Patent Application
20210002620
The invention relates to methods and materials for producing polyols and electron rich biofuels.
In Saccharomyces cerevisiae, more commonly known as budding yeast, there are about 900 uncharacterized open reading frames (ORFs). While the enzymatic function is postulated for some of these ORFs, it is typically only partially annotated, making further characterization essential for establishing gene functions. Advances in high-throughput screens have yielded transcriptomic and proteomic data that can prioritize candidate enzymes of unknown function for metabolic analysis. The metabolite profiles of uncharacterized ORFs selected based on sequence homology to known enzymes will inform functional genomics, metabolic engineering, and the development of new yeast strains of biotechnological utility.
There is a long standing interest in bioproduction of various chemicals, including biofuels and biomaterials. Although these processes have been achieved by engineering native or foreign enzymes into target microorganisms, the lack of a holistic understanding of metabolism limits the further optimization of the yield of biofuel and biomaterial production. Most of these biofuels and biomaterials are rich in electrons, rendering the reduction of the substrates important steps in their biosynthesis. Nicotinamide adenine dinucleotide reduced phosphate (NADPH) is the most important reducing power for various biofuels including biodiesel and isobutanol and biomaterials including monomers of polyolesters. To achieve a high yield of these chemicals, NADPH regeneration must be upregulated.
Polyols, the reduced forms of sugars, are an important natural family of carbohydrates (Shindou et al., 1988; Teo et al., 2006). Glycerol, the simplest polyol, is the backbone of phospholipids and is excreted by many microbes in response to stress (Nevoigt and Stahl, 1997; Pahlman et al., 2001). Longer chain polyols include erythritol, ribitol, xylitol, arabitol, sorbitol, and mannitol, all of which exist only in the D-form in nature and are usually found in plants. Due to the inability of most organisms to assimilate long chain polyols into glycolysis, they are often regarded as inert solutes with unclear physiological functions. While the biological function of polyols has remained obscure, they have gained commercial interest in the last decade due to their increased usage as a sugar substitute by the food industry (Bradshaw and Marsh, 1994).
Many fungi species are able to produce polyols via reduction of the corresponding sugar (Chang et al., 2007). To engineer polyol production, the standard approach is to first dephosphorylate the sugar phosphate and then reduce the resulting sugar to the polyol (Moon et al., 2010; Povelainen and Miasnikov, 2006, 2007; Toivari et al., 2010). This engineering approach differs from the natural glycerol excretion pathway, where (L)-glycerol-3-phosphate (a.k.a. sn-glycerol-3-phosphate) is first made from the reduction of dihydroxyacetone phosphate, followed by the dephosphorylation of the phosphorylated polyol (Albertyn et al., 1994; Norbeck et al., 1996). This natural approach avoids making a dephosphorylated sugar that might be consumed by or escape from the cell; however, it has not been feasible as an engineering strategy as a suitable polyol phosphatase has not previously been identified in any species.
Because many enzymes remain unannotated even in the best studied organisms such as Baker's yeast, it is possible that various polyol phosphate phosphatases exist but have yet to be characterized. One approach to metabolic enzyme annotation is to knock out the corresponding gene and assess the metabolome of the resulting strain. In general, substrates of the enzyme are likely to increase and products to decrease. This approach has been used to assign function to a variety of enzymes (Clasquin et al., Current protocols in bioinformatics/editoral board, Andreas D. Baxevanis . . . [et al.] Chapter 14, Unit 14 11, 2012; Clasquin et al., 2011; Fiehn et al., Nat Biotechnol 18, 1157-1161, 2000; Quanbeck et al., Frontiers in plant science 3, 15, 2012; Raamsdonk et al., Nat Biotechnol 19, 45-50, 2001; Saghatelian and Cravatt, ife sciences 77, 1759-1766, 2005; Su et al., J Am Chem Soc 134, 773-776, 2012; Xu et al., Mol Syst Biol 9, 665, 2013; Yonekura-Sakakibara et al., The Plant cell 20, 2160-2176, 2008).
Thus, there exists in the art a need for genetically engineered microorganisms with enhanced capacity for NADPH regeneration and generation of industrially important compounds, such as polyols.
In one aspect of the disclosure, there is provided a recombinant S. cerevisiae cell comprising a polynucleotide encoding Pyp1, ortholog thereof, or a variant of Pyp1 at least 70% identical to the amino acid sequence of SEQ ID NO: 1, wherein the polynucleotide is operably linked to a heterologous promoter.
In another aspect of the disclosure, there is provided a recombinant microorganism comprising a polynucleotide encoding Pyp1, ortholog thereof, or a variant of Pyp1 at least 70% identical to the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the microorganism is a fungus or a bacteria. In some embodiments, the fungus is S. cerevisiae.
In some embodiments, the microorganism further comprises a polynucleotide encoding an enzyme with sugar phosphate dehydrogenase activity operably linked to a heterologous promoter.
In another aspect of the disclosure, there is provided a method of producing a polyol comprising: (a) culturing a recombinant microorganism comprising a polynucleotide encoding Pyp1, ortholog thereof, or a variant of Pyp1 at least 70% identical to the amino acid sequence of SEQ ID NO: 1; and optionally (b) separating the polyol from the culture.
In some embodiments, the polynucleotide is operably linked to a heterologous promoter.
In some embodiments, the microorganism is a fungus or a bacteria. In some embodiments, the fungus is S. cerevisiae.
In some embodiments, the polyol is a four, five or six-carbon polyol. In some embodiments, the polyol is selected from the group consisting of erythritol, ribitol, arabitol, mannitol and sorbitol. In specific embodiments, the polyol is sorbitol.
In some embodiments, the recombinant microorganism further comprises a polynucleotide encoding an enzyme with sugar phosphate dehydrogenase activity operably linked to a heterologous promoter.
In another aspect of the disclosure, there is provided a recombinant microorganism comprising a mutation or deletion of the DET1 gene or ortholog thereof and a mutation or deletion of the PHO13 gene or ortholog thereof.
In some embodiments, the recombinant microorganism further comprises an engineered biosynthetic pathway for an electron rich compound.
In some embodiments, the engineered biosynthetic pathway comprises 2 or more enzymes for the production of isobutanol.
In some embodiments, the enzyme with sugar phosphate dehydrogenase activity is a ribitol-5-phosphate dehydrogenase, a xylitol-5-phosphate dehydrogenase, an arabitol-5-phosphate dehydrogenase, and erytritol-4-phosphate dehydrogenase, a sorbitol-6-phosphate dehydrogenase, or a sedoheptitol-7-phosphate dehydrogenase.
In some embodiments, the microorganism is a fungus or a bacteria. In some embodiments, the fungus is Saccharomyces cerevisiae.
In another aspect of the disclosure, there is provided a method of producing an electron rich compound comprising: (a) culturing a Saccharomyces cerevisiae cell comprising a deletion or mutation of the DET1 gene and/or PHO13 gene, and overexpressing one or both of (i) a polynucleotide encoding an enzyme with sugar phosphate dehydrogenase activity and a gene encoding a polyol phosphatase or (ii) and engineered biosynthetic pathway for the electron rich compounds; and optionally (b) separating the electron rich compound from the culture.
In some embodiments, the electron rich compound is selected from the group consisting of polyols, butanol, isobutanol, fatty acids, fatty acid esters, long-chain fatty alcohols, biodiesel and biogas.
In some embodiments, the polyol comprises 4 or more carbon atoms. In some embodiments, the polyol is selected from the group consisting of erythritol, ribitol, arabitol, mannitol and sorbitol.
In some embodiments, the polyol phosphatase is encoded by PYP1 or an ortholog thereof.
In some embodiments, the enzyme with sugar phosphate dehydrogenase activity is derived from a species other than Saccharomyces cerevisiae.
In some embodiments, the Saccharomyces cerevisiae cell is cultured in a medium comprising glucose, cellulose or hemicelluloses as a carbon source.
In certain embodiments, the disclosure provides a method to clean up or eliminate unnecessary and inhibitory polyol phosphates which are generated in metabolic engineering processes where cells possess high sugar phosphate pools under a reducing environment. In various embodiments the method comprises contacting the inhibitory polyolphosphates with a polyol phosphatase, such as Pyp1.
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The present disclosure shows that YNL010W, a gene conserved across all fungi species and some plants, encodes a polyol phosphatase (Pyp1) and demonstrates that the enzyme prevents polyol phosphate accumulation in yeast, and that this is physiologically important due to polyols being transition state analogues (Scheme 1) and therefore inhibitors of the essential glycolytic enzyme phosphoglucose isomerase. Thus, through assigning function to this previously unannotated yeast gene, herein both a new glycolytic regulatory mechanism and a promising new enzyme for polyol metabolic engineering are identified.
The term “sugar phosphate dehydrogenase” refers to an enzyme that catalyzes a chemical reaction that reduces NADP+ to NADPH or NAD+ to NADH and oxidizes a sugar phosphate molecule.
The term “polyol phosphatase” refers to an enzyme that catalyzes a dephosphorylation reaction of a polyol phosphate molecule, producing a polyol and free phosphate.
The term “operably linked” refers to a functional relationship between two or more polynucleotide segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter/enhancer sequence, including any combination of cis-acting transcriptional control elements, is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, silencers, insulators, and locus control regions, need not be located in close proximity to the coding sequences whose transcription they enhance
The term “ortholog” refers to genes in different species that evolved from a common ancestral gene by speciation. Typically, orthologs retain the same function in the course of evolution.
The term “electron rich compound” refers to a compound produced by a pathway comprising one or more enzymatic steps requiring a high-energy electron donor such as NADH or NADPH. Electron rich compounds include, but are not limited to, chemical building blocks, polymer starting materials, fine chemicals, and biofuels.
The term “NADPH-derived compound” refers to an electron rich compound produced by a pathway comprising one or more enzymatic steps that utilize NADPH as an electron donor.
The term “biofuel” refers to an electron rich compound, produced by living organism by biomass conversion, that can be burned as a fuel source. Biofuels include, but are not limited to, ethanol, butanol, isobutanol, propanol, isopropanol, methanol, methane, ethane, propane, terpenes, fatty acids, fatty acid esters, biodiesel and biogas. Electron rich biofuels, such as butanol, are important industrial chemicals, useful as a fuel additives, and as feedstock chemicals in the plastics industry, and as a food grade extractants in the food and flavor industry. Each year, 10 to 12 billion pounds of butanol, for example, are produced by petrochemical means and the need for this commodity chemical will likely increase.
The term “polyol” refers to an alcohol containing multiple hydroxyl groups. Polyols of the disclosure include, but are not limited to, glycerol, ribitol, xylitol, arabitol, erythritol, and sorbitol.
The term “variant” when used in reference to a polypeptide means a polypeptide that has one or more substitutions, deletions or insertions relative to a parent polypeptide and retains the desired activity. When used in reference to Pyp1, “functional variant” means a variant that retains the ability to catalyze a dephosphorylation reaction of a polyol phosphate molecule, producing a polyol and free phosphate.
As used herein, the term “conservative amino acid substitution” is the replacement of one amino acid with another amino acid having similar properties, e.g. size, charge, hydrophobicity, hydrophilicity, and/or aromaticity, and includes exchanges as indicated below:
Amino acid residues which share common side-chain properties are often grouped as follows.
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Polyols, the reduced forms of sugars, are an important natural family of carbohydrates. Glycerol, the simplest polyol, is the backbone of phospholipids and is excreted by many microbes in response to stress. Longer chain polyols include erythritol, ribitol, xylitol, arabitol, sorbitol, and mannitol, all of which exist only in the D-form in nature and are usually found in plants. Due to the inability of most organisms to assimilate long chain polyols into glycolysis, they are often regarded as inert solutes with unclear physiological functions. While the biological function of polyols has remained obscure, they have gained more commercial interest in the last decade due to their increased usage as a sugar substitute by the food industry because they cannot be readily catabolized by humans and do not contribute to a high blood sugar level.
To achieve a high yield of polyols, the standard approach is to first dephosphorylate a sugar phosphate and then reduce the resulting sugar to the polyol. This engineering approach differs, for example, from the natural glycerol excretion pathway, where sn-(L)-glycerol-3-phosphate is first made from the reduction of dihydroxyacetone phosphate, followed by the dephosphorylation of the phosphorylated polyol. This natural approach is preferable because it avoids making a dephosphorylated intermediate that might be consumed by other pathways or escape the cell; however, it has not been feasible as an engineering strategy because a suitable polyol phosphate phosphatase for most polyols other than glycerol has not previously been identified in any species.
Described herein is the discovery that the previously uncharacterized S. cerevisiae YNL010W gene (also referred to herein as PYP1), which has close orthologs in all fungi species and some plants, encodes a broad-spectrum polyol phosphatase termed Pyp1 herein. The Pyp1 enzyme catalyzes a dephosphorylation reaction with the substrates sorbitol-6-phosphate, mannitol-6-phosphate, ribitol-5-phosphate, xylitol-5-phosphate, arabitol-5-phosphate, erythritol-4-phosphate and glycerol-3-phosphate. It is also demonstrated herein that the Pyp1 enzyme prevents polyol phosphate accumulation in yeast, and that this activity is physiologically important due to polyols being transition state analogues, and therefore inhibitors, of the essential glycolytic enzyme phosphoglucose isomerase, which carries high flux in rapidly growing yeast. The enzyme Pyp1 and its variants and orthologs are of particular utility for their ability to hydrolyze polyol-phosphates containing 4 or more carbon atoms into the corresponding polyol.
A combinatory over-expression of the enzyme encoded by the PYP1 gene and a gene encoding a sugar phosphate dehydrogenase (which converts sugar phosphate to polyol phosphate) represents a novel engineering pathway for polyol production. This approach can be applied to the production of many polyols, by directing the carbon flow in central metabolism toward the production of the corresponding sugar phosphate.
In exemplary embodiments, a recombinant microorganism of the disclosure comprises a polynucleotide encoding Pyp1 or a functional variant of Pyp1 that retains the ability to catalyze a dephosphorylation reaction of a polyol phosphate molecule, producing a polyol and free phosphate. In some or any embodiments, the functional variant comprises an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to at least 50 amino acids of residues 1-242 of SEQ ID NO: 1 that retains the ability to catalyze a dephosphorylation reaction of a polyol phosphate molecule. In some embodiments, the Pyp1 variant has been mutated to alter specificity to various substrates. In some embodiments, the Pyp1 protein variant is specific for sorbitol-6-phosphate, mannitol-6-phosphate, ribitol-5-phosphate, xylitol-5-phosphate, arabitol-5-phosphate, erythritol-4-phosphate and/or glycerol-3-phosphate, or combinations thereof.
In some embodiments of the disclosure, a recombinant microorganism comprises a polynucleotide encoding Pyp1 or a variant thereof. The wild type polynucleotide sequence of the S. cerevisiae PYP1 gene is SEQ ID NO: 2. In some embodiments, the PYP1 variant polynucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the wild type PYP1 gene (SEQ ID NO: 2). In some embodiments, the PYP1 gene or a variant thereof is operably linked to a heterologous promoter.
In certain embodiments, the polyol phosphatase is utilized to clean up or eliminate unnecessary and inhibitory polyol phosphates which are generated in many metabolic engineering processes where cells possess high sugar phosphate pools under a reducing environment. In certain embodiments, the disclosure provides a method to clean up or eliminate unnecessary and inhibitory polyol phosphates which are generated in many other metabolic engineering processes where cells possess high sugar phosphate pools under a reducing environment. In various embodiments the method comprises contacting the inhibitory polyol phosphates with a polyol phosphatase, such as Pyp1.
Microorganisms for polyol or electron rich biofuel production are selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microorganism used for production is preferably tolerant to the particular product sought to be produced so that the yield is not limited by toxicity.
Suitable microorganisms with a tolerance for the particular product sought to be produced are identified by screening based on the intrinsic tolerance of the strain. The intrinsic tolerance of microbes to a product is measured by determining the concentration of product that is responsible for 50% inhibition of the growth rate (IC50) when grown in a minimal medium. The IC50 values is determined using methods known in the art. For example, the microorganisms of interest are, in various embodiment, grown in the presence of various amounts of product and the growth rate monitored by measuring the optical density at 600 nanometers. The doubling time is calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of product that produces 50% inhibition of growth is determined from a graph of the percent inhibition of growth versus the product concentration. In various embodiments, the host strain has an IC50 for product of greater than about 0.5%.
Based on the criteria described above, microbial hosts for the production of electron rich biofuels include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Exemplary host species include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae.
In some or any embodiments, the recombinant microorganism of the disclosure is a yeast from a genus selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida. In some or any embodiments, the fungal species is selected from the group consisting of S. cerevisiae, P. pastoris, H. polymorpha, S. pombe, K. lactis, Y. lipolytica, and C. albicans. These examples are illustrative rather than limiting.
Fermentation media in the present disclosure contain suitable carbon substrates. Suitable substrates include, but are not limited to, monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate is, in various aspects, a one-carbon substrate such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Thus, it is contemplated that the source of carbon utilized in the present disclosure encompasses a wide variety of carbon containing substrates and will only be limited by the choice of organism.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for electron rich biofuel and polyol production.
In some embodiments of the disclosure, there is provided a method of producing a polyol in a recombinant microorganism is provided comprising the step of culturing a recombinant microorganism comprising a polynucleotide encoding Pyp1, ortholog thereof, or a variant of Pyp1 at least 70% identical to the amino acid sequence of SEQ ID NO: 1. The method optionally comprises the step of separating the polyol from the culture.
In some embodiments, the polynucleotide encoding Pyp1 is overexpressed in S. cerevisiae. In some embodiments, the polynucleotide encoding Pyp1 is expressed in a host microorganism other than S. cerevisiae (see section II, Recombinant microorganisms). In some embodiments, the polynucleotide encoding Pyp1 is expressed in an organism known to produce high levels of sorbitol-6-phosphate, mannitol-6-phosphate, ribitol-5-phosphate, xylitol-5-phosphate, arabitol-5-phosphate, erythritol-4-phosphate and/or glycerol-3-phosphate, or combinations thereof. In some embodiments, the polynucleotide encoding Pyp1 is overexpressed in Lactobacillus plantarum, which has the capacity to convert fructose-6-phosphate into sorbitol-6-phosphate.
In some embodiments, the polynucleotide encoding Pyp1 is expressed in a microorganism engineered to express the enzymes necessary to produce sorbitol-6-phosphate, mannitol-6-phosphate, ribitol-5-phosphate, xylitol-5-phosphate, arabitol-5-phosphate, and/or erythritol-4-phosphate, or combinations thereof.
In some embodiments, recombinant microorganisms of the disclosure comprise a polynucleotide encoding a sugar phosphate dehydrogenase enzyme to increase production of a particular polyol phosphate. Such embodiments are used in conjunction with a recombinant microorganism comprising a polynucleotide encoding Pyp1, as described above. Thus, increasing production of a particular polyol phosphate provides more substrate for Pyp1 catalysis of the polyol phosphate dephosphorylation reaction. In various embodiments, the sugar phosphate dehydrogenase is a ribitol-5-phosphate dehydrogenase, a xylitol-5-phosphate dehydrogenase, an arabitol-5-phosphate dehydrogenase, an erytritol-4-phosphate dehydrogenase, a glycerol-3-phosphate dehydrogenase, a mannitol-6 phosphate dehydrogenase, or a sorbitol-6-phosphate dehydrogenase. For example, ribitol-5-phosphate dehydrogenase has been found in gram positive pathogens including Haemophilus influenza (accession number Q48230) and Staphylococcus aureus (accession number ADL64305). Mannitol-1-phosphate (same as mannitol-6-phosphate due to the symmetric structure) dehydrogenase (accession number YP_491834.1) and sorbitol-6-phosphate dehydrogenase (accession number YP_490914.1) have been found in various bacteria including Escherichia coli.
In various embodiments, the recombinant microorganism is engineered to overexpress a native gene encoding a sugar phosphate dehydrogenase enzyme or a gene encoding a sugar phosphate dehydrogenase enzyme derived from another species. Methods of obtaining and overexpressing desired genes from a microbial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries are created by restriction endonuclease digestion and are screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA is amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available. Some tools for codon optimization are available based on the GC content of the host organism.
The recent development of untargeted liquid chromatography-mass spectrometry (LC-MS) holds the potential in fully mapping microbial metabolism and more completely revealing the key regulatory events in redox balance and energy homeostasis. Using this technique, it was discovered that multiple genes whose knockout directly or indirectly contributes to an upregulated NADPH level, which in turn kinetically and thermodynamically favors production of electron rich bioproducts. Thus, yeast strains lacking these proteins advantageously have an increased cellular pool of NADPH available to generate electron rich compounds. Specifically, the deletion of a protein phosphatase, Pho13, or the deletion of a putative phosphatase, Det1, caused NADPH to accumulate in S. cerevisiae cells. The deletion of Pho13 resulted in an upregulation of NADPH regeneration (NADP+→NADPH) via the acetate production pathway. This upregulation resulted in accumulated NADPH, but not NADP+ (i.e., a higher NADPH/NADP+ratio). The deletion of Det1 resulted in a higher total pool of NADPH and NADP+, with a greater effect on NADPH and thus an increased NADPH/NADP+ratio. Both enzymes have orthologs among fungi species. Deletion of one or two of these enzymes would substantially contribute to a better reducing potential by elevating the pool size of NADPH and the ratio of NADPH/NADP+.
A. PHO13
Pho13 in S. cerevisiae is a protein phosphatase with unknown function. It was previously shown that pho13 deletion results in better utilization of xylose (Fujitomi et al., 2012; Ni et al., 2007; Van Vleet et al., 2008), but the mechanism behind the enhanced xylose utilization, however, is unknown. It was also shown that the pho13 deletion strain accumulates acetate in the media (Van Vleet et al., 2008). It was proposed that Pho13 was a protein phosphatase due to its ability to dephosphorylate model phosphopeptides in vitro (Tuleva et al., 1998).
Through the use of untargeted LC-MS measurements, it has been discovered that S. cerevisiae yeast with a pho13 deletion accumulate NADPH compared to a wild-type strain, but not NADP+, NADH or NAD+. Acetate excretion via Ald6 is one of the two major NADPH producing pathways (the other one is oxidative pentose phosphate pathway). Thus, observations described herein that NADPH accumulates are consistent with upregulated acetate excretion. It is also demonstrated herein that oxidative pentose phosphate pathway intermediates, 6-phosphogluconate, glucono-δ-lactone-6-phosphate accumulate in the pho13 deletion strain, while pentose-phosphates and sedoheptulose-7-phosphate in the non-oxidative pentose phosphate pathway decrease. These data indicate that the pho13 deletion strain also has an altered pentose phosphate pathway flux. Alteration of both NADPH producing pathways to enhance NADPH production through the deletion or alteration of Pho13 is fundamentally beneficial for metabolic engineering.
In some embodiments, a recombinant microorganism is provided comprising a partially or wholly deleted PHO13 gene or a nonfunctional PHO13 gene (SEQ ID NO: 3).
In some embodiments, partially or wholly deleting or inactivating the PHO13 gene results in an at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in cellular NADPH level compared to a wild type strain.
It is understood that reference to the PHO13 gene includes orthologs from species other than S. cerevisiae.
B. DET1
The Det1 (decreased ergosterol transport) protein encoded by the partially annotated ydr051c gene of S. cerevisiae is currently postulated in the literature to be an acid phosphatase involved in sterol transport between the endoplasmic reticulum and plasma membrane. Previous biochemical characterization of DET1 did not identified potential substrates.
The instant disclosure is based in part on discoveries using untargeted LC-MS to reveal that the partially annotated ydr051c gene (also referred to herein as DET1) of S. cerevisiae accumulates NADPH and NADP+ in a prototrophic deletion collection strain where the DET1 open reading frame was knocked out. In a comparable experiment using a yeast strain that overexpresses DET1, the level of NADPH was depleted compared to a wild type strain. Evidence presented herein indicates that the DET1 gene encodes a novel NADP phosphatase, the first of its kind identified in higher eukaryotes. The only previously known NADP phosphatase is from the archaea Methanococcus jannaschii. The enzyme encoded by the DET1 gene may contribute to the regulation of NADP, which is crucial to understanding the pentose phosphate pathway and of industrial importance. The regulatory mechanisms of NAD+/NADP+ metabolic flux have yet to be elucidated.
In some embodiments, a recombinant microorganism is provided comprising a partially or wholly deleted DET1 gene or a mutated (i.e., nonfunctional) DET1 gene. In some embodiments, a recombinant microorganism comprises a partially or wholly deleted DET1 gene or a mutated DET1 gene (SEQ ID NO: 4) and a partially or wholly deleted PHO13 gene or a mutated PHO13 gene.
In some embodiments, partially or wholly deleting or inactivating the DET1 gene results in an at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in cellular NADPH level compared to a wild type strain.
It is understood that reference to the DET1 gene includes orthologs from species other than S. cerevisiae.
C. Production of Electron Rich Compounds Including Biofuels
As noted above, an increased cellular NADPH level, obtained by inactivation of DET1 and/or PHO13, is advantageously utilized to increase the cellular pool of NADPH available to generate electron rich compounds including biofuels. Recombinant organisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a fermentable carbon substrate to a desired end product such as an electron rich biofuel is constructed using techniques well known in the art. For example, U.S. Pat. Nos. 7,851,188 and 7,993,889 (incorporated by reference) describe methods and materials to produce isobutanol in a microorganism. Pathways to a very large number of other value-added compounds are known in the art.
D. Elimination of Undesired Polyol Phosphates in Metabolic Engineering
Polyol phosphates, at high concentration, are also known to be inhibitory to primary metabolism because they structurally mimic the enediol intermediate of key enzymes (phosphoglucose isomerase, ribose-phosphate isomerase, triose-phosphate isomerase). Polyol phosphates are highly produced in many metabolic engineering processes where highly concentrated sugar phosphates are reduced to polyol phosphates.
In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has an enediol intermediate and is known to be inhibited by 2-carboxy-D-arabitol-1-phosphate (CA1P) (Andralojc et al., Biochem J. 304 (Pt 3):781-6, 1994). The corresponding 2-carboxy-D-arabitol-1-phosphatase is activated by light and controls cellular level of CA1P. Thus, to further improve RuBisCO activity in plants, maintaining a low intracellular level of CA1P and other polyol phosphates will be helpful. Pyp1 and its homologues can be engineered into plants and provide an alternative way to activate RuBisCO by dephosphorylating the inhibitory polyol phosphates into the inactivated polyol forms.
In metabolic engineering for various bioproducts, the cytosol of hosting cells is usually kept highly reduced to maintain a fast turnover rate from substrate to synthesize reduced products (for example, biodiesel, bioethanol, bioisoprene, fatty acids, other biofuels and biomaterials). Thus, cells are usually engineered to maintain a high NADPH/NADP+ or NADH/NAD+ ratio. High ratios of these cofactors can also occur if redox is not optimally balanced in the engineered cells. Irrespective of its cause, a high redox potential will cause the cells to generate some undesired byproducts, such as polyols (reduced from sugar) and polyol phosphates (reduced from sugar phosphates or phosphorylated from polyols). Polyol phosphates are highly inhibitory because they are transition state analogues of key sugar phosphate isomerases (phosphoglucose isomerase, triose phosphate isomerase and ribose phosphate isomerase).
Recombinant expression of a polyol phosphatase, such as Pyp1 or a homolog or derivative thereof, provides a useful method to avoid the accumulation of polyol phosphates, relieving the inhibition of the key nodes in glycolysis, the pentose phosphate pathway, or carbon fixation pathways. In some embodiments, expression of Pyp1 is useful in enhancing rates or yields of glucose- or xylose-based fermentation processes, carbon-fixation limited plant or microbial growth or biomass accumulation processes, or other processes requiring high glycolysis, pentose phosphate pathway, or carbon fixation fluxes.
The produced electron rich biofuel or polyol are isolated from the medium using methods known in the art. For example, in various embodiments, solids are removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the desired product is isolated from the medium, which optionally has been treated to remove solids as described above, using methods such as distillation, liquid-liquid extraction, or membrane-based separation. Because electron rich biofuels such as isobutanol form a low boiling point, azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation is, in various embodiments, used in combination with another separation method to obtain separation around the azeotrope. In various embodiments, methods that are used in combination with distillation to isolate and purify electron rich biofuels include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, electron rich biofuels are, in various embodiments, isolated using azeotropic distillation using an entrainer (see for example Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, N.Y., 2001).
An isobutanol-water mixture forms a heterogeneous azeotrope so that distillation is used in various embodiments in combination with decantation to isolate and purify the isobutanol. In this method, the isobutanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase is optionally returned to the first distillation column as reflux. The isobutanol-rich decanted organic phase is optionally further purified by distillation in a second distillation column.
Isobutanol is, in various embodiments, isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the isobutanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The isobutanol-containing organic phase is then distilled to separate the isobutanol from the solvent.
Distillation in combination with adsorption is also used in various embodiments to isolate isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).
Additionally, distillation in combination with pervaporation is also used in various embodiments to isolate and purify the isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).
The following examples are provided for illustration and are not in any way to limit the scope of the invention.
The study described herein was initiated via a metabolomics screen of yeast deletion strains with genes of unknown function. Yeast metabolome was measured by liquid chromatography-mass spectrometry (LC-MS). It was found that the deletion of PYP1, formerly known as the uncharacterized gene YNL010W, led to the accumulation of three metabolites in yeast grown on glucose (
Yeast strains were derived from prototrophic S288C. Prototrophic deletions were created by homologous recombination using the allele amplified by PCR from the synthetic genetic analysis (SGA) deletion set (Tong et al., 2001). A Pgi1-inducible strain was generated as described by McIsaac et al., Nucleic Acids Res 2013, 41, e57. Briefly, human hormone estradiol was used to induce PGI1 specifically via insertion of a synthetic promoter involving chimeric transcription factor-estrogen receptor in front of PGI1. 1 nM and 5 nM estradiol were added as low and high expression levels of yeast Pgi1, resulting in a 2.5-fold difference in Pgi1 protein level.
Cells were grown in minimal media comprising 6.7 g/L Difco Yeast Nitrogen Base without amino acids plus 2% (w/v) glucose or sorbitol. Glycerol medium was composed of 6.7 g/L Difco Yeast Nitrogen Base without amino acids, 0.79 g/L Complete Supplement Mixture (Sunrise Science, San Diego, Calif.) and 3% (v/v) glycerol. Trehalose minimal media were prepared by mixing 6.7 g/L Difco Yeast Nitrogen Base without amino acids and 1% (w/v) trehalose, and adjusting pH to 4.8 by adding succinic acid.
The metabolome of batch culture Saccharomyces cerevisiae was characterized as described by Xu et al., Molecular cell 2012, 48, 52. Briefly, saturated overnight cultures were diluted 1:30 and grown in liquid media in a shaking flask to A600 of −0.6. A portion of the cells (3 mL) was filtered onto a 50 mm nylon membrane filter (Millipore, Billerica, Mass.), which was immediately transferred into −20° C. extraction solvent (40:40:20 acetonitrile/methanol/water). For carbon upshift, 100 mL of cell culture grown on trehalose at A600 of −0.6 was poured onto a 100 mm cellulose acetate membrane filter (Sterlitech, Kent, Wash.) resting on a vacuum filter holder with a 1000 mL funnel (Kimble Chase, Vineland, N.J.) and was washed with 100 mL pre-warmed (30° C.) glucose minimal medium. Immediately after the wash media went through, the filter was taken off the holder and the cells were washed into a new flask containing 100 mL pre-warmed (30° C.) glucose minimal medium. Samples were then taken at the indicated time points after the switch and filtered and quenched as described above.
Cell extracts were analyzed by reversed phase ion-pairing liquid chromatography (LC) coupled by electrospray ionization (ESI) (negative mode) to a high-resolution, high-accuracy mass spectrometer (Exactive; Thermo Fisher Scientific, Waltham, Mass.) operated in full scan mode at 1 s scan time, 105 resolution at m/z 200. Peaks differing between wild-type and pyp1Δ strain were determined using the in-house developed, open-source software MAVEN (Lu et al., 2010; Melamud et al., 2010). Compounds' identities were verified by mass and retention time matched to authenticated standards. Isomers are reported separately only where they are fully chromatographically resolved. Differences in metabolome between wild type and pyp1Δ strains were tested for significance using Student's T-test. The resulting P-values were then corrected using the Benjamini-Yekutieli False Discovery Rate (FDR) model (Benjamini and Daniel, Ann Stat 20, 1165-1188, 2001)
Absolute intracellular metabolite concentrations in steadily growing S. cerevisiae were determined as described by Bennett et al., Nature Protocols 2008, 3, 1299. Metabolite concentrations after perturbations were computed based on fold-change in ion counts relative to steadily-growing cells (grown and analyzed in parallel) multiplied by the known absolute concentration in the steadily growing cells, as determined using an isotope ratio-based approach.
The number of C and N atoms in each accumulated compound was determined by the method described in Hegeman et al., Anal Chem 2007, 79, 6912. Yeast batch cultures were grown with uniformly labeled glucose or ammonium sulfate (Cambridge Isotopes, Andover, Mass.) for >20 generations to ensure complete labeling of the metabolome.
The formulae of the three metabolites identified in the PYP1 deletion strain were obtained by labeling cells with 13C and 15N20 and observing no shift from nitrogen labeling and a shift of +8, +5, or +4 daltons from carbon labeling (
The existence of octulose-8-phosphate (O8P) in yeast was reported previously. Indeed, the chromatographic retention time of the accumulated C8H17O11P1 exactly matched the synthesized O8P standard. To find the identity of the five- and four-carbon compounds, an isotope labeling experiment was performed that involved switching yeast cells from U-13C-glucose to unlabeled ribitol, arabitol, xylitol or erythritol (
While ribitol-5P and erythritol-4P are polyol phosphates, O8P is a sugar phosphate. The most common conformer of the seven carbon sugar sedoheptulose and its derivatives in solution is β-furanose. It is likely that O8P has a similar β-furanose ring structure (
To determine if the accumulated compounds were indeed the substrates of Pyp1, O8P and ribitol-5P were synthesized, and the biochemical activity of recombinant Pyp1 on these compounds was tested.
Synthesis of ribitol-5-phosphate was performed as described by Egan et al., Journal of the American Chemical Society 1982, 104, 2898. Synthesis of D-Glycero-D-Altro-Octulose-8-phosphate was performed enzymatically as described by Kapuscinski et al., Carbohydrate research 1985, 140, 69.
Incubation with Pyp1 led to the depletion of ribitol-5P and the accumulation of ribitol (
To test if Pyp1 is indeed a D-polyol phosphatase with no specificity for the length of the overall carbon chain, its activity against sorbitol-6-phosphate (sorbitol-6P), L-glycerol-3-phosphate (glycerol-3P, the common sn-glycerol-3-phosphate) and D-glycerol-3-phosphate (glycerol-1P, sn-glycerol-1-phosphate) was also measured. Incubation with Pyp1 led to the depletion of sorbitol-6P and glycerol-1P, but not glycerol-3P (
Although D-polyol phosphates were not thought to be present in baker's yeast, they have been discovered in other fungi species (see Jennings, Adv Microb Physiol 1984, 25, 149). There are two likely routes for the in vivo biosynthesis of these compounds. The first involves the reduction of the corresponding sugar phosphate and the second involves phosphorylation of polyols. Both of these activities were discovered in fungi but the responsible genes have yet to be discovered. As there are no detectable polyols except glycerol in baker's yeast, polyol phosphates in cells grown on glucose as the sole carbon source are more likely made from the reduction of sugar phosphates. In more distant organisms, ribitol-5-phosphate dehydrogenase has been found in gram positive pathogens including Haemophilus influenza and Staphylococcus aureus. Mannitol-1-phosphate (same as mannitol-6-phosphate due to the symmetric structure) dehydrogenase and sorbitol-6-phosphate dehydrogenase have been found in various bacteria, including Escherichia coli. None of these enzymes have homologs in yeast, so the responsible enzyme for polyol phosphate dehydrogenase activity remains unknown. It was demonstrated herein, however, that deletion of Zwf1, the first step in the oxidative PPP, resulted in the disappearance of ribitol-5-phosphate (
When polyols are present in the growth environment, polyol phosphates can be made from the phosphorylation of polyols. Of all the natural long-chain polyols, sorbitol is the only one known to be able to support yeast growth as the sole carbon source. Unlike bacteria, which use a phosphotransferase system, yeast utilize sorbitol by first oxidizing it into fructose. Because yeast could phosphorylate various polyols in the absence of glucose, it was hypothesized that sorbitol-6P could be made in yeast grown on sorbitol. To test this, both wild type and pyp1Δ strains were grown on minimal media containing sorbitol as the sole carbon source. It was determined that while wild type cells managed to grow after a long lag phase, pyp1Δ cells could not grow (
In the initial screening of Pyp1's phosphatase activity, the gene encoding Pyp1 was amplified by PCR using S. cerevisiae genomic DNA. The amplified fragments were cloned into a modified pET15b vector (Novagen, Darmstadt, Germany) and overexpressed in the E. coli BL21(DE3) Gold strain (Stratagene, La Jolla, Calif.) as previously described by Kuznetsova et al., J Biol Chem 2010, 285, 21049. The recombinant proteins were purified using metal ion affinity chromatography on nickel affinity resin (Qiagen, Hilden, Germany) to high homogeneity and stored at −80° C. Purified Pyp1 was screened for the presence of phosphatase activity against the general phosphatase substrate p-nitrophenyl phosphate (pNPP) and 90 phosphorylated metabolites as described previously by Kuznetsova et al., J Biol Chem 2006, 281, 36149.
For the determination of kinetic parameters (Km and kcat) of Pyp1, a yeast Open Reading Frame (ORF) strain with an expression vector containing C-terminal His-tagged Pyp1 (Open Biosystems, Thermo Fisher Scientific, San Jose, Calif.) was grown on galactose to induce Pyp1 expression. The resulting cells were lysed using glass beads and His-tagged Pyp1 was purified using Qiagen Ni-NTA spin columns follow the protocol provided by Qiagen. Phosphatase activity against ribitol-5-phosphate, sorbitol-6-phosphate, and octulose-8-phosphate was determined by monitoring the increase of ribitol, sorbitol or octulose using LC-MS. The reaction mixture contained 100 mM Tris-HCl at the PH of 8.0, 10 mM MgCl2 and a range of substrate concentrations (0.01 to 5 mM). Kinetic parameters were calculated by non-linear regression analysis of raw data to fit to the Hill equation using the GraphPad Prism Software (GraphPad Software, San Diego, Calif.).
For the determination of IC50 of sorbitol-6-phosphate and ribitol-5-phosphate on phosphoglucose isomerase, yeast Pgi1 was purchased from Sigma Aldrich (St. Louis, Mo.). Phosphoglucose isomerase activity was determined by adding fructose-6-phosphate and monitoring the appearance of glucose-6-phosphate using LC/MS. It was determined that the LC/MS-based assay is consistently more sensitive and accurate than the colometric-based assay, which involves coupling the Pgi1 activity with glucose-6-phosphate dehydrogenase activity and monitoring the appearance of NADH. The reaction mixture contained 100 mM Tris-HCl at a pH of 8.0, 10 mM MgCl2, 0.6 mM fructose-6-phosphate (concentration in cells grown exponentially on glucose), and a range of sorbitol-6-phosphate and ribitol-5-phosphate concentrations (0.05 to 5 mM). The resulting data were again fitted to the Hill equation using the GraphPad Prism Software.
To investigate the physiological effect of sorbitol-6P, apple aldose-6-phosphate reductase (A6PR), which converts fructose-6-phosphate (F6P) to sorbitol-6P, was expressed under a gal promoter on a high copy plasmid. The resulting cells (yA6PR) and cells transformed with the same plasmid but without the A6PR gene (control cells) were grown on galactose. It was determined that yA6PR cells showed a consistently slower growth rate than the control cells (
The mechanism of phosphofructokinase involves the phosphorylation of the β-furanose form of F6P into FBP without opening of the furanose ring. The mechanism of phosphoglucose isomerase involves the opening of the glucose pyranose ring, isomerization of glucose into fructose through an enediol intermediate, and the closing of the fructose ring. It was determined that the structure of sorbitol-6P mimics the structure of the enediol intermediate of phosphoglucose isomerase (
To test if the inhibition of glycolysis is conserved among other polyol species and if Pyp1 is also responsive for protecting glycolysis from other polyol phosphates, wild type and pyp1Δ cells were grown on glycerol or mannitol. Indeed, pyp1Δ cells grew defectively on glycerol and were unable to grow on mannitol, further confirming that Pyp1 is a D-polyol phosphatase in vivo.
Other polyols, such as erythritol, ribitol and xylitol also exist widely in nature. Because yeast cannot utilize these polyols as a sole carbon source, a condition where these polyols could be transported and phosphorylated at a high rate in growing cells was sought. Trehalose is a natural dimer of glucose that can be slowly utilized by yeast by its cleavage into two glucose monomers by the enzyme trehalase. The cleavage and growth rate is slow, so the growth is usually considered a glucose-limited culturing condition. It was determined that in the presence of ribitol, ribitol-5P accumulates, but does not affect the growth rate of wild type cells growing on trehalose (
Since all of the above results indicate that Pyp1 is essential for yeast in the presence of polyols in relatively poor growth conditions, Pyp1 expression levels were determined at different growth rates controlled by chemostats. Surprisingly, Pyp1's expression is highly correlated to growth rate, regardless of the limiting nutrient used (
To test this hypothesis, pyp1Δ cells and wild type cells were grown on trehalose in the presence of ribitol and then switched to glucose plus ribitol. This carbon upshift boosted the cell's growth rate. Although the transport of ribitol into the cell is repressed by glucose, the accumulated ribitol-5P could not be degraded instantly in pyp1Δ cells, resulting in an eight-fold higher concentration of ribitol-5P compared to wild type cells after one hour of switching to glucose (
NADPH and NADP+levels in S. cerevisiae cells lacking either DET1 or PHO13 were compared to NADPH and NADP+levels in wild type cells. The results showed that deletion of DET1 or PHO13 accumulates NADPH in S. cerevisiae cells. Deletion of DET1, but not PHO13, also accumulates NADP+ in S. cerevisiae cells (
Cellular NADPH and NADP+levels were quantitated by liquid chromatography-mass spectrometry. det1 and pho13 deletion strains were created by homologous recombination using the allele amplified by PCR from the synthetic genetic analysis (SGA) deletion set.47 Cells were grown in minimal media comprising 6.7 g/L Difco Yeast Nitrogen Base without amino acids plus 2% (w/v) glucose. The metabolome of batch culture Saccharomyces cerevisiae was characterized as described previously. Briefly, saturated overnight cultures were diluted 1:30 and grown in liquid media in a shaking flask to A600 of ˜0.6. A portion of the cells (3 mL) were filtered onto a 50 mm nylon membrane filter (Millipore, Billerica, Mass.), which was immediately transferred into −20° C. extraction solvent (40:40:20 acetonitrile/methanol/water).
LC/MS Metabolite measurement Cell extracts were analyzed by reversed phase ion-pairing liquid chromatography (LC) coupled by electrospray ionization (ESI) (negative mode) to a high-resolution, high-accuracy mass spectrometer (Exactive; Thermo Fisher Scientific, Waltham, Mass.) operated in full scan mode at 1 s scan time, 105 resolution. Relative concentration of NADPH and NADP+ between wild-type, det1 and pho13 strains were determined using the in-house developed, open-source software MAVEN. Compounds' identities were verified by mass and retention time matched to authenticated standards. Isomers are reported separately only where they are fully chromatographically resolved.
Additional experiments were run to confirm the results above, as well as investigate new characteristics of the gene.
Methods: Protein Purification and Enzymatic Assays
For Pyp1's polyol phosphatase activity assay, a yeast Open Reading Frame (ORF) strain with an expression vector containing C-terminal His-tagged PYP1 (Open Biosystems, Thermo Fisher Scientific, San Jose, Calif.) was grown on galactose to induce Pyp1 expression. The resulting cells were lysed using glass beads and His-tagged Pyp1 was purified using Qiagen Ni-NTA spin columns according to the manufacturer's instructions. Phosphatase activity against ribitol-5-phosphate, sorbitol-6-phosphate and octulose-8-phosphate was determined by monitoring the increase of ribitol, sorbitol or octulose using LC-MS. The reaction mixture contained 100 mM Tris-HCl at the pH 8.0, 10 mM MgCl2 and a range of substrate concentrations (0.01 to 5 mM).
Purified Pyp1 was also assayed against other compounds with similar structures and 90 common phosphorylated metabolites. Briefly, the gene encoding Pyp1 was amplified by PCR using S. cerevisiae genomic DNA. The amplified fragments were cloned into a modified pET15b vector (Novagen, Darmstadt, Germany) and overexpressed in the E. coli BL21 (DE3) Gold strain (Stratagene, La Jolla, Calif.) as previously described (Kuznetsova et al., 2010). The recombinant protein was purified using metal ion affinity chromatography on nickel chelate resin (Qiagen, Hilden, Germany) to high homogeneity and stored at −80° C. Purified Pyp1 was then screened for phosphatase activity as described previously (Kuznetsova et al., 2006). Due to the difficulty of obtaining (D)-glycerol-3P, the activity of Pyp1 on (D)-glycerol-3P is determined using racemic glycerol-3P as the substrate. Because (L)-glycerol-3P has very low activity, the resulting activity on racemic glycerol-3P was used as the activity on (D)-glycerol-3P. Compounds with specific activity higher than 0.1 μmol/mg/min are shown in
Results
To measure the inhibition of Pgi by sorbitol-6-phosphate and ribitol-5-hosphate, yeast Pgi1 was purchased from Sigma Aldrich (St. Louis, Mo.). Phosphoglucose isomerase activity was determined by adding fructose-6-phosphate and monitoring the appearance of glucose-6-phosphate using LC/MS. We found such LC/MS based assay is consistently more sensitive and accurate than the more typical colorimetric-based assay, which involves coupling the Pgi1 activity with glucose-6-phosphate dehydrogenase activity and monitoring the appearance of NADH. The reaction mixture contained 100 mM Tris-HCl at pH 8.0, 10 mM MgCl2, 0.6 mM fructose-6-phosphate (the physiological concentration in cells grown exponentially on glucose), and a range of sorbitol-6-phosphate and ribitol-5-phosphate concentrations (0.05 to 5 mM). The resulting data were fitted to the Hill equation using the GraphPad Prism Software.
Yeast strains with putative phosphatases of unknown function deleted for changes in metabolite concentrations were screened. Yeast were grown in glucose minimal media, and metabolites were extracted into 40:40:20 methanol: acetonitrile: water, followed by metabolome analysis by reversed-phase ion-pairing liquid chromatography—high resolution mass spectrometry (LC-MS). It was found that the deletion of PYP1, formerly known as the uncharacterized gene YNL010W, while not significantly altering the concentrations of most metabolites, led to the statistically significant (false discovery rate <0.05) accumulation of three compounds in negative ion mode (
To identify the five- and four-carbon polyol phosphates, we performed an isotope labeling experiment involving switching pyp1Δ cells from U-13C-glucose to unlabeled ribitol, arabitol, xylitol or erythritol. Although baker's yeast cannot effectively utilize these polyols as carbon sources, in the absence of glucose, they slowly transport and phosphorylate them. Results showed that feeding ribitol and erythritol resulted in the build-up of intracellular metabolites with exact mass and LC retention time matching the C5H13O8P1 and C4H11O7P1 that accumulated with Pyp1 deletion (
Ribitol-5P and erythritol-4P are polyol phosphates, but octulose-8P is a sugar phosphate. We were curious why Pyp1 deletion resulted in accumulation of metabolites from these different structural families. The most common conformer of the seven carbon sugar sedoheptulose and its derivatives in solution is β-furanose (Kuchel et al., 1990). Octulose-8P likely has a similar β-furanose ring structure (
Pyp1 is a Broad Spectrum (D)-Polyol Phosphatase
Although polyols are excretion products of fungi and plants, polyol phosphates were not previously thought to be an intermediate in this pathway (sugar phosphate 4 sugar 4 polyol). To determine if the accumulated compounds were indeed the substrates of Pyp1, the biochemical activity of the purified recombinant Pyp1 on these compounds was analyzed. Incubation with Pyp1 led to the depletion of ribitol-5P and the accumulation of ribitol (
Source of Ribitol-5P in Glucose-Grown Yeast
Although polyol phosphates have not been previously described in Baker's yeast, they have been noted in other fungi species (Jennings, 1984). There are two likely routes for the cellular biosynthesis of these compounds. The first involves the reduction of the corresponding sugar phosphate and the second involves phosphorylation of polyols. Both activities have been described in fungi but the responsible genes remain unknown (Jennings, 1984). In Baker's yeast grown on glucose, the only known polyol is glycerol, and thus polyol phosphates are likely made from the reduction of sugar phosphates (as in Haemophilus influenza, Staphylococcus aureus (Pereira and Brown, 2004), and Escherichia coli (Novotny et al., 1984)). Consistent with this, deletion of Zwf1, the first step in the oxidative pentose phosphate pathway (PPP), eliminated the endogenous peak for ribitol-5P, presumably due to decreased levels of ribose-5P and ribulose-5P (
Growth on Sorbitol Requires Pyp1
When polyols are present in the growth environment, polyol phosphates can be made by their phosphorylation. Of the natural long-chain polyols, sorbitol is the only one known to support yeast growth as the sole carbon source (Sarthy et al., 1994). The very long lag of growth begins with sorbitol dehydrogenase which converts sorbitol to fructose. Due to the structural similarity of sorbitol to glucose, we hypothesize that yeast might sometimes erroneously phosphorylate sorbitol into sorbitol-6P, which could be toxic in excess. To test this, both wild type and pyp1Δ strains were fed minimal media containing sorbitol as the sole carbon source. While wild type cells grew to saturation after a long lag phase, pyp1Δ cells never grew (
To investigate whether Pyp1 is important for growth on other polyol substrates, pyp1Δ yeast were fed glycerol or mannitol. The pyp1Δ cells grew poorly on glycerol. While wild type cells managed to reach saturation after a long lag phase on mannitol, pyp1Δ cells were unable to grow on mannitol (
To more directly ascertain whether the impaired growth was a result of polyol phosphate toxicity, yeast trehalose (a dimer of glucose that can be slowly utilized by yeast resulting in carbon-limited growth) (Jules et al., 2004; Walther et al., 2010)) were fed with or without addition of ribitol. In wild type yeast, addition of ribitol to the medium resulted in accumulation of intracellular ribitol-5P, without impacting growth rate (
Polyol Phosphates are Inhibitors of Phosphoglucoisomerase (Pgi)
One potential mechanism by which polyol phosphates could impair cell growth is through metabolic enzyme inhibition. Sorbitol-6P is structurally similar to the enediol transition state of the Pgi reaction (Scheme 1) and sorbitol-6P and other structural mimics of the enediol intermediate are known Pgi inhibitors (Milewski et al., 2006). It was confirmed that sorbitol-6P and less potently ribitol-5P inhibit Pgi (
It was then tested whether ribitol-5P accumulation results in these metabolic hallmarks of Pgi deficiency. Cells were initially grown in trehalose+ribitol and then switched to glucose+ribitol to enhance glycolytic flux. Relative to wild-type yeast, the pyp1Δ strain accumulated dramatically more ribitol-5P (
Sorbitol-6-phosphatase activity has been found in apple leaves (Zhou et al., 2003) and silk worms (Oda et al., 2005). The gene encoding this enzyme, however, has not been discovered. In engineered fungi, sorbitol production has been achieved by expressing bacterial sorbitol-6P dehydrogenase, but again the gene encoding the required phosphatase activity remained missing (Ladero et al., 2007). Here we identify the previously unannotated yeast gene YNL010W, which we now term PYP1, as a polyol phosphate phosphatase. Pyp1 dephosphorylates a variety of compounds with the common structural motif of a D-glycerol-3-phosphate tail, including D-glycerol-3P, erythrose-4P, ribitol-5P, and sorbitol-6P (
One functional role of Pyp1 is to limit the polyol phosphate concentrations in cells. High levels of polyol phosphates impair yeast growth, at least in part by inhibition of the upper glycolytic enzyme phosphoglucoisomerase (Pgi), for which sorbitol-6-phosphate is a transition state analogue. Other sugar phosphate isomerases, such as triose-phosphate isomerase and ribose-5-phosphate isomerase, have similar enediol reaction intermediates (Komives et al., 1991; Zhang et al., 2003), and thus are also expected to be inhibited by polyol phosphates. Similar to strong inhibition of Pgi by sorbitol-6 phosphate, triose-phosphate isomerase will likely be strongly inhibited by D-glycerol-3P, and ribose-5-phosphate isomerase by ribitol-5P or xylitol-5P. Flux through each of these enzymes tends to increase with faster yeast growth rate. For example, ribose-5-phosphate isomerase is required to feed ribosome biogenesis. Thus rapidly growing yeast cells may be particularly sensitive to enzyme inhibition by polyol phosphates. To determine whether Pyp1 function is associated with growth rate, we analyzed its expression as a function of growth rate across 25 different chemostat conditions (Brauer et al., 2008). Interestingly, Pyp1's expression was strongly positively correlated with growth rate, regardless of the limiting nutrient used (top 6% of all transcripts in genome) (
The transition state inhibition by polyol phosphates and their derivatives is not limited to sugar phosphate isomerase. In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) also has an enediol intermediate and is known to be inhibited by 2-carboxy-D-arabitol-1-phosphate (CA1P) (Andralojc et al., 1994). The corresponding 2-carboxy-D-arabitol-1-phosphatase is activated by light and controls cellular level of CA1P. In darkness, RuBisCO is inhibited and also protected by CA1P from proteolysis (Khan et al., FEBS 266, 840-847, 1999).
Pyp1 is conserved across fungi species and some plants and bacteria, including Ricinus communis, Dehalococcoides ethenogenes, and Bacillus anthracis (Gibney et al., Phylogenetic Portrait of the Saccharomyces cerevisiae Functional Genome. G3 (Bethesda) 3, 1335-1340, 2013). The highly conserved nature of Pyp1 in fungi indicates the importance of clearing polyol phosphates. It is likely that the homologous enzymes play the same role in bacteria and plants. Although we have not identified a Pyp1 homolog in mammals, nor are linear polyol phosphates longer than three carbons known mammalian metabolites, polyols are also produced in humans and can accumulate in disease (Lee et al., 1995). For example, during hyperglycemia (e.g., due to diabetes), sorbitol accumulates in a number of organs due to the action of aldose reductase on glucose, which may contribute to diabetic complications including diabetic retinopathy, peripheral neuropathy and diabetic kidney disease (Brownlee, 2001; Nishikawa et al., 2000; Schrijvers et al., Endocrine reviews 25, 971-1010, 2004). It is unclear whether such sorbitol sometimes becomes phosphorylated to sorbitol-6P and, if so, whether sorbitol-6P contributes to disease pathology.
Beyond their potential cellular toxicity, polyol phosphates may also have a productive regulatory role in central carbon metabolism. Pgi sits at the branch point between glycolysis and the oxidative pentose phosphate pathway. Despite being ideally situated to regulate the branching ratio between these pathways, Pgi is not an allosteric enzyme, with no physiological regulators known (Milewski et al., 2006; Noltmann, 1972). It is tempting to speculate that polyol phosphates might serve as endogenous Pgi regulators, rendering Pgi an enzyme controlled by active site competition (Fell, 1997, Understanding the Control of Metabolism (Portland Press).; Goyal et al., PLoS computational biology 6, e1000802, 2010; Heinrich and Rapoport, European Journal of Biochemistry 42, 89-95, 1974; Hofmeyr and Cornishbowden, European Journal of Biochemistry 200, 223-236, 1991; Kacser et al., Biochemical Society Transactions 23, 341-366, 1995; Kell and Westerhoff, Fems Microbiology Reviews 39, 305-320, 1986). It is hypothesized that polyol phosphates, and their hydrolysis by Pyp1 contribute to physiological metabolic flux control.
This application claims the benefit of U.S. Provisional Application No. 62/836,190, filed Apr. 19, 2019, which is herein incorporated by reference in its entirety.
This invention was made with government support under Grant No. CBET-0941143 and Grant No. MCB-0643859 awarded by the National Science Foundation; Grant No. GM-071508 awarded by the National Institutes of Health; Grant No. DE-SC0002077 awarded by the Department of Energy; and Grant No. FA9550-09-1-0580 awarded by the United States Air Force, Air Force Office of Scientific Research. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62836190 | Apr 2019 | US |
