PRIOR RELATED APPLICATIONS

[0001] This invention claims priority to US 61/331 ,427, filed on May 7, 2010 and incorporated by reference in its entirety herein. FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] This invention was made with government support under Grant No: BES 0000383 awarded by the NSF. The government has certain rights in the invention.

REFERENCE TO MICROFICHE APPENDIX [0003] Not applicable.

FIELD OF THE INVENTION

[0004] Engineered bacteria that produce higher levels of 4-carbon dicarboxylic acids from the Krebs Cycle, especially succinate, and methods and uses for same. BACKGROUND OF THE INVENTION

[0005] Succinate has many industrial uses. As a specialty chemical, it is a flavor and formulating ingredient in food processing, a pharmaceutical ingredient, and a surfactant. Succinate's greatest market potential, though, would be its use as an intermediary commodity chemical feedstock for producing bulk chemicals, stronger-than-steel plastics, ethylene diamine disuccinate (a biodegradable chelator), and diethyl succinate (a green solvent for replacement of methylene chloride). Along with succinic acid, other 4-carbon dicarboxylic acids from the Krebs Cycle, such as malic acid and fumaric acid, also have feedstock potential.

[0006] More than 17,000 tons of succinate are sold per year. It is currently sold in the U.S. for $2.70-4.00/lb, depending on its purity. Succinate is currently produced petrochemically from butane through maleic anhydride. It can also be made by fermentation from glucose at a production cost of about $1.00/lb, but for succinate to be competitive with maleic anhydride as a commodity chemical, its overall production cost should be lowered to approximately 15 cents/lb.

[0007] The production of succinate, malate, and fumarate from glucose, xylose, sorbitol, and other "green" renewable feedstocks (in this case through fermentation processes) is a way to supplant the more energy intensive methods of deriving such acids from nonrenewable sources.

[0008] Succinate is an intermediate for anaerobic fermentations by propionate producing bacteria (e.g., Actinobacillus succinogenes), but those processes result in low yields and concentrations and these bacteria are generally not cost effective to use.

[0009] It has long been known that mixtures of acids are produced from E. coli fermentation. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to desired product, and this greatly reduces the cost effectiveness of the method.

[0010] Metabolic engineering has the potential to considerably improve bacterial productivity by manipulating the throughput of metabolic pathways.

Specifically, manipulating enzyme levels through the amplification, addition, or deletion of a particular pathway can result in high yields of a desired product. Several examples of increasing succinate levels through metabolic engineering are known, including several patented examples from our own group. However, there is always room for continued improvement.

[0011] What is needed in the art is an improved bacterial strain that produces higher levels of succinate and other carboxylic acids than hereto for provided.

SUMMARY OF THE INVENTION

[0012] The present invention establishes an improved in vivo method for production of succinic acid that increases the yield, and the production rate of succinate and other components of the Krebs cycle, and reduces the byproduct formate.

[0013] Generally speaking, the invention includes recombinant bacteria engineered to produce fewer by products (acetate, lactate, EtOH), balancing the carbon flux through the fermentative pathway and the glyoxylate cycle

(which has less NADH requirement), as well as driving the Krebs cycle through increased expression of PYC and supplying increased NADH through overexpression of FDH, which also has the effect of reducing the byproduct formate.

[0014] Thus, genes encoding proteins essential for the production of lactate, acetate and ethanol are disrupted and the glyoxylate cycle is turned on. The glyoxylate cycle (aka glyoxylate shunt or bypass) like the citric acid cycle, begins with the condensation of acetyl CoA and oxaloacetate to form citrate, which is then isomerized to isocitrate. Instead of being decarboxylated, isocitrate is cleaved by isocitrate lyase into succinate and glyoxylate. The subsequent steps regenerate oxaloacetate from glyoxylate. Acetyl CoA condenses with glyoxylate to form malate in a reaction catalyzed by malate synthase, which resembles citrate synthase. Finally, malate is oxidized to oxaloacetate, as in the citric acid cycle. The sum of these reactions of the glyoxylate -TC A pathway is:

2 Acetvi CoA + NAD " 4 2 H,0— >

s¾cd»&te 4- 2 CoA + NADH. 4 2 H*

[0015] Thus, using the glyoxylate cycle, allows conversion of acetate to succinate and reduces the requirements for NADH. Additional NADH is produced with the added FDH, which also reduces formate byproduct.

[0016] Thus, such bacteria have disrupted alcohol dehydrogenase (thus producing less EtOH), disrupted lactate dehydrogenase (thus producing less lactate), disrupted acetate kinase or phosphotransacetylase or both ack-pta (thus producing less acetate), a disruption in iclR (thus allowing expression of aceBAK and operation of the glyoxylate shunt to utilize acetate to make succinate) and overexpression of NAD+ dependent FDH (thus producing needed NADH and reducing formate) and overexpressed PYC (further driving the Kreb's cycle).

[0017] In a preferred embodiment, the invention is E. coli comprising a disruption in adhE, a disruption in Idh, a disruption in ack-pta, a disruption in iclR and overexpression of both FDH and PYC.

[0018] The invention also includes methods employing such bacteria, including methods of making succinate and other components of the TCA cycle, such as citrate, alpha-ketoglutarate, fumarate, malate and oxaloacetate.

[0019] In more detail, one embodiment of the invention is a bacteria comprising reduced activity of alcohol hydrogenase (ADH), reduced activity of lactate dehydrogenase (LDH), reduced activity of acetate kinase or phosphotransacetylase or both, reduced activity of the aceBAK operon repressor (ICLR) and overexpression of NAD ⁺ -dependent formate dehydrogenase (FDH) and overexpression of pyruvate carboxylase (PYC). More preferred is E. coli bacteria comprising AadhE, Aldh, Aack-pta, AiclR and FDH ⁺ and PYC ⁺ .

[0020] The above bacteria can be used to make any four carbon dicarboxylic acid from the Krebs cycle simply by culturing the bacteria in a medium, and isolating a four carbon dicarboxylic acid from said bacteria or medium or both. In preferred embodiments, the four carbon dicarboxylic acid is succinate, and the medium is supplemented with 25-250 mM formate.

[0021] Succinic acid and succinate are used interchangeably herein, as are other acid/base nomenclature for organic acids.

[0022] As used herein, "FDH" means a protein having NAD ⁺ -dependent formate dehydrogenase activity. Many such proteins are available in GenBank and exemplary proteins are described in US7256016, incorporated herein in its entirety by reference. The gene encoding FHD in Candida is fdhl, but the gene may have other names in other species.

[0023] In the exemplified embodiment the FDH is from Candida boidinii, but obviously any functional FDH can be used from any source since by definition FDH is an NAD ⁺ formate dehydrogenase and will catalyze the same reaction. Thus, the FDH can selected from the group consisting of Candida boidinii FDH, Candida methylica FDH, Pseodomonas sp 101 FDH, Arabidopsis thaliana FDH, Staphylococcus aureus FDH, Saccharomyces bayanus FDH, Saccharomyces exiguus FDH, Saccharomyces servazzii FDH, Zygosaccharomyces rouxil FDH, Saccharomyces kluyveri FDH, Kluyveromyces thermotolerans FDH, Kluyveromyces lactis FDH, Kluyveromyces marxianus FDH, Pichia angusta FDH, Debaryomyces hansenii FDH, Pichia sorbitophila FDH, Candida tropicalis FDH, and Yarrowia lipolytica FDH, among others.

In a preferred embodiment, the FDH has SEQ ID NO. 1 (Figure 7), but variations having 65-75% identity are found in other species.

Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides that encode the same amino acid sequence. NCBI(TM) provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using such databases, a gene or cDNA may be "optimized" for expression in E. coli, or other bacterial species using the codon bias for the species in which the gene will be expressed.

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, 1 1 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 = 1 1 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(TM) (ncbi.nlm.nih.gov/BLAST ).

[0027] As used herein, "ADH" means a protein having alcohol dehydrogenase activity. Many such proteins are available in GenBank. The E. coli gene encoding this protein is adhE, but the gene may have other names in other species.

[0028] As used herein, "PYC" means pyruvate carboxylase. Many such proteins are available in GenBank. In a preferred embodiment, the pyruvate carboxylase (PYC) is from Lactococcus lactisis (SEQ ID NO: 2 of Figure 8), but other sequences having 60-75% identity are available in a variety of other species. The gene encoding PYC in Lactococcus is called pycA, but the gene may have other names in other species.

[0029] We have exemplified the invention herein using E. coli, but it can easily be performed in other bacteria, including Bacillus, Lactobacillus, Lactococcus, Clostridia and the like, provided the bacteria have the same or equivalent pathways to those diagrammed in Figure 1 such that the same modifications can be made therein. Since the Krebs cycle and the glyoxylate bypass are common in bacteria and plants, the invention is believed to be generally applicable.

[0030] As used herein, "LDH" means a protein having lactate dehydrogenase activity. Many such proteins are available in GenBank. The E. coli gene encoding this protein is IdhA, but it may have other names in other species.

[0031] As used herein, "ADH" means a protein having alcohol dehyrogenase activity. Many such proteins are available in GenBank. The E. coli gene encoding this protein is adhE, but it may have other names in other species.

[0032] As used herein the aceBAK operon repressor is ICLR and the gene encoding same is iclR, but it may have other names in other species.

[0033] The disruptions in ADH, LDH, ACK-PTA and ICLR can be derived as described in US7223567, incorporated herein in its entirety by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a diagram showing the genetically engineered central anaerobic metabolic pathway of E. coli strain SBS550MG (AadhEAldhAAiclRAack-pta) harboring pHL413KFl (PYC ⁺ , FDH ⁺ ).

[0035] Shaded X-boxes in Figure 1 represent inactivated pathways in E. coli strain SBS550MG (AadhEAldhAAiclRAack-pta) (Sanchez et al, 2005): lactate (LDH), acetate (PTA, ACK), ethanol (ADH), and glyoxylate bypass operon (aceBAK) repressor gene (iclR). The asterisks indicate the overexpressed heterologous enzymes pyruvate carboxylase (PYC) from Lactococcus lactis (Lin et al., 2004) and the NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii. The bold arrow in the dotted box represents the newly introduced NADH regenerating formate dehydrogenase (FDH) pathway demonstrated herein. The glyoxylate shunt, turned on by deleting iclR, is shown crossing the Kreb's cycle (see straight arrow).

[0036] Figure 2 Schematic diagram showing the construction of pHL413KFl ((PYC ⁺ , FDH ⁺ ). The 1.1 Kb fdhl (Candida biodinii) fragment was PCR amplified from pFDHl (Sakai et al. 1997), restriction digested with Pstl, and ligated to the 7.8 Kb Pstl restriction digested pHL413Kan (PYC ⁺ ). The newly constructed 8.8 Kb pHL413KFl (PYC ⁺ , FDH ⁺ ) co-expresses the heterologous pyruvate carboxylase gene (pycA) from Lactococcus lactis and the NAD+- dependent formate dehydrogenase gene (fdhl) from Candida boidinii.

[0037] Abbreviations in Fig. 2: Ptrc, trc promoter; PpycA, native promoter of the Lactococcus lactis pyruvate carboxylase (pycA) gene; Km, kanamycin resistance gene; pBR322 ori, origin of replication; laclq: lac operon repressor; rbs: ribosome binding site; restriction enzyme sites: Ncol, EcoRI, Sacl, Kpnl, Smal, Hindlll, Pstl, Bgll, Ndel, Apal, Narl.

[0038] Figure 3 is metabolite data from anaerobic shake flask fermentation experiments showing the relative succinate yield with the control strain normalized to 100% (A) and the residual formate (mM) (B) using the genetically engineered E. coli strain SBS550MG (AadhEAldhAAiclRAack- pta) harboring either pHL413Kan (PYC ⁺ overexpressing control vector) or vector pHL413KFl (PYC FDH ). The reported values represent averages of HPLC data from at least three independent experiments and error bars indicate the standard error of the means.

[0039] Figure 4 is metabolite data from the anaerobic phase of bioreactor experiments showing the glucose consumption (mM) (A) and the succinate production (mM) (B) over time by the same two mutants.

[0040] Figure 5 is metabolite data from the anaerobic phase of bioreactor experiments showing the succinate production rate (g/L/h) (A) and the residual formate (mM) (B) over time by the same two mutants.

[0041] Figure 6 is metabolite data from anaerobic shake flask fermentation experiments showing the relative succinate yield with the control strain at 0 mM formate normalized to 100% (A) and the residual formate concentration (mM) with and without 50 mM sodium formate supplementation (B). The reported values represent averages of HPLC data from two independent experiments using the same pair of mutants.

[0042] Figure 7. Exemplary FDH sequence SEQ ID NO. 1.

[0043] Figure 8. Exemplary PYC sequence SEQ ID NO. 2.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0044] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word

"comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

[0045] The term "about" means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.

[0046] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive. [0047] The terms "comprise", "have", "include" and "contain" (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.

[0048] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "cells" and similar designations include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations that arise after genetic engineering is concluded. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0049] The terms "operably associated" or "operably linked," as used herein, refer to functionally coupled nucleic acid sequences.

[0050] As used herein "recombinant" is relating to, derived from, or containing genetically engineered material. In other words, the genome was intentionally manipulated in some way.

[0051] "Reduced activity" or "inactivation" 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 (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, and the like.

[0052] Overexpression" or "overexpressed" is defined herein to be at least 150% of protein activity as compared with an appropriate control species. Overexpression can be achieved by mutating the protein to produce a more active 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 the gene to the cell, or upregulating the endogenous gene, and the like. An overexpressed gene can be represented by the ⁺ symbol, e.g., PYC ⁺ . [0053] The terms "disruption" 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 protein at least 90% over the wild type un-disrupted protein. A gene or protein can be completely (100%) reduced by knockout or removal of the entire genomic DNA sequence. A knockout mutant can be represented by the Δ symbol.

[0054] 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%)) gene product by completely preventing transcription and/or translation of active protein.

[0055] Generally speaking we have referenced protein names herein, but it is understood that a change in protein activity can of course be effected by changing the gene. This provides clarity since the gene nomenclature can be widely divergent in bacteria, but the proteins are defined by their activities and thus names .

[0056] The following abbreviations, plasmids and strains are used herein:

ABBREVIATION FULL NAME

PYC Pyruvate carboxylase

pycA Gene encoding PYC from Lactococcus lactis

PLASMID AND STRAINS

pHL413Kan - plasmid containing only PYC

pHL413KF1 - plasmid containing PYC and FDH (see figure 2)

SBS550MG - AadhEMdhAMclRAack-pta::CmR

SBS550MG pHL413KF1 - / adhE/ ldhA/ iclR/ ack-pta::CmR plus overexpressed PYC and FDH

[0057] The following examples are illustrative only, and are not intended to unduly limit the scope of the invention.

EXAMPLE 1: METHODS

[0058] Disrupted bacteria were prepared according to known techniques, essentially as described in US7223567. Overexpressed PYC and FDH was achieved by combining these two genes on one plasmid as shown in Figure 2, but the genes can also be added to the bacterial chromosome or added by any other vector or even via separate vectors.

[0059] Anaerobic shake flasks experiments were performed at 37°C with shaking at 250 rpm for 20-24 hours using C0 ₂ purged flasks containing 200 OD units of cells resuspended in 10 ml LB broth supplemented with 20 g/L glucose, 1 g/L NaHC0 ₃ , 50 g/L MgC0 ₃ , 50 μg/ml kanamycin (Figure 3), and 50 mM sodium formate (Figure 6).

[0060] Bioreactor experiments were performed using aerobically grown biomass using modified dual-phase medium with 4 g/L glucose unless noted otherwise followed by the anaerobic succinate production phase using 0.2 L/min C0 ₂ , 20 g/L glucose, 6.4 g/L MgC0 ₃ , 50 μg/ml kanamycin, and 14.3 M NH ₄ OH to maintain the pH at 7.0 (Figures 4 and 5). EXAMPLE 2: RESULTS

[0061] To demonstrate the utility and advantages of the invention, we used the previously engineered high succinate producing E. coli strain having AadhEAldhAAiclRAack-pta plus PYC ⁺ [strain SBS550MG pHL413Kan]. This strain was further enhanced by adding an NAD+-dependent formate dehydrogenase (FDH ⁺ ) to regenerate NADH in vivo and manipulate intracellular NADH availability (see dotted box in Figure 1). The performance of the two strains were then compared.

[0062] In previous studies, it was demonstrated that this NAD ⁺ -dependent FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide

(Berrios-Rivera et al., 2002a, b, 2004). Implementation of the NADH regeneration system thus doubled the maximum yield of NADH using glucose as a substrate from 2 to 4 mol NADH/mol of substrate consumed in complex medium.

[0063] In our experiments, this increase in NADH availability significantly changed the final metabolite concentration pattern under anaerobic conditions. The parent strain used in this demonstration was capable of producing succinate from glucose to a yield of about 1.6 mol/mol with an average anaerobic productivity rate of 10 mM/h (Sanchez et al., 2005), but adding in an NAD ⁺ -dependant FDH resulted in much faster average succinate production rate (15 mM/h).

[0064] Along with succinate as a major product, the parent strain also produced 12.7 mM of formate during the anaerobic production phase. The overexpressed biologically active NAD ⁺ -dependent formate dehydrogenase (FDH) from Candida boidinii (Figure 7) also served to reduce production of formate.

[0065] The newly introduced NADH regenerating formate dehydrogenase pathway will provide one mole of NADH from one mole of formate. In contrast, the native formate dehydrogenase converts formate to C0 ₂ and H ₂ with no cofactor involvement. The new system retains the reducing power of formate resulting in a more robust strain with a much faster average succinate production rate (15 mM/h) and reduced average byproduct formate concentration (3 mM), thereby leading to opportunities for reduced costs associated with downstream processing, purification, and waste disposal.

[0066] The functionality of the new succinate production system that regenerates NADH leading to an increase in succinate production rate and decrease in the byproduct formate, was successfully demonstrated in anaerobic shake flask fermentations (Figure 3, 6) and anaerobic bioreactor experiments (Figures 4- 5).

[0067] An additional feature of the new system was that this NAD+-dependent FDH resulted in an increase in the succinate production yield. As previously mentioned, the new pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide (Berrios-Rivera et al., 2002a, b, 2004). This conversion can be exploited by supplementing fermentations with external formate thereby providing an additional increase in the NADH availability. A demonstration of the increased succinate yield using externally supplemented formate was performed using shake flask fermentations (Figure 6). The percentage increase in molar yield is 2-5%.

[0068] In summary, the advantages of using the new improved system include:

[0069] 1. Increased rate of succinate production because of an increase in NADH availability.

[0070] 2. Reduced levels of byproduct formate due to overexpression of an NAD ⁺ -dependent formate dehydrogenase that converts 1 mole of formate into 1 moll of NADH and carbon dioxide.

[0071] 3. Increased succinate yields using externally supplemented formate because the NAD+-dependent formate dehydrogenase provides additional NADH availability.

[0072] The following references are incorporated by reference herein in their entirety.

[0073] Berrios-Rivera, S.J., Bennett, G.N., San, K.-Y., 2002a. Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+- dependent formate dehydrogenase. Metab. Eng. 4,

217-229. [0074] Berrios-Rivera, S.J., Bennett, G.N., San, K.-Y., 2002b. The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metab. Eng. 4, 230-237.

[0075] Berrios-Rivera, S.J., Sanchez, A.M., Bennett, G.N., San, K.-Y., 2004 Effect of different levels of NADH availability on metabolite distribution in

Escherichia coli fermentation in defined and complex media. App. Microbiol. Biotechnol. 65, 426-432.

[0076] Lin H., San, K.-Y., Bennett, G.N. 2005. Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of

Escherichia coli. Appl. Microbiol. Biotechnol. 67, 515-523.

[0077] Sakai, Y., Murdanato, A.P., Konishi, T., Iwamatsu, A., Kato, N., 1997.

Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1 -disrupted strain on methanol, methylamine, and choline. J. Bacteriol. 179, 4480-4485.

[0078] Sanchez, A.M., Bennett, G.N., San, K.-Y., 2005. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab. Eng. 7, 229-239.

[0079] Vemuri, G.N., Eiteman, M.A., Altman, E., 2002. Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl. Environ. Microbiol. 68, 1715- 1727.