WO2008147470A2 - Anaerobic synthesis of oxidized products by e. coli - Google Patents

Abstract

Engineered bacterial strains having reduced or inactive cytoplasmic nitrate reductase (NAR), nitrate/nitrite-dependent regulatory system (NAR-X), periplasmic nitrate reductase (NAP), format dehydrogenase (FDH), and/or lactate dehydrogenase (LDH) can be used for anaerobic production of oxidized products such as acetate and pyruvate. This Abstract is provided to comply with rules requiring an Abstract that allows a searcher or other reader to quickly ascertain subject matter of the technical disclosure. This Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. 37 CFR 1.72(b).

Description

ANAEROBIC SYNTHESIS OF OXIDIZED PRODUCTS BY E. COLI

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 60/870835 filed December 19, 2006, entitled "ANAEROBIC SYNTHESIS OF OXIDIZED PRODUCTS BY E. COLI," which is incorporated herein in its entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] The present invention may have been developed with funds from the United States

Government. Therefore, the United States Government may have certain rights in the invention.

REFERENCE TO A COMPACT DISK APPENDIX

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] The invention is a new process/method for the anaerobic (i.e., absence of oxygen) production of oxidized chemicals using engineered E. coli strains.

BACKGROUND OF THE INVENTION

[0005] E. coli, the workhorse of modern biotechnology, has been engineered for the production of a wide variety of products (Gonzalez, 2005). This organism is especially important for the production of bulk chemicals since it is able to metabolize low-priced sugars obtained from renewable sources such as plant biomass at high rates. In addition, E. coli can grow both aerobically and anaerobically, has low nutritional requirements, and there are well-established genetic tools that facilitate its genetic/metabolic modification. Further, E. coli has been used in large-scale fermentations and production on an industrial level and a large number of strains are considered safe organisms. [0006] E. coli can grow on many carbon sources using either fermentative or respiratory metabolism. Fermentative metabolism involves internally achieving redox balance by synthesizing reduced products such as ethanol, succinate, and lactate. Respiratory metabolism requires an exogenous electron acceptor such as oxygen, nitrate, nitrite, fumarate, trimethylamine N-oxide, or dimethylsulfoxide. During fermentation the cells only generate energy by substrate-level phosphorylation, but respiratory metabolism generates a proton motive force, which is used to drive the synthesis of larger amounts of ATP and other energy- requiring processes such as transport, motility, etc.

[0007] Anaerobic fermentation and aerobic respiration have been the two metabolic modes of interest for the industrial production of chemicals via fermentation (Table 1). Aerobic respiration offers very efficient cell growth (growth rate and yield) and converts a high percentage of the carbon source into carbon dioxide and cell mass. Anaerobic fermentation, on the other hand, results in poor cell growth and the synthesis of several fermentation products at high yields (e.g. lactate, formate, ethanol, acetate, succinate, etc.).

[0008] From a practical standpoint, the ideal fermentation process should have three main traits: the desired product should be synthesized at high levels (titer), yields, and rates (productivity). As discussed above, neither anaerobic fermentation nor aerobic respiration is able to completely support these traits. Furthermore, the production of oxidized chemicals at high yields requires the presence of an external electron acceptor to reoxidize the NADH to NAD+, thus maintaining the glycolytic flux.

[0009] FIG. 1 illustrates this need taking as example the engineering of a homoacetogenic pathway in E. coli. In such a case, oxygen represents the common electron acceptor used in industry. For example, several microorganisms have been engineered to produce acetic and pyruvic acids under aerobic conditions (Gonzalez, 2005). Producing chemicals via aerobic processes, however, is more costly than using anaerobic methods for two reasons (Table 1). First, aerobic fermenters are more expensive to build, due to both the higher cost per unit and the need for smaller fermenters with reduced economy of scale. Secondly, the aerobic fermenters are more costly to operate than their anaerobic counterpart due to low solubility of oxygen, which in turn requires high energy input to ensure appropriate supply of oxygen to the cells. This is especially relevant for the production of commodity chemicals, where fermentation costs can represent 50-90% of the total production cost. Therefore, the use of alternative electron acceptors could provide an ideal metabolic state where both efficient cell growth and product synthesis can be achieved and the aforementioned disadvantages overcome. Such a strategy could result in high yields and productivities of the desired product in a process with low capital and operational costs due to the absence of oxygen.

[0010] E. coli synthesizes three formate dehydrogenase enzymes, FDH-N, FDH-H, and FDH-O ^encoded by the genes fdnGHI, fdhF, and fdoGHI, respectively (Gennis, R. and Stewart.1996; Gunsalus, 1992). FDH-N is a membrane-bound enzyme that exhibits maximal expression during nitrate respiration (Cheryan et ah, 1997; Enoch and Lester, 1975). It functions in the formate-nitrate respiratory chain by coupling to the NAR-G nitrate reductase for reduction of nitrate to nitrite. FDHH is part of the formate-hydrogen lyase (FHL) complex and, for optimal synthesis, requires both anaerobic conditions and the presence of formate. Since nitrate suppresses FDH-H synthesis, this enzyme is thought to play a role in fermentation and is not believed to be actively involved in electron transfer to either of the respiratory nitrate reductases (Cole, 1996). The FHL complex catalyzes the disproportionation of formate to CO₂ and hydrogen. Induction of the FHL requires formate, molybdate, absence of e- acceptors (O₂ or nitrate), and acidic pH. The other component of the FHL complex is a hydrogenase (Hyd-3). There are two other hydrogenases in E. coli, Hyd-1 (coded for by hyaABC) and Hyd-2 (coded for by hybO and hybC), but these are involved in hydrogen oxidation rather than proton reduction (Sawers et at., 2004). The third formate dehydrogenase, FDH-O, is membrane bound and is structurally and immunologically related to FDH-N. However, FDH-O (coded for by f do GHI) is synthesized at relatively low levels independent of either oxygen or nitrate availability (Abaibou et al, 1995). It has been proposed that FDH-O couples with the NAR-Z membrane-bound nitrate reductase enzyme in a fashion similar to that used by the FDH-N/NAR-G formate dehydrogenase-nitrate reductase complex.

[0011] What is required is an engineered bacteria with modified nitrate and nitrite metabolic systems to create a platform for the anaerobic synthesis of oxidized products. By engineering nitrate and nitrite metabolic systems increased productivity can be achieved including substrate, products, and cell growth while simultaneously reducing nitrate to nitrite to ammonium. The engineered bacteria will efficiently used nitrate as both nitrogen source and electron acceptor. Benefits of an engineered nitrate/nitrite metabolic system include inducing anaerobic respiration with increase growth together with reduced capital cost as shown in TABLE 1. TABLE 1 RESPIRATORY VS FERMENTATIVE METABOLISM

Variable Anaerobic Anaerobic Aerobic

Fermentation Respiration Respiration

SUMMARY OF THE INVENTION

[0012] [Summarize claims here.j

[0013] Proteins are described by function and a GENBANK™ reference sequence, as is standard practice in the field. For every reference protein there is an associated reference nucleotide, those of ordinary skill in the art can use the reference protein sequence to retrieve the encoding nucleotides. Protein sequence and activity are associated with similar proteins in a variety of species. Thus, all formate dehydrogenase enzymes will catalyze the conversion of formic acid to carbon dioxide. All nitrate reductase enzymes will catalyze the interconversion of nitrate (NO₃ ⁺) to nitrite (NO₂ ^"). Finally all nitrate/nitrite transporters transfer nitrate and/or nitrite across cell membranes. Reference sequences are provided here as examples of multiple similar sequences that share enzymatic function and sequence similarity to the protein sequences described in the reference sequence.

[0014] As used herein formate dehydrogenase (fdh) is described herein to refer to any one of the formate dehydrogenase proteins FDH-N, FDH-H, and FDH-O encoded by the genes fdnGHI, fdhF, and fdoGHI, including active subunits of the FDH complexes and accessory proteins required for assembly, stability and activity of the FDH complexes. Several FDH proteins are known including fdhA (AP 002097), fdhB (AP 002098), fdhC (AP 002099), fdhD (AP_003913), fdhF (NP_756934), fdhL (AP_003917), fdhN (AP_002097), fdhO (AP 003914), fdhE (NP 290520), fdhG (YP 540698), fdhH (AP 004580), fdhJ(CAA37989),fdoG, fdoH (AP_0039\5), fdoI, fdnG (NP_4\599\), fdnH, and fdnl these GENBANK™ records are incorporated herein by reference. Reducing or eliminating activity of fdhA , fdhB, fdhC, fdhD, fdhF, fdhL, fdhN, fdhO, fdhE, fdhG, fdhH, fdhJ, fdoH oxfdnG will dramatically reduce or eliminate FDH complex activity because the components are required for the assembly and activity of the complex. Additionally, selenium and molybdenum are necessary at the active site of FDH-H protein, and if Se or Mb are removed or replaced, the activity of the FDH complex will be inhibited. An active FDH complex converts formate (HCOO^") to carbon dioxide (CO₂), therefore reduced activity is defined herein as a reduction of formate conversion to carbon dioxide. [0015] Periplasmic nitrate reductase (nap) is used herein to describe any one of the periplasmic nitrate reductase proteins including active subunits of the NAP complex {nap AB) as is common in the art. NAP protein references are available on GenBank™ for napC (AP 002798), napB (AP 002799), napH (AP 002800), napG (AP 002801), napA (AP 002802), napD (AP 002803), and napF (AP 002804), incorporated herein by reference. Reducing or eliminating activity of napC, napB, napH, napG, napA, napD, or napF will dramatically reduce or eliminate NAP complex activity because each component is required for the activity of the complex.

[0016] Cytoplasmic nitrate reductase (nar) is used herein as in the art to describe any one of the respiratory nitrate reductase proteins whose active sites are located in the cytoplasm, including active subunits of two nitrate reductase (NAR) complexes: NAR A (NAR-GHJI), encoded by the narGHJI operon, and NAR Z (NAR-ZYWV), encoded by the narZYWV operon. NAR protein references available on GenBank™ include nar G (AP OO 1852), rcαr/f (AP_001853), rcαrJ (AP_001854), rcαr/ (AP_001855), narZ, narY, narW, and narV incorporated herein by reference. By reducing or eliminating activity of nar G, narH, narl, or narJ will dramatically reduce or eliminate NAR complex activity because each component is required for the activity of the complex.

[0017] Nitrate transporter/ (nitrate/nitrite antiporter) (narK) is used herein as in the art to describe any one of the nitrate transporter or nitrate/nitrite antiporter proteins including NAR- K (narK) and NAR-U (narU).

[0018] NAR-X is used herein as in the art to describe any one of the nitrate/nitrite- dependent two-component regulatory systems including subunits of NAR-XL (narXL operon).

[0019] Nitrite transporter (nir) is used herein as in the art to describe any one of the nitrite transport proteins including NIR-C. NIR-C is a nitrite transporter which is a member of the FNT family of formate and nitrite transporters. It functions to import nitrite as a substrate for a NADH-dependent nitrite reductase, the latter coded for by other genes in the nir operon (nirBDC). The nir operon is anaerobically expressed and is repressed by oxygen.

[0020] Lactate dehydrogenase (Idh) is used herein as in the art to describe any of the lactate dehydrogenase proteins. These include three lactate dehydrogenase enzymes in E. coli that interconvert pyruvate and lactate. In one embodiment an NAD-linked fermentative D- lactate dehydrogenase, encoded by the ldhA gene, is inactivated. In another embodiment one or both of the two membrane-bound flavoproteins, (D-lactate dehydrogenase and L-lactate dehydrogenase, encoded by the did and HdD genes, respectively), each specific for the D- or L-isomer of lactate, are inactivated.

[0021] Fumarate reductase (frd) is used herein as in the art to describe any of the proteins in E. coli that convert fumarate to succinate, and includes FRD-ABCD.

[0022] Alcohol/acetaldehyde dehydrogenase (adh) is used herein as in the art to describe any of the proteins that converts acetyl-CoA to acetaldehyde and/or acetaldehyde to ethanol. Examples of ADH in E. coli include ADH-E (adhE) and ADH-P (adhP).

[0023] The terms "operably associated" or "operably linked," as used herein, refer to functionally coupled nucleic acid sequences.

[0024] "Reduced activity" is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated or "inactivated" (100%). Proteins activity can be reduced with inhibitors, by mutation, or by suppression of expression or translation, by removal of an essential co factor or activator, and the like. By "null mutant" or "null mutation" what is meant is that activity is completely removed by creating a non-functional gene. In one example, the control plasmid is inserted without the gene of interest. In another example the gene of interest is completely removed by recombination. Additionally, the gene of interest may be inactivated by point mutation, or truncation, which eliminates activity.

[0025] "Overexpression" or "overexpressed" is defined herein to be greater than wild type activity, preferably above 125% increase, more preferably above 150% increase in protein activity as compared with an appropriate control species. Preferably, the activity is increased 100-500%. Overexpression is achieved by mutating the protein to produce a more active form, a more stable form, or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of a gene to the cell, up-regulating an existing gene, adding an exogenous gene, and the like.

[0026] The terms "disruption" and "disruption strains," as used herein, refer to cell strains in which the native gene or promoter is mutated, deleted, interrupted, or down-regulated in such a way as to decrease the activity of the gene. A gene is completely (100%) reduced by knockout or removal of the entire genomic DNA sequence or by a "null mutation." Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can completely inactivate (100%) a gene and its product by completely preventing transcription and/or translation of active protein.

[0027] The term "exogenous" indicates that the protein or nucleic acid is a non-native molecule introduced from outside the organism or system, without regard to species of origin. For example, an exogenous peptide may be applied to the cell culture, an exogenous RNA may be expressed from a recombinant DNA transfected into a cell, or a native gene may be under the control of exogenous regulatory sequences.

[0028] As used herein "recombinant" is relating to, derived from, or containing genetically engineered material.

[0029] A gene or cDNA may be "optimized" for expression in E. coli, or other bacterial species using the genetic codes or codon bias for the species. Various nucleotides can encode a single peptide sequence. Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides which encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species (www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc. cgi).

[0030] An Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. As a system of enzyme nomenclature, every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number.

[0031] In calculating "% identity" the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity = number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250. The default parameters were used, except the filters were turned OFF. As of Jan. 1, 2001 the default parameters were as follows: BLASTN or BLASTP as appropriate; Matrix = none for BLASTN, BLOSUM62 for BLASTP; G Cost to open gap default = 5 for nucleotides, 11 for proteins; E Cost to extend gap [Integer] default = 2 for nucleotides, 1 for proteins; q Penalty for nucleotide mismatch [Integer] default = -3; r reward for nucleotide match [Integer] default = 1; e expect value [Real] default = 10; W word size [Integer] default = 11 for nucleotides, 3 for proteins; y Dropoff (X) for blast extensions in bits (default if zero) default = 20 for blastn, 7 for other programs; X dropoff value for gapped alignment (in bits) 30 for blastn, 15 for other programs; Z final X dropoff value for gapped alignment (in bits) 50 for blastn, 25 for other programs. This program is available online at NCBI™ (www.ncbi.nlm.nih.gov/BLAST/).

[0032] Common restriction enzymes and restriction sites are found at NEB™ (New ENGLAND BIOLABS™, www.neb.com) and INVITROGEN™ (www.invitrogen.com) as well as other commercial enzyme suppliers. ATCC™, American Type Culture Collection™ (www.atcc.org), DSMZ™, Deutsche Sammlung von Mikroorganismen und Zellkulturen™ (www.dsmz.de), KBIF™, Korean Biological Resource Center™ (kbif.kribb.re.kr), and WDCM™, World Data Centre for Microorganisms™ (wdcm.nig.ac.jp) have extensive collections of cell strains that are publicly available. NEB™, Invitrogen™, ATCC™, DSMZ™, KBIF™, and WDCM™ databases are incorporated herein by reference.

TABLE 2 ABBREVIATIONS

Abbr Term

Ap/Ap^κ ampicillin / Ap resistance

ATCC™ American Tissue-type Culture Collection

Cm/Cm^R chloramphenicol / Cm resistance

Cn/Cn^R carbenicillin / Cn resistance

CoIEl gram-negative origin of replication

Em/Em^R erythromycin / Em resistance fdh formate dehydrogenase

HPLC high performance liquid chromatography

Km/Km^R kanamycin / Km resistance

Mh lactate dehydrogenase nap respiratory nitrate reductase, periplasmic nar respiratory nitrate reductase, cytoplasmic

NCBI™ National Center for Biotechnology Information

OriII gram-positive origin of replication

Ox/OxR oxacillin / Ox resistance

Sm/Sm^R streptomycin or streptomycin resistance

Tc/Tc^R tetracycline / Tc resistance

Thi^R/Cm^R thiamphenicol/chloramphenicol resistance

Wt wild-type

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 : Synthesis of oxidized products requires an NADH/electron sink. The production of acetate from glucose in E. coli through a hypothetically engineered pathway is illustrated. Bars represent potential genetic modifications to block the synthesis of competing products thus generating a homoacetogenic pathway. However, this engineered pathway would not be viable unless an electron acceptor is present to regenerate the NAD+ reduced to NADH (represented as "H"; 2H=NADH) during the operation of the glycolytic pathway (i.e., dispose of the electron generated by the oxidation of glucose.

[0034] FIG. 2: (A) Anaerobic fermentation and nitrate respiration of glucose in wild-type cells. Metabolized glucose, cell growth, and products are shown. (B) Acetate production from glucose (20 g/L) in the presence (50 mM) and absence of nitrate. Data is shown for wild-type W3110 and recombinant strains lacking fumarate reductase (FRD: frdABCD mutant shown) and lactate dehydrogenase (LDH: ldhA mutant shown) activities. Acetate yields in recombinant strains represent higher than 90% of the theoretical maximum.

[0035] FIG. 3: Diauxic growth of wild-type cells during anaerobic respiration.

[0036] FIG. 4: Systems involved in the metabolism of nitrate and nitrite in E. coli. Arrows indicate examples of target for genetic modifications.

[0037] Error! Reference source not found.: Performance of triple mutant KS03. (A) Comparison of cell growth in triple mutant KS03 (ΔNAR-K/NAR-X/NAP) and wild-type. Maximum specific growth rate during the exponential phase (ΔM) is shown. (B) Metabolism of sugar, nitrate, and nitrite in KS03 (ΔNAR-K/NAR-X/NAP). The volumetric rate of glucose consumption was 0.37 g glucose/ L/h and 1.01 g glucose/ L/h for W3110 and KS03, respectively.

[0038] FIG. 6: Growth of wild-type W3110 and triple mutant KS03 (ΔNAR-K/NAR- X/NAP) in an ammonium-deprived medium.

[0039] FIG. 7: Performance of pentamutant strain KS05 (ΔNAR-K/NAR- X/NAP/FDH/LDH). (A) Efficient utilization of nitrate as electron acceptor and nitrogen source (compared to the triple mutant). (B) Growth of wild-type and the triple- and penta- mutants.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The following description of embodiments illustrates the invention, but is not meant to unduly limit the invention.

EXAMPLE 1 : MATERIALS AND METHODS

[0041] E. coli strain W3110 (ATCC 27325) was used as wild type. Deletion mutants lacking individual genes or entire operons were constructed using the one-step inactivation

10 method of Datsenko and Wanner (Datsenko and Wanner, 2000). In this method, a PCR insert was first created using plasmid pKD4 (Datsenko and Wanner, 2000) as template and primers with flanking regions homologous to sequences in the coding region(s) of the gene(s) to be inactivated, resulting in an insert with kanamycin resistance gene at the center, flanked by two FRT (flippase recognition target) sites, and outer homology regions. Wild type cells expressing λ Red recombinase (transformed with plasmid pKD46 (Datsenko and Wanner, 2000), grown at 3O⁰C) were transformed with the PCR insert, and the gene sequence between the two homology regions were replaced with the FRT::Km::FRT sequence through recombination.

[0042] To eliminate the kanamycin resistance gene, the mutants were transformed with pCP20 (Datsenko and Wanner, 2000), a temperature sensitive plasmid expressing flippase (FLP). FLP expressed from this plasmid remove the Km region from the FRT::Km::FRT site, leaving one FRT site behind. pCP20 was then removed by growing the cells at 43⁰C. All mutants were verified with genomic PCR after construction to ensure that the gene of interest had been disrupted. By using plasmid pKD4 as a template we ensured the creation of in- frame gene deletions, thus preventing polarity effects on the expression of downstream genes (Id.).

[0043] The disruption of multiple genes in a common host was achieved by sequentially implementing the procedure described above after eliminating the kanamycin resistance cassette introduced with the preceding mutation. Creation of strains with multiple gene disruptions could result in the recombination of the new PCR fragment at the scar of an earlier gene disruption or chromosomal rearrangements might result from FLP -promoted recombination events between FRT sites at different loci (Id.). To verify that these events did not occur in strains with multiple gene disruptions each strain was rechecked in all gene disruption sites by PCR on elimination of a resistance gene from a new locus in such multiple mutants.

[0044] Wild-type and mutant strains along with the relevant genotypes are shown in TABLE 3. Primers used for gene disruption and verification are shown in TABLE 4. During strain construction, cultures were grown in LB broth or on LB plates (1.5% agar). Standard recombinant DNA procedures were used for plasmid isolation, electroporation, and polymerase chain reaction. The strains were kept in 32.5% glycerol stocks at -8O⁰C. Plates were prepared using LB medium containing 1.5% agar. Antibiotics were included as needed

11 at the following concentrations: 100 μg/ml ampicillin, 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 12.5 μg/ml tetracycline.

TABLE 3 SELECTED STRAINS AND THEIR GENOTYPES

12 TABLE 4 PLASMIDS AND PRIMERS

*"f" and "r" indicate forward are reverse primer sequences (5' to 3'). Gene or operon inactivated is incorporated in the primer name.

[0045] Wild-type and recombinant strains were evaluated for their capacity to perform anaerobic nitrate respiration of glucose using a minimal media of the following composition KH2PO4, 3.5 g/L; K₂HPO₄, 5 g/L; (NH₄)₂HPO₄, 3.5 g/L; MgSO₄H₂O, 0.25 g/L; CaCl₂2H₂O, 0.015 g/L; and Thiamine, 5xlO^"4 g/L. Trace metals were separately prepared as a IOOX solution and added to achieve the following concentration in the final medium: FeCl₃, 1.6 mg/L; CoCl₂OH₂O, 0.2 mg/L; CuCl₂, 0.1 mg/L; ZnCl₂4H₂O, 0.2 mg/L; NaMoO₄, 0.2 mg/L; H3BO3, 0.05 mg/L. Molybdate and selenate were also added at a final concentration of 1 μM.

[0046] To ensure fully respiratory conditions, equimolar concentrations of nitrate and glucose were used; this represents the maximum concentration of nitrate required for the complete oxidation of glucose into acetic acid and CO₂ (considering the reduction of nitrate to nitrite to ammonium by using NADH and/or formate as electron donors). All experiments were conducted at 37⁰C under anaerobic conditions. Experiments in tubes were conducted in sealed anaerobic tubes (Hungate tubes) completely filled with media, initial pH of 6.8, and 3- [N-morpholino]propanesulfonic acid (0.1 M, pH 7.4) was added for pH control. Chemicals were obtained from FISHER SCIENTIFIC™ (PA) and SIGMA-ALDRICH CO.™ (MO).

[0047] Fermentations were conducted in a SIXFORS™ multi-fermentation system (INFORS HT™, Switzerland) with six 500ml working volume vessels and independent control of temperature, pH, and stirrer speed (200 rpm). The system was fully equipped and computer controlled using manufacturer IRIS NT™ software. Each vessel was fitted with a condenser to minimize evaporation, which was operated with a O⁰C cooling methanol-water supply. Anaerobic conditions were maintained by flushing the headspace with ultrahigh purity argon (MATHESON TRI-GAS,™ TX) at 0.01 LPM. An oxygen trap (ALLTECH ASSOCIATES, INC.,™ IL) was used to eliminate traces of oxygen from the gas stream. In some experiments (specified in each case) carbon dioxide or hydrogen were also included in the gas phase. Prior to use, the stock cultures (stored as glycerol stocks at -8O⁰C) were streaked onto LB plates and incubated overnight at 37°C in an OXOID™ anaerobic jar with the CO₂ gas generating kit (OXOID LTD,™ UK). A single colony was used to inoculate 17.5 mL Hungate tubes completely filled with medium. The tubes were incubated at 37⁰C until an

13 OD550 of ~0.4 was reached. An appropriate volume of this actively growing pre-culture was centrifuged and the pellet washed and used to inoculate 350 mL of medium in each fermenter, with the target starting optical density of 0.05 at 550 nm.

[0048] Four milliliters of culture were drawn using a syringe and chilled on ice-water bath to quench cellular metabolism. Optical density was measured at 550 nm and used as an estimate of cell mass (1 OΔ = 0.34 g dry weight/L). After centrifugation, the supernatant was stored at -20⁰C for High Performance Liquid Chromatography (HPLC) analysis. To quantify concentration of glucose, nitrate, organic acids, and ethanol, samples were analyzed with ion- exclusion HPLC using a Shimadzu Prominence SIL 20 system (SHIMADZU SCIENTIFIC INSTRUMENTS INC.™, MD) equipped with an HPX-87H organic acid column (BIO- RAD,™ CA). Operating conditions to optimize peak separation (30 mM H₂SO₄ in mobile phase, column temperature 42°C) were determined using a previously described approach. Nitrite concentrations were estimated colorimetrically as previously described (Gonzalez et al, 2003; Nicholas and Nason, 1957).

[0049] Specific rates for cell growth (μ), glycerol consumption, and product synthesis were estimated by plotting total cell, glucose, or product concentration versus the integral of cell concentration (ICC), and fitting these plots to polynomial functions. The slope of the curves thus obtained (a straight line during exponential growth) was used as the average specific rate.

EXAMPLE 2: SINGLE MUTANT CHARACTERIZATION

[0050] The production of oxidized chemicals from sugars requires the presence of an external electron acceptor to reoxidize the NADH to NAD+ and maintain the glycolytic flux. The use of oxygen as electron acceptor typically results in a high cost of the fermentation process due to the increase in capital and operational costs. The cost of an anaerobic fermentation process using nitrate as an alternative electron acceptor will be significantly lower than the cost of an aerobic process. This is very relevant for the production of commodity chemicals whose fermentation cost can represent 50-90% of the total production cost. In addition, the reduction of nitrate to nitrite to ammonium permits the use of nitrate as nitrogen source and reduces the associated cost. The same sequence of reactions alkalinizes the medium (Nitrate reduction: NADH + NO₃ ^" -> NAD + NO₂ ^" + H₂O; Nitrite reduction: NO₂ ^" + 3NADH -» 2OH^" + NH₄ ⁺ + 3NAD), thus reducing the use of base for pH control during the production of organic acids such as pyruvic and acetic. Ammonium produced will

14 also reduce the requirement of ammonium salts typically used to purify organic acids. Contamination of the environment by oxidized nitrogen species (nitrates and nitrites) arising from the use of fertilizers in agriculture and several by-product streams in the chemical industry also makes it highly desirable to develop a process for the reduction of nitrate. To eliminate the synthesis of competing by-products succinate and lactate the activities of lactate dehydrogenase and fumarate reductase were reduced by disrupting genes ldhA and operon frdABCD, respectively. Resulting strains produced acetate at yields approaching the theoretical maximum (i.e., around 0.6 g acetate/g glucose) (FIG. 2B).

[0051] Anaerobic fermentation of sugars by E. coli is a well-known process that generates a mixture of fermentation products including organic acids, ethanol, and CO₂. The most abundant fermentation product is lactic acid, followed by formic acid and almost equimolar amounts of acetic acid and ethanol along with minor amounts of succinic acid (FIG. T). The inclusion of nitrate in the culture medium results in a shift in the composition of fermentation products (FIG. 2), with reduced products being either absent (ethanol) or in much lower amounts (lactate and succinate). Acetic acid, however, becomes the main fermentation product. Drastic reduction in succinate production and lack of ethanol are known to be the result of the negative regulation of the expression and the activity of fumarate reductase (coded for by frdABCD) and alcohol/acetaldehyde dehydrogenase (coded for by adhE) enzymes by nitrate. Similarly, decreased production of formate is due to repression of the /?/7 operon by nitrate (Kaiser and Sawers, 1995) and the consumption of formate as electron donor for the reduction of nitrate and nitrite. Although providing an external electron acceptor (nitrate) results in a higher generation of energy via respiration, the accumulation of the toxic compound nitrite is detrimental for cell growth. The overall effect is a lower specific growth rate during nitrate respiration, although the final cell concentration was very similar to that of anaerobic fermentation (FIG. 2).

[0052] An important feature observed during the respiratory metabolism of glucose in the presence of nitrate was a diauxic growth (FIG. 3). Diauxic growth or diauxie is a phenomenon of bacterial growth in which an organism given a mixture of organic compounds first grows exclusively on one (i.e. nitrate) until that compound is exhausted, and then, after a lag during which it forms induced enzymes for utilizing the second compound, resumes growth on the latter (i.e. nitrite). Wild-type W3110 exhibited an initial phase of growth with a maximum specific growth rate of 0.66 h^"1. During this phase nitrite accumulated at a rate almost identical to the consumption of nitrate (FIG. 3): i.e. nitrate

15 respiration was the main metabolic mode. The repression exerted by nitrate over the enzymes/proteins/genes involved in the reduction of nitrite to ammonium prevented nitrite reduction during this first phase of growth. The depletion of nitrate from the medium triggered a diauxic lag followed by a second phase of growth. Nitrite was then used as electron acceptor and cell metabolism was based on nitrite respiration. This phenomenon has important implications for the potential use of nitrate as both electron acceptor and nitrogen source in the production of oxidized chemicals. The disruption of this diauxie should result in the simultaneous reduction of nitrate to nitrite and nitrite to ammonium, thus enabling the use of nitrate as N-source and improving cell growth by preventing nitrite accumulation.

[0053] Many proteins are involved in regulatory, transport, and enzymatic functions related to the reduction of nitrate to nitrite to ammonium. Among them are three nitrate reductases, two nitrite reductases, three nitrate/nitrite transporters, two two-component regulatory systems, two formate dehydrogenases, and two NADH dehydrogenases (FIG. 4). The three nitrate reductases catalyzing the reduction of nitrate into nitrite include the cytoplasmic, membrane-associated enzymes NAR-G and NAR-Z and a periplasmic nitrate reductase (NAP). The reduction of nitrite to ammonia is catalyzed by the cytoplasmic, NADH-dependent nitrite reductase NIR-BD and the periplasmic, cytocrome c nitrite reductase NRF. Nitrate and nitrite are transported in and out of the cells by two nitrate (NAR- K and NAR-U) and three nitrite (NAR-K, NAR-U, and NIR-C) transporters. Nitrite extrusion on the presence of nitrate mainly takes place through NAR-K, but nitrite uptake can be supported at similar rates by either NAR-K or NIR-C. Nitrate uptake can be equivalently supported by either NAR-K or NAR-U. The expression of nitrate- and nitrite-regulated genes is mediated by two environmental signals (the absence of oxygen and the presence of nitrate/nitrite ions in the culture medium), several global regulators (FNR, FIS, IHF, and H- NS, and CRA), and by the homologous two-component regulatory systems NAR-X/NAR-L and NAR-Q/NAR-P. Formate and NADH are among the electron donors for nitrate and nitrite reduction. Formate dehydrogenases (FDH-N and FDH-O) and NADH dehydrogenases deliver the electrons from formate and NADH to the quinone pools, which, in turn, pass the electrons to the nitrate and nitrite reductases. This conserves cellular energy and generates ATP via the proton-translocating ATPase.

[0054] A first group of genetic modifications was introduced to simultaneously reduce nitrate to nitrite and nitrite to ammonium, also avoiding the accumulation of nitrite in the medium. The strategy is based on preventing the extrusion of nitrite produced by the more

16 active membrane-associated nitrate reductase (NAR-G) while avoiding the production of nitrite via the periplasmic nitrate reductase (NAP). Using this approach, genes involved in these processes were disrupted, both singularly and in combinations, : i.e. napFDAGHBC, encoding periplasmic nitrate reductase NAP; narK, encoding a nitrite/nitrate transporter; and narX encoding sensor NAR-X which is part of the homologous two-component regulatory system NAR-X/NAR-L (in vitro molecular studies have shown that NAR-X negatively regulates the NAR-L protein by acting as a NAR-L-phosphate phosphatase; our goal on deleting narX is to prevent the decrease in NAR-G with the decay in nitrate concentrations as the fermentation proceed). The aforementioned strategies for genetic modifications are summarized in FIG. 4.

[0055] A second group of genetic modifications aiming at maximizing the use of nitrate and nitrate for the oxidation of glycolytic NADH were also designed. These include blocking the use of formate as electron donor and minimizing the production of NADH during the dissimilation of pyruvate. The first was achieved by disrupting the genes encoding FDH-N and FDH-O, fdnGHI and fdoGHI respectively. On the other hand, genes coding for pyruvate dissimilating enzymes PDH (aceEF) and POX-B (poxB) were also disrupted.

[0056] Assessing the effect of genetic modifications inevitably involves the analysis of how beneficial nitrate respiration is for cell growth. Formate-dependent nitrate respiration is known to generate significant amounts of ATP. This process, however, also generates a toxic compound (nitrite) whose metabolism is repressed by the presence of nitrate. Formate- independent nitrate respiration can have other beneficial effects. For example, the NADH- dependent reduction of nitrate and nitrite regenerates NAD+ and thus favor the glycolytic flux and the production of acetate, both pathways resulting in the generation of additional ATP via substrate-level phosphorylation (pathways involved in the synthesis of reduced products such as lactic acid and ethanol are not energy efficient). To investigate the metabolic effects of the aforementioned genetic modifications, wild-type and recombinant strains were evaluated (not shown) and discussed in what follows.

[0057] Wild-type W3110 consumed nitrate and produced nitrite during the first growth phase, which resulted in a nitrite accumulation in the medium that reached 40 mM at the onset of the transition phase, clearly demonstrating that only nitrate was used as electron acceptor until this point (nitrite concentration equals the concentration of nitrate in the initial medium). A second phase of growth was marked by the use of nitrite as electron acceptor,

17 which was then consumed. The introduced genetic modifications affected, to different extends, cell growth, production and consumption of nitrite, and glucose consumption.

[0058] Blocking the nitrate/nitrite transporter NAR-K resulted in a strain lacking the diauxie observed in the wild type; no transition phase was observed that separates the use of nitrate (first phase) and nitrite (second phase) as electron acceptors. This is due to the ability of W3l lOAnarK to simultaneously convert nitrate to nitrite to ammonium. Another effect of this modification was a more robust growth as can be seen from the higher maximum specific growth rate (0.81 h, - 23% higher than the wild type) and a higher cell concentration (4.18 OD550, -30% higher than the wild type). The overall effect was a reduction on the fermentation time from 14 to 10 hours (-30%), which also resulted in an increase in glucose consumption rate. These changes appear to be caused by a decrease in the export of intracellularly produced nitrite (produced by the membrane bound nitrate reductases NAR-G and NAR-Z), which in turn activates nitrite detoxification by the cytoplasmic, NADH- dependent nitrite reductase (NIR-BD).

[0059] The benefits from higher activity of NIR-BD are two-fold. First, it directly increases metabolic activity by reducing the levels of the toxic metabolite nitrite. Secondly, it results in a higher NADH oxidation rate, with the subsequent increase in glycolytic flux, energy generation by substrate level phosphorylation, and cell growth. Since NAR-K is a nitrate/nitrite antiporter (nitrate import and nitrite export), an alternative explanation could be that the observed changes are due to a decrease in nitrate import. However, a decrease in nitrate import would result in a decrease in nitrate reduction since the periplasmic nitrate reductase, NAP, is known to be repressed by high nitrate concentration (Choe and Reznikoff, 1993; Rabin and Stewart, 1993; Wang et al, 1999). However, the disruption of narK gene did not affect nitrate utilization (data not shown), supporting the hypothesis that the decrease in nitrite export and not the decrease in nitrate imported caused the observed changes. Also, it is known that nitrate uptake can be equivalently supported by either NAR-K or NAR-U. Taking together, these results show that the narK mutation did increase metabolic activities by improving any respiratory process but rather by accelerating nitrite detoxification and improving redox balancing.

[0060] It has been reported that nitrite has been as effective as nitrate in inducing the expression of genes encoding FDH-N and NAR-G in narX deletion mutants (i.e. deletion of the gene encoding response regulator NAR-X) (Rabin and Stewart, 1993). The same mutation negatively affected the expression of genes encoding FRD-ABCD, NAP, and NRF in the

18 presence of nitrite (Rabin and Stewart, 1993). Our goal on deleting this gene was to improve nitrate reduction (NAR-G) and prevent the formate dependent reduction of nitrate (NAP) and nitrite (NRF). After blocking the synthesis of the sensor protein NAR-X a diauxic growth was still observed with two respiratory phases; a first phase representing nitrate respiration and a second phase nitrite respiration. Nitrite accumulation at the end of the nitrate respiratory phase was at levels very similar to the wild-type strain. The maximum specific growth rate during nitrate respiration phase was slightly higher than wild-type but the maximum cell concentration was lower and the fermentation time longer. Therefore, we could expect to see a faster nitrate respiration phase and a lower accumulation of formate during this first phase in a narX null mutant, characteristic that are observed in W31 lOAnarX (higher specific growth rate than wild-type and zero formate). The higher specific growth rate could be related to a significant use of formate as electron acceptor increasing energy generation. Lower levels of succinate were observed in both phases in agreement with predicted effect of narX mutation. Slightly higher concentration of formate (2.04 in narXvs 1.72 in WT) and similar nitrite concentrations were observed at the stationary phase. Lower activity of NRF could explain higher formate concentration and larger duration of the second phase.

[0061] NAP is optimally induced under limiting concentrations of nitrate, induced weakly during growth in the presence of nitrite, and repressed by high nitrate concentration (Choe, 1993; Rabin, 1993; Wang, 1999). Deletion of the genes encoding NAP resulted in a nitrate respiratory phase very similar to the wild-type (μ = 0.68 h^"1) in agreement with a minor role of this enzyme at high concentrations of nitrate. However, there were major differences with wild-type and other recombinant strains. ΔNAR-X only exhibited the first phase of growth, i.e. nitrate respiration. The final concentration of cells at the end of this phase was lower than the wild-type (1.64 OD550 vs 2 OD550). Nitrite accumulated in the medium in stoichiometric proportions respect to nitrate and was not used as electron acceptor. Although there is no report about a possible role of NAP in the transition mediating the two phases of a nitrate- nitrite diauxie, it has been shown that one physiological role of the periplasmic nitrate reductase of E. coli is to enable bacteria to scavenge nitrate in nitrate-limited environments (Potter et al, 1999). In a sense, the transition phase is a nitrate limited environment on which the role of NAP would be supporting growth and/or maintenance while the cells start using nitrite as electron acceptor.

19 EXAMPLE 3: MULTIPLE MUTANTS CHARACTERIZATION

[0062] Single mutations studied above brought about metabolic changes that resulted in dramatically change the use metabolism of nitrate and nitrite. We now study the behavior of strains containing multiple mutations, namely all possible double mutants (strains ΔNAR-K/NAR-X, ΔNAP/NAR-X, and ΔNAP/NAR-K and the triple mutant strain KS03 (ΔNAR-K/NAR-X/NAP). The double mutants behaved as expected in most cases exhibiting additive effects of changes observed in single mutants (data not shown). Surprisingly, the narX mutation reverted the negative effect of the napF- C mutation: i.e., strain ΔNAP/NAR-X recovered the wild-type phenotype (diauxic growth) that had been lost by the napF-C mutation. The volumetric rate of glucose consumption was 0.37 g glucose/ L/h for wild type and 1.01 g glucose/ L/h for KS03 (ΔNAR-K/NAR-X/NAP). Thus, the best performance was observed with the triple mutant (FIG. 5).

[0063] E. coli cultures can use nitrate as a nitrogen source, although its feasibility has only been demonstrated in N-limited continuous culture (Cole et ah, 1974). The triple-mutant constructed in this work should be able to simultaneously convert nitrate to nitrite to ammonium, thus using nitrate as nitrogen source. To test this hypothesis, we cultivated KS03 (ΔNAR-K NAR-X and NAP) in a modified medium containing 1/100 of the original amount of ammonium (i.e. 0.035 g/L of ammonium sulfate/phosphate). The cells grew as efficiently as they did in the full strength ammonium media (FIG. 6). As a reference, the growth of wild- type W3110 in the low-ammonium media was greatly impaired reaching stationary phase at 8 hours and a maximum OD of 0.5 (FIG. 6). Thus, the triple mutant can efficiently use nitrate.

[0064] Formate and NADH are among the electron donors for nitrate and nitrite reduction. Formate is produced by the enzyme PFL during the conversion of pyruvate into acetyl-CoA (FIG. 1). Pyruvate is also dissimilated by PDH and POX-B (FIG. 1; only PDH shown), which generate additional reducing equivalents. Formate dehydrogenase (FDH) and NADH dehydrogenase deliver the electrons from formate and NADH to the quinone pools, which, in turn, pass the electrons to the nitrate and nitrite reductases (FIG. 4). This conserves cellular energy and generates ATP via the proton-translocating ATPase. E. coli synthesizes three formate dehydrogenase enzymes: respiratory FDH-N and FDH-O encoded by the operons fdnGHI and fdoGHI, respectively (FIG. 4), and fermentative FDH-H, encoded by the gene fdhF (Gennis, R. and Stewart.1996; Gunsalus, 1992). The optimum utilization of nitrate and nitrite as electron acceptors in the synthesis of oxidized chemicals requires first maximizing

20 the dissimilation of pyruvate via PFL (i.e. preventing the production of extra reducing equivalents through PDH and POX-B) and second decoupling formate oxidation from the reduction of nitrate and nitrite. This would result in maximizing the use of nitrate and nitrite in the oxidation of NADH generated by the glycolytic pathway. We created strains devoid of the genes coding for the two respiratory formate dehydrogenases (strains W3110 AfdnGHI and W3110 AfdoGHI) and the two pyruvate dissimilating enzymes PDH and POX-B (strains W3l lOAaceEF and W3110ApoxB). Disruption of FDH-N was sufficient to achieve equimolar concentrations of acetate and formate. This is in agreement with previous reports that FDH-N exhibits maximal expression during nitrate respiration (Chaudhry and MacGregor, 1983; Enoch and Lester, 1975) while FDH-O is synthesized at relatively low levels independent of nitrate availability (Abaibou et al, 1995). These results also indicate little or no involvement of PDH and POX-B in the dissimilation of pyruvate.

[0065] To further channel carbon toward the production of acetate/formate, we blocked the synthesis of lactate by disrupting the gene idhA. The performance of the pentamutant KS05 (ΔNAR-K NAR-X NAP FDH and LDH) thus created is shown in FIG. 7. This strain exhibited healthy growth and converted more than 90% of the carbon source into equimolar amounts of acetate and formate. It was also able to efficiently use nitrate as both electron acceptor and nitrogen source. All the characteristics observed in previous mutants are found in this strain: (1) simultaneous reduction of nitrate to nitrite to ammonium, (2) no diauxic growth, (3) decreased accumulation of nitrite, (4) more robust growth, (5) efficient use of nitrate as both electron and nitrogen source (i.e. medium with low ammonium and high sugar concentration).

[0066] Taken together these results show that KS03 (ΔNAR-K NAR-X NAP) especially KS05 (ΔNAR-K NAR-X NAP FDH and LDH) represent an excellent platform for the cost- effective production of oxidized chemicals under anaerobic conditions. Chemicals that can be effectively synthesized using these mutants include lactate, formate, ethanol, acetate, succinate, citrate, pyruvate and related organic acids or amino acids.

[0067] All references are listed herein for the convenience of the reader. Each is incorporated by reference in its entirety: Abaibou, et al, Expression and characterization of the Escherichia coli fdo locus and a possible physiological role for aerobic formate dehydrogenase. J. Bacteriol, 177: 7141-7149(1995); Cheryan, et al, Production of acetic acid by Clostridium thermoaceticum. Adv. App. Microbiol, 43: 1-33(1997); Chaudhry and

21 MacGregor, Cytochrome b from Escherichia coli nitrate reductase; its properties and association with the enzyme complex. J. Biol. Chem., 258: 5819-5827(1983); Choe and Reznikoff, Identification of the regulatory sequence of anaerobically expressed locus aeg- 46.5. J Bacteriol., 175: 1165-72(1993); Cole, et al., Nitrite and ammonia assimilation by anaerobic continuous cultures of Escherichia coli. J. Gen. Microbial., 85: 11-22(1974); Cole, Nitrate reduction to ammonia by enteric bacteria: redundancy, or a strategy for survival during oxygen starvation? FEMS Microbiol. Lett., 136: 1-11(1996); Datsenko and Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A., 97:6640-6645(2000); Enoch and Lester, The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli. J. Biol. Chem., 250: 6693-6705(1975); Gennis and Stewart, Respiration, p. 217-261. In Neidhardt, et al. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., ASM Press, Washington, DC(1996); Gonzalez, et al., Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: Comparison of KOl 1 (parent) to LYOl (resistant mutant). Biotechnol. Prog. 19:612-23(2003); Gonzalez, Metabolic Engineering of Bacteria for Food Ingredients. In: Food Biotechnology: Second Edition, pp. 111-130. Shetty, et al. (ed.), CRC Press, Boca Raton, FL(2005); Gunsalus, Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes., J Bacteriol. 174:7069-74(1992); Kaiser and Sawers, Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP. J. Bacteriol., 177:3647-55(1995); Rabin and Stewart, Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12. J. Bacteriol., 175:3259-68(1993); Sawers, R. G., Blokesch and Bock, Anaerobic formate and hydrogen metabolism. In Neidhardt, et al. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, Web Edition. ASM Press, Washington, DC. (http://www.ecosal.org) (2004); Tishkov and Popov, Catalytic mechanism and application of formate dehydrogenase. Biochemistry (Mosc). 69:1252-67(2004); Wang, et al., The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite. J. Bacteriol., 181 : 5303-8(1999); and Yoshida, et al., Enhanced Hydrogen Production from Formic Acid by Formate Hydrogen Lyase-Overexpressing Escherichia coli Strains. Appl. Environ. Microbiol. 71 : 6762-8(2005)

22

Claims

CLAIMSWhat is claimed is:

1. An engineered bacteria comprising inactivation of a cytoplasmic nitrate reductase (nar) or periplasmic nitrate reductase (nap) and inactivation of a nitrate/nitrite-dependent regulatory system (narX) and inactivation of a nitrate/nitrite antiporter (narK) gene wherein said bacteria simultaneously reduces nitrate to nitrite to ammonium.

2. The bacteria of claim 0, comprising inactivation of one or more genes selected from nar, nap, formate dehydrogenase (fdh),fdh,fdo,fdn,, lactate dehydrogenase (Idh), idhA, fumarate reductase (frdABCD),frdA,frdB,frdC,frdD, alcohol dehydrogenase (adh), adhE, adhP, pyruvate dehydrogenase (aceEF-lpdA), aceEF ΑnάpoxB.

3. The bacteria of claim 0, wherein said bacteria is E. coli.

4. The bacteria of claim 0, wherein the bacteria is W311 OA(narK_narXC_napF-C).

5. The bacteria of claim 0, wherein the bacteria is W311 QA(narK_narXC_napF-CJdnG).

6. The bacteria of claim 0, wherein the bacteria is W3110A(narK_narXC_napF- CJdnGJdhA).

7. A method of producing oxidation products comprising:

(A) culturing the bacteria of claim 0 under anaerobic conditions with nitrate or nitrite and a carbon source under conditions that produce an oxidation product, and

(B) purifying said oxidation product.

8. The method of claim 7, wherein in the oxidation product is selected from the group consisting of lactate, formate, ethanol, acetate, succinate, citrate, pyruvate, and acetone.

9. The method of claim 7, wherein in the oxidation product is formate.

10. A method of producing oxidation products from engineered bacteria, comprising:

23 (A) growing engineered bacteria comprising reduced activity of a cytoplasmic nitrate reductases (nar) or periplasmic nitrate reductase (nap) and reduced activity of a nitrate/nitrite-dependent regulatory system (narX) and reduced activity of a nitrate/nitrite antiporter (narK) gene under anaerobic conditions with nitrate or nitrite and a carbon source under conditions that produce an oxidation product, and

(B) purifying said oxidation product.

11. The method of claim 10, wherein in the oxidation product is selected from the group consisting of lactate, formate, ethanol, acetate, succinate, citrate, pyruvate, and acetone.

12. The method of claim 10, wherein in the oxidation product is formate.

13. The method of claim 10, wherein the engineered bacteria comprises inactivation of one or more genes selected from nar, nap, formate dehydrogenase (fdh),fdh,fdo,fdn,, lactate dehydrogenase (Idh), UhA, fumarate reductase (frdABCD),frdA,frdB,frdC,frdD, alcohol dehydrogenase (adh), adhE, adhP, pyruvate dehydrogenase (aceEF-lpdA), aceEF and poxB.

14. The method of claim 10, wherein the engineered bacteria is E. coli.

15. The method of claim 10, wherein the engineered bacteria is A(narK_narXC_napF-C).

16. The method of claim 15, wherein the engineered bacteria is E. coli.

17. The method of claim 10, wherein the engineered bacteria is A(narK_narXC_napF- CJdnG).

18. The method of claim 17, wherein the engineered bacteria is E. coli.

19. The method of claim 10, wherein the engineered bacteria is A(narK_narXC_napF- CJdnGJdhA).

20. The method of claim 19, wherein the engineered bacteria is E. coli.

24