Materials and methods for differential biosynthesis in species of the genera Ralstonia and Cupriavidus and organisms related thereto

Information

  • Patent Grant
  • 11053287
  • Patent Number
    11,053,287
  • Date Filed
    Tuesday, April 30, 2019
    5 years ago
  • Date Issued
    Tuesday, July 6, 2021
    2 years ago
Abstract
Methods for increasing carbon-based chemical product yield in an organism by increasing carbon uptake and/or altering a pathway to or from an overflow metabolite in the organism, nonnaturally occurring organisms having increased carbon-based chemical product yield with increased carbon uptake and/or an altered pathway to or from an overflow metabolite, and methods for producing a carbon-based chemical product with these organisms are provided.
Description
FIELD

The present invention relates to methods for increasing carbon-based chemical product yield in an organism by increasing carbon uptake and/or altering a pathway to or from an overflow metabolite in the organism, nonnaturally occurring organisms having increased carbon-based chemical product yield with increased carbon uptake and/or an altered pathway to or from an overflow metabolite, and methods for producing a carbon-based chemical product with these organisms.


BACKGROUND

Under conditions of nutrient limitation, a phenomenon known as overflow metabolism (also known as energy spilling uncoupling or spillage) occurs in many bacteria (Schlegel and Vollbrecht Microbiology 1980 117:475-481). The range and quantity of overflow metabolites produced in a particular fermentation can depend upon the limitation applied (e.g. nitrogen, phosphate, oxygen), the extent of the limitation, the carbon source provided and the fermentation conditions such as, but not limited to, pH, source of phosphates or ammonia. See (Vollbrecht et al. European Journal of Applied Microbiology and Biotechnology 1978 6(2):145-155; Vollbrecht and Schlegel European Journal of Applied Microbiology and Biotechnology 1978 6(2):157-166; and Vollbrecht et al. European Journal of Applied Microbiology and Biotechnology 1979 7(3):267-276). Such overflow metabolites represent a net loss of carbon which could otherwise be converted into one or more desired compounds.


Tripartite tricarboxylate transporters (TTT) are carbon transporter proteins that use ion-electrochemical gradients to move substrates in a symporter mechanism (Rosa et al. Front Cell Infect. Microbiol. 2018 8:33; Winnen et al. Res. Microbiol. 2003 154(7):457-65).


Replacement of traditional chemical production processes relying on, for example fossil fuels and/or potentially toxic chemicals, with environmentally friendly and/or sustainable solutions is being considered, including work to identify suitable building blocks for such use in the manufacturing of such chemicals. Methods and organisms are needed which channel carbon into selected biosynthetic pathways by blocking undesirable pathways, thereby improving production of one or more desired compounds.


SUMMARY

Methods for increasing product yield of organisms and organisms capable of increased product yield are provided.


An aspect of the present invention relates to methods for increasing carbon-based chemical product yield in an organism. These methods comprise modifying an organism selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto to increase carbon uptake and/or alter a pathway to or from an overflow metabolite. Organism are modified in accordance with the present invention by modulating activity of one or more polypeptides or functional fragments thereof functioning to increase carbon uptake and/or in a pathway to or from an overflow metabolite, thereby increasing carbon-based chemical product yield in the organism as compared to an organism without said modulated polypeptide activity.


In one nonlimiting embodiment, the organism is modified to increase carbon uptake via one or more modifications to alter expression and/or activity of a carbon transporter protein or functional fragment thereof.


In one nonlimiting embodiment, modifications to alter expression and/or activity of a carbon transporter protein or functional fragment thereof comprise altering expression and/or activity of one or more genes encoding a TctA, a TctB or a TctC.


In one nonlimiting embodiment, one or more genes as listed in Table 2 are modified.


In one nonlimiting embodiment, the organism is modified to alter a pathway to or from an overflow metabolite by disrupting one or more genes associated with the production of lactate, hydroxybutyrate, acetate and/or 2,3 butandiol, as shown in Table 3.


Another aspect of the present invention relates to nonnaturally occurring organisms capable of yielding a carbon-based chemical product. These nonnaturally occurring organisms are selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto and are modified to increase carbon uptake and/or alter a pathway to or from an overflow metabolite.


In one nonlimiting embodiment, the nonnaturally occurring organisms are modified to increase carbon uptake via one or more modifications to alter expression and/or activity of a carbon transporter protein or functional fragment thereof.


In one nonlimiting embodiment, one or more modifications to alter expression and/or activity of a carbon transporter protein or functional fragment thereof comprise altering expression and/or activity of one or more genes encoding a TctA, a TctB or a TctC is made to the organism.


In one nonlimiting embodiment, one or more genes as listed in Table 2 or Table 3 are modified.


Yet another aspect of the present invention relates to methods for producing a carbon-based chemical product. In these methods, a nonnaturally occurring organism of the present invention is fermented with a carbon source.


In one nonlimiting embodiment, the carbon source is derived from a biological or nonbiological feedstock. In one nonlimiting embodiment, the feedstock fed to the fermentation process comprises a gaseous or liquid stream. In one nonlimiting embodiment, the feedstock is selected from gases, sugars, sugar acids, carboxylic acids, aromatics, and alcohols. In one nonlimiting embodiment, the carbon source is derived from a by-product or waste stream of a food or agricultural industry.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 is a schematic of enzymatic production of PHB from fructose. Examples of enzymes and/or their genes which may be interfered with and pathways which may be blocked are indicated by strikes.





DETAILED DESCRIPTION

The present invention provides methods of increasing carbon-based chemical product yield in an organism as well as nonnaturally occurring organisms modified to exhibit increased product yield as compared to unmodified organisms.


The methods of the present invention comprise modifying an organism selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto to increase carbon uptake and/or alter a pathway to or from an overflow metabolite. Organism are modified in accordance with the present invention by modulating activity of one or more polypeptides or functional fragments thereof functioning to increase carbon uptake and/or in a pathway to or from an overflow metabolite, thereby increasing carbon-based chemical product yield in the organism as compared to an organism without said modulated polypeptide activity. In one nonlimiting embodiment, genes in these pathways to or from an overflow metabolite may be modified. Such modifications may comprise overexpressing an endogenous or exogenous nucleic acid sequence in the organism. Alternatively, such modification may comprise downregulating, deleting or mutating an endogenous or exogenous nucleic acid sequence in the organism. The modified organisms are then cultured under conditions suitable for biosynthesis of the one or more products.


In certain aspects, the organism is modified by altering, engineering, or introducing one or more nucleic acid sequences within the organism to have one or more different characteristic properties relative to those of the corresponding unmodified wild type organism. The altering of modifying of the nucleic acid sequences can be, for example and without limitation, via genetic engineering, by adaptive mutation, or by selective isolation of naturally occurring mutant strains.


In some nonlimiting embodiments, one or more enzymes or nucleic acids of the organism are modified via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co-factor specificity. In some embodiments, the enzymes in the pathways outlined herein can be gene dosed (i.e., overexpressed by having a plurality of copies of the gene in the host organism), into the resulting genetically modified organism via episomal or chromosomal integration approaches. In some nonlimiting embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux. Attenuation strategies include, but are not limited to, the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors, and RNA interference (RNAi). In some embodiments, fluxomic, metabolomic and transcriptomal data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome-scale attenuation or knockout strategies in directing carbon flux. In some embodiments, the tolerance of the host microorganism to high concentrations of the extracellular product can be improved through continuous cultivation in a selective environment.


The modified nucleic acid sequences of the organism can include, for example, one or more enzymes, one or more promoters, one or more transcription factors, or combinations thereof. The modifications can be to nucleic acids encoding polypeptides functioning as a transhydrogenase, reductase, dehydrogenase, or hydrogenase enzyme or functional fragments thereof. The modifications can be to nucleic acids not directly involved in encoding polypeptides functioning as a transhydrogenase, reductase, dehydrogenase, or hydrogenase enzyme or functional fragments thereof, but indirectly affecting the polypeptides through the interconnected metabolic network and metabolic control strategy of the organism. The modification of the nucleic acid sequences can include one or more deletions, one or more substitutions, one or more insertions, or combinations thereof.


Enzymes with substitutions will generally have not more than 50 (e.g., not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, not more than 25, not more than 30, not more than 35, not more than 40, or not more than 50) amino acid substitutions (e.g., conservative or non-conservative substitutions). This applies to any of the enzymes described herein and functional fragments thereof. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. In contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics. Deletion variants can, for example, lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids.


In one nonlimiting embodiment, modification of the organism is carried out by allele exchange. In this embodiment, genome edits are made in a Cupriavidus or Ralstonia organism with perturbed PHB synthesis or an organism with properties similar thereto by allele exchange (also referred to as allelic exchange). In one non-limiting embodiment, the organism is a ΔphaCAB H16 C. necator strain generated using allele exchange.


The term ‘allele’ is often used interchangeably with the term ‘gene’ more generally, and refers to a defined genomic locus. In allele exchange, a specific run of DNA sequence (i.e., the native allele) in a genome of an organism is literally exchanged for a recombinant, mutant, or synthetic run of DNA sequence (i.e., the recombinant allele). Depending on the nature of the recombinant allele, this allele exchange can result in a gene deletion, a gene substitution, or a gene insertion.


In one nonlimiting embodiment, recombinant/synthetic alleles can be constructed via gene synthesis and/or standard molecular biology techniques. These alleles are then cloned into a plasmid vector for transfer into the organism and execution of the allele exchange procedure.


In some nonlimiting embodiments, the organism is modified to include one or more exogenous nucleic acid sequences.


The term “exogenous” as used herein with reference to a nucleic acid (or a protein) and an organism refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.


In contrast, the term “endogenous” as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell “endogenously expressing” a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host “endogenously producing” or that “endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.


In certain aspects, the organism is modified to include one or more functional fragments of enzymes, other polypeptides, or nucleic acids. The phrase “functional fragment” as used herein refers to a peptide fragment of a polypeptide or a nucleic acid sequence fragment encoding a peptide fragment of a polypeptide that has at least 25%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the activity of the corresponding mature, full-length, polypeptide. The functional fragment can generally, but not always, be comprised of a continuous region of the polypeptide, wherein the region has functional activity.


Tripartite tricarboxylate transporters (TTT) are carbon transporter proteins that use ion-electrochemical gradients to move substrates in a symporter mechanism. Each transporter is comprised of two transmembrane proteins and a periplasmic solute-binding protein. TctA is a well-conserved 12 transmembrane protein while TctB is a poorly conserved smaller transmembrane protein with 4 putative TMs domains. TctC is a periplasmic tricarboxylate-binding receptor. The TctC protein is highly represented in the protobacteria genomes possibly providing specificity to the transporter. Recent genome analysis of Ralstonia has shown an overrepresentation of tctC homologs. In one nonlimiting embodiment of the present invention, one or more modifications to increase carbon uptake comprise altering expression and/or activity of one or more of carbon transporter proteins or functional fragments thereof.


Within biotechnology the approach of redirecting carbon flux to a desired product by utilizing nutrient limitation is a well-established process. With Cupriavidus, it is the principle methodology for obtaining high polyhydroxyalkanoate (PHA) titers, as it exploits the organism's natural mechanism for intracellular storage of carbon and energy. To utilize Cupriavidus or Ralstonia for the purposes of generating other chemicals however, this natural mechanism is detrimental for obtaining high productivity and/or yields. Elimination or significant attenuation of PHA synthesis is therefore required, in order to maximize the efficiency of generating the desired product.


An unintended consequence of such an approach is that there is a cascade of effects upon metabolism due to high amounts of reducing equivalents and build-up of key central metabolites including pyruvate and acetyl-CoA. These manifestations include among others metabolic bottlenecks, heightened generation of overflow metabolites and a redox imbalance.


Furthermore, under conditions of nutrient limitation, a phenomenon known as overflow metabolism (also known as energy spilling uncoupling or spillage) occurs in many bacteria (Schlegel & Vollbrecht Microbiology 1980 117:475-481). In growth conditions in which there is a relative excess of carbon source and other nutrients (e.g. phosphorous, nitrogen and/or oxygen) are limiting cell growth, overflow metabolism results in the use of this excess energy (or carbon), not for biomass formation but for the excretion of metabolites, typically organic acids. Such overflow metabolites represent a net loss of carbon which could otherwise be converted into one or more desired compounds. Accordingly, in one nonlimiting embodiment of the method of the present invention, one or more modifications in a pathway to/from an overflow metabolite are made to increase carbon-based chemical product yield.


By “carbon-based chemical product” as used herein, it is meant to include C3 to C12 alkenes, alcohols, diols, monoacids, diacids, hydroxyacids, amino acids and diamines. In one nonlimiting embodiment, the carbon-based chemical product may be any C6-C12 difunctional aliphatic fatty acid or derivative thereof including, but not limited to, C6-C12 amino acids, C6-C12 diamines, C6-C12 hydroxyacids, C6-C12 diols, and C6-C12 diacids. Nonlimiting examples of carbon-based chemical products produced in accordance with this disclosure include 1,3-propanediol, 1,2-propanediol, methionine, threonine, lysine, glutamic acid, tryptophan, aspartic acid, leucine, isoleucine, valine, citric acid, maleic acid, succinic acid, isoprene, linalool, limonene, 3-hydroxypropanoic acid, malonic acid, lactic acid, n-butanol, 2-butanone, butadiene, 2-3 butanediol, 1-3 butanediol, benzoic acid, 1,4-benzenediamine, benzeneamine, pyridine, vanillin, hydroquinone, 1,4-diaminobutane, 2-hydroxyisobutyric acid, itaconic acid, 3-hydroxybutyrate and nylon intermediates.


In some nonlimiting embodiments, the organism has been modified to exhibit an increased synthesis of the extracellular product relative to that of the corresponding wild type organism.


In some nonlimiting embodiments, the carbon-based chemical product includes pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 1,7-heptanediol, or a combination thereof. Additional descriptions of the synthesis of these carbon-based chemical products with Ralstonia, Cupriavidus, or an organism similar thereto can be found in U.S. Pat. No. 10,196,657, the disclosure of which is incorporated by reference herein in its entirety for all purposes.


In some nonlimiting embodiments, the carbon-based chemical product includes 1,4-butanediol, putrescine, 4-hydroxybutyrate, 4-aminobutyrate, or a combination thereof. Additional descriptions of the synthesis of these carbon-based chemical products with Ralstonia, Cupriavidus, or an organism related thereto can be found in U.S. Pat. Nos. 10,072,150 and 9,637,764, the disclosures of which are incorporated by reference herein in their entirety for all purposes.


In some nonlimiting embodiments, the carbon-based chemical product includes glutaric acid, 5-aminopentanoic acid, cadaverine (also known as 1,5 pentanediamine), 5-hydroxypentanoic acid, 1,5-pentanediol, glutarate semialdehyde (also known as 5-oxopentanoate), or a combination thereof. Additional descriptions of the synthesis of these carbon-based chemical products with Ralstonia, Cupriavidus, or an organism related thereto can be found in U.S. Pat. No. 9,920,339, the disclosure of which is incorporated by reference herein in its entirety for all purposes.


In some nonlimiting embodiments, the carbon-based chemical product includes isoprene. Additional descriptions of the synthesis of this carbon-based chemical product with Ralstonia, Cupriavidus, or an organism related thereto can be found in U.S. Pat. No. 9,862,973, the disclosure of which is incorporated by reference herein in its entirety for all purposes.


In some nonlimiting embodiments, the carbon-based chemical product includes adipic acid, 6-aminohexanoic acid, hexamethylenediamine, caprolactam, 1,6-hexanediol, or a combination thereof. Additional descriptions of the synthesis of these carbon-based chemical products with Ralstonia, Cupriavidus, or an organism related thereto can be found in U.S. Pat. No. 9,580,733, the disclosure of which is incorporated by reference herein in its entirety for all purposes.


For products of the present invention containing carboxylic acid groups such as organic monoacids, hydroxyacids, aminoacids and dicarboxylic acids, these products may be formed or converted to their ionic salt form when an acidic proton present in the parent product either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and/or bicarbonate, sodium hydroxide, ammonia and the like. The salt can be isolated as is from the system as the salt or converted to the free acid by reducing the pH to below the lowest pKa through addition of acid or treatment with an acidic ion exchange resin.


For products of the present invention containing amine groups such as but not limited to organic amines, aminoacids and diamine, these products may be formed or converted to their ionic salt form by addition of an acidic proton to the amine to form the ammonium salt, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid or muconic acid. Acceptable inorganic bases are known in the art and include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and/or bicarbonate, ammonia, sodium hydroxide, and the like. The salt can be isolated as is from the system as a salt or converted to the free amine by raising the pH to above the lowest pKa through addition of base or treatment with a basic ion exchange resin.


For products of the present invention containing both amine groups and carboxylic acid groups such as but not limited to aminoacids, these products may be formed or converted to their ionic salt form by either 1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid or muconic acid. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and/or bicarbonate, ammonia, sodium hydroxide, and the like or 2) when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases are known in the art and include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. The salt can be isolated as is from the system or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatment with an acidic ion exchange resin.


Nonnaturally occurring organism produced and used in accordance with the present invention are selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto.


For purposes of the present invention, by “diminishing” or “diminished” polyhydroxybutyrate synthesis, it is meant that the organism is altered to synthesize less polyhydroxybutyrate as compared to an unaltered wild-type organism of the same species. Organisms used in this disclosure can exhibit at least 20%, 25%, 30%, 40%, 50% or even greater decreased polyhydroxybutyrate synthesis as compared to an unperturbed wild-type organism of the same species.


Nonlimiting examples of species of Cupriavidus or Ralstonia useful in accordance with this disclosure include Cupriavidus necator, Cupriavidus metallidurans, Cupriavidus taiwanensis, Cupriavidus pinatubonensis, Cupriavidus basilensis and Ralstonia pickettii.



C. necator (also referred to as Hydrogenomonas eutrophus, Alcaligenes eutropha, Ralstonia eutropha, and Wautersia eutropha) is a Gram-negative, flagellated soil bacterium of the Betaproteobacteria class. This hydrogen-oxidizing bacterium is capable of growing at the interface of anaerobic and aerobic environments and easily adapts between heterotrophic and autotrophic lifestyles. Sources of energy for the bacterium include both organic compounds and hydrogen. Additional properties of C. necator include microaerophilicity, copper resistance (Makar, N. S. & Casida, L. E. Int. J. of Systematic Bacteriology 1987 37(4): 323-326), bacterial predation (Byrd et al. Can J Microbiol 1985 31:1157-1163; Sillman, C. E. & Casida, L. E. Can J Microbiol 1986 32:760-762; Zeph, L. E. & Casida, L. E. Applied and Environmental Microbiology 1986 52(4):819-823) and polyhydroxybutyrate (PHB) synthesis. In addition, the cells have been reported to be capable of either aerobic or nitrate dependent anaerobic growth. A nonlimiting example of a C. necator organism useful in the present invention is a C. necator of the H16 strain. In one nonlimiting embodiment, a C. necator host of the H16 strain with at least a portion of the phaC1AB1 gene locus knocked out (ΔphaCAB) is used. In one nonlimiting embodiment, the organism is further modified to eliminate phaCAB, involved in PHBs production and/or H16-A0006-9 encoding endonucleases thereby improving transformation efficiency as described in U.S. patent application Ser. No. 15/717,216, teachings of which are incorporated herein by reference. However, other means of eliminating PHB synthesis are included within the scope of the invention.


By “an organism with properties similar thereto” it is meant an organism having one or more of the above-mentioned properties of C. necator.


In the process described herein, a fermentation strategy can be used that entails anaerobic, micro-aerobic or aerobic cultivation coupled with nutrient limitation such as iron, sulphate, nitrogen, potassium, oxygen, phosphorus, carbon and/or or NADP limitations, gradients thereof and any combinations thereof.


A cell retention strategy using a ceramic hollow fiber membrane can also be employed to achieve and maintain a high cell density during fermentation.


The principal carbon source fed to the fermentation can derive from a biological or non-biological feedstock. In one nonlimiting embodiment, the feedstock is fed to the fermentation as a gaseous or liquid stream.


Feedstocks for fermentation may be gases such as carbon dioxide or hydrogen; sugars such as glucose, xylose or fructose; sugar acids such as gluconate; fatty acids or fats/oils, carboxylic acids such as propionic acid, lactic acid, and formic acid; amino acids, aromatics such as phenol and benzoic acid and/or alcohols such as glycerol.


The feedstocks may be carbon sources derived from by-product or waste streams such as brewing, dairy, plant oil, ethanol, corn, soy, fish, or sugar industries or any other food or agricultural waste such as used cooking oil.


The biological feedstock can be, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, paper-pulp waste, black liquor, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, thin stillage, condensed distillers' solubles or waste streams from the food processing or dairy industries municipal waste such as fruit peel/pulp or whey. The non-biological feedstock can be, or can derive from, natural gas, syngas, CO2/H2, CO, H2, O2, methanol, ethanol, waste streams from processes to produce monomers for the Nylon-66 and Nylon-6 industries such as but not limited to non-volatile residues (NVRs) and caustic wash waste streams from the cyclohexane oxidation process used to manufacture adipic acid or caprolactam or waste stream from other chemical industry processes such as, but not limited to a carbon black industry or a hydrogen-refining industry, or petrochemical industry, a nonlimiting example being a PTA-waste stream.


In one nonlimiting embodiment, at least one of the enzymatic conversions of the production method comprises gas fermentation within the modulated Ralstonia or Cupriavidus organism or other organism with properties similar thereto. In this embodiment, the gas fermentation may comprise at least one of natural gas, syngas, CO, H2, O2, CO2/H2, methanol, ethanol, non-volatile residue, caustic wash from cyclohexane oxidation processes, or waste stream from a chemical industry such as, but not limited to a carbon black industry or a hydrogen-refining industry, or petrochemical industry. In one nonlimiting embodiment, the gas fermentation comprises CO2/H2.


The methods of the present invention may further comprise recovering produced product from the organism. Once produced, any method can be used to isolate these products or derivatives or compounds related thereto.


The isolation of at least one product can involve any one or more downstream processes generally known to be suitable for the at least partial separation and/or isolation of material from a reaction or bioprocess. The collection can, for example, involve centrifugations, cell disruptions, concentrations, precipitations, extractions, filtrations, crystallizations, distillations, chemical conversions, or combinations thereof. One or more biosynthetic products can be collected from the liquid or solid phase of the culture, or from the gas phase present in the headspace of a bioreactor or the off-gas.


The present invention also provides nonnaturally occurring organisms and methods for producing the nonnaturally occurring organisms modified to comprise one or more modifications to increase carbon uptake and/or one or more modifications in a pathway to/from an overflow metabolite. Such modification may comprise overexpressing an endogenous or exogenous nucleic acid sequence in the organism. Alternatively, such modification may comprise downregulating, deleting or mutating an endogenous or exogenous nucleic acid sequence in the organism. These nonnaturally occurring organisms exhibit increased product yield as compared to product yield in the same organism without modification to comprise one or more modifications to increase carbon uptake and/or one or more modifications in a pathway to/from an overflow metabolite. The nonnaturally occurring organisms are selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto.


Nonlimiting examples of species of Cupriavidus or Ralstonia useful in accordance with this disclosure include Cupriavidus necator, Cupriavidus metallidurans, Cupriavidus taiwanensis, Cupriavidus pinatubonensis, Cupriavidus basilensis and Ralstonia pickettii.


In one nonlimiting embodiment, the present invention relates to a substantially pure culture of the nonnaturally occurring organism modified to comprise one or more modifications to increase carbon uptake and/or one or more modifications in a pathway to/from an overflow metabolite.


As used herein, a “substantially pure culture” of an altered organism is a culture of that microorganism in which less than about 40% (i.e., less than about 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%; 0.01%; 0.001%; 0.0001%; or even less) of the total number of viable cells in the culture are viable cells other than the altered microorganism, e.g., bacterial, fungal (including yeast), mycoplasmal, or protozoan cells. The term “about” in this context means that the relevant percentage can be 15% of the specified percentage above or below the specified percentage. Thus, for example, about 20% can be 17% to 23%. Such a culture of nonnaturally occurring microorganisms includes the cells and a growth, storage, or transport medium. Media can be liquid, semi-solid (e.g., gelatinous media), or frozen. The culture includes the cells growing in the liquid or in/on the semi-solid medium or being stored or transported in a storage or transport medium, including a frozen storage or transport medium. The cultures are in a culture vessel or storage vessel or substrate (e.g., a culture dish, flask, or tube or a storage vial or tube).


In addition, the present invention provides bio-derived, bio-based, or fermentation-derived products produced using the methods and/or nonnaturally occurring organisms disclosed herein. Examples of such products include, but are not limited to, compositions comprising at least one bio-derived, bio-based, or fermentation-derived compound or any combination thereof, as well as molded substances, formulations and semi-solid or non-semi-solid streams comprising one or more of the bio-derived, bio-based, or fermentation-derived compounds or compositions, combinations or products thereof.


While the invention has been described in detail, in some instances making reference to a specific aspect thereof, it is apparent to one of skill in the art that various changes and modifications can be made thereto without departing from its spirit and scope. The following section provides further illustration of the methods and materials of the present invention. These Examples are illustrative only and are not intended to limit the scope of the invention in any way.


EXAMPLES
Example 1: Identifying Tcts Proteins that Alter Assimilation of Different Carbon Sources

In one nonlimiting embodiment, R. eutropha is grown in different carbon sources and RNAseq and/or proteomic experiments are performed to identify Tcts that are affected on different carbon sources. The Tcts are then validated by altering expression and/or activity and carbon utilization assessed. By modifying particular tcts, assimilation of different carbon sources can be altered. Such modifications may comprise overexpressing an endogenous or exogenous nucleic acid sequence in the organism. Alternatively, such modification may comprise downregulating, deleting or mutating an endogenous or exogenous nucleic acid sequence in the organism. For example, tctC genes H16_A1360, H16_A3350, H16_B1846 and H16_B1848 are induced through growth on pimelate, whilst H16_B0202 and H16_B1474 are induced through growth on adipate. TctC genes H16_A0036, H16_A1555, H16_B1078 and H16_B1448 are induced by growth on adipate and pimelate. TctC genes H16_A2384 and H16_B2116 are induced on fructose relative to pimelate and adipate. In addition, tctA gene H16_A2775 is induced on fructose relative to pimelate and adipate whilst H16_B2053 is induced on pimelate and adipate. TctB gene H16_B2054 is induced on pimelate and adipate whilst H16_A2774 is induced on fructose. RNA expression levels of selected C. necator H16 genes encoding Tct family transport components upon growth with fructose, adipate and pimelate as sole carbon sources are shown in Table 1. Gene expression levels were measured by RNAseq and are expressed as relative expression units (REU) that are normalized to total transcript levels.













TABLE 1







Expression
Expression
Expression




on fructose
on adipate
on pimelate


Gene locus
Description
(REU)
(REU)
(REU)



















H16_A0036
TctC-type
3
17
17


H16_A1360
TctC-type
60
19
234


H16_A1555
TctC-type
7
58
23


H16_A2384
TctC-type
278
8
18


H16_A2774
TctB-type
99
13
10


H16_A2775
TctA-type
162
22
23


H16_A3350
TctC-type
11
10
164


H16_B0202
TctC-type
41
422
43


H16_B1078
TctC-type
8
179
40


H16_B1448
TctC-type
13
154
113


H16_B1474
TctC-type
6
40
7


H16_B1846
TctC-type
1
1
10


H16_B1848
TctC-type
2
3
9


H16_B2053
TctA-type
27
605
436


H16_B2054
TctB-type
8
180
123


H16_B2116
TctC-type
272
6
5









Example 2: Putative TTT Transporter Genes Present in R. Eutropha Genome

Nonlimiting examples of putative TTT transporter genes that can be altered in accordance with the present invention are set forth in Table 2.











TABLE 2





tctA
tctB
tctC



















h16_A2775
h16_B2054
H16_A0036 SEQ ID
H16_A3375 (SEQ
H16_B0924 (SEQ


h16_B2053
h16_A3719
NO: 1)
ID NO: 53)
ID NO: 105)


h16_A3720
h16_A2774
H16_A0079 (SEQ ID
H16_A3385 (SEQ
H16_B0925 (SEQ


PHG080

NO: 2)
ID NO: 54)
ID NO: 106)


PHG079

H16_A0091 (SEQ ID
H16_A3428 (SEQ
H16_B0983 (SEQ




NO: 3)
ID NO: 55)
ID NO: 107)




H16_A0098 (SEQ ID
H16_A3650 (SEQ
H16_B0991 (SEQ




NO: 4)
ID NO: 56)
ID NO: 108)




H16_A0102 (SEQ ID
H16_A3718 (SEQ
H16_B1006 (SEQ




NO: 5)
ID NO: 57)
ID NO: 109)




H16_A0144 (SEQ ID
H16_B0053 (SEQ
H16_B1030 (SEQ




NO: 6)
ID NO: 58)
ID NO: 110)




H16_A0198 (SEQ ID
H16_B0066 (SEQ
H16_B1033 (SEQ




NO: 7)
ID NO: 59)
ID NO: 111)




H16_A0266 (SEQ ID
H16_B0088 (SEQ
H16_B1040 (SEQ




NO: 8)
ID NO: 60)
ID NO: 112)




H16_A0337 (SEQ ID
H16_B0128 (SEQ
H16_B1078 (SEQ




NO: 9)
ID NO: 61)
ID NO: 113)




H16_A0404 (SEQ ID
H16_B0129 (SEQ
H16_B1280 (SEQ




NO: 10)
ID NO: 62)
ID NO: 114)




H16_A0563 (SEQ ID
H16_B0202 (SEQ
H16_B1370 (SEQ




NO: 11)
ID NO: 63)
ID NO: 115)




H16_A0592 (SEQ ID
H16_B0206 (SEQ
H16_B1440 (SEQ




NO: 12)
ID NO: 64)
ID NO: 116)




H16_A0622 (SEQ ID
H16_B0215 (SEQ
H16_B1445 (SEQ




NO: 13)
ID NO: 65)
ID NO: 117)




H16_A1110 (SEQ ID
H16_B0284 (SEQ
H16_B1448 (SEQ




NO: 14)
ID NO: 66)
ID NO: 118)




H16_A1115 (SEQ ID
H16_B0302 (SEQ
H16_B1450 (SEQ




NO: 15)
ID NO: 67)
ID NO: 119)




H16_A1238 (SEQ ID
H16_B0312 (SEQ
H16_B1474 (SEQ




NO: 16)
ID NO: 68)
ID NO: 120)




H16_A1254 (SEQ ID
H16_B0331 (SEQ
H16_B1509 (SEQ




NO: 17)
ID NO: 69)
ID NO: 121)




H16_A1293 (SEQ ID
H16_B0335 (SEQ
H16_B1527 (SEQ




NO: 18)
ID NO: 70)
ID NO: 122)




H16_A1360 (SEQ ID
H16_B0346 (SEQ
H16_B1542 (SEQ




NO: 19)
ID NO: 71)
ID NO: 123)




H16_A1396 (SEQ ID
H16_B0349 (SEQ
H16_B1618 (SEQ




NO: 20)
ID NO: 72)
ID NO: 124)




H16_A1497 (SEQ ID
H16_B0363 (SEQ
H16_B1752 (SEQ




NO: 21)
ID NO: 73)
ID NO: 125)




H16_A1555 (SEQ ID
H16_B0368 (SEQ
H16_B1754 (SEQ




NO: 22)
ID NO: 74)
ID NO: 126)




H16_A1646 (SEQ ID
H16_B0369 (SEQ
H16_B1793 (SEQ




NO: 23)
ID NO: 75)
ID NO: 127)




H16_A1674 (SEQ ID
H16_B0391 (SEQ
H16_B1814 (SEQ




NO: 24)
ID NO: 76)
ID NO: 128)




H16_A1717 (SEQ ID
H16_B0392 (SEQ
H16_B1820 (SEQ




NO: 25)
ID NO: 77)
ID NO: 129)




H16_A1781 (SEQ ID
H16_B0398 (SEQ
H16_B1821 (SEQ




NO: 26)
ID NO: 78)
ID NO: 130)




H16_A1787 (SEQ ID
H16_B0399 (SEQ
H16_B1822 (SEQ




NO: 27)
ID NO: 79)
ID NO: 131)




H16_A1883 (SEQ ID
H16_B0418 (SEQ
H16_B1846 (SEQ




NO: 28)
ID NO: 80)
ID NO: 132)




H16_A1890 (SEQ ID
H16_B0480 (SEQ
H16_B1848 (SEQ




NO: 29)
ID NO: 81)
ID NO: 133)




H16_A1928 (SEQ ID
H16_B0482 (SEQ
H16_B1906 (SEQ




NO: 30)
ID NO: 82)
ID NO: 134)




H16_A2075 (SEQ ID
H16_B0486 (SEQ
H16_B1937 (SEQ




NO: 31)
ID NO: 83)
ID NO: 135)




H16_A2080 (SEQ ID
H16_B0512 (SEQ
H16_B1973 (SEQ




NO: 32)
ID NO: 84)
ID NO: 136)




H16_A2131 (SEQ ID
H16_B0513 (SEQ
H16_B1999 (SEQ




NO: 33)
ID NO: 85)
ID NO: 137)




H16_A2140 (SEQ ID
H16_B0532 (SEQ
H16_B2001 (SEQ




NO: 34)
ID NO: 86)
ID NO: 138)




H16_A2146 (SEQ ID
H16_B0537 (SEQ
H16_B2005 (SEQ




NO: 35)
ID NO: 87)
ID NO: 139)




H16_A2153 (SEQ ID
H16_B0557 (SEQ
H16_B2116 (SEQ




NO: 36)
ID NO: 88)
ID NO: 140)




H16_A2162 (SEQ ID
H16_B0606 (SEQ
H16_B2125 (SEQ




NO: 37)
ID NO: 89)
ID NO: 141)




H16_A2303 (SEQ ID
H16_B0613 (SEQ
H16_B2155 (SEQ




NO: 38)
ID NO: 90)
ID NO: 142)




H16_A2384 (SEQ ID
H16_B0678 (SEQ
H16_B2274 (SEQ




NO: 39)
ID NO: 91)
ID NO: 143)




H16_A2419 (SEQ ID
H16_B0695 (SEQ
H16_B2308 (SEQ




NO: 40)
ID NO: 92)
ID NO: 144)




H16_A2420 (SEQ ID
H16_B0697 (SEQ
H16_B2439 (SEQ




NO: 41)
ID NO: 93)
ID NO: 145)




H16_A2597 (SEQ ID
H16_B0705 (SEQ
H16_B2441 (SEQ




NO: 42)
ID NO: 94)
ID NO: 146)




H16_A2772 (SEQ ID
H16_B0726 (SEQ
H16_B2477 (SEQ




NO: 43)
ID NO: 95)
ID NO: 147)




H16_A2779 (SEQ ID
H16_B0742 (SEQ
H16_B2492 (SEQ




NO: 44)
ID NO: 96)
ID NO: 148)




H16_A2866 (SEQ ID
H16_B0748 (SEQ
H16_B2523 (SEQ




NO: 45)
ID NO: 97)
ID NO: 149)




H16_A2980 (SEQ ID
H16_B0766 (SEQ
H16_B2533 (SEQ




NO: 46)
ID NO: 98)
ID NO: 150)




H16_A3051 (SEQ ID
H16_B0775 (SEQ
PHG382 (SEQ ID




NO: 47)
ID NO: 99)
NO: 151)




H16_A3191 (SEQ ID
H16_B0822 (SEQ
PHG392 (SEQ ID




NO: 48)
ID NO: 100)
NO: 152)




H16_A3193 (SEQ ID
H16_B0844 (SEQ
PHG396 (SEQ ID




NO: 49)
ID NO: 101)
NO: 153)




H16_A3203 (SEQ ID
H16_B0851 (SEQ
PHG400 (SEQ ID




NO: 50)
ID NO: 102)
NO: 154)




H16_A3285 (SEQ ID
H16_B0908 (SEQ
PHG402 (SEQ ID




NO: 51)
ID NO: 103)
NO: 155)




H16_A3350 (SEQ ID
H16_B0912 (SEQ
PHG403 (SEQ ID




NO: 52)
ID NO: 104)
NO: 156)









Example 3: Modifications in a Pathway to/from an Overflow Metabolite

Nonlimiting examples of pathways that can be blocked to increase yield of the desired product are set forth in FIG. 1 and Table 3.









TABLE 3





A. Prevention or limitation of formation and/or accumulation of overflow metabolites



















By-products which






formation and/or
Enzymes that are targeted
Reactions

Genes in


accumulation are
for altering their
potentially
EC

Cupriavidus necator



prevented or limited:
expression and/or activity
catalyzed
numbers
encoding the enzymes





lactate
L-Lactate dehydrogenase
pyruvate <-> L-
EC:1.1.1.27
H16_A0666




lactate



D-lactate dehydrogenase
pyruvate <-> D-
EC:1.1.1.28
H16_A1681,




lactate

H16_A1682



Propionate CoA-
Lactoyl-CoA <->
EC:2.8.3.1
H16_A2718 (pct)



transferase
Lactate


acetate
Acetyl-CoA
acetyl-CoA −>
EC:3.1.2.1
H16_B1368



hydrolase/transferase
acetate



Acetyl-CoA hydrolase
acetyl-CoA <->
EC:2.8.3.18
H16_A1358




acetate



Propionate CoA-
acetyl-CoA <->
EC:2.8.3.1
H16_A2718 (pct)



transferase
acetate



Acyl-CoA synthetase
Acetyl-CoA <->
EC:6.2.1.1
H16_A1197




acetyladenylate




<-> acetate



Acetyl-coenzyme A
Acetyl-CoA <->
EC:6.2.1.1
H16_A1616,



synthetase
acetyladenylate

H16_A2525,




<-> acetate

H16_B0386,






H16_B1102



Acetate-CoA ligase
Acetyl-CoA <->
EC:6.2.1.1
H16_B0834




acetyladenylate




<-> acetate



Acetaldehyde
Acetyl-CoA <->
EC:1.2.1.10
H16_A1806,



dehydrogenase
acetaldehyde

H16_B0551,






H16_B0596



NAD-dependent
acetaldehyde <->
EC:1.2.1.3
H16_A0745,



aldehyde dehydrogenase
Acetate

H16_A1495,






H16_A3345,






H16_B0737,






H16_B0833,






H16_B1534,






H16_B1735,






H16_B1751,






H16_B1835,






H16_B2444



Aldehyde
acetaldehyde <->
EC:1.2.1.3
H16_B0421



dehydrogenase, NAD(P)-
Acetate



dependent



Aldehyde dehydrogenase
acetaldehyde <->
EC:1.2.1.—
H16_B1960




Acetate



Phosphotransacetylase
acetyl-CoA <->
EC:2.3.1.8
H16_B1631 (pta1),




Acetyl-P

H16_B1871 (pta2)



Acylphosphatase
Acetyl-P <->
EC:3.6.1.7
H16_A3325




Acetate



Acetate kinase
Acetyl-P <->
EC:2.7.2.1
H16_A0670.




Acetate

H16_B1630


butanoate
Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,



acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771



3-hydroxyacyl-CoA
Acetolacetyl-CoA
EC:1.1.1.35
H16_A0461



dehydrogenase
<-> (S)-3-




hydroxybutanoyl-




CoA



Enoyl-CoA
Acetolacetyl-CoA
EC:1.1.1.35
H16_A1526



hydratase/Delta(3)-cis-
<-> (S)-3-



delta(2)-trans-enoyl-CoA
hydroxybutanoyl-



isomerase
CoA



Enoyl-CoA
Acetolacetyl-CoA
EC:1.1.1.35
H16_B0724



hydratase/isomerase
<-> (S)-3-



family
hydroxybutanoyl-




CoA



3-Hydroxyacyl-CoA
Acetolacetyl-CoA
EC:1.1.1.157
H16_A0282 (paaH1),



dehydrogenase
<-> (S)-3-

H16_A1102 (paaH2)




hydroxybutanoyl-




CoA



Enoyl-CoA hydratase
(S)-3-
EC:4.2.1.17
H16_A0142,




hydroxybutanoyl-

H16_A1889,




CoA <->

H16_A2291,




Crotonoyl-CoA

H16_A3307,






H16_B0657,






H16_B0659



Propionate CoA-
Butyryl-CoA −>
EC:2.8.3.1
H16_A2718 (pct)



transferase
Butyrate



Phosphotransacetylase
Butyryl-CoA <->
EC:2.3.1.8
H16_B1631 (pta1),




Butyryl-

H16_B1871 (pta2)




Phosphate



Acetate kinase
Butyryl-
EC:2.7.2.1
H16_A0670,




Phosphate <->

H16_B1630




Butyrate


branched
Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035,


chain amino

aceto-2-

H16_A1036,


acid (leucine,

hydroxybutyrate

H16_A2231,


isoleucine,



H16_B0313,


valine)



H16_B0589,






H16_B0735,






H16_B2452



Ketol-acid
2-aceto-2-
EC:1.1.1.86
H16_A1037 (ilvC)



reductoisomerase
hydroxybutyrate




<-> <-> 2,3-




dihydroxy-3-




methylpentanoate



Dihydroxy-acid
2,3-dihydroxy-3-
EC:4.2.1.9
H16_A2987,



dehydratase
methylpentanoate −>

H16_B0280




3-methyl-2-




oxopentanoate



Branched-chain amino
3-methyl-2-
EC:2.6.1.42
H16_A0561



acid aminotransferase
oxopentanoate −>




L-isoleucine



Leucine dehydrogenase
3-methyl-2-
EC:1.4.1.9
H16_B0449




oxopentanoate −>




L-isoleucine



Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035,




acetolactate

H16_A1036,






H16_A2231,






H16_B0313,






H16_B0589,






H16_B0735,






H16_B2452



Ketol-acid
2-acetolactate <->
EC:1.1.1.86
H16_A1037 (ilvC)



reductoisomerase
<-> 2,3-




dihydroxy-3-




methylbutanoate



Dihydroxy-acid
2,3-dihydroxy-3-
EC:4.2.1.9
H16_A2987,



dehydratase
methylbutanoate −>

H16_B0280




2-oxoisovalerate



Branched-chain amino
2-oxoisovalerate −>
EC:2.6.1.42
H16_A0561



acid aminotransferase
L-valine



Leucine dehydrogenase
2-oxoisovalerate −>
EC:1.4.1.9
H16_B0449




L-valine



Aspartate/tyrosine/
2-oxoisovalerate
EC:2.6.1.66
H16_A2267



aromatic aminotransferase
<-> L-Valine



Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035,




acetolactate

H16_A1036,






H16_A2231,






H16_B0313,






H16_B0589,






H16_B0735,






H16_B2452



Ketol-acid
2-acetolactate <->
EC:1.1.1.86
H16_A1037 (ilvC)



reductoisomerase
<-> 2,3-




dihydroxy-3-




methylbutanoate



Dihydroxy-acid
2,3-dihydroxy-3-
EC:4.2.1.9
H16_A2987,



dehydratase
methylbutanoate −>

H16_B0280




2-oxoisovalerate



2-Isopropylmalate
2-oxoisovalerate −>
EC:2.3.3.13
H16_A1041 (leuA1),



synthase
(2s)

H16_B0081 (leuA2)




isopropylmalate



3-Isopropylmalate
(2s)
EC:4.2.1.33
H16_A1236 (leuC1)



dehydratase
isopropylmalate

large subunit,




<-> <-> (2R,3S)-3-

H16_A1237 (leuD1)




isopropylmalate

small subunit,






H16_A1549 (leuC2)






large subunit,






H16_A1550, (leuD2)






small subunit,






H16_A2620 (leuD3)






small subunit,






H16_A2621 (leuC3)






large subunit,






H16_B0051 (leuD4)






small subunit,






H16_B0052 (leuC4)






large subunit,






H16_B2275 (leuC5)






large subunit,






H16_B2276 (leuD5)






small subunit



3-Isopropylmalate
(2R,3S)-3-
EC:1.1.1.85
H16_A2619 (leuB3)



dehydrogenase
isopropylmalate




<-> 2-isopropyl-3-




oxosuccinate



Branched-chain amino
4-methyl-2-
EC:2.6.1.42
H16_A0561



acid aminotransferase
oxopentanoate <->




L-Leucine



Leucine dehydrogenase
4-methyl-2-
EC:1.4.1.9
H16_B0449




oxopentanoate <->




L-Leucine


isobutyrate
Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035,



(AlsA)
acetolactate

H16_A1036,






H16_A2231,






H16_B0313,






H16_B0589,






H16_B0735,






H16_B2452


acetoin
Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035 (ilvB),



(AlsA)
acetolactate

H16_A1036 (ilvH),






H16_A2231,






H16_B0313,






H16_B0589,






H16_B0735,






H16_B2452



Acetoin dehydrogenase
acetoin <->
EC:1.1.1.—]
H16_B0144 (AcoA)



E1 component alpha-
acetaldehyde +



subunit
acetyl-CoA



Acetoin dehydrogenase
acetoin <->
EC:1.1.1.—]
H16_B0145 (AcoB)



E1 component beta-
acetaldehyde +



subunit
acetyl-CoA


2,3 butanediol
Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035,



(AlsA)
acetolactate

H16_A1036,






H16_A2231,






H16_B0313,






H16_B0589,






H16_B0735,






H16_B2452



Alcohol dehydrogenase
production of
EC:1.1.1.1
H16_A0757 (adh)




ethanol and/or




2,3-butanediol



Butanediol
acetoin <-> 2,3
EC:1.1.1.4



Dehydrogenase [4]
butanediol


3-hydroxy-
Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,


butyrate
acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771



Acetoacetyl-CoA
Acetoacetyl-CoA
EC:1.1.1.36
H16_A1439 (phaB1),



reductase
<-> 3-

H16_A2002 (phaB2),




Hydroxybutyryl-

H16_A2171 (phaB3)




CoA



Poly(3-hydroxybutyrate)
3-
EC:2.3.1.—
H16_A1437 (phaC1),



polymerase
Hydroxybutyryl-

H16_A2003 (phaC2)




CoA <->




Polyhydroxybutyrate




(PHB)



Intracellular poly(3-
Polyhydroxybutyrate
EC:3.1.1.75
H16_A1150 (phaZ1),



hydroxybutyrate)
(PHB) <-> 3,3-

H16_A2862 (phaZ2),



depolymerase
hydroxybutanoyl

H16_B0339 (phaZ3),




oxybtanoate

H16_B1014 (phaZ5),






H16_B2073 (phaZ6),






H16_B2401 (phaZ7)



D-(−)-3-hydroxybutyrate
3,3-
EC:3.1.1.22
H16_A2251 (phaY1)



oligomer hydrolase
hydroxybutanoyl




oxybtanoate <->




3-




hydroxyburyrate



Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,



acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771



Succinyl-CoA: 3-ketoacid-
Acetoacetyl-CoA
EC:2.8.3.5
H16_A1331



coenzyme A transferase
<-> Acetoacetate

(subunit A),






H16_A1332






(subunit B)



D-beta-hydroxybutyrate
Acetoacetate <->
EC:1.1.1.30
h16_A1334,



dehydrogenase
3-

h16_A1814




hydroxyburyrate


Acetaldehyde
Acetaldehyde
Acetyl-CoA <->
EC:1.2.1.10
H16_A1806,


and ethanol
dehydrogenase
Acetaldehyde

H16_B0551,






H16_B0596



Alcohol dehydrogenase,
Acetaldehyde <->
EC:1.1.1.1
H16_A0861



class IV
Ethanol



Zn-dependent alcohol
Acetaldehyde <->
EC:1.1.1.1
H16_B0517,



dehydrogenase
Ethanol

H16_B1433 (adhP),






H16_B1699,






H16_B1745



Alcohol dehydrogenase,
Acetaldehyde <->
EC:1.1.1.1
H16_B1195 (adhC),



class III
Ethanol

H16_B2470



Alcohol dehydrogenase
Acetaldehyde <->
EC:1.1.2.8
H16_B1047 (quiA)



(cytochrome c)
Ethanol



Alcohol dehydrogenase
production of
EC:1.1.1.1
H16_A0757 (adh)




ethanol and/or




2,3-butanediol


Acetone
Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,



acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771



Succinyl-CoA: 3-ketoacid-
Acetoacetyl-CoA
EC:2.8.3.5
H16_A1331



coenzyme A transferase
<-> Acetoacetate

(subunit A),






H16_A1332






(subunit B)


Methanol
Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,



acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771


2-propanol
Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,



acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771










B. Re-direction of flux from an overflow metabolite


Metabolites:


malate, citrate, succinate, cis-aconitate, 2-oxoglutarate and isocitrate (TCA cycle intermediates).


Potential strategy:


In an aspect of the invention, an excess of TCA cycle intermediates such as malate, citrate, succinate,


cis-aconitate, 2-oxoglutarate and isocitrate is prevented by funneling the carbon through the TCA


cycle towards an intermediate of interest and/or tuning down the utilization of that same


intermediate in the TCA (resulting in driving flux out of the TCA cycle)












Enzymes that are targeted
Reactions

Genes in



for altering their
potentially
EC

Cupriavidus necator



Exemplification
expression and/or activity
catalyzed
numbers
encoding the enzymes





Under N-limiting
Aconitate hydratase
citrate <-> cis-
EC:4.2.1.3
H16_A2638 (acnA),


conditions cis-

aconitate <->

H16_B0568 (acnB)


aconitate

isocitrate


accumulates. To
Isocitrate
isocitrate <-> 2-
EC:1.1.1.41
H16_B1016 (icd3)


increase
dehydrogenase [NAD]
oxoglutarate


production of
Isocitrate
isocitrate <->
EC:1.1.1.42
H16_A3056 (icd1),


arginine one
dehydrogenase [NADP]
oxalusuccinate <->

H16_B1931


could accelerate

2-oxoglutarate


the flux from cis-
Aspartate/tyrosine/aromatic
2-oxoglutarate −>
EC:2.6.1.2
H16_A2267


aconitate to 2-
aminotransferase
Glutamate


oxoglutarate


(alter expression


and/or activity


of EC4.2.1.3,


1.1.1.41,


1.1.1.42) which


could then be


converted into


glutamate (EC


2.6.1.2) and


enter the


arginine


metabolism.





See Voldina et al. Appl Microbiol Biotechnol. 2014 98(8): 3579-89; Steinbüchel and Schlegel Eur J Biochem. 1984 141(3): 555-64; Raberg et al. PLoS One. 2014; 9(5): e95907; and Schlegel and Vollbrecht Microbiology 1980 117: 475-481













TABLE 3







A. Prevention or limitation of formation and/or accumulation of overflow metabolites











By-products which






formation and/or
Enzymes that are targeted
Reactions

Genes in


accumulation are
for altering their
potentially
EC

Cupriavidus necator



prevented or limited:
expression and/or activity
catalyzed
numbers
encoding the enzymes





lactate
L-Lactate dehydrogenase
pyruvate <-> L-
EC:1.1.1.27
H16_A0666




lactate



D-lactate dehydrogenase
pyruvate <-> D-
EC:1.1.1.28
H16_A1681,




lactate

H16_A1682



Propionate CoA-
Lactoyl-CoA <->
EC:2.8.3.1
H16_A2718 (pct)



transf erase
Lactate


3-hydroxy-
Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,


butyrate
acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771



Acetoacetyl-CoA reductase
Acetoacetyl-CoA
EC:1.1.1.36
H16_A1439 (phaB1),




<-> 3-

H16_A2002 (phaB2),




Hydroxybutyryl-

H16_A2171 (phaB3)




CoA



Poly(3-hydroxybutyrate)
3-
EC:2.3.1.—
H16_A1437 (phaC1),



polymerase
Hydroxybutyryl-

H16_A2003 (phaC2)




CoA <->




Polyhydroxybutyrate




(PHB)



Intracellular poly(3-
Polyhydroxybutyrate
EC:3.1.1.75
H16_A1150 (phaZ1),



hydroxybutyrate)
(PHB) <-> 3,3-

H16_A2862 (phaZ2),



depolymerase
hydroxybutanoyl

H16_B0339 (phaZ3),




oxybtanoate

H16_B1014 (phaZ5),






H16_B2073 (phaZ6),






H16_B2401 (phaZ7)



D-(−)-3-hydroxybutyrate
3,3-
EC:3.1.1.22
H16_A2251 (phaY1)



oligomer hydrolase
hydroxybutanoyl




oxybtanoate <->




3-




hydroxyburyrate



Acetyl-CoA
Acetyl-CoA <->
EC:2.3.1.9
H16_A0170,



acetyltransferase
Acetoacetyl-CoA

H16_A0867,






H16_A0868,






H16_A0872,






H16_A1297,






H16_A1438 (phaA),






H16_A1445,






H16_A1528,






H16_A1713,






H16_A1887,






H16_A1720,






H16_A2148,






H16_B0380,






H16_B0381,






H16_B0406,






H16_B0662,






H16_B0668,






H16_B0759,






H16_B1369,






H16_B1771



Succinyl-CoA: 3-ketoacid-
Acetoacetyl-CoA
EC:2.8.3.5
H16_A1331



coenzyme A transferase
<-> Acetoacetate

(subunit A),






H16_A1332






(subunit B)



D-beta-hydroxybutyrate
Acetoacetate <->
EC:1.1.1.30
h16_A1334,



dehydrogenase
3-

h16_A1814




hydroxyburyrate


acetate
Acetyl-CoA
acetyl-CoA −>
EC:3.1.2.1
H16_B1368



hydrolase/transferase
acetate



Acetyl-CoA hydrolase
acetyl-CoA <->
EC:2.8.3.18
H16_A1358




acetate



Propionate CoA-
acetyl-CoA <->
EC:2.8.3.1
H16_A2718 (pct)



transferase
acetate



Acyl-CoA synthetase
Acetyl-CoA <->
EC:6.2.1.1
H16_A1197




acetyladenylate




<-> acetate



Acetyl-coenzyme A
Acetyl-CoA <->
EC:6.2.1.1
H16_A1616,



synthetase
acetyladenylate

H16_A2525,




<-> acetate

H16_B0386,






H16_B1102



Acetate-CoA ligase
Acetyl-CoA <->
EC:6.2.1.1
H16_B0834




acetyladenylate




<-> acetate



Acetaldehyde
Acetyl-CoA <->
EC:1.2.1.10
H16_A1806,



dehydrogenase
acetaldehyde

H16_B0551,






H16_B0596



NAD-dependent aldehyde
acetaldehyde <->
EC:1.2.1.3
H16_A0745,



dehydrogenase
Acetate

H16_A1495,






H16_A3345,






H16_B0737,






H16_B0833,






H16_B1534,






H16_B1735,






H16_B1751,






H16_B1835,






H16_B2444



Aldehyde dehydrogenase,
acetaldehyde <->
EC:1.2.1.3
H16_B0421



NAD(P)-dependent
Acetate



Aldehyde dehydrogenase
acetaldehyde <->
EC:1.2.1.—
H16_B1960




Acetate



Phosphotransacetylase
acetyl-CoA <->
EC:2.3.1.8
H16_B1631 (pta1),




Acetyl-P

H16_B1871 (pta2)



Acylphosphatase
Acetyl-P <->
EC:3.6.1.7
H16_A3325




Acetate



Acetate kinase
Acetyl-P <->
EC:2.7.2.1
H16_A0670.




Acetate

H16_B1630


2,3
Acetolactate synthase
Pyruvate −> 2-
EC 2.2.1.6
H16_A1035,


butanediol
(AlsA)
acetolactate

H16_A1036,






H16_A2231,






H16_B0313,






H16_B0589,






H16_B0735,






H16_B2452



Alcohol dehydrogenase
production of
EC:1.1.1.1
H16_A0757 (adh)




ethanol and/or




2,3-butanediol



Butanediol Dehydrogenase
acetoin <-> 2,3
EC:1.1.1.4




butanediol








Claims
  • 1. A method for increasing carbon-based chemical product yield in an organism, said method comprising modifying an organism selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis by modulating activity of one or more polypeptides functioning to increase carbon uptake comprising one or more carbon transporter proteins selected from TctA, TctB or TctC, thereby increasing carbon-based chemical product yield in the organism as compared to an organism without said modulated polypeptide activity.
  • 2. The method of claim 1, wherein modulating the activity of one or more polypeptides comprises overexpressing or mutating an endogenous or exogenous nucleic acid sequence in the organism.
  • 3. The method of claim 1, wherein modulating the activity of one or more polypeptides thereof comprises, deleting or mutating an endogenous or exogenous nucleic acid sequence in the organism.
Parent Case Info

This patent application claims the benefit of priority from U.S. Provisional Application Ser. No. 62/665,800 filed May 2, 2018, teachings of which are herein incorporated by reference in their entirety.

US Referenced Citations (24)
Number Name Date Kind
7384783 Kunas et al. Jun 2008 B2
8809027 Lynch et al. Aug 2014 B1
8986960 Sichwart Mar 2015 B2
9580733 Botes et al. Feb 2017 B2
9637764 Botes et al. May 2017 B2
9862973 Botes et al. Jan 2018 B2
9920339 Kadi et al. Mar 2018 B2
10072150 Conradie et al. Sep 2018 B2
10196657 Pearlman et al. Feb 2019 B2
20120003706 Hickey Jan 2012 A1
20120064622 Fischer et al. Mar 2012 A1
20130034884 Burgard et al. Feb 2013 A1
20130065285 Sefton Mar 2013 A1
20130323714 Cheng et al. Dec 2013 A1
20150315599 Shetty et al. Nov 2015 A1
20170218406 Conradie et al. Aug 2017 A1
20180023103 Foster et al. Jan 2018 A1
20180023104 Cartman et al. Jan 2018 A1
20180100160 Bawdon et al. Apr 2018 A1
20190124947 Pearlman et al. May 2019 A1
20190300838 Smith et al. Oct 2019 A1
20190300839 Smith et al. Oct 2019 A1
20190316072 Smith et al. Oct 2019 A1
20190338320 Foster et al. Nov 2019 A1
Foreign Referenced Citations (30)
Number Date Country
0995490 Apr 2000 EP
1728853 Dec 2006 EP
1938892 Jul 2008 EP
3399015 Nov 2018 EP
2009225662 Oct 2009 JP
2013179909 Sep 2013 JP
2008094282 Aug 2008 WO
2010003007 Jan 2010 WO
2010069313 Jun 2010 WO
2013090769 Jun 2013 WO
2013152051 Oct 2013 WO
2013186340 Dec 2013 WO
2014093505 Jun 2014 WO
2014105793 Jul 2014 WO
2014105797 Jul 2014 WO
2017115855 Jul 2014 WO
2015117019 Aug 2015 WO
2015195654 Dec 2015 WO
2017165244 Sep 2017 WO
2018005770 Jan 2018 WO
2018022595 Feb 2018 WO
2018022633 Feb 2018 WO
2018106549 Jun 2018 WO
2019191761 Oct 2019 WO
2019191763 Oct 2019 WO
2019191767 Oct 2019 WO
2019191770 Oct 2019 WO
2019191772 Oct 2019 WO
2019213108 Nov 2019 WO
2019213118 Nov 2019 WO
Non-Patent Literature Citations (148)
Entry
Uniprot database, entry A0A0U2WHG0, Mar. 2016.
Non-final office action received for U.S. Appl. No. 16/399145, dated Aug. 12, 2020, 16 pages.
Brandt et al. “Elevated poly(3-hydroxybutyrate) synthesis in mutants of Ralstonia eutropha H16 defective in lipopolysaccharide biosynthesis” Appl Microbiol. Biotechnol. 2012 95:471-483.
Byrd et al. “Bacterial Control of Agromyces ramosus in soil” Can J Microbiol 1985 31:1157-1163.
Doberstein et al. “Polythioester synthesis in Ralstonia eutropha H16: novel insights into 3,3'-thiodipropionic acid and 3,3'-dithiodipropionic acid catabolism” Journal of Biotechnology 2014 184:187-198.
Grousseau et al. “Isopropanol production with engineered Cupriavidus necator as bioproduction platform” Appl Microbiol Biotechnol 2014 98:4277-4290.
Lu et al. “Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha” Appl Microbiol Biotechnol 2012 96:283-297.
Makkar, N.S. & Casida, L.E. “Cupriavidus necator gen. nov., sp. nov.: a Nonobligate Bacterial Predator of Bacteria in Soil” Int. J. of Systematic Bacteriology 1987 37(4): 323-326.
Orita et al. “Identification of mutation points in Cupriavidus necator NCIMB 11599 and genetic reconstitution of glucose-utilization ability in wild strain H16 for polyhydroxyalkanoate production” Journal of Bioscience and Bioengineering 2012 113(1):63-69.
Pohlmann et al. “Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralsonia eutropha H16” Nature Biotechnology 2007 1-6.
Raberg et al. “A closer look on the polyhydroxybutyrate—(PHB-) negative phenotype of Ralstonia eutropha PHB-4” PLoS One. 2014; 9(5): e95907.
Rosa et al. “Tripartite ATP-Independent Periplasmic (TRAP) Transporters and Tripartite Tricarboxylate Transporters (TTT): From Uptake to Pathogenicity” Front Cell Infect. Microbiol. 2018 8:33.
Schlegel and Vollbrecht “Formation of the Dehydrogenases for Lactate, Ethanol and Butanediol in the Strictly Aerobic Bacterium Alcaligenes eutrophus” Microbiology 1980 117:475-481.
Sillman, C. E. & Casida, L. E. “Isolation of nonobligate bacterial predators of bacteria from soil” Can J Microbiol 1986 32:760-762.
Steinbüchel and Schlegel “A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties” Eur J Biochem. 1984 141(3):555-64.
Vollbrecht and Schlegel “Excretion of Metabolites by Hydrogen Bacteria I. Autotrophic and Heterotrophic Fermentations” European Journal of Applied Microbiology and Biotechnology 1978 6(2):145-155.
Vollbrecht and Schlegel “Excretion of Metabolites by Hydrogen Bacteria II. Influence of Aeration, pH, Temperature, and Age of Cells” European Journal of Applied Microbiology and Biotechnology 1978 6(2):157-166.
Vollbrecht and Schlegel “Excretion of Metabolites by Hydrogen Bacteria IV. Respiration Rate-Dependent Formation of Primary Metabolites and of Poly-3-hydroxybutanoate” European Journal of Applied Microbiology and Biotechnology 1979 7(3):267-276.
Volodina et al. “Characterization of propionate CoA-transferase from Ralstonia eutropha H16” Appl Microbiol Biotechnol. 2014 98(8):3579-89.
Winnen et al. “The tripartite tricarboxylate transporter (TTT) family” Res. Microbiol. 2003 154(7):457-65.
Zeph, L.E. & Casida, L.E. “Gram-negative versus gram-positive (actinomycete) nonobligate bacterial predators of bacteria in soil” Applied and Environmental Microbiology 1986 52(4):819-823.
Invitation to Pay Additional Fees and, Where Applicable, Protest Fee in PCT/US2019/029798 dated Jul. 22, 2019.
Alagesan, S., et al., “13C-assisted metabolic flux analysis to investigate heterotrophic and mixotrophic metabolism in Cupriavidus necator H16”, Metabolomics, 2018, vol. 14, Issue 9, pp. 9.
Alagesan, S., et al., “Functional genetic elements for controlling gene expression in Cupriavidus necator H16”, Applied and Environmental Microbiology,vol. 84, Oct. 2018 (Oct. 2018), pp. 1-17.
Anderson, A.J., et al., “Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates”, Microbiology Review, 1990, vol. 54, pp. 450-472.
Atlic et al., “Continuous Production of Poly([R]-3-Hydroxybutyrate) by Cupriavidus Necator in a Multistage Bioreactor Cascade”, Appl Microbial Biotechnology, vol. 91, 2011, pp. 295-304.
Bramer, C.O., “The malate dehydrogenase of Ralstonia eutropha and functionality of the C(3)/C(4) metabolism in a Tn5-induced mdh mutant”, FEMS Microbiol Letters, Jul. 2, 2002, vol. 212, Issue 2, pp. 159-164.
Brigham, C.J., et aL, “Correction for Whole-genome microarray and gene deletion studies reveal regulation of the polyhydroxyalkanoate production cycle by the stringent response in Ralstonia eutropha H16”, Appl Environ Microbiol., 2017, vol. 83, Issue 15, pp. 1-2.
Brigham, C.J., et al., “Whole-genome microarray and gene deletion studies reveal regulation of the polyhydroxyalkanoate production cycle by the stringent response in Ralstonia eutropha HI6”, Appl Environ Microbial., 2012, vol. 78, Issue 22, pp. 8033-8044.
Brown, D.R., et al., “Nitrogen stress response and stringent response are coupled in Escherichia coli”, Nature Communications, 2014, vol. 5, 4115, pp. 8.
Chae, T.U., et al., “Metabolic engineering of Escherichia colifor the production of four-, five- and six-carbon lactarns Metabolic Engineering”, Academic Press, US, vol. 41 ,Apr. 5, 2017, pp. 82-91.
Chakravarty, J., et al., “Solvent production by engineered Ralstonia eutropha: channeling carbon to biofuel”, Applied Microbiology and Biotechnology, vol. 102, Apr. 29, 2018 (Apr. 29, 2018), pp. 5021-5031.
Chen, R., et al., “A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity”, PNAS, 1996, vol. 92, Issue 25, pp. 11666-11670.
Chen, R., et al. “Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehyrogenase” PNAS, 1996, vol. 93, pp. 12171-12176.
Choi, J.C., et al. “Modulation of 3-hydroxyvalerate molar fraction in poly(3-hydroxybutyrate-3-hydroxyvalerate) using Ralstonia eutropha transformant co-amplifying phbC and NADPH generation-related zwf genes”, Enzyme and Microbial Technology, 2003, vol. 32, Issue 1, pp. 178-185.
Cramm, R. J. “Genomic view of energy metabolism in Ralstonia eutropha HI6”, Journal of Molecular Microbiology and Biotechnology, 2009, vol. 16, pp. 38-52.
Darani, K.K., et al., “Simulation of bioreactors for poly(3-hydroxybutyrate) production from natural gas”, Iranian Journal of Chemistry and Chemical Engineering, vol. 39, 2018, pp. 1-24.
Ding, H., et al., “Glycerol utilization by Rhizobium leguminosarum requires an ABC transporter and affects competition for nodulation”, Microbiology, 2012, vol. 158, pp. 1369-1378.
Du et al., “Effects of Environmental Conditions on Cell Growth and Poly-B-Hydroxybutyrate Accumulation in Alcaligenes Eutrophus”, World Journal of Microbiology & Biotechnology, vol. 16, 2000, pp. 9-13.
Eggers et al., “Impact of Ralstonia Eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli”, Applied and Environmental Microbiology, vol. 80, No. 24,Dec. 2014, pp. 7702-7709.
Frng, Y., et al. “Tuning of acyl-ACP thioesterase activity directed for tailored fatty acid synthesis”, Applied Microbiology and Biotechnology, Springer, De, vol. 102, No. 7 ,Feb. 22, 2018, pp. 3173-3182.
Gao, C., et al. “Lactate utilization is regulated by the FadR-type regulator LldR in Pseudomonas aeruginosa”, Journal of Bacteriology, 2012, vol. 194, pp. 2687-2692.
Girdhar, A., et al., “Process Parameters for Influencing Polyhyroxyalkanoate Producing Bacterial Factories: An Overview”, Petroleum & Environmental Biotechnology, 2013, vol. 4, Issue 5, pp. 9.
Gyaneshwar et al., “Sulfur and Nitrogen Limitation in Escherichia coli K-12: Specific Homeostatic Responses”, Journal of Bacteriology, vol. 187, No. 3, Feb. 2005, pp. 1074-1090.
Hanko, E.K.R., et al., “Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator”, Scientific Reports, vol. 7, 2017, pp. 1-12.
Hauryliuk, V. et al.“Recent functional insights into the role of (p)ppGpp in bacterial physiology”, Nature Reviews Microbiology, 2015, vol. 13, pp. 298-309.
Haushalter, R.W., et al., “Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin-Fatty Acid Biosynthetic Pathway” Journal of the American Chemical Society, vol. 139, No. 13 ,Mar. 21, 2017, pp. 4615-4618.
Horvat et al., “Mathematical Modelling and Process Optimization of a Continuous 5-Stage Bioreactor Cascade for11 Production of Poly[-(R)-3-Hydroxybutyrate] by Cupriavidus Necator”, Bioprocess Biosyst Eng, vol. 36, 2013, pp. 1235-1250.
Hun-Suk Song et al: Enhanced isobutanolproduction from acetate by combinatorialoverexpression of acetyl-CoA synthetaseand anaplerotic enzymes in engineeredEscherichia coli, Biotechnology and Bioengineering,vol. 115, May 2, 2018 (May 2, 2018), pp. 1971-1978.
Lenczak, J.L., et al., “High cell density strategy for poly(3-hydroxybutyrate) production by Cupriavidus necator”, Brazilian Journal of Chemical Engineering, 2011, vol. 28, Issue 4, pp. 585-596.
Inoue, H., et al., “Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotrophAcidithiobacillus thiooxidans”, FEMS Microbial Letters, 2002, vol. 214, Issue 1, pp. 127-132.
International Search Report and Written Opinion for International Application Serial No. PCT/US2019/025189, dated Jul. 2, 2019, pp. 12.
International Search Report and Written Opinion for International Application Serial No. PCT/US2019/025194, dated Aug. 22, 2019, pp. 24.
International Search Report and Written Opinion for International Application Serial No. PCT/US2019/025202, dated Jul. 30, 2019, pp. 15.
International Search Report and Written Opinion for International Application Serial No. PCT/US2019/025211, dated Jul. 29, 2019, pp. 16.
International Search Report and Written Opinion for International Application Serial No. PCT/US2019/025218, dated Sep. 5, 2019, pp. 17.
International Search Report and Written Opinion for International Application Serial No. PCT/US2019/029973 dated Jul. 23, 2019, Jul. 23, 2019, 5 pgs.
International Search Report and Written Opinion in PCT/US2019/029795 dated Jul. 11, 2019, pp. 10.
International Search Report and Written Opinion in PCT/US2019/029798 dated Sep. 12, 2019, p. 19.
International Search Report and Written Opinion in PCT/US2019/029817 dated Sep. 23, 2019.
International Search Report and Written Opinion in PCT/US2019/029827 dated Sep. 23, 2019.
International Search Report and Written Opinion in PCT/US2019/029956 dated Aug. 13, 2019,.
Invitation to Pay Additional Fees and, WhereApplicable, Protest Fee in PCT/US2019/029817 dated Aug. 1, 2019.
Invitation to Pay Additional Fees and, WhereApplicable, Protest Fee in PCT/US2019/029827 datedJul. 23, 2019.
Jhonson, A., et al., “An engineered constitutive promoter set with broad activity range for Cupriavidus necator H16”, ACS Synthetic Biology, vol. 7, Jun. 27, 2018 (Jun. 27, 2018), pp. 1918-1928.
Joris, Beld, et al., “Evolution of acyl-ACP thioesterases and [beta]-ketoacyl-ACP synthases revealed by protein-protein interactions”, Journal of Applied Phycology, vol. 26, No. 4 ,Nov. 22, 2013, pp. 1619-1629.
Juengert, Jr, et al., “Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha HI6” Applied and Environmental Microbiology, 2017, vol. 83, Issue 13, pp. e00755-17.
Justyna Mozejko-Ciesielska et al: “Bacterial polyhydroxyalkanoates: Still fabulous?”, Microbiological Research, vol. 192, 2016, pp. 271-282.
Kaddor, C., et al., “Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransf erase system proteins on carbohydrate uptake and poly(3-ydroxybutyrate) accumulation in Ralstonia eutropha HI6”, Appl. Environ. Microbiol., 2011, vol. 77, pp. 3582-3590.
Kaddor, C., et al., “Implications of various phosphoenolpyruvate-carbohydrate phosphotransf erase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16”, AMB Express, 2011, vol. 1, pp. 16.
Karstens, K., et al., “Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha HI6”, Microbiology, 2014, vol. 160, pp. 711-722.
Bruland et al. “Unravelling the C3/C4 carbon metabolism in Ralstonia eutropha H16” Journal of Applied Microbiology 2010 109:79-90.
Tan, Z., et al. “Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improvement of succinate production” Appl. Environ. Microbiol, 2013, vol. 79, Issue 16, pp. 4838-4844.
Kazakov, A.E., et al., “Comparative genomics of regulation of fatty acid and branched-chain amino acid utilization in proteobacteria”, Journal of Bacteriology, 2009, vol. 191, pp. 52-64.
Kim et al. “Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli” Applied and Environmental Microbiology, 2004, vol. 70, Issue 2, pp. 1238-1241.
Kluge, J., et al., “Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi”, Applied Microbiology and Iotechnology, vol. 102, Jun. 2, 2018 (Jun. 2, 2018), pp. 6357-6372.
Koller et al., “Potential and Prospects of Continuous Polyhydroxyalkanoate (PHA) Production”, Bioengineering,May 29, 2015, pp. 94-121.
Koller, M., “A review on established and emerging fermentation schemes for microbial production of polyhydroxyalkanoate (PHA) biopolyesters”, Fermentation,vol. 4, Apr. 23, 2018 (Apr. 23, 2018), pp. 1-30.
Koller, M., et al., “Continuous production mode as a viable process-engineering tool for efficient poly (hydroxyalkanoate) (PHA) bio-production”, Chemical and Biochemical Product Engineering, vol. 28, Issue 1, 2014, pp. 65-77.
Krausse et al., “Essential role of the hprK gene inRalstonia eutropha HI6”, J Mol Microbiol Biotechnol, 2009, vol. 17, pp. 146-152.
Kunasundari et al., “Revisiting the Single Cell Protein Application of Cupriavidus Necator H16 and Recovering Bioplastic Granules Simultaneously”, Plos One, vol. 8, No. 10, Oct. 2013, 15 pages.
Lardi M. et al., “o54-Dependent Response to Nitrogen Limitation and Virulence in Burkholderia cenocepacia Strain H111” Appl. Environ. Microbiol., 2015, vol. 81, Issue 12, pp. 4077-4089.
Lee, J.N., et al., “Metabolic Engineering of Pentose Phosphate Pathway in Ralsonia eutropha for Enhanced Biosynthesis of Poly- -hydroxybutyrate”, Biotechnology Progress, 2003, vol. 19, Issue 5, pp. 1444-1449.
Lee, et al., “Regulation of poly- -hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligene eutrophus” FEMS Microbiological letters, 1995, vol. 131, pp. 35-39.
Lee, et al. “Microbial Production of Ethanol from Acetate by Engineered Ralstonia Eutropha”, Biotechnology and Bioprocess Engineering, vol. 21, 2016, pp. 402-407.
Leyn et al., “Control of proteobacterial centralcarbon metabolism by the HexR transcriptionalregulator: a case study in Shewanella oneidensis”, Journal of Biological Chemistry, 2011, vol. 286, Issue 41, pp. 35782-35794.
Leyn, S.A., et al.“Comparative genomics and evolution of transcriptional regulons in Proteobacteria”, Microbial Genomics, 2016, pp. 1-15.
Li, Z.J., et al. “Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production”, Appl Microbial Biotechnol., 2009, vol. 83, Issue 5, pp. 939-947.
Liu, X. et al., “Comparative analysis of genes frequently regulated by drugs based on connectivity map transcriptome data” PLoS One, 2017, vol. 12, Issue 6, e0179037.
Marc, J., et al., “Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production”, Metabolic Engineering,vol. 42, 2017, pp. 74-84.
March, J.C., et al., “Expression of an anaplerotic enzyme, pyruvate carboxylase, improves recombinant protein production in Escherichia coli” Applied and Environmental Microbiology, 2002, vol. 68, Issue 11, pp. 5620-5624.
Martin, Koller, et al., “Continuous production mode as a viable process-engineering tool for efficient poly (hydroxyalkanoate) (PHA) bio-production”, Chemical and Biochemical Engineering Quarterly, vol. 28, XP002792820 ,2014, pp. 65-77.
McKinlay, J.B., et al., “Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria” PNAS, 2010, vol. 107, Issue 26, pp. 11669-11675.
Meng, J., et al. “High-yield anaerobic succinate production by strategically regulating multiple metabolic pathways based on stoichiometric maximumin Escherichia coli” Microbial Cell Factories, vol. 15, 2016, pp. 13.
Montiel-Jarillo, G., et al., “Enrichment of a mixed microbial culture for polyhydroxyalkanoates production: Effect of pH and N and P concentrations”, Science of the Total Environment, vol. 583, 2017, pp. 300-307.
Nguyen, C., et al., “Trapping the dynamic acyl carrier protein in fatty acid biosynthesis”, Nature, vol. 505, No. 7483 ,Dec. 22, 2013, pp. 427-431.
Obruca, S., et al. “Application of random mutagenesis to enhance the production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil”, World J Microbiol Biotechnol, 2013, vol. 29, pp. 2417-2428.
Olaya-Abril et al., “Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222”, FEMS Microbiology Letters, 2008, vol. 365:fnx251, pp. 8.
Papagiani, M., “Recent advances in engineering the central carbon metabolism of industrially important bacteria”, Microbial Cell Factories, 2012, vol. 11, pp. 13.
Park, J-S., et al., “Metabolic Characteristics of Isocitrate Dehydrogenase Leaky Mutant of Alcaligene eutrophus and its Utilization for Poly-Hydroxybutyrate Production” Journal of Fermentation and Bioengineering, 1996, vol. 81, Issue 3, pp. 197-205.
Park, S., et al., “Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina”, Bioprocess Biosyst Eng., 2013, vol. 36, Issue 1, pp. 127-131.
Persuhn, D.C., et al. “The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae” FEMS Microbiology Letters, 2000, vol. 192, pp. 217-221.
Pryzbylski, D., et al., “Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator HI6”, Appl. Microbial. Biotechnol., 2013, vol. 97, 20, pp. 8875-8885.
Qi et al., “Model-driven redox pathway manipulation for improved isobutanol production in Bacillus subtilis complemented with experimental validation and metabolic profiling analysis” PLoS ONE, 2014, vol. 9, Issue 4, : e93815, pp. 1-11.
Raberg, M., “Ralstoni a eutropha H16 in progress: applications beside PHAs and establishment as production platform by advanced genetic tools”, Critical Reviews in Biotechnology, vol. 38, Dec. 12, 2017 (Dec. 12, 2017), pp. 494-510.
Russell, J.B., “The Energy Spilling Reactions of Bacteria and Other Organisms”, Journal of Molecular Microbiology Biotechnology, vol. 13, No. 1, 2007, pp. 1-11.
Sacamboio, E.N.M., et al. “The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae” Scientific Reports, 2017, vol. 7, Article No. 13546, pp. 1-12.
Sanchez, A.M., et al., “Effect of overexpression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the production of poly(3-hydroxybutyrate) in Escherichia coli” Biotechnol Prog., 2006, vol. 22, Issue 2, pp. 420-425.
Saur, U., et al., “The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria”, FEMS Microbiology Reviews, 2005, vol. 29, Issue 4, pp. 765-794.
Schlegel, H.G., et al., “Formation of the Dehydroganses for Lactate, Ethanol and Butanediol in the Strictly Aerobi Bacterium Alcaligene eutrophus” Microbiology, 1980, vol. 117, pp. 475-481.
Schobert, P., et al., “Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus” Journal of Bacterialogy, 1984, vol. 159, Issue 1, pp. 167-172.
Schramke, h., et al., “Revisiting Regulation of Potassium Homeostasis in Escherichia coli: The Connection toPhosphate Limitation”, Wiley Microbiologyopen, vol. 6, No. 3, 2017, pp. 1-16.
Schwartz, E., et al., “A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha HI6” Proteomics, 2009, vol. 9, Issue 22, pp. 5132-5142.
Segura, D., et al., “Inactivation of pycA, encoding pyruvate carboxylase activity, increases polybeta-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium” Appl Microbial Biotechnol, 2004, pp. 65, Issue 4, pp. 414-418.
Sekar, B.S., et al., “Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose somerase (pgi) and overexpression of glucose-6-phosphate lehydrogenase (zwf) and 6-phosphogluconate dehydrogenase ( gnd)”, Biotechnology for Biofuels, 2017, vol. 10, 85, pp. 12.
Shang et al., “Poly(3-hydroxybutyrate) Synthesis in Fed-batch Culture of Ralstonia Eutropha with Phosphate Limitation Under Different Glucose Concentrations”, Biotechnology Letters, vol. 25, Issue 17, 2003, pp. 1415-1419.
Shively, J.M., et al., “Something From Almost Nothing: Carbon Dioxide Fixation in Chemoautotrophs”, Annu. Rev. Microbiol., vol. 52 ,1998, pp. 191-230.
Silva, F., et al., “Impact of nitrogen feeding regulation on polyhydroxyalkanoates production by mixed microbial cultures”, New Biotechnology, vol. 37, 2017, pp. 90-98.
Stokke, R., et al., “Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme” Arch Microbiol., 2007, vol. 187, Issue 5, pp. 361-370.
Sun, J., et al., “Involvement of glnB, glnZ, and glnD genes in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in Azospirillum brasilense Sp7”, Appl. Environ. Microbiol, 2002, vol. 68, Issue 2, pp. 985-988.
Sun, J., et al., “The ntrB and ntrC genes are involved in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in Azospirillum brasilense Sp7”, Appl. Environ. Microbiol., 2000, vol. 66, Issue 1, pp. 113-117.
Tanaka, K, et al., Production of Poly (D-3-Hydr0xybutyrate) From CO2, H2, and O2 by High Cell Density Autotropic Cultivation of Alcaligenes Eutrophus Biotechnology and Bioengineering, Wiley, vol. 45, No. 3, (Feb. 5, 1995), XP000489583 ,Feb. 5, 1995, 268-275.
Valderrama, J.A., et al., “AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB” Journal of Biological Chemistry, 2014, vol. 289, Issue 4, pp. 1892-1904.
Vemuri, G.N., et al., “Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase” Biotechnology and Bioengineering, 2005, vol. 90, Issue 1 pp. 64-76.
Vollbrecht, D., et al., “Excretion of Metabolites by hydrogen Bacteria III. D(-)-3-hydroxybutanoate”, European J. Appl. Microbiol. Biotechnol., 1979, vol. 7, pp. 259-266.
Wang, F., et al., “Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Bath Culture of Alcaligene lat us under Nitrogen Limitation”, Applied and Environmental Microbiology, 1997, vol. 63, No. 9, pp. 3703-3706.
Wang, R., et al., “Isocitrate dehydrogenase from Streptococcus mutans: biochemical properties and evaluation of a putative phosphorylation site at Ser102” PLoS One, 2013, vol. 8, Issue 3, e58918.
Weiden et al., “Cation Transport in Escherichia coli Vii. Potassium Requirement for Phosphate Uptake”, The Journal of General Physiology, vol. 50, No. 6, 1967, pp. 1641-1661.
Weinberg, Z., et al. “Identification of 22 candidate structured RNAs in bacteria using the Cmfinder comparative genomics pipeline” Nucleic Acids Research, 2007, vol. 35, pp. 4809-4819.
Wu, M.C., et al. “A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71” PLoS One., 2015, vol. 10, Issue 5, pp. 1-17.
Youngquist et al., “Functional Genomics Analysis of Free Fatty Acid Production under Continuous PhosphateLimiting Conditions”, J. Ind. Microbial. Biotechnol., vol. 44, May 2017, pp. 759-772.
Zhu, J., et al., “Factors for promoting polyhydroxyalkanoate (PHA) synthesis in bio-nutrient-removal and recovery system”, 4th International Conference on nvironmental Systems Research (ICESR 2017) Conference paper, 2018, pp. 1-4.
Ziesack, M., et al., “Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression”, Applied and Environmental Microbiology, vol. 84, No. 10 ,Mar. 16, 2018, pp. 12.
International Preliminary Report on Patentability in PCT/US2019/029798 dated Nov. 3, 2020.
Kizer L et al. Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production. Applied and Environmental Microbiology. 2008. vol. 74, No. 10. p. 3229-3241. (Year: 2008).
Non-final office action received for U.S. Appl. No. 16/398,351, dated Feb. 1, 2021, 24 pages.
Non-final office action received for U.S. Appl. No. 16/398,401 , dated Feb. 16, 2021, 29 pages.
Prather KLJ et al. De nova biosynthetic pathways: Rational design of microbial chemical factories. Current Opinion in Biotechnology, 2008. 19:468-474 (Year: 2008).
Singh RK et al. Protein Engineering Approaches in the Post-Genomic Era. 2017. Current Protein and Peptide Science. 18, 1-11. (Year: 2017).
Zhang M et al. Propagated Perturbations from a Peripheral Mutation Show Interactions Supporting WW Domain Thermostablity, 2018, Structure. 26, 1474-1485. (Year: 2018).
Devos et al., “Practical Limits of Function Prediction”, PROTEINS: Structure, Function and Genetics, vol. 41, pp. 98-107 (2000).
International Preliminary Report on Patentability in PCT/US2019/029817 dated Nov. 3, 2020, 14 pages.
International Preliminary Report on Patentability in PCT/US2019/029795, dated Nov. 3, 2020, 7 pages.
International Preliminary Report on Patentability in PCT/US2019/029827, dated Nov. 3, 2020, 13 pages.
Kisselev, “Polypeptide Release Factors in Prokaryotes and Eukaryotes: Same Function, Different Structure”, Structure, vol. 10, pp. 8-9 (2002).
Non-Final office action received for U.S. Appl. No. 16/398,384, dated Oct. 23, 2020, 13 pages.
Whisstock et al., “Prediction of protein function from protein sequence and structure”, Quarterly Reviews of BioPhysics, vol. 36, Issue 3, pp. 307-340 (2003).
Witkowski et al., “Conversion of β-Ketoacyl Synthase to a Malonyl Decarboxylase by Replacement of the Active-Site Cysteine with Glutamine”, Biochemistry, vol. 38, pp. 11643-11650 (1999).
Related Publications (1)
Number Date Country
20190337995 A1 Nov 2019 US
Provisional Applications (1)
Number Date Country
62665800 May 2018 US