mqo gene, did gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

9. The genetically engineered bacterium according to any one of claims 1 - 4 and 6, wherein the genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mdh gene, did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

10. The genetically engineered bacterium according to any one of claims 1 - 4 and 6, wherein genetically engineered bacterium comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mdh gene, did gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

11. The genetically engineered bacterium according to any one of the claims 1 - 10, wherein the genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, preferably ubiquinone-8.

12. The genetically engineered bacterium according to any one of the claims 1 - 11 , wherein the genetically engineered bacterium is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

13. The genetically engineered bacterium according to any one of the claims 1 - 12, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

14. The genetically engineered bacterium according to any one of the claims 1 - 13, wherein the genetically engineered bacterium does not comprise a deletion of an ubi gene.

15. The genetically engineered bacterium according to any one of the claims 1 - 14, wherein the genetically engineered bacterium does not comprise a deletion of a cyoABCD gene, cydAB genes and / or appBC genes.

16. A method for producing sugar acids comprising the following steps: a) providing a genetically engineered bacterium comprising: a deletion of ndh gene, wherein the ndh gene encodes NADH:quinone oxidoreductase II (NDH-2); a deletion of one or more nuo genes, wherein the nuo genes encode NADH:ubiquinone oxidoreductase I (NDH-1 ); a deletion of IdhA gene; wherein the one or more nuo genes are selected from nuoA, nuoB, nuoC, nuoD, nuoE, nuoF, nuoG, nuoH, nuol, nuoJ, nuoK, nuoL, nuoM, and nuoN; wherein the genetically engineered bacterium is capable of biosynthesizing a quinone species endogenously; and wherein the genetically engineered bacterium is capable of expressing one or more ubiquinol oxidases (I -transporting) and / or quinol oxidases (electrogenic, proton-motive force generating) endogenously, wherein the genetically engineered bacterium expresses a quinone-dependent dehydrogenase, b) providing a culture medium comprising a carbon source and a substrate of the quinone-dependent dehydrogenase, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b), wherein the carbon source is selected from the group comprising glucose, xylose, arabinose, ribose, lyxose, allose, fucose, mannose, galactose, and meliobiose, or a combination thereof.

17. The method according to claim 16, wherein the quinone-dependent dehydrogenase is Quinoprotein glucose dehydrogenase (gcd), and wherein the substrate is pyrroloquinoline quinone (PQQ).

18. The method according to claim 16 or 17, wherein genetically engineered bacterium further comprises a deletion of mqo gene or mdh gene.

19. The method according to any one of claims 16 - 18, wherein genetically engineered bacterium further comprises a deletion of one or more genes selected from the group comprising: did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

20. The method according to any one of claims 16 - 19, wherein genetically engineered bacterium further comprises a deletion of one or more genes selected from the group comprising: did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN genes, glpABC genes, , wrbA gene, yieF gene, fadE gene, and gpsA gene.21 . The method according to any one of claims 16 - 20, wherein genetically engineered bacterium further comprises wherein the genetically engineered bacterium is an E. coli.

22. The method according to any one of claims 16 - 21 , wherein genetically engineered bacterium further comprises a deletion of ndh gene, nuoEFG genes, IdhA gene.

23. The method according to any one of the claims 16 - 22, wherein genetically engineered bacterium further comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mqo gene, did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

24. The method according to any one of the claims 16 - 22, wherein genetically engineered bacterium further comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mqo gene, did gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

25. The method according to any one of the claims 16 - 22, wherein genetically engineered bacterium further comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mdh gene, did gene, poxB gene, sdhABCD genes, glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and glpD gene.

26. The method according to any one of the claims 16 - 22, wherein genetically engineered bacterium further comprises a deletion of ndh gene, nuoEFG genes, IdhA gene, mdh gene, did gene, poxB gene, sdhABCD genes; glcDEF genes, IhgO gene, putA gene, IldD gene, dadA gene, kefF gene, mdaB-ygiN gene, glpABC genes, wrbA gene, yieF gene, fadE gene, and gpsA gene.

27. The method according to any one of the claims 16 - 26, wherein genetically engineered bacterium is capable of biosynthesizing ubiquinone endogenously, preferably ubiquinone-8.

28. The method according to any one of the claims 16 - 27, wherein genetically engineered bacterium further is capable of expressing one or more of cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

29. The genetically engineered bacterium according to any one of the claims 16 - 28, wherein the genetically engineered bacterium is capable of expressing cytochrome bO3 oxidase (CyoABCD), cytochrome bd-\ oxidase (CydAB) and cytochrome bd-\\ oxidase (AppBC) endogenously.

30. The genetically engineered bacterium according to any one of the claims 16 - 29, wherein the genetically engineered bacterium does not comprise a deletion of an ubi gene.

31. The genetically engineered bacterium according to any one of the claims 16 - 30, wherein the genetically engineered bacterium does not comprise a deletion of cyoABCD gene, cydAB genes and / or appBC genes.

32. A method for growth-coupled selection of NADH consuming enzymes comprising the following steps: a) providing a genetically engineered bacterium of any one of the claims 1 - 15, wherein the genetically engineered bacterium expresses an enzyme preferably selected from NADH oxidase, monooxygenase, phosphoketolase, alanine dehydrogenase, lactate dehydrogenase, and / or sarcosine oxidase and methanol dehydrogenase; b) providing a culture medium comprising a carbon source and a substrate, c) culturing the genetically engineered bacterium under aerobic conditions in the culture medium of b),wherein the growth of the strain used is absolutely dependent on the conversion of the substrate.

Images