WO2008147470A2 - Synthèse anaérobique de produits oxydés par e. coli - Google Patents

Synthèse anaérobique de produits oxydés par e. coli Download PDF

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WO2008147470A2
WO2008147470A2 PCT/US2007/088254 US2007088254W WO2008147470A2 WO 2008147470 A2 WO2008147470 A2 WO 2008147470A2 US 2007088254 W US2007088254 W US 2007088254W WO 2008147470 A2 WO2008147470 A2 WO 2008147470A2
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nitrate
nitrite
bacteria
nar
formate
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WO2008147470A3 (fr
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Ramon Gonzalez
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Rice University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C12P7/28—Acetone-containing products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/48—Tricarboxylic acids, e.g. citric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention may have been developed with funds from the United States
  • the invention is a new process/method for the anaerobic (i.e., absence of oxygen) production of oxidized chemicals using engineered E. coli strains.
  • 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.
  • 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.
  • an exogenous electron acceptor such as oxygen, nitrate, nitrite, fumarate, trimethylamine N-oxide, or dimethylsulfoxide.
  • 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.
  • 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.).
  • 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.
  • FIG. 1 illustrates this need taking as example the engineering of a homoacetogenic pathway in E. coli.
  • oxygen represents the common electron acceptor used in industry.
  • 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).
  • 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.
  • 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.
  • 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 CO 2 and hydrogen. Induction of the FHL requires formate, molybdate, absence of e- acceptors (O 2 or nitrate), and acidic pH.
  • the other component of the FHL complex is a hydrogenase (Hyd-3). There are two other hydrogenases in E.
  • FDH-O The third formate dehydrogenase, FDH-O, is membrane bound and is structurally and immunologically related to FDH-N.
  • 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).
  • 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.
  • an engineered bacteria with modified nitrate and nitrite metabolic systems to create a platform for the anaerobic synthesis of oxidized products.
  • 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
  • Proteins are described by function and a GENBANKTM 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 (NO 3 + ) to nitrite (NO 2 " ). 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.
  • formate dehydrogenase 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.
  • FDH proteins 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 GENBANKTM 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.
  • 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 GenBankTM 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.
  • 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-GHJI active subunits of two nitrate reductase
  • NAR Z NAR-ZYWV
  • NAR protein references available on GenBankTM include nar G (AP OO 1852), rc ⁇ r/f (AP_001853), rc ⁇ rJ (AP_001854), rc ⁇ r/ (AP_001855), narZ, narY, narW, and narV incorporated herein by reference.
  • 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).
  • 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).
  • Nitrite transporter 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.
  • 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.
  • 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.
  • 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.
  • ADH in E. coli include ADH-E (adhE) and ADH-P (adhP).
  • operably associated or “operably linked,” as used herein, refer to functionally coupled nucleic acid sequences.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NCBITM provides codon usage databases for optimizing DNA sequences for protein expression in various species (www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc. cgi).
  • An Enzyme Commission 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.
  • % 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.
  • NEBTM New ENGLAND BIOLABSTM, www.neb.com
  • INVITROGENTM www.invitrogen.com
  • ATCCTM American Type Culture CollectionTM (www.atcc.org)
  • DSMZTM Deutsche Sammlung von Mikroorganismen und ZellkulturenTM (www.dsmz.de)
  • KBIFTM Korean Biological Resource CenterTM (kbif.kribb.re.kr)
  • WDCMTM World Data Centre for MicroorganismsTM (wdcm.nig.ac.jp) have extensive collections of cell strains that are publicly available.
  • NEBTM, InvitrogenTM, ATCCTM, DSMZTM, KBIFTM, and WDCMTM databases are incorporated herein by reference.
  • Tc/Tc R tetracycline / Tc resistance
  • FIG. 1 Synthesis of oxidized products requires an NADH/electron sink.
  • 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.
  • FDD fumarate reductase
  • LDH lactate dehydrogenase
  • FIG. 3 Diauxic growth of wild-type cells during anaerobic respiration.
  • FIG. 4 Systems involved in the metabolism of nitrate and nitrite in E. coli. Arrows indicate examples of target for genetic modifications.
  • FIG. 6 Growth of wild-type W3110 and triple mutant KS03 ( ⁇ NAR-K/NAR- X/NAP) in an ammonium-deprived medium.
  • FIG. 7 Performance of pentamutant strain KS05 ( ⁇ NAR-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.
  • 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
  • Wild type cells expressing ⁇ Red recombinase transformed with plasmid pKD46 (Datsenko and Wanner, 2000), grown at 3O 0 C) 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.
  • 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 43 0 C. 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.).
  • 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.
  • 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 -8O 0 C. Plates were prepared using LB medium containing 1.5% agar. Antibiotics were included as needed
  • 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; K 2 HPO 4 , 5 g/L; (NH 4 ) 2 HPO 4 , 3.5 g/L; MgSO 4 H 2 O, 0.25 g/L; CaCl 2 2H 2 O, 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: FeCl 3 , 1.6 mg/L; CoCl 2 OH 2 O, 0.2 mg/L; CuCl 2 , 0.1 mg/L; ZnCl 2 4H 2 O, 0.2 mg/L; NaMoO 4 , 0.2 mg/L; H3BO3, 0.05 mg/L. Molybdate and selenate were also added at a final concentration of 1 ⁇ M.
  • EXAMPLE 2 SINGLE MUTANT CHARACTERIZATION
  • Anaerobic fermentation of sugars by E. coli is a well-known process that generates a mixture of fermentation products including organic acids, ethanol, and CO 2 .
  • 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 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.
  • 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.
  • nitrate provides 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).
  • 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
  • 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).
  • 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
  • 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.
  • 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
  • NAR-G active membrane-associated nitrate reductase
  • NAP periplasmic nitrate reductase
  • napFDAGHBC encoding periplasmic nitrate reductase NAP
  • narK encoding a nitrite/nitrate transporter
  • narX encoding sensor NAR-X which is part of the homologous two-component regulatory system NAR-X/NAR-L
  • 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.
  • 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.
  • 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,
  • 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).
  • NIR-BD 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.
  • 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.
  • NAR-G nitrate reduction
  • NAP formate dependent reduction of nitrate
  • NAF nitrite
  • 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.
  • Nitrite accumulated in the medium in stoichiometric proportions respect to nitrate and was not used as electron acceptor.
  • strains containing multiple mutations namely all possible double mutants (strains ⁇ NAR-K/NAR-X, ⁇ NAP/NAR-X, and ⁇ NAP/NAR-K and the triple mutant strain KS03 ( ⁇ NAR-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).
  • the narX mutation reverted the negative effect of the napF- C mutation: i.e., strain ⁇ NAP/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 ( ⁇ NAR-K/NAR-X/NAP).
  • the best performance was observed with the triple mutant (FIG. 5).
  • 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.
  • KS03 ⁇ NAR-K NAR-X and NAP
  • 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).
  • 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).
  • the triple mutant can efficiently use nitrate.
  • 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).
  • respiratory FDH-N and FDH-O encoded by the operons fdnGHI and fdoGHI, respectively (FIG. 4
  • fermentative FDH-H encoded by the gene fdhF
  • FDH-N 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.
  • KS03 ⁇ NAR-K NAR-X NAP
  • KS05 ⁇ NAR-K NAR-X NAP FDH and LDH
  • Chemicals that can be effectively synthesized using these mutants include lactate, formate, ethanol, acetate, succinate, citrate, pyruvate and related organic acids or amino acids.

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Abstract

L'invention concerne des souches bactériennes manipulées ayant une réductase de nitrate cytoplasmique réduite ou inactive (NAP), un système régulateur en fonction du nitrate/nitrite (NAR-X), une réductase de nitrate périplasmique (NAP), une déshydrogénase de format (FDH), et/ou une déshydrogénase de lactate (LDH), lesquelles souches peuvent être utilisées pour une production anaérobique de produits oxydés tels que l'acétate et le pyruvate. Le présent abrégé est donné en conformité avec les réglementations nécessitant qu'un abrégé permette à un chercheur ou à un autre lecteur de déterminer rapidement le sujet de la description technique. Cet abrégé est soumis en précisant bien qu'il ne devra pas être utilisé aux fins d'interpréter ou de limiter la portée ou la signification des revendications. 37 CFR 1.72 (b).
PCT/US2007/088254 2006-12-19 2007-12-19 Synthèse anaérobique de produits oxydés par e. coli WO2008147470A2 (fr)

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WO2014089025A1 (fr) * 2012-12-04 2014-06-12 Genomatica, Inc. Rendements accrus de produits de biosynthèse

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COLE: 'Nitrate reduction to ammonia by enteric bacteria: redundancy, or a strategy for survival during oxygen starvation?' FEMS MICROBIOLOGY LETTERS vol. 136, no. 1, February 1996, pages 1 - 11, XP002306920 *
ZHOU ET AL.: 'Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110' APPL. ENVIRON. MICROBIOL. vol. 69, no. 1, January 2003, pages 399 - 407 *

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WO2014089025A1 (fr) * 2012-12-04 2014-06-12 Genomatica, Inc. Rendements accrus de produits de biosynthèse

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