Patent Application
20160230198
This disclosure generally relates to microbiology and biochemical technology. This disclosure also relates to non-natural microorganisms for producing biochemicals from carbon sources. And this disclosure relates to methods for biological synthesis of biochemicals such as methylmalonic acid and its derivatives, from carbon substrates.
Methylmalonic acid (“MMA”) is used as an indicator of vitamin B deficiency. However, MMA is naturally produced in only very small quantities in cells in response to a deficiency in Vitamin B12 or metabolic aciduria through the citrate cycle or branched-chain amino acid (valine, leucine and isoleucine) degradation pathways.
More specifically, Kovachy et al. (1983 and 1988) investigated the origin of biologically-derived methylmalonic acid in rats and claimed that a protein of molecular weight 35 kDa catalyzes the hydrolysis of (S)-methylmalonyl-CoA, but not (R)-methylmalonyl-CoA, into methylmalonate along with having promiscuous activity on malonyl-CoA, propionyl-CoA, acetyl-CoA and palmitoyl-CoA (Kovachy et al., Recognition, isolation, and characterization of rat liver D-methylmalonyl coenzyme A hydrolase, J Biol Chem, 1983, 258(18), 11415-21; Kovachy et al., D-methylmalonyl-CoA hydrolase, Methods in Enzymol, 1988, 166: 393-400). Indeed this gene is believed to be responsible for the production of methylmalonic acid in biological samples such as urine, in response to vitamin B12 deficiency (see e.g. Kwok T, Cheng G, Lai W K, Poon P, Woo J, Pang C P: Use of fasting urinary methylmalonic acid to screen for metabolic vitamin B12 deficiency in older persons. Nutrition 2004, 20(9):764-768) or metabolic aciduria (see e.g. Rosenberg L E, Lilljeqvist A C, Hsia Y E: Methylmalonic aciduria. An inborn error leading to metabolic acidosis, long-chain ketonuria and intermittent hyperglycinemia. The New England journal of medicine 1968, 278(24):1319-1322). However, the observations of Kovachy et al., 1983 and Kovachy et al., 1988 were due to the promiscuity of 3-hydroxyisobutyryl-CoA hydrolase, which was demonstrated to act on (S)-methylmalonyl-CoA as well (Shimomura, Y. et al. 3-hydroxyisobutyryl-CoA hydrolase. Methods in enzymology, 2000, 324, 229-240). The rat 3-hydroxyisobutyryl-CoA hydrolase catalyzed the hydrolysis of other CoA compounds such as 3-hydroxypropionyl-CoA, 3-hydroxybutyryl-CoA, acetoacetyl-CoA, isobutyryl-CoA, etc, although with much lower specificity (Shimomura, Y. et al. Purification and partial characterization of 3-hydroxyisobutyryl-coenzyme A hydrolase of rat liver. The J Biol Chem, 1994, 269, 14248-14253). The corresponding gene from yeast was modified to hydrolyze malonyl-CoA in WO2013134424. The size of the product of the gene that encodes for 3-hydroxyisobutyryl-coenzyme A is approximately 35 kDa, misleading Kovachy et al., (1983 and 1988) to wrongly believe that this gene product was methylmalonyl-CoA hydrolase.
US 2009/0186358 allegedly discloses the engineering of cells to up-regulate or down-regulate the genes or proteins of the valine, leucine and isoleucine degradation pathway to increase methylmalonate production. However, because the genes encoding for these enzymes are not known, and there are no methods disclosed for identifying the same, it was not possible to engineer cells to increase methylmalonate production from either methylmalonyl-CoA or methylmalonate semialdehyde.
US20100190224 allegedly describes the production of 3-hydroxyisobutyric acid from methylmalonyl-CoA and allegedly describes the use of an enzyme that can hydrolyze methylmalonyl-CoA. However, the sequence of the corresponding gene and the enzyme related to methylmalonyl-CoA hydrolase activity are not disclosed and are only hypothetical.
The present disclosure relates to engineered microorganisms configured to produce methylmalonic acid, biological methods of making methylmalonic acid using the said microorganisms, and methylmalonic acid compositions produced by the said biological methods.
In some embodiments, the engineered microorganism is configured to produce or overproduce a target chemical chosen from methylamlonic acid or its esters. The microorganism may be a bacteria, yeast, or filamentous fungus. In some embodiments, the microorganism is also engineered to secrete the target chemical. In some embodiments, the microorganism comprises at least one exogenous nucleic acid sequence encoding at least one polypeptide for converting a metabolic intermediate into a target chemical. In further embodiments, at least one polypeptide encodes for at least one enzyme capable of facilitating a step in a pathway for producing methylmalonic acid from methylmalonyl-CoA. In some embodiments, methylmalonyl-CoA is produced from propionyl-CoA and/or succinyl-CoA. In some embodiments, wherein the microorganism has a cytoplasm, the microorganism is further engineered to produce the target chemical in the cytoplasm.
In some embodiments, propionyl-CoA is produced from propanoate, by the reduction of acryloyl-CoA, by the oxidative decarboxylation of 2-oxobutanoate or the oxidation of odd-chain fatty acids. In some embodiments, 2-oxobutanoate is produced by the deamination of amino acids such as threonine or methionine. In some embodiments, acryloyl-CoA is produced from lactoyl-CoA or 3-hydroxypropanoyl-CoA.
In some embodiments, succinyl-CoA is produced from succinate or by the oxidative decarboxylation of α-ketoglutarate.
In some embodiments, the microorganism is engineered to increase the carbon flux to propionyl-CoA and/or succinyl-CoA.
In some embodiments, the methods involve using an engineered microorganism, such as described herein, to produce a target chemical chosen from methyl malonic acid and esters of methylmalonic acid. In some embodiments, the method also involves secreting the target chemical from the microorganism. In some embodiments, the target chemical is produced in a fermenter by the engineered microorganism, and the target chemical is optionally purified. In some embodiments, the method involves contacting an engineered microorganism with a carbon substrate wherein the microorganism is engineered to produce enzymes in a metabolic pathway (such as described herein) that produces methylmalonic acid and/or its esters from the carbon substrate. In further embodiments, the method involves culturing the microorganism under conditions whereby methylmalonic acid is produce and harvesting the methylmalonic acid. In some embodiments, the microorganism is further engineered to minimize competing metabolic pathways.
The present disclosure relates to the design of non-natural microorganisms with an engineered metabolism to enable the production of biochemicals, such as industrial biochemicals, from carbon sources, including cheap carbon sources. More specifically, the engineered metabolic network facilitates the conversion of carbon substrates into methylmalonic acid and/or derivatives thereof. Carbon sources include, but not limited to, sugars such as glucose, fructose, sucrose, xylose and arabinose or their polymers, propanoate, fatty acids, glycerol, amino acids such as valine, leucine, and isoleucine, keto acids such as 2-oxobutanoic acid and pyruvate, and C1 substrates such as methane, carbon monoxide and carbon dioxide.
The present disclosure therefore provides means to engineer microorganisms with the capability to produce methylmalonic acid and/or esters thereof from carbon substrates such as those listed above by virtue of introducing nucleotide sequences encoding for one or more polypeptides that catalyze the enzymatic reactions in metabolic pathways that convert substrates to the desired products (“target chemicals”).
As used herein, the terms “polypeptide”, “peptide”, “protein” or “enzyme” are used interchangeably.
The sequences, including those naturally occurring as well as engineered, disclosed herein are intended to endow the microorganism with the ability to catalyze the desired reaction. It is understood that other enzymes that can catalyze the desired reactions are also within the scope of the disclosure. The skilled person will readily recognize that such enzymes may have a sequence identity of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% and will understand that they are not excluded from this disclosure.
As used herein, the acid and its conjugated base are used interchangeably, and refer to the molecule in context. For example, “methylmalonic acid” and “methylmalonate” refer to the same chemical, unless specifically distinguished.
As used herein, an engineered microorganism is one that is genetically modified from its corresponding wild-type. For example, the genetic modification could be one or more of: (i) introduction of exogenous nucleic acid sequences; (ii) introduction of additional copies of endogenous sequences; (iii) deletion of endogenous sequences and (iv) alteration of promoter or terminator sequences.
In some embodiments, wherein the microorganism has a cytoplasm, the microorganism may be further engineered to produce at least a portion, or at least a majority, or at least almost entirely, the target chemical in the cytoplasm. Identification and deletion of mitochondrial signal sequence to direct proteins into the cytosol is well-documented in the art (Strand M K, Stuart G R, Longley M J, Graziewicz M A, Dominick O C, Copeland W C (2003) POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA. Eukaryot Cell 2:809-820; http://www.cbs.dtu.dk/services/; http://ihg.gsf.de/ihg/mitoprot.html).
Those skilled in the art will understand that the herein disclosed pathways are described in relation to, but are not limited to, species-specific genes and proteins and that the invention encompasses homologs and orthologs of such gene and protein sequences. Homolog and ortholog sequences possess a relatively high degree of sequence identity (i.e. from about 65% to about 100% sequence identity) when aligned using methods known in the art. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 65% to 100% sequence identity. In some embodiments, useful polypeptide sequences have at least 65%, at least 75%, at least 85%, at least 90%, or at least 95% or at least 99% identity to the amino acid sequence of the reference enzyme of interest.
In some embodiments, a metabolic pathway is provided for the production of methylmalonic acid from (S)-methylmalonyl-CoA as illustrated in
Despite the publications of Kovachy et al., (1983 and 1988), genes for Methylmalonyl-CoA hydrolase (E.C. 3.1.2.17) have not been identified and are not known to exist. To the inventor's knowledge, this specification discloses such agene for the first time. In some embodiments, the catalytic promiscuity of some enzymes, such as enzymes listed in Table 1, may be combined with protein engineering to modify the protein such that it may be exploited in novel metabolic pathways and biosynthesis applications. In some embodiments, and as shown in Example 5, the catalytic promiscuity of 3-hydroxyisobutyryl CoA hydrolase is exploited to modify its function using protein engineering to produce an enzyme that is more consistent with a Methylmalonyl-CoA hydrolase.
For example, in some embodiments, the non-natural microorganism contains an engineered gene that encodes for a modified (S)-methylmalonyl-CoA hydrolase with higher specificity for (S)-methylmalonyl-CoA than the naturally occurring enzyme. Based on the crystal structure (PDB ID: 3BPT) of the human 3-hydroxyisobutyryl-CoA hydrolase, the mechanism of action of the enzyme was hypothesized which was validated by a series of site-directed mutagenesis (Rouhier, M. F., Characterization of YDR036C from Saccharomyces cerevisiae, PhD Thesis, 2011, Miami University, Oxford, Ohio, USA). The amino acids that were deemed important for the activity of 3-hydroxyisobutyryl-CoA hydrolase in yeast are Glutamate-124 (interacts with the β-hyroxyl group of 3-hydroxyisobutyric acid), Phenylalanine-177 (responsible for the substrate specificity of the enzyme) and Serine-328 (subject to post-translational regulation via phosphorylation). In the examples below, the present disclosure demonstrates that these amino acids are also relevant increasing the substrate-specificity for (S)-methylmalonyl-CoA. In some embodiments, the mitochondrial signal sequence is removed in the engineered gene to allow for cytosolic localization, as described in the examples. In some embodiments, the non-natural microorganism contains an engineered gene that encodes for a modified (S)-methylmalonyl-CoA hydrolase with higher specificity for (S)-methylmalonyl-CoA than the naturally occurring enzyme and comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 10, 32, 34, 36 or 38. In some embodiments, the amino acids at the positions Glu-124, Phe-177 and Ser-328 with respect to the sequence of the EHD3 gene from S. cerevisiae (UniProt ID: P28817) are altered in the (S)-methylmalonyl-CoA hydrolase. In some embodiments, the engineered enzyme also has (R)-methylmalonyl-CoA hydrolase activity.
As another example, thioesterases such as CoA hydrolases catalyze the removal of the CoA moiety. Thioesterases can be promiscuous (Zhuang, et al., Divergence of function in the hot dog fold enzyme superfamily: the bacterial thioesterase YciA, 2008, Biochemistry, 47(9):2789-96). In some embodiments, the promiscuity of thioesterases is exploited by engineering the protein sequence to increase the specificity to the desired substrate. In some embodiments, the (S)-methylmalonyl-CoA hydrolase is a thioesterase comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 19, 21 or 43 and at least one amino acid difference at a position relative to SEQ ID 19 selected from I39, M45, V60, K71 and V125. In some embodiments, the engineered enzyme has (R)-methylmalonyl-CoA hydrolase activity.
A person of ordinary skill in the art should appreciate that if the crystal structure of an enzyme or of a similar enzyme is known, then the properties of the enzyme may be modified by rational design or by directed evolution (see, for example, Recent advances in rational approaches for enzyme engineering, Comput Struct Biotechnol J. 2012; 2(3) e201209010, US20060160138, WO2003023032, US20080287320 and WO1999029902). For example, WO2013134424 modifies a yeast 3-hydroxyisobutyryl-CoA hydrolase into malonyl-CoA hydrolase to produce malonic acid. Such a modification or improvement in the enzyme properties may arise from the alteration in the structure-function of the enzyme and/or its interaction with other molecules. The interaction of an enzyme with other molecules such as for example the substrate can be quantified by the Michaelis constant (Km), which can be quantified using prior art (see for example, Stryer, Biochemistry, 4th edition, W.H.Freeman, Nelson and Cox, Lenhinger Principles of Biochemistry, 6th edition, W.H. Freeman). The rate of enzymatic activity is defined by kcat, which is the enzyme turnover number. As defined herein, an improvement in the enzyme is to increase the affinity between the enzyme and its substrate, as indicated by lower Km and/or to increase the kcat and/or to increase kcat/Km. Several examples of exploiting the promiscuity of enzymes for synthesizing biochemicals exist in the art. See, for example the description in US20130017593 A1 or WO2009135074 A2 or WO 2010071697 A1. These and other techniques can be used to modify enzymes as suggested herein, for example to enhance the activity of certain enzymes and/or increase the specificity of certain enzymes.
Referring now to
For example, as shown in
As another example, in some embodiments, the metabolic pathway includes step 13 in addition to step 12 such that the source of (S)-methylmalonyl-CoA is succinyl-CoA. As is shown in
(R)-methylmalonyl-CoA, thus produced from succinyl-CoA is converted into the S-epimer by methylmalonyl-CoA epimerase (EC 5.1.99.1). The gene encoding for this enzyme is characterized in multi-cellular organisms such as mold and mammals and the protein is localized in the mitochondria of the cells. Some UniProt IDs of exemplary methylmalonyl-CoA epimerases are Q2KIZ3, Q553V2, Q96PE7 and Q9D1I5. Therefore, in some embodiments, this step of the metabolic pathway is facilitated by an enzyme in which the signal sequence that directs the enzyme into the mitochondria is deleted in order to enable the localization of methylmalonyl-CoA epimerase in the cytosol of higher microorganisms. Expression of these genes in Escherichia coli result in an active enzyme, indicating that the enzyme can be constituted in a different host (Dayem et al., Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.” Biochemistry, 2002, 41(16): 5193-5201; Zhang, et al., Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli, Biotechnol Bioeng 2010, 105(3): 567-573; US20040185541 A1). In some embodiments, the metabolic pathway is implemented by a non-natural microorganism which harbors at least one gene encoding for methylmalonyl-CoA mutase comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID 14. In some embodiments, the non-natural microorganism harbors at least one gene encoding for methylmalonyl-CoA epimerase comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID 39. Methods to create such a non-natural microorganism are described in the examples.
In some embodiments, in which propionyl-CoA serves as the source of (S)-methylmalonyl-CoA, the metabolic pathway includes a process in which propionyl-CoA is produced from one or more of propionate, threonine, methionine or pyruvate, as shown in
Where propionate serves as the source of propionyl-CoA, propionate is converted into propionyl-CoA (Step 15) by propionyl-CoA synthase (EC 6.2.1.17). To the inventor's knowledge, this gene (and enzyme) have never been expressed in yeast before. In Escherichia coli, this enzyme is encoded by the prpE gene. However, the native enzyme is subjected to feedback inhibition by propionylation by propionyl-CoA at lysine 592 (Garrity et al., N-lysine propionylation controls the activity of propionyl-CoA synthetase, J Biol Chem. 2007 Oct. 12; 282(41):30239-45). In some embodiments, therefore the metabolic pathway is implemented in a non-natural microorganism which harbors at least one gene encoding for propionyl-CoA synthase comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID 8 or SEQ ID 41. Methods to create a non-natural microorganism harboring propionyl-CoA synthase are illustrated in examples.
Where threonine serves as the source of propionyl-CoA, threonine is dehydrated/deaminated by threonine dehydratase (Step 6, EC 4.3.1.19), which converts threonine into 2-oxobutanoate. In Escherichia coli, this enzyme is encoded by the catabolic tdcB (b3117) or biosynthetic ilvA (b3772) genes. Threonine is produced from aspartate and the first step in this pathway, aspartate kinase, is subject to feedback inhibition by threonine. The mechanism for feedback inhibition is well-studied and in yeast (Arevalo-Rodriguez et al., Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae, Yeast, 1999, 1(13): 1331-1345) and bacteria (Yoshida A, Tomita T, Kuzuyama T, Nishiyama M: Mechanism of concerted inhibition of alpha2beta2-type hetero-oligomeric aspartate kinase from Corynebacterium glutamicum. The Journal of biological chemistry 2010, 285(35):27477-27486). In some embodiments, the metabolic pathway is implemented by a non-natural microorganism created by enhancing the activity of EC 4.3.1.19 by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 44 SEQ ID 45 or SEQ ID 46.
Where methionine serves as the source of propionyl-CoA, the metabolic pathway may involve synthesizing 2-oxobutanoate from methionine by the action of methionine-γ-lyase (Step 7, EC 4.4.1.11). While there are some reports of this enzyme from archaea and eukaryota, this enzyme is more common in bacteria. For example, mdeA gene from Pseudomonas putida encodes for this enzyme and catalyzes the α,γ-elimination and γ-replacement reactions of L-methionine and its S-substituted derivatives. In some embodiments, the metabolic pathway is implemented in a microorganism which is engineered with genes that encode for threonine hydratase/deaminase or methionine-γ-lyase to render the conversion of threonine or methionine into 2-oxobutanoate. In some embodiments, the native aspartate kinase in the microorganism is replaced with feedback-resistant aspartate kinase to decouple threonine/methionine production from regulation.
2-oxobutanoate produced from step 6 or step 7 is oxidatively decarboxylated to propanoyl-CoA. This reaction is catalyzed by 2-oxobutanoate formate-lyase (Step 9, EC 2.3.1.-). In Escherichia coli, this enzyme is encoded by the tdcE (b3114) gene, which encodes for the inactive enzyme that is activated by pflA (b0902) gene product. The functioning of this enzyme is similar to that of pyruvate formate-lyase. Since pyruvate formate lyase is sensitive to oxygen, the grcA (b2579) gene from Escherichia coli replaces an oxidatively damaged pyruvate formate-lyase subunit. The auxiliary genes needed to sustain the activity of 2-oxobutanoate formate-lyase, pflA and grcA, are co-expressed with tdcE and the ensuing formate is oxidized to carbon dioxide by formate dehydrogenase such as for example, EC 1.2.1.2. In some embodiments, the metabolic pathway is implemented in a non-natural microorganism which is created by enhancing the activity of 2-oxobutanoate formate-lyase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 47, 48 or 49.
2-oxobutanoate is decarboxylated by the ydbK (b1378) gene product in E. coli, 2-oxobutanoate synthase (Step 8, EC 1.2.7.1). Based on sequence similarity, YdbK is predicted to function as 2-oxoacid:flavodoxin oxidoreductase synthase (Nakayama et al., 2013, Escherichia coli pyruvate:flavodoxin oxidoreductase, YdbK—regulation of expression and biological roles in protection against oxidative stress. Genes Genet Syst. 2013; 88(3):175-88). Oxidative decarboxylation of 2-oxobutanoate is also catalyzed by branched-chain 2-oxo acid dehydrogenases (Step 10, EC 1.2.4.4). In some embodiments, the metabolic pathway is implemented in a non-natural microorganism which is created by enhancing the activity of 2-oxobutanoate synthase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 56 or SEQ ID 57.
In bacteria, the pyruvate dehydrogenase enzyme complex is also able to recognize 2-oxobutanoate as the substrate, and decarboxylate it to propanoyl-CoA. In some embodiments, the metabolic pathway is implemented in a microorganism, which is engineered with the genes that encode for at least one of 2-oxobutanoate synthase, 2-oxobutanoate formate-lyase and 2-oxo acid dehydrogenase enzymes.
In some embodiments, propanoyl-CoA is produced from pyruvate according to the sequence of reactions shown in FIG.2. Pyruvate is reduced to (R)-lactate by the action of D-lactate dehydrogenase (Step 1, EC 1.1.1.28) commonly using NADH as the reducing agent. An example of a gene that encodes for D-lactate dehydrogenase is ldhD from Lactobacillus plantarum (UniProt ID of the corresponding enzyme: Q88VJ2) or the ldhA (b1380) from Escherichia coli (UniProt ID of the corresponding enzyme: P52643). (R)-lactate is also produced by the hydrolysis of methylglyoxal for example by glyoxylase III (EC 4.2.1.130) or by glyoxylase I (EC 4.4.1.5). In some embodiments, the metabolic pathway is implemented by the non-natural microorganism which is created by enhancing the activity of D-lactate dehydrogenase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 61 or SEQ ID 62. Pyruvate is reduced to (S)-lactate by the action of (S)-lactate dehydrogenase (EC 1.1.1.27) commonly using NADH as the reducing agent. An example of a gene that encodes for (S)-lactate dehydrogenase is ldh gene of Lactobacillus casei (UniProt ID of the corresponding enzyme: P00343). In some embodiments, the metabolic pathway is implemented in the non-natural microorganism which is created by enhancing the activity of (S)-lactate dehydrogenase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 63. Methods to engineer (R)-lactate dehydrogenase activity or (S)-lactate dehydrogenase activity in an organism are described in the examples.
(R)-lactate or (S)-lactate formed in Step 1 is converted into (R)-lactoyl-CoA or (S)-lactoyl-CoA, respectively by the action of lactate CoA transferase (Step 2, EC 2.8.3.-). Lactate CoA-transferase is one of the key enzymes of the propionate fermentation pathway in anaerobic microorganisms such as Clostridium propionicum and Megasphaera elsdenii. When using lactate as a substrate the enzyme catalyzes an early step in the pathway yielding lactyl-CoA. The pct gene from Clostridium propionicum encodes for lactoyl-CoA transferase. This enzyme can use propanoyl-CoA as well as acetyl-CoA as the donor of Coenzyme A. The pct gene from Megasphaera elsdenii was shown to have a lower Km for (R)-lactate than for (S)-lactate and was used to produce 1,2-propanediol by engineering E. coli (Niu and Guo, 2015, Stereospecific Microbial Conversion of Lactic Acid into 1,2-Propanediol, ACS Synthetic Biology, 4(4): 378-382). In some embodiments, the metabolic pathway is implemented by the non-natural microorganism which is created by enhancing the activity of lactate-CoA transferase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 64 or SEQ ID 65 or SEQ ID 66.
The CoA donating entity is acetyl-CoA, which is converted to acetate. Acetate is recycled back to acetyl-CoA by the action of acetyl-CoA synthetase (EC 6.2.1.1). In some embodiments, the metabolic pathway is implemented in a non-natural microorganism which is created by enhancing the activity of acetyl-CoA synthetase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 67 or SEQ ID 68. Acetyl-CoA is also produced from acetaldehyde by the action of acetaldehyde dehydrogenase (Step 5, EC 1.2.1.10). This enzyme can catalyze the reversible reaction shown by step 5. An example of a gene that encodes for acetaldehyde dehydrogenase is adhE (b1241) in Escherichia coli (UniProt ID of the corresponding enzyme: P0A9Q7). The CoA donating entity is propionyl-CoA, which is converted to propionate. Propionate is recycled back to propionyl-CoA by the action of propionyl-CoA synthase. In some embodiments, the metabolic pathway is implemented in a non-natural microorganism which is created by enhancing the activity of propionyl-CoA synthetase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 8 or SEQ ID 41 or SEQ ID 42.
Lactoyl-CoA is dehydrated to acryloyl-CoA by the action of lactoyl-CoA dehydratase (Step 3, EC 4.2.1.54). Lactyl-CoA dehydratase is one of the enzymes in the propionate fermentation pathway. The enzyme complex is composed of two proteins, EI (encoded by lcdC) is the activator protein and EII (lcdAB) is the actual dehydratase (Schweiger and Buckel, 1984, On the dehydration of (R)-lactate in the fermentation of alanine to propionate by Clostridium propionicum, FEBS 171(1): 79-84; Hofmeister and Buckel, 1992, (R)-Lactyl-CoA dehydratase from Clostridium propionicum, Eur J Biochem, 206:547-552). The three genes provide for activity and the genes from Clostridium propionicum were shown to function heterologously in Escherichia coli and participate in fermenting lactate to propanoate (Kandasamy et al., 2013, Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation, Appl Microbiol Biotechnol., 97(3):1191-1200). Similarly, EP1343874 B1 and related patents teaches the expression of the genes that encode for lactoyl-CoA dehydratase from M. elsdenii in yeast. The engineered yeast was used to produce 3-hydroxypropionic acid. In some embodiments, the non-natural microorganism is created by enhancing the activity of lactate-CoA dehydratase by introducing enzymes comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequences selected from SEQ ID 69, SEQ ID 70 and SEQ ID 71.
Acryloyl-CoA is reduced to propanoyl-CoA by the action of acryloyl-CoA reductase (Step 4, EC 1.3.1.95). This heterohexadecameric enzyme from C. propionicum catalyzes the irreversible, NADH-dependent conversion of acrylyl-CoA (acryloyl-CoA) to propionyl-CoA. It is a complex of acryloyl-CoA reductase (encoded by acrC) and an electron-transfer flavoprotein (encoded by acrA and acrB). These genes, from Clostridium propionicum were shown to function heterologously in Escherichia coli and participate in fermenting lactate to propanoate (Kandasamy et al., 2013). Another class of acryloyl-CoA reductase is from Rhodobacter sphaeroides and Ruegeria pomeroyi which uses NADPH as the reducing agent (Asao and Alber, 2013, Acrylyl-coenzyme A reductase, an enzyme involved in the assimilation of 3-hydroxypropionate by Rhodobacter sphaeroides, J. Bacteriology, 195(20):4716-4725). In some embodiments, the non-natural microorganism is created by enhancing the activity of acryloyl-CoA reductase by introducing enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequences selected from SEQ ID 72 and SEQ ID 73.
In some embodiments, the metabolic pathway is implemented (and methylmalonic acid is produced) in a microorganism engineered with genes encoding for at least one or more of the enzymes described above, including one or more of D-lactate dehydrogenase, L-lactate dehydrogenase, lactate CoA transferase, lactoyl-CoA dehydratase, acryloyl-CoA reductase, acetyl-CoA synthase, propionyl-CoA synthase, propionyl-CoA carboxlase and methylmalonyl-CoA hydrolase.
In some embodiments, the metabolic pathway involves production of methylmalonic acid from L-glutamate, according to
In some embodiments, methylmalonic acid is reduced to methylmalonic semialdehyde by the action of methylmalonic semialdehyde dehydrogenase. The reducing agent in this conversion is NADH or NADPH. Methylmalonic semialdehyde is also converted to 2-methylpropane-1,3-diol by the action of methylmalonic semialdehyde dehydrogenase and alcohol dehydrogenase. In some embodiments, methylmalonic semialdehyde is converted to 2-methylpropane-1,3-diamine by the action of transaminases (EC 2.6.1.-). In some embodiments of the invention, 2-methylpropane-1,3-diol is converted to the corresponding ester by the action of alcohol acyl transferases (EC. 2.3.1.-).
Embodiments according to the specification encompass microorganisms such as yeast and bacteria that are engineered to include one or more of the aforementioned enzymes and produce methylmalonic acid via a metabolic pathway for example according to one or more of the pathways provided herein. In some embodiments, one or more of the aforementioned enzymes is engineered to have a Km that is less than the Km of the corresponding wild type enzyme. In some embodiments, one or more of the aforementioned enzymes is engineered to have a Km that is less than about half of the Km of the corresponding wild type enzyme. In some embodiments, the microorganism is engineered by introducing heterologous genes either from a plasmid or by integrating in the chromosome. In some embodiments, the microorganism is a bacteria chosen from one or more of: Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Flavobacterium, Lactobacillus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas. In some embodiments, the microorganism is a yeast chosen from one or more of: Candida, Pichia, Kluyveromyces, Saccharomyces, Debaromyces, Hansenula, Pachysolen and Yarrowia. In some embodiments, the microorganism is a methanogenic archaea such as Methanococcus maripaludis. In some embodiments, the microorganism is a filamentous fungus chosen from one or more of: Aspergillus, Penicillium, Acremonium, Fusarium, Neospora and Mucor.
In addition, or in the alternative (if the microorganism produces methylmalonic acid), to including one or more of the metabolic pathway enzymes described above in a bacteria, the yield (efficiency of conversion) of methylmalonate from substrates may be increased by eliminating pathways that compete for the substrate to produce by-products. In some embodiments, the genes that encode for enzymes that catalyze the conversion of pyruvate into by-products such as lactate, acetate and formate is minimized in the bacterial microorganism. For example, in Escherichia coli, the conversion of pyruvate to lactate is catalyzed by lactate dehydrogenase and is encoded by lldD gene (b3605) and ldhA gene (b1380). The conversion of pyruvate to formate is catalyzed by pyruvate formate-lyase. This enzyme is encoded by pflB gene (b0903) and is activated by pflA gene (b0902) in Escherichia coli. The conversion of pyruvate to acetate occurs by the two routes. The first pathway utilizes a single step conversion catalyzed by pyruvate oxidase (EC 1.2.5.1), encoded by the poxB gene (b0871) in Escherichia coli. The second pathway uses acetyl-CoA as an intermediate. Acetyl-CoA is converted to acetylphosphate by phosphotransacetylase (EC 2.3.1.8), which is encoded by the pta gene in (b2297) Escherichia coli. Acetylphosphate is converted to acetate by liberating phosphate by acetate kinase (EC 2.7.2.1) and is encoded by ackA gene (b2296) in Escherichia coli. In order to enhance the availability of succinyl-CoA for methylmalonate production, the conversion of succinate to succinyl-CoA is enhanced by overexpressing succinyl-CoA synthase. In Escherichia coli this enzyme is encoded by the b0728 and b0729 genes.
Further, the transport of methylmalonic acid in bacteria is mediated by a dicarboxylic acid transporter. Examples of several dicarboxylic acid transporters are reported in literature. The genes in Escherichia coli that catalyze the transport are encoded by the genes listed in Table 3.
In some embodiments, the bacterial microorganism is in addition or in the alternative engineered by down-regulating at least one of the genes that encode for the enzymes that catalyze the conversion of pyruvate into acetate, lactate or formate. In some embodiments, the bacterial microorganism is engineered by the introduction of anaplerotic enzymes such as pyruvate carboxylase and ATP-generating phosphoenolpyruvate carboxykinase (Uniprot ID: A6VKV4). In some embodiments, the non-natural microorganism is created by enhancing the activity of ATP-generating phosphoenolpyruvate carboxykinase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 50.
In some embodiments, wherein the microorganism utilizes the phosphoenolpyruvate-dependent phosphotransferase system for the uptake of hexose, the bacterial microorganism is engineer or further engineered to have an inactivated phosphotransferase system and enhanced hexokinase activity. In some embodiments, the bacterial microorganism is engineered with enhanced dicarboxylic acid transporter activity.
Eukaryotic metabolism is compartmentalized and therefore, the regulatory mechanisms significantly differ from those in bacteria. In addition, or in the alternative (if the microorganism produces methylmalonic acid), to including one or more of the metabolic pathway enzymes described above in yeast, in order to increase the yield of methylmalonate, the conversion of pyruvate to ethanol is minimized by deleting at least one of pyruvate decarboxylase or alcohol dehydrogenase reactions. Since there are multiple genes that encode for each of these reactions, the activity of these enzymes is minimized by down-regulating the gene expression either by deletion of or by decreasing the promoter strength of the genes. In eukaryotes, pyruvate is transported from cytosol into mitochondria. The transport is mediated by pyruvate transporter. The activity of the pyruvate transporter can be attenuated by decreasing the expression of the gene that encodes for it. For example in S. cerevisiae, a gene that encodes for the pyruvate transport into the mitochondria could be YIL006W.
Anaplerotic reactions in eukaryotes are predominantly in the mitochondria. Expressing ATP-generating phosphoenolpyruvate carboxykinase (EC 4.1.1.49) in the cytosol will provide oxaloacetate for threonine/methionine synthesis along with the generation of ATP. An example of a gene encoding for this enzyme is pckA from Actinobacillus succinogenes (UniProt ID: A6VKV4). In some embodiments, the non-natural microorganism is created by enhancing the activity of ATP-generating phosphoenolpyruvate carboxykinase by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 50. Methods to create a microorganism with enhanced ATP-generating phosphoenolpyruvate carboxykinase activity are described in the examples.
In some embodiments, the non-natural microorganism is created by enhancing the activity of pyridine nucleotide transhydrogenase (EC 1.6.1.2 or EC 1.6.1.3) by introducing an enzyme comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from SEQ ID 52 or SEQ ID 53. Methods to create a microorganism with enhanced transhydrogenase activity are described in the examples.
The excretion of methylmalonate out of the cell is mediated by dicarboxylic acid transporters. The first dicarboxylic acid transporter in yeast was reported in Kluyveromyces lactis, which transported malate, succinate, fumarate and α-ketoglutarate. Several transporters have been described since then (Casal M, Paiva S, Queiros O, Soares-Silva I: Transport of carboxylic acids in yeasts. FEMS microbiology reviews 2008, 32(6):974-994; Grobler J, Bauer F, Subden R E, Van Vuuren H J: The mael gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids. Yeast 1995, 11(15):1485-1491, both of which are herein incorporated by reference in their entirety). For example, the malic acid permease (MAE1) from Schizosaccharomyces pombe encodes for a permease for dicarboxylic acids, including malonic acid. Physiological characterization of S. cerevisiae strain transformed with S. pombe MAE1 gene (GenBank ID: NM_001020205) enabled the transport of monoanionic form of acids.
Detailed information on the transporters identified is reviewed by Casal et al (supra) and also thoroughly documented in Saccharomyces cerevisiae at http://genolevures.org/yeti.html. Exemplary dicarboxylic acid transporters are shown in Table 4.
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Candida utilis
Kluyveromyces marxianus
In some embodiments, a eukaryotic microorganism is engineered by down-regulating the transport of pyruvate into the mitochondria by attenuating the expression of the transporter gene. In some embodiments, the conversion of pyruvate to ethanol is minimized by down-regulating the activity of pyruvate decarboxylase/alcohol dehydrogenase enzymes. In some embodiments, the energy efficiency of the production of aspartate is enhanced by introducing ATP-generating phosphoenolpyruvate carboxykinase. In some embodiments, the eukaryotic microorganism is engineered by enhancing the dicarboxylic acid export activity.
The following examples are provided only as a means to further illustrate the invention and not to restrict it in any manner.
LC-MS analysis was conducted on an ultrahigh pressure LC system (Shimadzu UFLC XR) online with a triple stage quadrupole mass spectrometer (5500 QTRAP, AB Sciex, Washington, DC, USA) equipped with a 100×2.1 mm inner diameter, 5 μm, HYPERCARB column. The column temperature was maintained at 35° C. An injection volume of 10 μL was chosen. A linear binary gradient at a flow rate of 0.3 mL/min with water and acetonitrile as solvents were used, with each containing 0.1% formic acid. The initial gradient concentration was 2% acetonitrile, which was kept constant for 1 min, linearly increased to 98% in 3.50 min, kept constant for 1 min, and followed by column equilibration steps. The LC column eluate entered the electrospray ionization (ESI) interface of the mass spectrometer operating in the negative ion mode. The MS parameters were sheath gas (N2 99.99%, flow rate=25 units) with vaporization temperature of 350° C. and collision cell exit potential of −9 V, spray voltage of 4.5 kV, entrance potential of −10 V and declustering potential of −30 V. Acquisition was carried out in the MRM mode to achieve maximal sensitivity and reliable quantitation over several orders of magnitude of compound abundance. Q1, precursor molecule, of 116.9 with a Q3 transition of 73 (CE-15) and 55 (CE-30) were selected and conditions optimized using Analyst software. Concentrations of were calculated based on peak areas integrated by MultiQuant (version 2.0.2) compared to a standard curve of known concentration using authentic methylmalonic acid. Liquid chromatography retention time was used to distinguish methylmalonic acid from succinate by using standards under the above conditions.
This example describes yeast cells that are engineered to produce methylmalonic acid. DNA was synthesized de novo (GenScript, Piscataway, N.J.) according to sequence ID 1 and Sequence ID 2 and was cloned into yeast/E. coli shuttle vector with ampicillin resistance, leucine marker and bidirectional Gal1/Gal10 promoters for expressing the genes in yeast. The de novo synthesized DNA according to sequence ID 1 contained restriction sites for BamHI and XhoI and the de novo synthesized DNA according to sequence ID 2 contained restriction sites for SpeI and SacI. The shuttle vector also contained these restriction sites after Gal1 and Gal10 promoter regions, respectively. The de novo synthesized DNA and the plasmid were digested with the corresponding restriction enzymes to the construct the plasmid pGC203 shown in
DNA encoding for propanoyl-CoA synthase was amplified from the genomic DNA of E. coli using the primers with the sequence ID 5 and sequence ID 6. The resulting DNA fragment was restriction digested with EcoRI and SacI enzymes and ligated into a yeast/E. coli shuttle vector with ampicillin resistance, uracil marker and Gal10 promoter for expressing the gene in yeast. The DNA encoding for propanoyl-CoA synthase corresponds to Sequence ID 7.
The mitochondrial signal sequence was identified by TargetP1.1 (Emanuelsson O, Nielsen H, Brunak S, von Heijne G: Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of molecular biology 2000, 300(4):1005-1016) to be the first 31 amino acids, which was replaced with ATG. DNA corresponding to Sequence ID 9 and was amplified from the genome of Saccharomyces cerevisiae using the primers with Sequence ID 11 and Sequence ID 12. This amplified DNA fragment was digested with BamHI and XhoI and ligated into the yeast/E. coli shuttle vector with ampicillin resistance, uracil marker and Gal1 promoter, which was also digested with the same enzymes. The resulting plasmid contains the two genes is shown in
The two plasmids, pGC203 and pGC314 were transformed into S. cerevisiae strain BY4741 using standard protocols (R. D. Gietz and R. A. Woods, Methods Enzymol., 2002, 350, 87). The transformed yeast strain (Y6) containing the two plasmids was grown in synthetic defined medium lacking leucine and uracil. As a control, BY4741 transformed with the two shuttle vectors without the genes of interest (Y1) was also grown in synthetic defined medium lacking leucine and uracil. In this manner, only the gene corresponding to Sequence ID 1 (Y2), genes corresponding to Sequence ID 1 and Sequence ID 2 (Y3), genes corresponding to Sequence ID 1 and Sequence ID 9 (Y4) and genes corresponding to Sequence ID 1, Sequence ID 7 and Sequence ID 9 (Y5) were introduced into yeast. The six yeasts were grown in 250 mL shake flasks at 30° C. in 25 mL of synthetic defined lacking uracil and leucine supplemented with 10 g/L of galactose as carbon source and inducer. The flasks were shaken at 200 rpm for 55.5 h. The wild-type and Y1 control produced small quantities of methylmalonic acid, which is attributed to the promiscuous, residual activity of 3-hydroxyisobutyryl-CoA. As indicated in
This example demonstrates the production of methylmalonic acid by engineered bacteria.
DNA sequence corresponding to Sequence ID 13 was amplified from the genomic DNA of Escherichia coli using primers corresponding to Sequence ID15 and Sequence ID 16. The amplified DNA was digested with BglII and XhoI restriction enzymes and was ligated into pUC-based plasmid which was also digested with the same enzymes. This plasmid, designated pGC406 and shown in
DNA sequence corresponding to Sequence ID 17 was synthesized as gBlocks (Integrated DNA Technologies, Coralville, Iowa) and was restriction digested with BamHI and XhoI restriction enzymes. The DNA fragment was ligated into pUC-based plasmid which was also digested with the same restriction enzymes such that the DNA is expressed under the control of lac promoter. The plasmid is designated pGC412 and is shown in
DNA corresponding to Sequence ID 9 and was amplified from the genome of Saccharomyces cerevisiae using the primers with Sequence ID 11 and Sequence ID 12. This DNA was restriction digested with BamHI and XhoI and ligated into pACYC-based plasmid which was also digested with the same restriction enzymes. The plasmid was designated pGC532 and is shown in
The two plasmids, pGC432 and pGC532, were transformed into Escherichia coli BW25113 using electroporation and the resulting strain is called B5. In this manner, only the gene corresponding to Sequence ID 9 (B4), genes corresponding to Sequence ID 13 and Sequence ID 17 (B3) and the gene corresponding to sequence ID 13 (B2) were introduced into the bacterium and compared against the control which contained empty plasmids (B 1) for methylmalonic acid production. The bacteria were grown in a medium that contained M9 minimal medium (50%) and LB broth (50%) at a starting pH of 7. The plasmids were maintained by adding 100 mg/L of Ampicillin and 50 mg/L of chloramphenicol. 18 g/L of glucose was used as the carbon source. The bacterial cultures were grown in 250 mL shakeflasks with 25 mL working volume at 37° C. by shaking at 200 rpm. The concentration of methylmalonic acid was detected in all the strains at the beginning of the experiment. While there was no significant change in the methylmalonic acid concentration in the strains B1-B4, E. coli containing Steps 13, 14 and 12 produced methylmalonic acid (
This example demonstrates the functional expression of methylmalonyl-CoA hydrolases in bacteria.
DNA sequence corresponding to Sequence ID 18 was de novo synthesized using gBlocks (Integrated DNA Technologies, Coralville, Iowa) and restriction digested by BglII and
XhoI and ligated into pACYC-based plasmid which was also digested with the same enzymes. The resulting plasmid is designated pGC588 and is shown in
This example demonstrates the functional expression of methylmalonyl-CoA hydrolases in yeast.
465 bp fragment from pGC588 was liberated by digestion with BamHI and XhoI and was ligated into pGC314 which was also digested with the same enzymes to create pGC617 (
This example demonstrates how the activity of methylmalonyl-CoA hydrolase could be improved by engineering the protein. The sequence corresponding to Sequence ID 10 was able to catalyze the hydrolysis of (S)-methylmalonyl-CoA into methylmalonic acid. In order to improve the activity of the enzyme, critical amino acids were altered using Q5 Site-Directed Mutagenesis kit (New England Biolabs, Ipswich, Mass.). The mutations were introduced by PCR using primers described below and the plasmid pGC532 as a template. Using the primers indicated by Sequence ID 24 and Sequence ID 25, the glutamate 94 of Sequence ID 10 was mutated to serine. The resulting DNA sequence is shown in Sequence ID 31 and the corresponding sequence of the engineered protein is shown in Sequence ID 32. The resulting plasmid is designated pGC711 (
The plasmids pGC711 and pGC432 (B8), pGC712 and pGC432 (B9), pGC713 and pGC432 (B10) and pGC712 and pGC432 (B11) were transformed into BW25113 and these transformants along with B1, empty plasmid control, and B5, harboring the plasmids pGC543 and pGC432 were grown on medium that contains M9 mineral salts (50%) and LB (50%) and ampicillin (100 mg/L) and chloramphenicol (50 mg/L) and glucose as the carbon source. After growth for 8 h, the cells were harvested and resuspended in 0.1 M Tris-HCl (pH 8). Cell extract from these bacterial cells were prepared by sonication and was used to assay for methylmalonyl-CoA hydrolase activity. The assay was performed as described above with 0 μM, 50 μM, 100 μM, 150 μM or 200 μM of (S)-methylmalonyl-CoA in the enzyme mixture. One unit (U) of mmCoA hydrolase activity is defined as the amount of enzyme required to produce 1 μmole of CoA in one minute. The activity as a function of the substrate concentration was plotted as Lineweaver-Burk plot (D L Nelson, M M Cox, Lehninger Principles of Biochemistry WH Freeman Publishing, 2012) to calculate the Michaelis constant (Km). B1 did not have any detectable activity. The value of Km was high for the unengineered methylmalonyl-CoA hydrolase. However, it decreased significantly for the engineered enzymes (see
This example demonstrates the use of engineered enzyme in bacteria for methylmalonic acid production.
The bacterial cells described in the previous example, B1, B5, B8, B9, B10 and B11 were grown on medium that contains M9 mineral salts (50%) and LB (50%) and ampicillin (100 mg/L) and chloramphenicol (50 mg/L) and glucose as the carbon source. The supernatant was analyzed for methylmalonic acid production. While B1 did not produce any methylmalonic acid, the recombinant bacteria containing the engineered methylmalonyl-CoA hydrolases produced methylmalonic acid.
This example demonstrates the engineering of yeast cells by the introduction of ATP-generating phosphoenolpyruvate carboxykinase.
DNA sequence corresponding to SEQ ID 51 is synthesized de novo and is digested with BamHI and XhoI restriction enzymes and is cloned into a yeast/E. coli shuttle vector with ampicillin resistance, histidine marker and Gall promoter for expressing the gene in yeast, which is also digested with the same enzymes. The resulting plasmid is designated pGC756 and is illustrated in
This example demonstrates the engineering of yeast cells by the introduction of a NADH transhydrogenase.
DNA corresponding to SEQ ID 54 and 55 is de novo synthesized with restriction sites for EcoRI and Sad at the 5′ and 3′ ends and is restriction digested with the enzymes. The fragment is cloned into a yeast/E. coli shuttle vector with ampicillin resistance, histidine marker and Gal10 promoter for expressing the gene in yeast which is also digested with the same enzymes. The resulting plasmids are designated pGC781 (
The Saccharomyces cerevisiae strain IMX581 (Mans, R., H. M. van Rossum, et al. (2015). CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res 15(2)) has Cas9 nuclease integrated in its chromosome such that it can be used as the host strain for manipulating the genome using CRISPR technology (US20140068797 A1). The guide RNA (gRNA) is expressed from either pMEL or pROS series of plasmids. The genes of the methylmalonic acid pathway are integrated in the chromosome of IMX581 using this technology. The gRNA sequences are designed using Yeastriction online tool (http://yeastriction.tnw.tudelft.nl/#!/). The gRNA sequence is introduced into pMEL plasmid using complementary primers that have 50 bp of homology that are PAGE-purified. The primers are dissolved in distilled water to a final concentration of 100 μM and the primers are mixed in 1:1 molar ratio and the mixture is heated to 95° C. for 5 min and annealed by cooling to room temperature. The primers are mixed with pMEL10 as template and is amplified using Q5 High Fidelity 2X Master Mix (New England BioLabs (Ipswich, Mass.). The PCR product is digested with DpnI for 30 minutes and the PCR product purified on an agarose gel. The protocol for simultaneous integration and deletion is described in Mans et al (supra). Using the protocol, genes that encode for the proteins listed in the table below are integrated into the loci in the S. cerevisiae chromosome. The terminator and promoters that are used to express the genes are also listed in the table. The table also provides metabolic alterations in yeast that are conducive to increased methylmalonic acid production.
The engineered yeast hosting the genes of the methylmalonic acid pathway is grown in 250 mL shake flasks at 30° C. in 25 mL of synthetic defined supplemented with 10 g/L of glucose as carbon source. The flasks are shaken at 200 rpm for 24 h. Methylmalonic acid is measured in the supernatant.
A number of embodiments have been described but a person of skill understands that still other embodiments are encompassed by this disclosure. It will be appreciated by those skilled in the art that changes could be made to the embodiments described herein without departing from the broad inventive concepts thereof. It is understood, therefore, that this disclosure and the inventive concepts are not limited to the particular embodiments disclosed, but are intended to cover modifications within the spirit and scope of the inventive concepts including as defined in the appended claims. Accordingly, the foregoing description of various embodiments does not necessarily imply exclusion. For example, “some” embodiments or “other” embodiments may include all or part of “some,” “other,” “further,” and “certain” embodiments within the scope of the invention. Non-exclusive examples of additional embodiments are provided below.
This application is a continuation of International Application No. PCT/US16/17218, filed Feb. 9, 2016, which claims the benefit of U.S. Provisional Application Number 62/114541, filed Feb. 10, 2015, and entitled “Microorganisms for the Synthesis of Methylmalonic Acid and Derivatives.” Each of the above-identified applications are herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62114541 | Feb 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US16/17218 | Feb 2016 | US |
| Child | 15019963 | US |
