KR102611978B1 - A microorganism having enhanced productivity of succinate and a method for producing succinate using the same - Google Patents

Abstract

The present invention relates to a microorganism of the Escherichia genus containing an FdrA variant or a polynucleotide encoding the variant, and a method for producing succinic acid using the microorganism.

Description

Microorganism with enhanced succinic acid productivity and method for producing succinate using the same {A microorganism having enhanced productivity of succinate and a method for producing succinate using the same}

The present invention relates to a microorganism of the Escherichia genus containing an FdrA variant or a polynucleotide encoding the variant, and a method for producing succinic acid using the microorganism.

Succinic acid is an intermediate metabolite of the TCA cycle and is the simplest dicarboxylic organic acid. Since succinic acid can be used to produce various polyester compounds, succinic acid is used as an industrial derivative in various fields such as foods, clothing, medicine, and plastics, and its uses are so diverse that it plays an important role in petrochemical technology (Willke et al., Appl. Microbiol. Biotechnol., 66:131, 2004). The global market for succinic acid is currently worth about 20 trillion won, and most of the production platforms so far have been production through chemical synthesis, but the proportion of succinic acid production through biological processes is increasing significantly, and biotechnology methods are being used to produce succinic acid. Platforms for mass production of succinic acid are becoming increasingly important for various reasons, such as utilization of renewable energy resources and prevention of environmental pollution.

As a method for producing succinic acid, there is a method of producing succinic acid by varying the components of the culture medium, and there is a method of producing succinic acid using whey (Korean Patent No. 10-0301960), which uses whey, a waste product from the dairy industry. Because it was used directly as a culture raw material without pretreatment, the culture efficiency was low, and as a result of using expensive yeast extract as a nitrogen source, there was a problem in that it was not possible to completely achieve an economical medium.

Accordingly, as the demand for succinic acid increases, research is still needed to increase effective succinic acid production capacity.

KR 10-2020-0038377 A

One object of the present invention is to produce succinic acid, which includes an FdrA variant consisting of the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to position 296 of SEQ ID NO: 2, is replaced with tyrosine, or a polynucleotide encoding the variant. The goal is to provide microorganisms of the Escherichia genus with productive capacity.

Another object of the present invention is to provide an FdrA variant consisting of the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to position 296 of SEQ ID NO: 2, is replaced with tyrosine, or a polynucleotide encoding the variant. To provide a method for producing succinic acid comprising culturing microorganisms of the Escherichia genus in a medium.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered to be limited by the specific description described below. Additionally, numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

One aspect of the present invention is an FdrA variant consisting of the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to position 296 of SEQ ID NO: 2, is replaced with tyrosine, or a polynucleotide encoding the variant, S Provides microorganisms of the Escherichia genus.

In the present invention, the term "FdrA (putative acyl-CoA synthetase FdrA or Putative acyl-CoA synthetase with NAD(P)-binding Rossmann-fold domain)" is an enzyme whose function is unknown. Specifically, it can be used interchangeably with terms such as ECK0511, G6287, b0518, and ylbD. The protein and gene sequences of FdrA can be obtained by searching in NCBI Genbank, a known database. For example, the FdrA may be a polypeptide having FdrA activity encoded by a gene consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto.

For the purposes of the present invention, any FdrA variant of the present invention may be included as long as it can enhance the ability to produce succinic acid. Specifically, the FdrA variant may be derived from a microorganism of the Escherichia genus, and more specifically, may be derived from Escherichia coli , but is not limited thereto.

The FdrA variant of the present invention may have and/or include, or consist essentially of, the amino acid sequence shown in SEQ ID NO: 1.

In addition, in the FdrA variant of the present invention, the amino acid corresponding to position 296 based on the amino acid sequence of SEQ ID NO: 2 in the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1, It may include an amino acid sequence having more than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity. In addition, as long as the amino acid sequence has such homology or identity and shows efficacy corresponding to the FdrA variant of the present invention, the FdrA variant having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added is also within the scope of the present invention. It is self-evident that it is included within.

In the present invention, it is described as 'polypeptide or protein containing an amino acid sequence described in a specific sequence number', 'polypeptide or protein composed of an amino acid sequence described in a specific sequence number', or 'polypeptide or protein having an amino acid sequence described in a specific sequence number'. Even if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number, a protein with an amino acid sequence in which part of the sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in the present invention. is self-explanatory. For example, the amino acid sequence may have a sequence added to the N-terminus and/or C-terminus that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution. .

For example, addition or deletion of sequences, naturally occurring mutations, silent mutations, or This is the case with conservative substitutions.

As used herein, the term “conservative substitution” refers to replacing one amino acid with another amino acid having similar structural and/or chemical properties. These amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; Negatively charged (acidic) amino acids include glutamic acid and aspartate; Aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In addition, amino acids can be classified into amino acids with electrically charged side chains and amino acids with uncharged side chains. Amino acids with electrically charged side chains include astratic acid, glutamic acid, and lysine. , arginine, and histidine, and amino acids with uncharged side chains can be further classified as nonpolar amino acids or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, and isoleucine. Methionine, phenylalanine. Tryptophan, proline, and polar amino acids can be classified to include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

In the present invention, the term "variant" means that one or more amino acids are different from the amino acid sequence before the mutation due to conservative substitution and/or modification of the variant, but have functions or properties. This refers to the polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Additionally, some variants may include variants in which one or more parts, such as the N-terminal leader sequence or transmembrane domain, have been removed. Other variants may include variants with portions removed from the N- and/or C-terminus of the mature protein. The term "variant" is a mixed use of terms such as variant, modified, variant polypeptide, mutated protein, protein variant, mutation and mutant (English expressions include modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) It can be used as a term, and is not limited thereto if the term is used in a modified sense. For the purpose of the present invention, the variant may be a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to the 296th position of the amino acid sequence in SEQ ID NO: 2, is replaced with tyrosine.

Additionally, FdrA variants may contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a polypeptide can be conjugated with a signal (or leader) sequence at the N-terminus of a protein that is involved in protein transfer, co-translationally or post-translationally. The polypeptide may also be conjugated with other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.

In the present invention, the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and may be used with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or part of a sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with a polynucleotide containing a common codon or a codon taking codon degeneracy into account.

Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: It can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Or, as performed in the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ed.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073. For example, BLAST from the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity or identity of polynucleotides or polypeptides is defined in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, e.g., Needleman et al. (1970), J Mol Biol. This can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing values 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

As an example of the present invention, the variant of the present invention may have FdrA activity. Additionally, the mutant of the present invention may have an activity that increases the ability to produce succinic acid compared to the wild-type polypeptide having FdrA activity.

In the present invention, the term “corresponding to” refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in a polypeptide. Identifying the amino acid at the corresponding position may be determining the specific amino acid in the sequence that refers to the specific sequence. As used herein, “corresponding region” generally refers to a similar or corresponding position in a related or reference protein.

For example, an arbitrary amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. . For example, sequence alignment algorithms such as those described herein can identify the positions of amino acids or where modifications such as substitutions, insertions, or deletions occur compared to a query sequence (also referred to as a “reference sequence”).

Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), etc. may be used, but are not limited thereto, and sequence alignment programs and pairwise sequence comparison algorithms known in the art may be appropriately used.

In the present invention, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or more, which is a polymer of nucleotides in which nucleotide monomers are connected in a long chain by covalent bonds, and more specifically, codes for the FdrA variant. refers to a polynucleotide fragment that

The polynucleotide is a polynucleotide encoding an FdrA variant of the invention or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% of the FdrA variant of the invention. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity. The polynucleotide encoding the FdrA variant of the present invention may include a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1.

The polynucleotide of the present invention has a coding region within the range that does not change the amino acid sequence of the FdrA variant of the present invention, taking into account codon degeneracy or preferred codons in organisms intended to express the FdrA variant of the present invention. Various modifications may be made to .

In addition, the polynucleotide of the present invention includes, without limitation, any probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present invention. You can. The “stringent condition” refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, among polynucleotides with high homology or identity, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, Or, conditions in which polynucleotides with 99% or more homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize with each other, or 60°C, which is the washing condition of normal southern hybridization, Washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, more specifically 68°C, 0.1ХSSC, 0.1% SDS. Conditions can be listed.

Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases may be possible depending on the stringency of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, polynucleotides of the invention may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

Specifically, polynucleotides having homology or identity with the polynucleotide of the present invention can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. Additionally, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art depending on the purpose.

The appropriate stringency to hybridize the polynucleotide depends on the length of the polynucleotide and the degree of complementarity, variables that are well known in the art (e.g., J. Sambrook et al., supra).

In the present invention, the term "vector" refers to a DNA product containing the base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) to enable expression of the target polypeptide in a suitable host. may include. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable microorganism, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors can be used. , pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. The selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of the target nucleic acid molecule, and to display selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers that provide may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

In the present invention, the term “transformation” refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or microorganism so that the polypeptide encoded by the polynucleotide can be expressed within the microorganism. As long as the transformed polynucleotide can be expressed in the microorganism, it can include both of these, regardless of whether they are inserted into the chromosome of the microorganism or located outside the chromosome. Additionally, the polynucleotide includes DNA and/or RNA encoding the polypeptide of interest. The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a microorganism. For example, the polynucleotide can be introduced into a microorganism in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

In addition, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the FdrA variant of the present invention.

The microorganism of the present invention may contain an FdrA variant of the present invention, a polynucleotide encoding the FdrA variant, or a vector containing the polynucleotide of the present invention.

In the present invention, the term "microorganism" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may undergo specific mechanisms due to causes such as insertion of foreign genes or enhanced or inactivated activity of endogenous genes. A weakened or enhanced microorganism may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In the present invention, “microorganism,” “strain,” and “microorganism” have the same meaning and can be used interchangeably without limitation.

The microorganism of the present invention is a strain containing any one or more of the FdrA variant of the present invention, the polynucleotide of the present invention, and the vector containing the polynucleotide of the present invention; A strain modified to express an FdrA variant of the invention or a polynucleotide of the invention; Strains (e.g., recombinant strains) expressing FdrA variants of the invention, or polynucleotides of the invention; Alternatively, it may be a strain (eg, a recombinant strain) having the activity of the FdrA variant of the present invention, but is not limited thereto.

The microorganism of the present invention may be a microorganism with the ability to produce succinic acid.

For example, the microorganism with the ability to produce succinic acid may be a microorganism with enhanced FdrA enzyme activity.

The strain of the present invention is a microorganism that naturally has the ability to produce FdrA or succinic acid, or the mutant of the present invention or a polynucleotide encoding the same (or a vector containing the polynucleotide) is introduced into the parent strain without the ability to produce FdrA or succinic acid. and/or may be a microorganism endowed with the ability to produce succinic acid, but is not limited thereto.

As an example, the strain of the present invention is a cell or microorganism that is transformed with a vector containing a polynucleotide encoding a polynucleotide of the present invention or a variant of the present invention and expresses the variant of the present invention. The strain of the invention may include all microorganisms capable of producing succinic acid, including variants of the invention. For example, the strain of the present invention may be a recombinant strain in which the FdrA variant is expressed by introducing a polynucleotide encoding the variant of the present invention into a natural wild-type microorganism or a microorganism that produces succinic acid, thereby increasing the ability to produce succinic acid. The recombinant strain with increased succinic acid production ability is compared to a natural wild-type microorganism or an FDRA non-modified microorganism (i.e., a microorganism that expresses wild-type FDRA (SEQ ID NO: 2) or a microorganism that does not express a mutant (SEQ ID NO: 1) protein. It may be a microorganism with increased succinic acid production ability, but is not limited thereto. As an example, the microorganism with an increased succinic acid production ability of the present invention may be a microorganism with an increased succinic acid production ability compared to an Escherichia genus microorganism containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same, but is not limited thereto.

As an example, the recombinant strain with increased production ability is about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, and about 10% compared to the succinic acid production ability of the parent strain or unmodified microorganism before mutation. or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more or more, about 60.5% or more, about 61% or more, or about 61.1% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 90% or less, about 80% or less , may be about 70% or less or about 65% or less), but is not limited thereto as long as it has a + value increase compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain with increased production capacity has a succinic acid production capacity of about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, 1.4 times or more, 1.5 times or more, and 1.55 times more than the parent strain or unmodified microorganism before mutation. It may be increased by more than two times, more than 1.6 times, or more than 1.61 times (the upper limit is not particularly limited, and may be, for example, about 10 times less, about 9 times less, about 8 times less, or about 7 times less). Not limited. The term "about" is a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range that are equivalent or similar to the value that appears after the term "about." Not limited.

In the present invention, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and is either a wild-type strain or a natural strain itself, or a strain that has a genetic mutation due to natural or artificial factors. It may refer to the strain before change. For example, the unmodified microorganism may mean a strain in which the FDRA variant described herein is not introduced or before it is introduced. The “non-modified microorganism” may be used interchangeably with “pre-transformed strain”, “pre-transformed microorganism”, “non-mutated strain”, “non-modified strain”, “non-mutated microorganism” or “reference microorganism”.

As another example of the present invention, the microorganism of the present invention may be a microorganism with an increased oxamate production ability compared to a microorganism of the Escherichia genus containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same, but is limited thereto. no.

Specifically, in one embodiment of the present invention, the present inventors discovered for the first time that fdrA can encode an enzyme that decomposes allantoin (oxalurate) into oxamate. Accordingly, it was confirmed that the FdrA (D296Y) variant of the present invention improves succinic acid production by accelerating the conversion of allantoin to oxamate (Example 2).

More specifically, the variant prevents oxalate production from glyoxylate by accelerating the production of oxamate, an effective inhibitor of lactate dehydrogenase (LDH). As a result, more glyoxylate is exposed to malate synthase G, thereby increasing the succinic acid production capacity (Figure 2).

The present inventors first discovered that fdrA encodes an enzyme that decomposes allantoin (oxalurate) into oxamate. Accordingly, it was confirmed that the FdrA (D296Y) variant of the present invention improves succinic acid production by accelerating the conversion of allantoin to oxamate, and the pathway is shown in Figure 2.

As another example of the present invention, the succinic acid may be produced by consuming glycerol through glycerol fermentation, but is not limited thereto. Specifically, the succinic acid may be produced through the metabolic pathway of PEP, but is not limited thereto.

Wild-type Escherichia coli produces complex fermentation products under anaerobic conditions. This fermentation complex product is a mixed acid-fermentation type containing acetic acid, lactic acid, formic acid, succinic acid, and alcohol. However, glycerol fermentation in wild-type E. coli does not occur well under anaerobic conditions.

The glycerol may be pure glycerol or waste glycerol generated from the biodiesel production process. The waste glycerol may be generated during the biodiesel production process (using an alkaline catalyst and methanol or ethanol) using soy milk, waste cooking oil, etc. Additionally, the waste glycerol can be sterilized without purification and diluted to an appropriate glycerol concentration in the culture medium. Alternatively, it may be pretreated waste glycerol in which the black impurity layer generated by adding an acid such as HCl to waste glycerol has been removed.

As another example of the present invention, the microorganism may be a microorganism of the genus Escherichia, specifically Escherichia coli , but is not limited thereto.

In the present invention, the term “enhancement” of polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase may include showing an activity that it did not originally have, or showing improved activity compared to the intrinsic activity or activity before modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the change in trait when the trait changes due to genetic mutation due to natural or artificial factors. This may be used interchangeably with “activity before modification.” “Enhanced,” “upregulated,” “overexpressed,” or “increased” in the activity of a polypeptide compared to its intrinsic activity means the activity and/or concentration (expression) of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation. It means an improvement compared to the amount).

The enhancement can be achieved by introducing an exogenous polypeptide or enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by increasing the activity level of the polypeptide, the expression level, or the amount of product released from the polypeptide.

Enhancement of the activity of the polypeptide can be done by applying various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Specifically, genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology, may be used, but are not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the enhancement of the polypeptide activity of the present invention is

1) Increased intracellular copy number of the polynucleotide encoding the polypeptide;

2) Replacement of the gene expression control region on the chromosome encoding the polypeptide with a highly active sequence;

3) Modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

4) modifying the amino acid sequence of the polypeptide to enhance its activity;

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) Codon optimization of the polynucleotide encoding the polypeptide;

8) Analyzing the tertiary structure of the polypeptide, select exposed sites and modify or chemically modify them; or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited.

More specifically,

Above 1) the increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by the introduction into the host cell of a vector capable of replicating and functioning independently of the host to which the polynucleotide encoding the polypeptide is operably linked. It may be possible. Alternatively, this may be achieved by introducing one or two or more copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell into the host cell, but is not limited to this. The vector is the same as described above.

2) Replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a highly active sequence, for example, by deletion, insertion, non-conservative or Sequence variation may occur due to conservative substitution or a combination thereof, or replacement may occur with a sequence with stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but the method is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13. (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) The base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base sequence encoding another start codon with a higher polypeptide expression rate than the internal start codon. It may be a substitution, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) includes deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide. The combination of these may result in a mutation in the sequence, or a replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to check whether chromosome insertion has occurred. The selection marker is as described above.

6) Introduction of a foreign polynucleotide showing the activity of the polypeptide may be introduction into the host cell of a foreign polynucleotide encoding a polypeptide showing the same/similar activity as the polypeptide. There are no restrictions on the origin or sequence of the foreign polynucleotide as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by a person skilled in the art, and by expressing the introduced polynucleotide in the host cell, a polypeptide can be produced and its activity can be increased.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon optimization of the native polynucleotide to increase transcription or translation within the host cell, or optimized transcription and translation of the foreign polynucleotide within the host cell. It may be that the codons have been optimized to allow this.

Above 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to modify or chemically modify the sequence information, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins to determine the degree of sequence similarity. Accordingly, a template protein candidate may be determined, the structure confirmed based on this, and an exposed site to be modified or chemically modified may be selected and modified or modified.

Such enhancement of polypeptide activity means that the activity or concentration of the corresponding polypeptide is increased based on the activity or concentration of the polypeptide expressed in the wild type or unmodified microbial strain, or the amount of the product produced from the polypeptide is increased. However, it is not limited to this.

Modification of part or all of the polynucleotide in the microorganism of the present invention is (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) It may be induced by, but is not limited to, light and/or chemical treatment, such as ultraviolet rays and radiation. The method of modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the gene of interest is injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

Another aspect of the present invention is an FdrA variant consisting of the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to position 296 of SEQ ID NO: 2, is replaced with tyrosine, or an S comprising a polynucleotide encoding the variant. A method for producing succinic acid is provided, comprising culturing microorganisms of the Escherichia genus in a medium.

In the production method of the present invention, the FdrA variant, polynucleotide, microorganism, succinic acid, etc. are as described in the other embodiments above.

The method for producing succinic acid of the present invention may include, but is not limited to, the step of culturing Escherichia microorganisms containing the variant of the present invention or the polynucleotide of the present invention or the vector of the present invention in a medium.

As an example of the present invention, the microorganism of the present invention may be a microorganism with an increased succinic acid production ability compared to an Escherichia microorganism containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same, but is not limited thereto.

As another example of the present invention, the microorganism of the present invention may be a microorganism with an increased oxamate production ability compared to a microorganism of the Escherichia genus containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same, but is limited thereto. no.

In the present invention, the term “culture” means growing the microorganism of the present invention under appropriately controlled environmental conditions. The culture process of the present invention can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.

In the present invention, the term "medium" refers to a material that is mainly mixed with nutrients necessary for cultivating the microorganisms of the present invention, and supplies nutrients and growth factors, including water, which are essential for survival and growth. . Specifically, the medium and other culture conditions used for cultivating the microorganism of the present invention can be any medium used for cultivating ordinary microorganisms without particular limitation, but the microorganism of the present invention can be grown with an appropriate carbon source, nitrogen source, personnel, and inorganic substances. It can be cultured under aerobic conditions in a typical medium containing compounds, amino acids, and/or vitamins, while controlling temperature, pH, etc.

In the present invention, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamic acid, methionine, lysine, etc. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e. converted to reducing sugars). Carbohydrates such as molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.

The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used These nitrogen sources may be used individually or in combination of two or more types, but are not limited thereto.

The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and/or appropriate precursors. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.

Additionally, during cultivation of the microorganism of the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited thereto. That is not the case.

In the culture of the present invention, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

Succinic acid produced by the culture of the present invention may be secreted into the medium or remain within the cells.

As another example of the present invention, the succinic acid may be produced by consuming glycerol through glycerol fermentation, but is not limited thereto.

The succinic acid production method of the present invention includes preparing a microorganism of the Escherichia genus of the present invention, preparing a medium for culturing the strain, or a combination thereof (in any order), for example. For example, before the culturing step, it may be additionally included.

The method for producing succinic acid of the present invention may further include the step of recovering succinic acid from the culture medium (medium in which the culture was performed) or Escherichia genus microorganisms. The recovering step may be additionally included after the culturing step.

The recovery may be to collect the desired succinic acid using a suitable method known in the art according to the method of cultivating the microorganism of the present invention, for example, a batch, continuous or fed-batch culture method. For example, centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatographies such as chromatography, HPLC, or a combination of these methods can be used, and the desired succinic acid can be recovered from the medium or microorganism using a suitable method known in the art.

Additionally, the method for producing succinic acid of the present invention may additionally include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the method for producing succinic acid of the present invention includes both a recovery step and a purification step, the recovery step and the purification step are performed continuously or discontinuously regardless of the order, or are performed simultaneously or integrated into one step. It may be, but is not limited to this.

Another aspect of the present invention is a variant of the present invention, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a microorganism of the genus Escherichia containing the polynucleotide of the present invention; The medium in which it was cultured; Alternatively, a composition for producing succinic acid comprising a combination of two or more of these is provided.

The composition of the present invention may further comprise any suitable excipients commonly used in compositions for producing amino acids, such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, or tonicity agents. However, it is not limited to this.

In the composition of the present invention, variants, polynucleotides, vectors, strains, media, succinic acid, etc. are as described in the other embodiments above.

When cultivating a microorganism of the Escherichia genus containing a variant of FdrA of the present invention, it is possible to produce succinic acid at a higher yield compared to microorganisms with existing unmodified polypeptides.

Figure 1 shows the results showing the relative fitness of E. coli into which a single nucleotide mutation was introduced.
Figure 2 shows the succinic acid production pathway through the glyoxylate pathway during glycerol fermentation.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are merely preferred embodiments for illustrating the present invention and are therefore not intended to limit the scope of the present invention thereto. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by anyone skilled in the technical field of the present invention or a similar technical field.

Example 1. Construction of a succinic acid producing strain into which a novel FdrA variant was introduced

Example 1-1. Confirmation of FdrA mutation that increases succinic acid production ability

In order to create microorganisms with increased succinic acid production ability, genetic changes in E. coli KCTC 13592BP (KR 10-2128031 B1) strain, which has been adaptively evolved through co-culture with methane-producing bacteria and has improved succinic acid production ability, were investigated and Transient Mutator Multiplex Automated Genome Through Engineering (TM-MAGE) (Lennen RM et al. 2016. Nucleic Acids Res 44:e36.), seven single base mutations (dnaK (chaperone Hsp70, A586E), ybbP (putative ABC transporter permease) affecting succinic acid production were identified. , W612R), fdrA (putative NAD(P)-binding acyl-CoA synthetase, D296Y), pgpC (phosphatidylglycero-phosphatase C, R91L), yfjI (uncharacterized protein, I384V), cysN (sulfate adenylyl-transferase subunit 1, D171E) , rob (right oriC-binding transcriptional activator, R156S)) was selected.

Next, referring to a known method (KR 10-2128031 B1), in order to adapt and evolve wild-type E. coli (39 ^th population) and E. coli into which each of the above seven single nucleotide mutations were introduced for succinic acid production using glycerol, E .coli and M.formicicum were jointly subcultured anaerobically for each round up to 39 rounds.

For the control group (Ancestor), E.coli and M.formicicum were co-cultured anaerobically for 96 hours. The results are shown in Table 1 below.

As shown in Table 1, it was confirmed that the seven single base mutations increased the succinic acid production ability of E. coli. In particular, the FdrA variant (D296Y) was confirmed to have the greatest effect on increasing the succinic acid production ability. .

Additionally, the relative fitness of the E. coli strain was evaluated by measuring competitive growth when the strain and the parent strain (Ancestor) were co-cultured. Compatibility was assessed under three conditions (anaerobic coculture of E. coli and M. formicicum in glycerol (90mM), anaerobic monoculture of E. coli in glycerol (90mM) and DMSO (50mM), and aerobic monoculture of E. coli in LB medium). Anaerobic cultivation was performed on fermentation medium (100 ml) for 96 hours. Aerobic culture in LB (10 ml) was used as a control.

After culturing, the bacterial cells were diluted to form colonies ranging from 50 to 100 on X-Gal (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside) agar plates. The two competing strains were distinguished by white/blue colonies. The ancestral E. coli (DlacZ) strain was white and the competing strain was blue. Relative fitness (W) was calculated using the ratio of Malthusian parameters of the two strains. W = ln(A _f /A _i )/ln(B _f /B _i ), where A represents the cell density of the evolving or mutant population and B represents cells. Density of the ancestral population, and the subscripts i and f denote the initial and final densities, respectively (1, 30, 31). A W value >1.0 indicates that the fitness of the E. coli population has increased compared to its ancestor, and a W value <1.0 indicates a decrease in fitness.

The results are shown in Figure 1.

As shown in Figure 1, it was confirmed that no significant increase in relative fitness was found in E. coli into which the seven single nucleotide mutations were introduced compared to the parent strain.

Example 1-2. Construction of a succinic acid producing strain into which a novel FdrA variant was introduced

Transformant E. coli into which the FdrA variant (D296Y), which showed the highest succinic acid production in Example 1-1, was introduced, was prepared.

Specifically, it was produced using the known TM-MAGE (transient mutator multiplex automated genome engineering) method (Lennen RM et al. 2016. Nucleic Acids Res 44:e36.). Oligonucleotides for TM-MAGE were designed using MODEST (Bonde MT et al. 2014. Nucleic Acids Res 42:W408-W415.) and were set to 90 bp in length with 15 bp minimum end homology and 50 bp long insert end homology. . Ancestral E. coli analyzed by next-generation sequencing (NGS) was used as a starter strain for TM-MAGE, and the ancestral E. coli was transformed using plasmid pMA7-SacB (Addgene, USA). E. coli carrying pMA7-SacB was grown overnight in LB (Lennox, Sigma-Aldrich, USA) containing 100 mg/ml ampicillin and 0.5mM MgSO ₄ at 37°C with shaking at 180 rpm. 1% overnight culture was inoculated into the same medium and cultured under the same conditions. When the OD600 reached 0.4 to 0.6, Larabinose was added to a final concentration of 0.2% (wt/vol), and the incubation was continued for 10 minutes, followed by incubation on ice for 15 minutes. Cells were collected at 4,000 ㎍ using a Combi 514-R centrifuge (Hanil, Korea) for 5 minutes at 4°C, washed three times with cooled distilled water (30ml, 1ml, 1ml), and finally resuspended in 200ml of cooled distilled water. Concentrated. Then, a mixture of competent cells (50 ml) and MAGE oligonucleotides (0.5 ml, 50 pmol) were electroporated using MicroPulser (Bio-Rad, USA) and incubated in LB containing 100 mg/ml ampicillin and 0.5 mM MgSO4. It was resuspended in Lennox (Sigma-Aldrich, USA) and plated on agar plates. Individual colonies were selected by multiple allele specific colony (MASC) PCR. For plasmid curing, positive colonies were streaked on sucrose plates (10 g/liter tryptone, 5 g/liter yeast extract, 15 g/liter agar, and 5% [wt/vol] sucrose). Successful point mutations in the genome were then confirmed by DNA sequencing (Cosmogenetech, Korea).

The transformed E. coli into which the FdrA variant (D296Y) was introduced was named Escherichia coli fdrAD296Y, and was internationally deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC), an international depository under the Budapest Treaty, with the deposit number as of November 30, 2021. Was granted KCTC 14806BP.

Example 2. Evaluation of succinic acid production ability of succinic acid producing strain into which a novel FdrA variant was introduced

The effect of the function and genetic mutation of the FdrA variant (D296Y) of the present invention on succinic acid production was analyzed using transformed E. coli into which the FdrA variant (D296Y) prepared in Example 1-2 was introduced.

First, the promoter region of fdrA was fused with the lacZ reporter gene, and possible effectors (inducers) were searched by β-galactosidase assay.

Specifically, a fusion of the fdrA promoter region with lacZ was constructed using the reporter gene fusion plasmid pJL29. The fdrA promoter region was amplified from E. coli MG1655 genomic DNA using primers fdrA_SalI (59-CGG CAT TGT CGA CAT GTA AA-39) and fdrA_HindIII (59-CCT CTT CAA GCT TCT GCA TA-39). The corresponding fragment covered the complete upstream regulatory region of fdrA and was cloned into the corresponding restriction site of the reporter gene fusion plasmid pJL29. The fdrA-lacZ gene fusion was used to transform E. coli MC4100, a lacZ-deficient strain.

The transformed E. coli MC4100 was grown in M9 medium containing glycerol (50mM) and DMSO (50mM) under anaerobic conditions (Kim OB, Unden G. 2007. J Bacteriol 189:1597-1603.). The culture contains 20mM allantoin, oxamate, NH ₄ Cl or oxalate| as effectors. β-Galactosidase activity was measured by a known method (Miller JH. 1992. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY.). The β-galactosidase activity of empty lacZ fusion plasmid pJL2 was measured under each culture condition and used as a blank value. The results are shown in Table 2 below.

As shown in Table 2, it was confirmed that allantoin significantly increased the transcription of fdrA under anaerobic conditions, but oxamate and oxalate had no effect on gene expression. Therefore, it was confirmed that FdrA plays a role in anaerobic allantoin degradation, but oxamate, succinic acid, and oxalate do not serve as substrates.

Next, the effect of genetic variation on succinic acid production in the FdrA mutant was analyzed.

Specifically, each of the E. coli MG1655 (wild-type strain (ancestor), fdrA deletion (ΔfdrA), fdrA deletion and pNTR-SD::fdrA (ΔfdrApNTR-SD::fdrA), and fdrA mutant (fdrA _D296Y )) is anaerobic. It was grown for 72 hours in M9 medium containing glycerol (50mM), DMSO (50mM), and allantoin (20mM). Allantoin was used as the sole nitrogen source. The results are shown in Table 3 below.

As shown in Table 3 and Figure 2, most of the allantoin in the wild type strain is decomposed into oxamate (~80%) and some is decomposed into oxalate (~20%), and in the △fdrA strain, oxamate is not produced. It was confirmed that the oxalic acid fraction increased. In addition, it was confirmed that the fdrA cloned plasmid (pNTR-SD::fdrA) can complement oxamate production while reducing oxalate production. This suggests that FdrA catalyzes the oxamate generation step and that oxalate is linked to the alternative pathway of oxamate generation in the metabolic context.

In particular, in the fdrA mutant (D296Y), succinic acid production increased by about 61% compared to the non-mutant, and almost all allantoin was confirmed to be decomposed into oxamate. This suggests that the D296Y mutation improves enzyme activity to produce oxamate or increase substrate affinity, and it was confirmed that the 25% increase in oxamate production is the direct cause of the increased succinic acid production ability in the mutant of the present invention.

In summary, the present inventors discovered for the first time that fdrA encodes an enzyme responsible for decomposing allantoin (oxalurate) into oxamate. Accordingly, it was confirmed that the FdrA (D296Y) variant of the present invention improves succinic acid production by accelerating the conversion of allantoin to oxamate, and the pathway is shown in Figure 2.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology KCTC14806BP 20211130

<110> Ewha University - Industry Collaboration Foundation <120> A microorganism having enhanced productivity of succinate and a method for producing succinate using the same <130> KPA211391-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 555 <212> PRT <213> Unknown <220> <223> FdrA variant <400> 1 Met Ile His Ala Phe Ile Lys Lys Gly Cys Phe Gln Asp Ser Val Ser 1 5 10 15 Leu Met Ile Ile Ser Arg Lys Leu Ser Glu Ser Glu Asn Val Asp Asp 20 25 30 Val Ser Val Met Met Gly Thr Pro Ala Asn Lys Ala Leu Leu Asp Thr 35 40 45 Thr Gly Phe Trp His Asp Asp Phe Asn Asn Ala Thr Pro Asn Asp Ile 50 55 60 Cys Val Ala Ile Arg Ser Glu Ala Ala Asp Ala Gly Ile Ala Gln Ala 65 70 75 80 Ile Met Gln Gln Leu Glu Glu Ala Leu Lys Gln Leu Ala Gln Gly Ser 85 90 95 Gly Ser Ser Gln Ala Leu Thr Gln Val Arg Arg Trp Asp Ser Ala Cys 100 105 110 Gln Lys Leu Pro Asp Ala Asn Leu Ala Leu Ile Ser Val Ala Gly Glu 115 120 125 Tyr Ala Ala Glu Leu Ala Asn Gln Ala Leu Asp Arg Asn Leu Asn Val 130 135 140 Met Met Phe Ser Asp Asn Val Thr Leu Glu Asp Glu Ile Gln Leu Lys 145 150 155 160 Thr Arg Ala Arg Glu Lys Gly Leu Leu Val Met Gly Pro Asp Cys Gly 165 170 175 Thr Ser Met Ile Ala Gly Thr Pro Leu Ala Phe Ala Asn Val Met Pro 180 185 190 Glu Gly Asn Ile Gly Val Ile Gly Ala Ser Gly Thr Gly Ile Gln Glu 195 200 205 Leu Cys Ser Gln Ile Ala Leu Ala Gly Glu Gly Ile Thr His Ala Ile 210 215 220 Gly Leu Gly Gly Arg Asp Leu Ser Arg Glu Val Gly Gly Ile Ser Ala 225 230 235 240 Leu Thr Ala Leu Glu Met Leu Ser Ala Asp Glu Lys Ser Glu Val Leu 245 250 255 Ala Phe Val Ser Lys Pro Pro Ala Glu Ala Val Arg Leu Lys Ile Val 260 265 270 Asn Ala Met Lys Ala Thr Gly Lys Pro Thr Val Ala Leu Phe Leu Gly 275 280 285 Tyr Thr Pro Ala Val Ala Arg Tyr Glu Asn Val Trp Phe Ala Ser Ser 290 295 300 Leu Asp Glu Ala Ala Arg Leu Ala Cys Leu Leu Ser Arg Val Thr Ala 305 310 315 320 Arg Arg Asn Ala Ile Ala Pro Val Ser Ser Gly Phe Ile Cys Gly Leu 325 330 335 Tyr Thr Gly Gly Thr Leu Ala Ala Glu Ala Ala Gly Leu Leu Ala Gly 340 345 350 His Leu Gly Val Glu Ala Asp Asp Thr His Gln His Gly Met Met Leu 355 360 365 Asp Ala Asp Ser His Gln Ile Ile Asp Leu Gly Asp Asp Phe Tyr Thr 370 375 380 Val Gly Arg Pro His Pro Met Ile Asp Pro Thr Leu Arg Asn Gln Leu 385 390 395 400 Ile Ala Asp Leu Gly Ala Lys Pro Gln Val Arg Val Leu Leu Leu Asp 405 410 415 Val Val Ile Gly Phe Gly Ala Thr Ala Asp Pro Ala Ala Ser Leu Val 420 425 430 Ser Ala Trp Gln Lys Ala Cys Ala Ala Arg Leu Asp Asn Gln Pro Leu 435 440 445 Tyr Ala Ile Ala Thr Val Thr Gly Thr Glu Arg Asp Pro Gln Cys Arg 450 455 460 Ser Gln Gln Ile Ala Thr Leu Glu Asp Ala Gly Ile Ala Val Val Ser 465 470 475 480 Ser Leu Pro Glu Ala Thr Leu Leu Ala Ala Ala Leu Ile His Pro Leu 485 490 495 Ser Pro Ala Ala Gln Gln His Thr Pro Ser Leu Leu Glu Asn Val Ala 500 505 510 Val Ile Asn Ile Gly Leu Arg Ser Phe Ala Leu Glu Leu Gln Ser Ala 515 520 525 Ser Lys Pro Val Val His Tyr Gln Trp Ser Pro Val Ala Gly Gly Asn 530 535 540 Lys Lys Leu Ala Arg Leu Leu Glu Arg Leu Gln 545 550 555 <210> 2 <211> 555 <212> PRT <213> Escherichia coli <400> 2 Met Ile His Ala Phe Ile Lys Lys Gly Cys Phe Gln Asp Ser Val Ser 1 5 10 15 Leu Met Ile Ile Ser Arg Lys Leu Ser Glu Ser Glu Asn Val Asp Asp 20 25 30 Val Ser Val Met Met Gly Thr Pro Ala Asn Lys Ala Leu Leu Asp Thr 35 40 45 Thr Gly Phe Trp His Asp Asp Phe Asn Asn Ala Thr Pro Asn Asp Ile 50 55 60 Cys Val Ala Ile Arg Ser Glu Ala Ala Asp Ala Gly Ile Ala Gln Ala 65 70 75 80 Ile Met Gln Gln Leu Glu Glu Ala Leu Lys Gln Leu Ala Gln Gly Ser 85 90 95 Gly Ser Ser Gln Ala Leu Thr Gln Val Arg Arg Trp Asp Ser Ala Cys 100 105 110 Gln Lys Leu Pro Asp Ala Asn Leu Ala Leu Ile Ser Val Ala Gly Glu 115 120 125 Tyr Ala Ala Glu Leu Ala Asn Gln Ala Leu Asp Arg Asn Leu Asn Val 130 135 140 Met Met Phe Ser Asp Asn Val Thr Leu Glu Asp Glu Ile Gln Leu Lys 145 150 155 160 Thr Arg Ala Arg Glu Lys Gly Leu Leu Val Met Gly Pro Asp Cys Gly 165 170 175 Thr Ser Met Ile Ala Gly Thr Pro Leu Ala Phe Ala Asn Val Met Pro 180 185 190 Glu Gly Asn Ile Gly Val Ile Gly Ala Ser Gly Thr Gly Ile Gln Glu 195 200 205 Leu Cys Ser Gln Ile Ala Leu Ala Gly Glu Gly Ile Thr His Ala Ile 210 215 220 Gly Leu Gly Gly Arg Asp Leu Ser Arg Glu Val Gly Gly Ile Ser Ala 225 230 235 240 Leu Thr Ala Leu Glu Met Leu Ser Ala Asp Glu Lys Ser Glu Val Leu 245 250 255 Ala Phe Val Ser Lys Pro Pro Ala Glu Ala Val Arg Leu Lys Ile Val 260 265 270 Asn Ala Met Lys Ala Thr Gly Lys Pro Thr Val Ala Leu Phe Leu Gly 275 280 285 Tyr Thr Pro Ala Val Ala Arg Asp Glu Asn Val Trp Phe Ala Ser Ser 290 295 300 Leu Asp Glu Ala Ala Arg Leu Ala Cys Leu Leu Ser Arg Val Thr Ala 305 310 315 320 Arg Arg Asn Ala Ile Ala Pro Val Ser Ser Gly Phe Ile Cys Gly Leu 325 330 335 Tyr Thr Gly Gly Thr Leu Ala Ala Glu Ala Ala Gly Leu Leu Ala Gly 340 345 350 His Leu Gly Val Glu Ala Asp Asp Thr His Gln His Gly Met Met Leu 355 360 365 Asp Ala Asp Ser His Gln Ile Ile Asp Leu Gly Asp Asp Phe Tyr Thr 370 375 380 Val Gly Arg Pro His Pro Met Ile Asp Pro Thr Leu Arg Asn Gln Leu 385 390 395 400 Ile Ala Asp Leu Gly Ala Lys Pro Gln Val Arg Val Leu Leu Leu Asp 405 410 415 Val Val Ile Gly Phe Gly Ala Thr Ala Asp Pro Ala Ala Ser Leu Val 420 425 430 Ser Ala Trp Gln Lys Ala Cys Ala Ala Arg Leu Asp Asn Gln Pro Leu 435 440 445 Tyr Ala Ile Ala Thr Val Thr Gly Thr Glu Arg Asp Pro Gln Cys Arg 450 455 460 Ser Gln Gln Ile Ala Thr Leu Glu Asp Ala Gly Ile Ala Val Val Ser 465 470 475 480 Ser Leu Pro Glu Ala Thr Leu Leu Ala Ala Ala Leu Ile His Pro Leu 485 490 495 Ser Pro Ala Ala Gln Gln His Thr Pro Ser Leu Leu Glu Asn Val Ala 500 505 510 Val Ile Asn Ile Gly Leu Arg Ser Phe Ala Leu Glu Leu Gln Ser Ala 515 520 525 Ser Lys Pro Val Val His Tyr Gln Trp Ser Pro Val Ala Gly Gly Asn 530 535 540 Lys Lys Leu Ala Arg Leu Leu Glu Arg Leu Gln 545 550 555 <210> 3 <211> 1668 <212> DNA <213> Escherichia coli <400> 3 atgatccacg cctttattaa aaaagggtgt tttcaggatt cggtcagttt aatgattatt 60 tcacgaaaac tcagcgaatc agaaaatgtt gatgatgttt ccgtaatgat gggtacgccc 120 gccaataaag cgttattaga taccacaggt ttctggcatg acgattttaa taacgccacg 180 ccgaacgata tttgcgtggc aattcgtagc gaagcggcgg atgcggggat cgcgcaggcg 240 attatgcagc agcttgaaga ggcgctaaaa caactggcgc aggggtcagg cagcagccag 300 gcgttgacgc aggtgcgtcg ctgggacagt gcctgtcaga aattacccga tgccaatctg 360 gcgctgattt cagtggctgg cgagtatgcg gcggagctgg caaaccaggc gctggatcgc 420 aacctcaacg tgatgatgtt ctccgataac gtcacgctgg aagatgaaat ccaacttaaa 480 acccgcgcgc gggaaaaagg cttgctggtg atggggccgg actgcggtac gtcgatgatt 540 gccggcacac cgctggcttt tgctaacgtg atgccggaag gcaatattgg cgtcattggc 600 gcttccggta ccgggattca ggagctgtgt tcgcagattg cgctggcagg ggagggaatt 660 actcacgcga ttggccttgg cgggcgcgac ctcagccgtg aagtgggcgg catcagtgcg 720 ctaacagcgc tggaaatgct cagtgcagac gagaaaagcg aagtgctggc atttgtttca 780 aaaccacctg ccgaagctgt gcgtctgaaa attgttaatg ccatgaaagc aaccggcaaa 840 ccgacggtgg cgctgttttt aggttatacc ccggcggtgg cccgcgacga gaatgtctgg 900 tttgcctcct cgctggatga ggccgcacgc ctggcttgcc tgctttcacg cgtcacggcg 960 cgacgtaacg caatagcgcc tgtcagcagc ggatttattt gcggtttgta taccggcggt 1020 acgctggctg ccgaagcggc gggattactt gccggacacc ttggcgtgga agccgacgat 1080 acccatcaac atggcatgat gctggacgcc gatagccacc agattattga cctcggcgat 1140 gatttctaca ccgtcgggcg tccccatccg atgatcgacc caaccttacg caaccagtta 1200 attgccgatc tcggcgctaa accgcaagtg cgcgtgttgc tgcttgatgt cgtgattggc 1260 ttcggtgcga ccgccgatcc tgccgcctcg ctggtgagcg cctggcaaaa agcctgtgcc 1320 gcgcgtttag ataatcaacc actgtatgcc attgccacgg tgacaggcac tgaacgtgac 1380 ccgcaatgcc gctcgcagca aatcgccacg ctggaagatg cggggattgc ggtcgtgagt 1440 tcgctaccgg aagccacctt gctggcggca gcgttaattc atccgctctc gcctgccgca 1500 cagcaacaca caccgtcatt actggaaaac gtcgccgtga ttaacatcgg attacgcagc 1560 tttgcgctgg agctacaaag cgccagcaaa ccggttgtgc attaccaatg gtcgccagtc 1620 gccggtggca ataaaaaaact ggctcgttta ttagaacgtt tgcaataa 1668

Claims (10)

Escherichia having the ability to produce succinic acid, comprising an FdrA variant consisting of the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to the 296th position of SEQ ID NO: 2, is replaced with tyrosine, or a polynucleotide encoding the variant. ( Escherichia ) genus microorganisms.
The microorganism according to claim 1, wherein the microorganism has an increased succinic acid production ability compared to a microorganism of the Escherichia genus containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same.
The microorganism according to claim 1, wherein the microorganism has an increased oxamate production ability compared to a microorganism of the Escherichia genus containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same.
The microorganism according to claim 1, wherein the succinic acid is produced by consuming glycerol through glycerol fermentation.
The microorganism according to any one of claims 1 to 4, wherein the microorganism of the Escherichia genus is Escherichia coli .
A microorganism of the Escherichia genus containing an FdrA variant consisting of the amino acid sequence shown in SEQ ID NO: 1, in which aspartic acid, the amino acid corresponding to the 296th position of SEQ ID NO: 2, is replaced with tyrosine, or a polynucleotide encoding the variant. A method for producing succinic acid comprising culturing in a medium.
The method of claim 6, wherein the microorganism has an increased succinic acid production ability compared to a microorganism of the Escherichia genus containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same.
The method of claim 6, wherein the microorganism has an increased oxamate production ability compared to a microorganism of the Escherichia genus containing the polypeptide of SEQ ID NO: 2 or a polynucleotide encoding the same.
The method of claim 6, wherein the succinic acid is produced by consuming glycerol through glycerol fermentation.
The method according to any one of claims 6 to 9, further comprising recovering succinic acid from the cultured microorganism or culture.