Patent Grant
11788055
The present invention relates to methods for increasing carbon-based chemical product yield in an organism by perturbing redox balance within the organism, nonnaturally occurring organisms having increased carbon-based chemical product yield, and methods for production of these organisms by modulating activity of one or more polypeptides functioning as a transhydrogenase, reductase, dehydrogenase or hydrogenase enzyme in the organisms.
When organisms, such as Cupriavidus necator, are grown under conditions in which there is a relative excess of a carbon source and a limitation of other nutrients (e.g. phosphorous, nitrogen and/or oxygen), excess carbon is sunk intracellularly into the storage carbohydrate polyhydroxybutyrate (PHB). PHB has a key role in intracellular carbon and energy storage.
Importantly, PHB is used as a mechanism for redox balancing to enable cell survival during periods of starvation and other stress conditions.
The enzymes citrate synthase and isocitrate dehydrogenase are known to be significantly inhibited by NADH and NADPH and therefore, the redox balance is considered a major regulatory factor for diverting carbon flux from either the TCA cycle or PHB biosynthetic pathway (Lee et al. FEMS Microbiological letters 1995 131:35-39).
It is known that PHB production rate increases with increased ratio of intracellular NADH/NAD and NADPH/NADP, which are highly dependent on the limiting nutrient (Wang and Lee Applied and Environmental Microbiology 1997 370-376; Pryzbylski et al. Appl. Microbiol. Biotechnol. 2013 97(20):8875-85). Under normal conditions the conversion of NADPH to NADP by acetoacetyl-CoA reductase (PhaB) reduces this effect, alleviating the inhibitory effect upon TCA cycle enzymes. However, when the PHB pathway is absent, the NADPH/NADP ratio continues to limit entry to the TCA pathway leading to the overflow of carbon to pyruvate (Lee et al. FEMS Microbiological letters 1995 131:35-39; Grousseau et al. Appl Microbiol Biotechnol. 2014 98(9):4277-90).
Under conditions of oxygen limitation in the absence of terminal electron acceptors, Cupriavidus generates a variety of NAD-linked dehydrogenases. These enzymes prevent ‘over-reduction’ of the cytoplasm, along with reversible cytoplasmic soluble hydrogenases by regenerating NAD+ (Cramm, R. J. Mol. Microbiol. Biotechnol. 2009 16(1-2):38-52). The consequence is partial fermentation products as observed by Schlegel & Vollbrecht (Microbiology 1980 117:475-481), reporting the accumulation of ethanol, succinate, formate, acetate, and 2-oxoglutarate.
Within biotechnology, the approach of redirecting carbon flux to a desired product utilizing nutrient limitation is well-established. With Cupriavidus, it is the principle methodology for obtaining high PHI titres, as it exploits the organism's natural mechanism of intracellular storage of carbon and energy. To utilize Cupriavidus or Ralstonia to generating other chemicals however, this natural mechanism is detrimental for obtaining high productivity and/or yields. Attenuation or elimination 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.
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 and biosynthetic systems for use in the manufacturing of a range of products.
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 perturbing redox balance in an organism selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto by modulating activity of one or more polypeptides functioning as a transhydrogenase, reductase, dehydrogenase or hydrogenase enzyme or functional fragment thereof.
In one nonlimiting embodiment, modulating the activity of one or more polypeptides comprises overexpressing an endogenous or exogenous nucleic acid sequence in the organism.
In another nonlimiting embodiment, modulating the activity of one or more polypeptides comprises downregulating, deleting or mutating an endogenous or exogenous nucleic acid sequence in the organism.
In one nonlimiting embodiment, the perturbation in redox balance increases yield of a carbon-based chemical product derived from a NADPH dependent dehydrogenase or reductase enzyme pathway.
In one nonlimiting embodiment, the perturbation in redox balance increases yield of a carbon-based chemical product derived from a NADH dependent dehydrogenase or reductase enzyme pathway.
In one nonlimiting embodiment, the organism expresses a native or exogenous transhydrogenase or functional fragment thereof.
Another aspect of the present invention relates to nonnaturally occurring organisms capable of yielding a carbon-based chemical product. These nonnaturally occurring organisms comprise a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto modified to perturb redox balance by modulating activity of one or more polypeptides functioning as a transhydrogenase, reductase, dehydrogenase or hydrogenase enzyme or functional fragment thereof.
In one nonlimiting embodiment, the nonnaturally occurring organism exhibits increased carbon-based chemical product yield as compared to an organism without modulated polypeptide activity.
In one nonlimiting embodiment, the nonnaturally occurring organism exhibits increased yield of a carbon-based chemical product derived from a NADPH dependent dehydrogenase or reductase enzyme pathway.
In one nonlimiting embodiment, the nonnaturally occurring organism exhibits increased yield of a carbon-based chemical product derived from a NADH dependent dehydrogenase or reductase enzyme pathway.
In one nonlimiting embodiment, the organism expresses a native or exogenous transhydrogenase or functional fragment thereof.
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, feedstock fed to the fermentation process comprises a gaseous or liquid stream.
PHB has a key role in intracellular carbon and energy storage. Importantly, PHB is also used as a mechanism for redox balancing to enable cell survival during periods of starvation and other stress conditions. Upon formation of PHB, NADP+ is generated. When the PHB pathway is perturbed or deleted, NADP+ is no longer generated and there is consequently an NADP+ imbalance referred to as a redox imbalance. With NADP+ formation blocked as a result of the absence of PHB synthesis, the cell is forced to utilize other mechanisms to balance NADPH/NADP+.
The present invention relates to further manipulation of these mechanisms to increase or decrease the NADPH/NADH to NADP+/NAD+ ratios within an organism, in order to increase product yield in the organism.
In the present invention carbon-based chemical product yield is increased in an organism via perturbing redox balance within the organism by modulating the activity of one or more polypeptides functioning as a transhydrogenase, reductase, dehydrogenase, or hydrogenase enzyme or functional fragment thereof in the organism.
In one nonlimiting embodiment, the activity of one or more polypeptides functioning as a transhydrogenase, reductase, dehydrogenase, or hydrogenase enzyme or functional fragment is genetically modulated to have one or more different characteristic properties relative to those of the corresponding unmodified wild type organism. In certain aspects, the organism is modified by altering, engineering, or introducing one or more nucleic acid sequences within the 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 cofactor 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 nonlimiting 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.
Nonlimiting examples of enzymes in the NADPH dependent dehydrogenase or reductase enzyme pathway which may be altered in redox balance in accordance with this disclosure include aldehyde reductases or alcohol dehydrogenases for alcohol synthesis, 3-ketoacyl-ACP reductases, enoyl-ACP reductases for fatty acid derived products, HMG-CoA reductases or 4-hydroxy-3-methylbut-2-enyl diphosphate reductases for isoprene or isoprenoid-derived products, alcohol dehydrogenases for isopropanol synthesis, aspartate dehydrogenases for aspartate synthesis-derived products and oxoglutarate dehydrogenases for glutarate-derived products.
Nonlimiting examples of enzymes in the NADH dependent dehydrogenase or reductase enzyme pathway which may be altered in redox balance in accordance with this disclosure include alcohol dehydrogenases, 3-hydroxybutyryl-CoA dehydrogenases, or aldehyde dehydrogenases for butanol synthesis; lactate dehydrogenases for lactate synthesis; methylglyoxal dehydrogenases or alcohol dehydrogenases for propanol, 1,2 propandiol, or 1,3 propandiol synthesis; alcohol dehydrogenases for ethanol synthesis; and butanediol dehydrogenases for 2,3, butandiol, 1,3 butandiol and butadiene synthesis.
In one nonlimiting embodiment, genetic modulation comprises deleting/reducing the expression of terminal oxidases to increase the NAD(P)H/NAD(P)+ ratio by limiting the organism's capacity to use oxygen as a terminal electron acceptor for the regeneration of NAD+. In one nonlimiting embodiment, genetic modulation comprises targeting hydrogenases (EC 1.12.1.2 and EC 1.12.5.1) that can release or take up hydrogen, including the overexpression of a soluble, oxygen tolerant NiFe hydrogenase (SH, encoded by hoxFUYH in C. necator H16) to either increase NADH production under lithotrophic or mixotrophic cultivation with molecular hydrogen or increase the rate of NAD+ recycling in heterotrophically grown cultures when the intracellular redox state is over-reduced by high NAD(P)H levels (e.g. microaerobic or anaerobic fermentations). Further genetic modulation of the hydrogenases may include changing the ratio of the soluble (SH) and membrane-bound (MBH, EC 1.12.5.1 encoded by hoxKGZ in C. necator) hydrogenase enzymes to alter the ratio of ATP and NADH molecules produced from the oxidation of molecular hydrogen. In one nonlimiting embodiment, genetic modulation comprises targeting transhydrogenase (EC 1.6.1.1, EC 1.6.1.2, or EC 7.1.1.1)) genes which shuttle hydrogen between NADP (H) and NAD(H). Modulations of the transhydrogenase genes include the deletion, reduced expression or overexpression of either subunit encoding transhydrogenases PntA1, PntA2, PntA3, PntA4 in C. necator H16 in order to alter the ratio of NADPH to NADH. In one nonlimiting embodiment, genetic modulation comprises deleting or mutating one or more NAD(P)+ alcohol dehydrogenases. In one nonlimiting embodiment, genetic modulation comprises deleting or mutating NAD(P)+ Lactate dehydrogenase enzymes (EC 1.1.1.27 and EC 1.1.1.28). In one nonlimiting embodiment, genetic modulation comprises deleting or mutating NAD+-dependent formate dehydrogenase enzymes (EC 1.2.1.2). In one nonlimiting embodiment, genetic modulation comprises exchange of NADPH with various NADH enzyme equivalents. In one nonlimiting embodiment, genetic modulation comprises oxygen limitation. In one nonlimiting embodiment, genetic modulation comprises using a defined oxygen respiration rate. In one nonlimiting embodiment, genetic modulation comprises exchanging or augmenting native NAD+ dependent enzymes with equivalent enzymes able to perform similar reactions. In one nonlimiting embodiment, genetic modulation comprises deleting or mutating terminal oxidases thereby hindering the capacity to use oxygen as a terminal electron acceptor. Nonlimiting examples of terminal oxidases which can be deleted include coxMNOP, ctaABCDE, cyoABC, cyo123, and ccoNOPQ. In one nonlimiting embodiment, genetic modulation comprises deletion or mutation of reversible cytoplasmic soluble hydrogenases (EC 1.12) including, but not limited to, HoxH, HoxY, HoxF and Hox. In one nonlimiting embodiment, genetic modulation comprises overexpressing, mutating or deleting one or more NADP (EC 1.6.1.2 and EC 1.6.1.1) genes which shuttle hydrogen between NADP (H) and NAD(H). Nonlimiting examples of transhydrogenase genes include PntA1, namely H16_A0850, H16_A0851, and H16_A0852, PntA2, namely H16_A1264, H16_A1265 and H16_A1266, PntA3, namely H16_A3128, H16_A3130 and H16_A3131 and PntA4, namely H16_B1714 and H16_B1715. In one nonlimiting embodiment, further manipulation comprises the overexpression of triosephosphate isomerase (EC 5.3.1.1, H16_A1047). In one nonlimiting embodiment, genetic modulation comprises replacement of isocitrate dehydrogenase with an NAD+ dependent version from such as those found in Methylophilus methylotrophus, Acidithiobacillus thiooxidans (Inoue et al. FEMS Microbiol Lett 2002 214(1):127-32), Methylococcus capsulatus (Stokke et al. Arch Microbiol. 2007 187(5):361-70), and Streptococcus mutans (Wang et al. PLoS One. 2013 8(3):e58918). In one nonlimiting embodiment, genetic modulation comprises deleting or mutating one or more NAD(P) alcohol dehydrogenases (EC 1.1.1.1 and EC 1.1.1.2). Nonlimiting examples include H16_A0171, H16_A0602, H16_A0757, H16_A0849, H16_A0861, H16_A0931, H16_A1168, H16_A1591, H16_A3330, H16_B0517, H16_B0663, H16_B0713, H16_B0831, H16_B1417, H16_B1433, H16_B1699, H16 81745, H16_B1834, H16_B1960, H16_B2470 and PHG229. In one nonlimiting embodiment, genetic modulation comprises deleting or mutating a NAD(P) lactate dehydrogenase (EC 1.1.1.27 and EC 1.1.1.28) enzymes. Nonlimiting examples include H16_A0666, H16_B0460, H16_B1817, H16_A1681 and H16_A1682. In one nonlimiting embodiment, genetic modulation comprises overexpression of heterologous Zwf from Zymomonas mobilis or Leuconostoc mesenteroides to prevent NADPH feedback inhibition on glycolysis. In one nonlimiting embodiment, genetic modulation comprises overexpression of Gnd from Gluconobacter oxydans or Corynebacterium glutamicum to prevent NADPH feedback inhibition on glycolysis. In one nonlimiting embodiment, genetic modulation comprises molecular hydrogen production via the reverse reaction of the membrane bound and soluble NiFe hydrogenases (EC 1.12.1.2 and EC 1.12.5.1).
In one nonlimiting embodiment of the present invention, at least one exogenous nucleic acid excluding Gnd, tktA and zwf of E. coli is introduced into the organism.
Additional redox balancing strategies which can be preturbed in accordance with the present invention are set forth herein in the Examples and Table A.
In some nonlimiting embodiments, the methods for increasing product yield further comprise reducing NADPH/NADP ratio under limiting conditions such as phosphate, carbon, nitrogen, and/or oxygen by increasing activity or introducing select NADPH dependent enzymes. It is important dissipate excess reducing potential so that the reactions are not subject to inhibition and carbon flux can be maintained.
In one nonlimiting embodiment, this is achieved via overexpression or mutation of an alcohol dehydrogenase, EC 1.1.1.2, or aldehyde reductase, EC 1.1.1.21, to generate NADP from NADPH by converting either formaldehyde to methanol, propionaldehyde to propanol, 3-hydroxypropanal to 1-3 propanediol, butyraldehyde to butanol, isobutanal to isobutanol, glucose to sorbitol, C5 to C18 fatty aldehydes to corresponding C5 to C18 fatty alcohols, succinate semialdehyde to 4-hydroxybutanoate, oxaloacetate to L-aspartate, 2-oxoglutaric acid to glutamate, L-lactaldehyde to propane-1,2-diol, C4-C18 enoyl-ACP to a corresponding C4-C18 acyl-ACP, C4-C18 enoyl-CoA to a corresponding C4-C18 acyl-CoA, C6-C18 acyl-ACP to a corresponding C6-C18 fatty aldehyde, C6-C18 acyl-CoA to corresponding C6-C18 fatty aldehyde, 4-hydroxybenzoate to protocatechuate, phenol to catechol, acetaldehyde to ethanol, acetone to 2-propanol, pyruvate to 2-hydroxypropanoic acid, D-glyceraldehyde to glycerol or acetoin to butanediol.
In one nonlimiting embodiment, this is achieved by diverting carbon flux enzymes such as EC 1.1.1.61, EC 1.1.1.79, EC 1.1.1.2 or EC 1.1.1.21 to convert succinate semialdehyde to 4-hydroxybutanoate.
In one nonlimiting embodiment, this is achieved via overexpression of aspartate dehydrogenase or glutamate dehydrogenase to convert oxaloacetate to L-aspartate or 2-oxoglutaric acid to glutamate.
In one nonlimiting embodiment, this is achieved via conversion of malonyl-CoA by EC 1.2.1.18 to malonate-semialdehyde and/or subsequently 3-hydroxypropionate by EC 1.1.1.298.
In one nonlimiting embodiment, this is achieved via conversion of L-lactaldehyde to propane-1,2-diol by EC 1.1.1.55.
In one nonlimiting embodiment, this is achieved via introduction of EC 1.1.1.36, EC 1.3.1.38 and/or EC 1.1.1.100 to convert C4-C18 enoyl-ACP to a corresponding C4-C18 acyl-ACP or to convert C4-C18 enoyl-CoA to a corresponding C4-C18 acyl-CoA.
In one nonlimiting embodiment, this is achieved via introduction of EC 1.2.1.80 to convert C6-C18 acyl-ACP to a corresponding C6-C18 fatty aldehyde or to convert C6-C18. Acyl-CoA to a corresponding C6-C18 fatty aldehyde.
In one nonlimiting embodiment, this is achieved via introduction of an enzyme belonging to EC 1.14.13.X which converts 4-hydroxybenzoate to protocatechuate or phenol to catechol.
Additional NADPH balancing strategies which can be used in accordance with the present invention are set forth herein in the Examples.
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, amino acids 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, cyclopentylpropionic 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. 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. 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.
For products of the present invention containing both amine groups and carboxylic acid groups such as but not limited to amino acids, 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, cyclopentylpropionic 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, 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. 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 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.
Nonnaturally occurring organisms 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.
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.
Accordingly, 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-6,6 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 suitable method can be used to isolate these products or derivatives or compounds related thereto.
The present invention also provides nonnaturally occurring organisms and methods for producing the nonnaturally occurring organisms with the redox balance perturbed to increase product yield as compared to product yield in the same organism without the redox balance perturbed. The nonnaturally occurring organisms are selected from a species of Cupriavidus or Ralstonia with diminished polyhydroxybutyrate synthesis or an organism with properties similar thereto. Redox balance is perturbed in the organism by modulating activity of one or more polypeptides having the activity of any one of a transhydrogenase, reductase, dehydrogenase, or hydrogenase enzyme or a functional fragment thereof.
In some embodiments, the organism is modulated by increasing activity or introducing select NADPH dependent enzymes. These nonnaturally occurring organisms exhibit increased product yield as compared to product yield in the same organism without pertubation of the redox balance and/or increasing activity or introducing select NADPH dependent enzymes.
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 of the nonnaturally occurring organisms of the present invention, at least one exogenous nucleic acid excluding gnd, tktA and zwf of E. coli is introduced.
In one nonlimiting embodiment, the present invention relates to a substantially pure culture of the nonnaturally occurring organism with a redox balance perturbed to increase product yield.
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.
Redox Rebalancing Strategies
For each CO2 molecule fixed, 1 molecule of NADH is oxidized to NAD+ by the GAPDH (EC 1.2.1.12)-mediated reduction of 1,3 biphosphoglycerate to glyceradehyde-3-phosphate. In C. necator, the two CBB cycle operons (on chromosome 2 and plasmid pHG1) are induced by nitrogen limitation (Brigham et al. Appl Environ Microbiol. 2012 78(22):8033-44; Brigham et al. Appl Environ Microbiol. 2017 83(15):1-2), suggesting that the rate of CO2 fixation is limited by the intrinsic activity of the CBB pathway enzymes and not CO2 availability. Under certain growth conditions it may be possible to regenerate more NAD+ molecules, and rebalance the cellular redox state, by overexpressing the CBB pathway enzymes. In support of this notion, the CO2 fixation pathway has been shown to have a central redox cofactor recycling role in Rhodopseudomonas palustris when cultured under photoheterotrophic growth conditions (McKinlay and Harwood PNAS 2010 107 (26) 11669-11675).
Under conditions where molecular oxygen is not limiting the rate of NADH oxidation, the NAD+ to NADH ratio can be increased by the overexpression of components of the respiratory electron transport chain that limit the rate of electron transfer between NADH and oxygen. The core C. necator respiratory chain consists of an NADH dehydrogenase complex, succinate dehydrogenase, a bc1 complex, and at least three terminal oxidases that either receive electrons directly from ubiquinone Q8/menaquinone electron carriers or cytochrome C. In a comparative proteomic study, two isoforms (menG2 and menG3) of demethylmenaquinone methytransfererase, an enzyme involved in ubiquinone/menaquinone biosynthesis, were shown to be induced to higher levels under nitrogen limitation in a PHB-deficient strain, PHB-4, than wildtype H16 (Raberg et al. PLoS ONE 2014 9(5):1-11). This is consistent with an adaptive response to increased NADH production as a result of more carbon flux entering the TCA cycle, rather than polyhydroxyalkanoate synthesis, and suggests that a component of the respiratory electron transport chain can limit the rate of NADH to NAD+ recycling under certain conditions.
Under conditions where the rate of NAD+ regeneration is limited by the aerobic respiratory chain, C. necator has the capacity to use organic substrates (as well as molecular oxygen) as final electron acceptors, by expressing a variety of NAD(P)-linked dehydrogenases to produce partial fermentation products such as ethanol, malate, citrate, isocitrate, lactate, succinate, formate, acetate, 2-oxoglutarate, cis-aconitate and 3-Hydroxybutyrate. Table 1 contains a list of NAD(P)-dependent dehydrogenases that can be overexpressed/heterologously expressed in C. necator to rebalance the redox state of C. necator strains and increase the production of specific overflow metabolites.
One such partial fermentation product is 3-hydroxybutyrate, which has been shown to accumulate in PHB-deficient strains, especially under nitrogen and oxygen limitation (Vollbrecht and Schlegel European J. Appl. Microbiol. Biotechnol. 1979 7:259-266). In the absence of polyhydroxyalkanoate synthase activity, the overflow metabolism of the PHB pathway can be channeled towards the (R)-3-Hydroxybutyrate production by the co-expression of an (R)-3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.36, encoded by phaB1, phaB2 or phaB3), with either a thioesterase (E.C. 3.2.1) or CoA-transferase (E.C. 2.8.3.8) that is able to remove the CoA moiety from the (R)-3-hydroxybutyryl-CoA intermediate (
Instead of NADP+, 1 NAD+ molecule can be generated for each acetoactetyl-CoA molecule reduced, by one of two alternative 3-hydroxybutyrate biosynthetic pathways (
In the first NADH-dependent pathway, flux is channeled into the production of (S)-3-hydroxybutyrate by either deleting the (R)-3-hydroxybutyryl-CoA dehydrogenase activities encoded by phaB1, phaB2 and phaB3 and/or the expression of NADH-dependent (S)-3-hydroxybutyryl-CoA dehydrogenases, encoded by C. necator paaH1 (H16_A0281) or paaH2 (H16_A1103).
In the second NADH-dependent pathway, (R)-3-hydroxybutyrate is generated in two enzymatic steps involving first the removal of the CoA moiety from acetoacetyl-CoA by succinyl-CoA:3-ketoacid CoA transferase (E.C. 2.8.3.5) and then the reduction of the acetoacetate intermediate by (R)-3 hydroxybutyrate dehydrogenase (E.C. 1.1.1.30).
Succinyl-CoA:3-ketoacid CoA transferase contains 2 subunits encoded in C. necator H16 by H16_A1331 and H16_A1332. These ORFs are organized into a bicistronic operon, whose transcription is preferentially induced in the C. necator H16 ΔphaC1AB1 mutant, compared to wildtype H16, under nitrogen limitation conditions (see RNAseq data in Table 2).
The gene product encoded by H16_A1331 has also been shown to be overexpressed in the PHB-negative strain, C. necator PHB−4, compared to wild type H16 (Raberg et al. PLoS ONE 2014 9(5):1-11).
(R)-3 hydroxybutyrate dehydrogenase (E.C. 1.1.1.30), is a homotetrameric enzyme involved in the degradation of PHBs, by oxidizing depolymerized (R)-3 hydroxybutyrate monomers back to acetoacetate (see
Modifications of the TCA Cycle to Increase the NAD+ to NADH and/or NADP+ to NADPH Ratios
The glyoxylate pathway produces 1 NADH molecule for each acetyl-CoA molecule, compared with 2 NADH molecules and 1 NADPH molecule with the complete oxidation of acetyl-CoA by the TCA cycle. Isocitrate lyase (ICL, EC 4.1.3.1) is a homotetramer with two gene copies encoded in the C. necator H16 genome, H16_A2211 (iclA) and H16_A2227 (iclB).
One of the two C. necator H16 isocitrate lyase genes, iclA (H16_A2211) is preferentially induced in the ΔphaC1AB1 genetic background compared to wild type H16, especially under nitrogen limitation (see Table 3).
Elevated ICL polypeptide levels have also been observed in a proteomic comparison of a similar PHB-negative strain, C. necator PHB−4, with wild type H16 (Raberg et al. PLoS ONE 2014 9(5):1-11). These observations suggest that C. necator compensates for the loss of NADP+ recycling, via the PHB pathway, by redirecting flux through the glyoxylate pathway and away from some of the NAD(P)H generating steps of the TCA cycle.
IDH catalyzes the decarboxylation of isocitrate to 2-oxoglutarate (α-ketoglutarate), with the release of CO2 and either NADPH or NADH. Only an NADP+-dependent IDH activity has been measured so far in C. necator cell-free extracts (Wang et al. PLoS One. 2013 8(3):e58918), although a putative NAD+-dependent IDH is encoded by icd3. By switching the nucleotide specificity of the native C. necator IDH activity to NAD+, 3 NADH molecules can be formed by the complete oxidation of acetyl-CoA, instead of 2 NADH molecules and 1 NADPH molecule. This has the net effect of increasing the NADH to NAD+ ratio but reducing the NADPH to NADP+ ratio.
NAD+-dependent IDH enzymes putatively include C. necator icd3 (H16_B1016), and the IDH from Congregibacter litoralis KT71 ClIDH, EAQ96042 (Wu et al. PLoS One. 2015 10(5):1-17), Acidithiobacillus thiooxidans (Inoue et al., FEMS Microbiol Lett. 2002 214(1):127-32) and Streptococcus mutans (Wang et al. PLoS One. 2013 8(3):e58918).
A mutant version of the E. coli IDH (K44D/Y345I/V351A/Y391K/R395S) with an altered nucleotide specificity for NAD+ (Chen et al. PNAS 1996 92 (25):11666-11670) could also be used to replace the native C. necator NADP+ dependent IDH enzyme activity.
Aspergillus itaconicus cis-aconitate decarboxylase (EC 4.1.1.6) catalyzes the decarboxylation of cis-aconitate to produce itaconic acid and CO2. By heterologously expressing this enzyme in C. necator, excess flux can be channeled out of the TCA cycle before reaching the oxidative enzymatic steps that produce 1 NADPH molecule and 2 NADH molecules per acetyl-CoA molecule oxidised.
Strategies for Increasing the NADPH Pool
In C. necator, the catabolism of fructose, glucose and gluconate proceeds by the Entner-Doudoroff (ED) pathway, where the oxidation of 1 mol of fructose/glucose to pyruvate yields 1 mol of NADPH and 1 mol of NADH. However, 2 mols of NADPH can theoretically be produced from each mol of fructose/glucose catabolized to pyruvate, by engineering a C. necator strain that utilizes the oxidative pentose phosphate (oxPP) pathway rather than the ED pathway. The C. necator H16 genome encodes all the enzymes of the oxPP pathway, with the exception of 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44 and EC 1.1.1.351) which performs an essential oxidative decarboxylation step to convert 6-phospho-D-gluconate to D-ribose 5-phosphate. The preparatory phase of the Embden-Meyerhoff-Parnas (EMP) pathway, for the conversion of one hexose sugar molecule into two glyceraldehyde 3-phosphate (GAP) molecules, also appears to be not functional in C. necator, as a result of there being no gene coding for phosphofructokinase (EC 2.7.1.11). Lee et al. (Biotechnology Progress 2003 19(5):1444-49) has exemplified the basic principle of using the oxPP pathway to increase the NADPH to NADH ratio in C. necator, by heterologously expressing the E. coli gnd and tktA genes encoding 6PGDH and the oxPP transketolase (TK), respectively. Measurable increases in NADPH levels were observed for the expression of E. coli gnd and tktA. However, the expression of E. coli gnd led to reduced cell growth and lower PHB titers. Although Lee et al. 2003 putatively detected endogenous 6-phosphogluconate dehydrogenase activity from C. necator cell free extracts, the subsequent publication of the whole C. necator H16 genome sequence revealed that C. necator is unlikely to possesses a 6PGDH enzyme. The performance of the oxPP pathway for increasing the NADPH pool in C. necator may be improved by:
(1) the overexpression of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with 6PGDH to overcome feedback inhibition by NADPH. The activities of both G6PDH and 6PGDH are sensitive to feedback inhibition by NADPH (Sekar et al. Biotechnol. Biofuels 2017 10:85). In the case of G6PDH, this can lead to flux through both the ED and oxPP pathways being negatively affected. However, the overexpression of G6PDH has been shown to increase the flux through ED and oxPP pathways, resulting in higher NADPH production rates. The E. coli zwf gene encoding G6PDH has been expressed in C. necator, resulting in an altered molar ratio of 3-hydroxyvalerate to 3-hydroxybutyrate in biopolymers and increased NADPH levels (Choi et al. Enzyme and Microbial Technology 2003 32(1):178-185);
(2) eliminating futile cycles between the Calvin Benson Bassham and oxPP pathways by removing or attenuating one or more enzymatic steps that are unique to the CBB cycle. The oxidative pentose phosphate pathway and the Calvin Benson Bassham (CBB) CO2 fixation pathway share some of the same enzymatic steps that operate in opposing directions (see
(3) co-expressing phosphofructokinase with 6PGDH to create a C. necator strain with complete EMP, ED and oxPP pathways. The EMP, ED and oxPP pathways produce different ratios of NADH, NADPH and ATP molecules for each hexose sugar oxidized (see Table 4). This enables the fine turning of the molecular stoichiometry between NADH, NADPH and ATP for optimal NADPH and biomass production, by manipulating the relative fluxes through these competing pathways.
Estimated molar ratios of NADPH, NADH and ATP produced from the oxidation of 1 mol of glucose, gluconate and fructose to pyruvate by either the Embden-Meyerhoff-Parnas (EMP), Entner-Doudoroff (ED) or oxidative pentose phosphate (oxPP) pathways, and 1 mol of glycerol by either the aerobic or fermentative glycerol utilization pathways are shown in Table 4.
Glycerol catabolism proceeds by one of two routes to dihydroxyacetone-phosphate (DHAP), which is then isomerized to glyceraldehyde 3-phosphate and converted to pyruvate by the lower EMP pathway (
The glycerol-utilization pathway can be optimized for the production of NADH by a similar strategy used to optimize NADPH production. 2 mols NADH can be produced for each mol of glycerol oxidized to pyruvate by expressing the ‘fermentative’ glycerol utilization pathway consisting of an NAD+-dependent glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29), in a C. necator strain deficient in glycerol kinase (EC 2.7.1.30) activity (e.g. C. necator ΔH16A2507 and ΔH16B1199).
For each mol of pyruvate produced, the classical lower EMP pathway generates 1 mol of NADH from the oxidation of glyceraldehyde 3-phosphate (GAP) to 1,3 biphosphoglycerate by an NAD+-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). By deleting the native NAD+-dependent GAPDH activity in C. necator (H16_A3146, H16_B1386 and PHG418) and recombinantly expressing a NADP+-dependent GAPDH enzyme (EC 1.2.1.13), 1 mol of NADPH can instead be synthesized from the lower EMP pathway. When combined with measures to maximize NADPH production from the upstream glycerol utilization pathway, a maximum of 2 mols of NADPH, and no NADH, can be synthesized from the oxidation of 1 mol of glycerol to pyruvate. Depending on whether the ED, EMP or oxPP pathways are utilized, a maximum of 2 to 3.67 mols of NADPH can be produced from hexose sugars using an NADP+-dependent GAPDH enzyme.
In the CBB cycle, GAPDH catalyzes the reverse reaction to the lower EMP pathway by reducing 1,3 biphosphoglycerate to glyceraldehyde 3-phosphate, with the result that 1 NAD(P)H molecule is consumed for every CO2 molecule fixed. The flux through this NAD(P)H-consuming reaction can be minimized either by redirecting 1,3 biphosphoglycerate from the CBB cycle into the lower EMP pathway through the overexpression of phosphoglycerate kinase (EC 2.7.2.3), or by deleting/attenuating the activity of an upstream CBB enzyme, such as phosphoribulokinase (EC 2.7.1.19), to limit the carbon flux entering the CBB cycle.
Modifications of the TCA Cycle to Increase the NADPH Synthesis
A maximum of 1 mol of NADPH is generated from 1 mol of acetyl-CoA entering the citric acid cycle. However, the enzyme performing this NADPH-generating reaction, isocitrate dehydrogenase (EC 1.1.1.42), resides at a branch point in the TCA cycle where flux can either enter the glyoxylate pathway, for the conversion of acetyl CoA into succinate or malate via isocitrate lyase (ICL, EC 4.1.3.1), or remain in the TCA cycle for the complete oxidation of 1 mol of acetyl-CoA to 2 mols of NADH and 1 mol of NADPH. The TCA cycle can be manipulated to achieve the maximum theoretical yield of NADPH, by deleting the C. necator genes coding for isocitrate lyase (iclA, H16_A2211 and iclB, H16_A2227) and/or the overexpression of a NADP+-dependent isocitrate dehydrogenase.
The anaplerotic node of the TCA cycle mediates the interconversion between C3 and C4 molecules for the maintenance of TCA cycle intermediates and metabolism of C3/C4 substrates. For instance, the C3 molecule phosphoenolpyruvate is converted by either phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) or phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) to the C4 molecule oxaloacetate. In a similar carboxylation reaction, pyruvate is converted to oxaloacetate by pyruvate carboxylase (PC, EC 6.4.1.1). Under microaerobic or anaerobic conditions, oxaloacetate can be recycled back to pyruvate by malic enzyme (ME, EC1.1.1.40), which catalyzes the NADP+-dependent oxidative decarboxylation of malate to pyruvate (see
The pyridine nucleotide transhydrogenases (EC 1.6.1.1 EC 1.6.1.2, and EC 7.1.1.1) catalyze the reversible hydride transfer between NAD(H) and NADP(H). These enzymes can be divided into two groups, energy-independent soluble transhydrogenases (STH, EC 1.6.1.1) and the energy-dependent, proton-translocating, membrane-bound transhydrogenases (H+-TH, EC 1.6.1.2, EC 7.1.1). Although both isoforms theoretically catalyze reversible hydride exchange between NAD(H) and NADP(H). The membrane-bound transhydrogenases couple electron transfer between pyridine nucleotides with proton import from the periplasmic space into the cytosol, with the result that the transfer of electrons between NADH and NADP+ is strongly favored thermodynamically. The C. necator H16 genome codes for four putative transhydrogenases composed of two alpha-beta subunits, PntA1, PntA2, PntA3 and PntA4. The overexpression of a transhydrogenase may directly increase the NADPH pool in C. necator. In some circumstances, the overexpression of a soluble transhydrogenase can actually increase productive yields of NADPH-dependent biosynthetic pathways. For instance, the overexpression of STH increased NADPH-dependent poly(3-hydroxybutyrate) production by 82% in a recombinant E. coli strain (Sanchez et al. Biotechnol Prog. 2006 22(2):420-5).
NAD+ kinase (NADK, EC 2.7.1.23) plays an essential role in de novo NADP+ biosynthesis, by catalyzing the conversion of NAD+ to NADP+. Most NADKs can also phosphorylate NADH to directly produce NADPH, but the enzymes generally have a substrate preference for NAD+. The overexpression of NADKs have been shown to increase the production yields of a number of NADPH-dependent pathways, including a recombinant polyhydroxyalkanoate pathway expressed in E. coli. (Li et al. Appl Microbiol Biotechnol. 2009 83(5):939-47). Higher NADPH yields may be achieved in C. necator by combining the overexpression of NADK with strategies to increase the NADH pool, such as limiting the capacity of the respiratory electron transport chain to oxidize NADH under aerobic conditions or the ability of oxidoreductases to use organic substrates as terminal electron acceptors under oxygen-limited culture conditions.
In addition to heterotrophic and lithotrophic modes of growth, C. necator is capable of adopting a mixotrophic lifestyle by simultaneously deriving carbon and energy from organic and inorganic substrates, such as fructose and CO2/H2. A mixotrophic cultivation strategy can provide a means to independently manipulate biomass accumulation and ATP/NAD(P)H production, allowing a greater degree of in-process control of the intracellular NADH/NAD+ ratio during fermentations. One example of a mixotrophic fermentation strategy able to control NADH levels consists of an organic carbon source supplying all the carbon and most of the energy requirements, supplemented with molecular hydrogen for additional NADH/ATP synthesis. The redox state of a fermentation can be monitored in real time by measuring the CO2 evolution rate and H2/O2 uptake rates, allowing the intracellular NADH pool to be maintained at constant level by adjusting the hydrogen supply.
Cupriavidus necator H16 NAD(P)+ oxidoreductases
Escherichia coli
Saccharomyces
cerevisiae
Escherichia coli
Homo sapiens
Saccharomyces
cerevisiae
Homo sapiens
Saccharomyces
cerevisiae
Monoraphidium
neglectum
Homo sapiens
Saccharomyces
cerevisiae
Escherichia coli
Aquifex aeolicus
Sulfolobus
acidocaldarius
Homo sapiens
Crinalium
epipsammum
Pseudopedobacter
saltans
This patent application claims the benefit of priority from U.S. Provisional Patent Application Ser. No. 62/665,814 filed May 2, 2018, teachings of which are herein incorporated by reference in their entirety.
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| Number | Date | Country | |
|---|---|---|---|
| 20190338375 A1 | Nov 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62665814 | May 2018 | US |
