# Patent

## Title
Enzyme used in biosynthesis of 1, 4-BDO and screening method of the same

## Abstract

Recombinant butyraldehyde dehydrogenases (Blds) with improved production of 1,4-BDO, as well as recombinant microorganisms comprising polynucleotides encoding the recombinant Blds, and methods of producing 1,4-BDO by using the recombinant microorganisms.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Korean Patent Application No. 10-2012-0083513, filed on Jul. 30, 2012 and Korean patent application no. 10-2013-0117595, filed on Oct. 1, 2013, in the Korean Intellectual Property Office, and is a continuation-in-part of co-pending U.S. patent application Ser. No. 13/954,696, filed on Jul. 30, 2013, the entire disclosures of which are hereby incorporated by reference.


INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 93,422 Byte ASCII (Text) file named 718229 _ST25-Revised created on Mar. 11, 2016.
BACKGROUND
1. Field
The present disclosure relates to butyraldehyde dehydrogenases, which are improved for efficient production of 1,4-BDO, and transformed strains containing the same, and methods of producing 1,4-BDO with high-efficiency by using the transformed microorganism.
2. Description of the Related Art
1,4-utanediol(1,4-BDO), as a solvent used annually about 130 million tons worldwide, is produced from petroleum-based substances such as acetylene, butane, propylene, and butadiene.
1,4-BDO is used throughout the chemical industry as a polymer, a solvent, or a fine chemical intermediate of a variety of chemicals. Currently, most chemical substances composed of 4 carbon atoms are derived from 1,4-BDO, maleic anhydride, or the like and are synthesized. However, as oil prices are increasing, the cost of production is also increasing, bringing attention to develop a complementary and an alternative process of chemical production. Herein, a biological process using a microorganism is presented as an alternative to the chemical production process.
Unlike the existing chemical methods, Genometica built a biosynthetic pathway of 1,4-BDO in 2011 by using succinyl-CoA synthetase gene (sucCD) from Clostridium kluyveri, CoA-dependent succinate semialdehyde dehydrogenase gene (sucD) from Porphyromonas gingivalis, NAD dependent 4-hydroxybutyrate dehydrogenase gene (4hbd) from P. gingivalis, 4-hydroxybutyryl CoA:acetyl-CoA transferase gene (cat2) from P. gingivalis, and alcohol dehydrogenase gene (adhE2) from Clostridium acetobutylicum within Escherichia coli (E. coli).
According to an embodiment, pathways that are already identified in E. coli are modified to construct a new biosynthetic pathway. For example, a microorganism is developed for efficient production of 1,4-BDO with a Bld mutant that is appropriate for the new pathway.
SUMMARY
Provided are recombinant butyraldehyde dehydrogenases (Blds) used to produce 1,4-BDO with high-efficiency. In particular, provided is an isolated, non-naturally occurring (e.g., synthetic or recombinant) polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising a substitution of at least one amino acid selected from the group consisting of Asn409, Arg361, Ala467, Met371, Ala176, Leu273, and Lys279 in the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide catalyzes the conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde.
Provided are transformed (recombinant) microorganisms comprising polynucleotides encoding the recombinant Bld to produce 1,4-BDO with high-efficiency.
Provided are transformed microorganisms comprising polynucleotides encoding the recombinant Bld and butanol dehydrogenase (Bdh) for use in producing 1,4-BDO with high-efficiency.
Provided are transformed microorganisms comprising polynucleotides encoding sucCD (or cat1), sucD, 4hbd, cat2, recombinant Bld, and bdh for use in producing 1,4-BDO with high-efficiency.
Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1A depicts a biosynthetic pathway of 1,4-BDO built in E. coli. 
FIG. 1B depicts a biosynthetic pathway of 1,4-BDO built in E. coli. 
FIG. 2 shows the results of an aldehyde reaction when the supernatant obtained by incubating selected colonies reacts with Schiff's reagent. In FIG. 2, (a) shows the result of E. Coli TOP10 in which a foreign gene is not introduced therein; (b) shows the result of E. Coli TOP in which pSTV-cs4c and pUCM are introduced therein; and (c) shows the result of E. Coli TOP10 in which pSTV-cs4c and pUCM-bld are introduced therein.
FIG. 3 is an absorbance graph obtained at a wavelength of 540 nm after 1 hour of the reaction showing the results of an aldehyde reaction when the supernatant obtained by incubating selected colonies reacts with Schiff's reagent. A bld with a good activity results in the production of a lot of 4-hydroxybutyraldehydes, which is confirmed to be useful in a screening method using Schiff's reagent.
FIG. 4 is a graph showing the yield of 1,4-BDO according to a bld mutant by introducing cs4c (cat1, sucD, 4hbd, and cat2 genes) and a bdh to Bld-WT and various Bld mutants (Bld-M1 to Bld-M5) in a microorganism. As a positive control, cs4c and adhE were introduced to a microorganism and the yield of 1,4-BDO was determined.
FIG. 5 is a graph of 1,4-BDO concentration for different Bld mutants, which illustrates which mutation position selected from Bld-M1 to Bld-M5 shown in Table 3 induces the most activity. Bld-S1 to Bld-S6 mutants were produced, and the yield of 1,4-BDO was confirmed by using the produced butyraldehyde dehydrogenase mutants. As a result, it was confirmed that 1,4-BDO was substantially produced in the case of Bld-S2 mutant, and a mutant having a substitution at the 273rd position of Bld-WT (see SEQ ID NO: 1) had the greatest 1,4-BDO productivity.
FIG. 6 shows common sequences by comparing a butyraldehyde dehydrogenase with sequences of proteins that are predicted to have a similar activity with the butyraldehyde dehydrogenase.
FIG. 7A is an image of the three-dimensional structure of Bld.
FIG. 7B is a drawing depicting the catalytic site of the Bld, and its substrate, NADPH.
FIG. 7C is a drawing depicting the three-dimensional structure of Bld, an amino acid that affects the activity of the Bld, and NADPH as the substrate.
FIG. 7D is a drawing depicting the three-dimensional structure of Bld and an amino acid that affects the activity of the Bld.
FIG. 8 is a graph displaying the activity levels of six L273X mutations, which shows relative titers of 1,4-BDO with respect to E. coli that expresses Bld-WT, such as CS4C and Bdh, and 6 mutations.
FIG. 9A is a graph displaying specific activities measured in purified Bld-WT, L273I, and L273T, by measuring concentration of NADH in a reaction mixture including butyryl-CoA as a substrate.
FIG. 9B is a graph displaying specific activities measured in purified Bld-WT, L273I, and L273T, by measuring concentration of NADH in a reaction mixture including purified Bdh and butyl-CoA as a substrate.



DETAILED DESCRIPTION
One aspect of the present invention provides a recombinant butyraldehyde dehydrogenase (Bld).
According to an aspect of the prevention invention, a butyraldehyde dehydrogenase or a butyraldehyde dehydrogenase mutant having a catalytic activity of converting 4-hydroxybutyryl-CoA into 4-hydroxybutyraldehyde is provided.
The bld may be a gene derived from Clostridium saccharoperbutylacetonicum. The Bld may have a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
The term “polynucleotide” used in the specification comprehensively refers to DNA (gNDA and cDNA) and RNA molecules. A nucleotide, the basic building unit in a polynucleotide, includes not only a natural nucleotide, but also an analogue wherein sugar or a base is modified.
The butyraldehyde dehydrogenase may comprise the amino acid sequence of SEQ ID NO: 1. In addition, the butyraldehyde dehydrogenase mutant may comprise a variant of SEQ ID NO: 1, wherein Leu273 is substituted with lle, Cys, Met, Ser, Thr, or Val. Additionally, the butyraldehyde dehydrogenase mutant may comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7
For example, the butyraldehyde dehydrogenase mutant may have a substitution as detailed below:
Asn409 with Thr, Arg361 with Ser, and Ala467 with Ser, in the amino acid sequence of SEQ ID NO: 1;
Arg361 with Ser and Ala467 with Ser in the amino acid sequence of SEQ ID NO: 1;
Met371 with Arg, Arg361 with Ser, and Ala467 with Ser in the amino acid sequence of SEQ ID NO:1;
Ala176 with Thr, Leu273 with lle, Lys279 with Arg, Arg361 with Ser, and Ala467 with Ser in the amino acid sequence of SEQ ID NO: 1;
Ala176 with Thr in the amino acid sequence of SEQ ID NO: 1;
Leu273 with lle in the amino acid sequence of SEQ ID NO: 1;
Lys279 with Arg in the amino acid sequence of SEQ ID NO: 1;
Arg361 with Ser in the amino acid sequence of SEQ ID NO: 1;
Ala467 with Ser in the amino acid sequence of SEQ ID NO: 1;
Asn409 with Thr in the amino acid sequence of SEQ ID NO: 1; and/or
Met371 with Arg in the amino acid sequence of SEQ ID NO: 1.
Also, the catalytic site of the mutant may have a substitution of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) amino acid selected from the group consisting of Thr43, Asn144, Ala241, Gly242, Ala243, Gly244, Pro246, Leu273, Pro274, lle276, Ala277, Lys279, Glu368, His398, Val432, and Thr441 in the amino acid sequence of SEQ ID NO: 1.
For example, the catalytic site of the mutant may have a substitution of Thr43 with Asp, Asn144 with Asp, Ala241 with Val, Gly242 with Ser, Ala243 with Gly, Gly244 with Ser, Pro246 with Tyr, Leu273 with lle, Cys, Ser, Thr, or Val, Pro274 with Tyr, lle276 with Leu, Ala277 with Val, Lys279 with Arg, Glu368 with Gln, His398 with Lys, Val432 with Leu, and Thr441 with Asp in the amino acid sequence of SEQ ID NO: 1.
Also, the mutant may have a substitution of at least one amino acid selected from the group consisting of Met91, lle139, Thr140, Pro141, Ser142, Thr143, Asn166, Gly167, His168, Pro169, Gly170, Asn201, Pro202, Thr203, Met204, Leu207, Asp208, lle210, Lle211, Lys212, Thr222, Gly223, Gly224, Pro225, Met227, Thr230, Leu231, Ala241, Gly242, Ala243, Gly244, Leu273, Pro274, Cys275, Ser326, lle327, Asn328, Lys329, Val332, Thr367, Glu368, Leu369, Met370, and Arg396 in the amino acid sequence of SEQ ID NO: 1 with other amino acid.
For example, the mutant may have a substitution of Met91 with Asp, lle139 with Leu, Thr140 with Lys, Pro141 with Tyr, Ser142 with Gly, Thr143 with Lys, Asn166 with Asp, Gly167 with Ser, His168 with Lys, Pro169 with Tyr, Gly170 with Ser, Asn201 with Asp, Pro202 with Tyr, Thr203 with Lys, Met204 with Asp, Leu207 with lle, Asp208 with Asn, lle210 with Leu, lle211 with Leu, Lys212 with Thr, Thr222 with Lys, Gly223 with Ser, Gly224 with Ser, Pro225 with His, Met227 with Lys, Thr230 with Lys, Leu231 with Val, Ala241 with Val, Gly242 with Ser, Ala243 with Val, Gly244 with Ser, Leu273 with lle, Cys, Met, Thr or Val, Pro274 with His, Cys275 with Met, Ser326 with Gly, lle327 with Leu, Asn328 with Asp, Lys329 with Thr, Val332 with Leu, Thr367 with Lys, Glu368 with Gln, Leu369 with lle, Met370 with Lys, and Arg396 with Lys in the amino acid sequence of SEQ ID NO: 1.
Thus, provided is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with a substitution of at least one amino acid selected from the group consisting of Asn409, Arg361, Ala467, Met371, Ala176, Leu273, and Lys279 in the amino acid sequence of SEQ ID NO: 1 and, optionally, (a) a substitution of at least one amino acid selected from the group consisting of Thr43, Asn144, Ala241, Gly242, Ala243, Gly244, Pro246, Leu273, Pro274, lle276, Ala277, Lys279, Glu368, His398, Val432, and Thr441 in the amino acid sequence of SEQ ID NO: 1 and/or (b) a substitution of at least one amino acid selected from a group consisting of Met91, lle139, Thr140, Pro141, Ser142, Thr143, Asn166, Gly167, His168, Pro169, Gly170, Asn201, Pro202, Thr203, Met204, Leu207, Asp208, Lle210, Lle211, Lys212, Thr222, Gly223, Gly224, Pro225, Met227, Thr230, Leu231, Ala241, Gly242, Ala243, Gly244, Leu273, Pro274, Cys275, Ser326, lle327, Asn328, Lys329, Val332, Thr367, Glu368, Leu369, Met370, and Arg396 in the amino acid sequence of SEQ ID NO: 1.
In one embodiment, the butyraldehyde dehydrogenase mutant may be a polypeptide having a sequence set forth in the amino acid sequence of SEQ ID NO: 2, which is a variant of SEQ ID NO: 1 in which Leu273 has been substituted with lle.
In another embodiment, the butyraldehyde dehydrogenase mutant may be a polypeptide having a sequence set forth in SEQ ID NO: 3, which is a variant of SEQ ID NO: 1 in which Leu273 has been substituted with Cys.
In another embodiment, the butyraldehyde dehydrogenase mutant may be a polypeptide having a sequence set forth in SEQ ID NO: 4, which is a variant of SEQ ID NO: 1 in which Leu273 has been substituted with Met.
In another embodiment, the butyraldehyde dehydrogenase mutant may be a polypeptide having a sequence set forth in SEQ ID NO: 5, which is a variant of SEQ ID NO: 1 in which Leu273 has been substituted with Ser.
In another embodiment, the butyraldehyde dehydrogenase mutant may be a polypeptide having a sequence set forth in SEQ ID NO: 6, which is a variant of SEQ ID NO: 1 in which Leu273 has been substituted with Thr.
In another embodiment, the butyraldehyde dehydrogenase mutant may be a polypeptide having a sequence set forth in SEQ ID NO: 7, which is a variant of SEQ ID NO: 1 in which Leu273 has been substituted with Val.
According to another aspect of the present invention, a polynucleotide that encodes the butyraldehyde dehydrogenase or butyraldehyde dehydrogenase mutant is provided. Herein, the polynucleotide may be derived from Clostridium saccharoperbutylacetonicum. 
According to another aspect of the present invention, a recombinant microorganism that comprises the above-described polynucleotide and is capable of producing 1,4-BDO is provided.
The recombinant microorganism may further include a polynucleotide encoding butanol dehydrogenase (Bdh) having a catalytic activity of converting 4-hydroxybutyraldehyde into 1,4-butanediol. Herein, the polynucleotide encoding bdh may comprise the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8. A polynucleotide encoding the butanol dehydrogenase may comprise the nucleotide sequence of SEQ ID NO: 9.
The recombinant microorganism that is capable of producing 1,4-BDO may include an enzyme that converts succinate into succinyl-CoA, an enzyme that converts succinyl-CoA into succinate semialdehyde, an enzyme that converts succinate semialdehyde into 4-hydroxybutyrate, and/or an enzyme that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA.
The recombinant microorganism may include a polynucleotide encoding succinyl-CoA:coenzyme A transferase (e.g., cat1) or succinyl-CoA synthetase gene (e.g., sucCD) that converts succinate into succinyl-CoA, a polynucleotide encoding CoA-dependent succinate semialdehyde dehydrogenase (e.g., SucD) that converts succinyl-CoA into succinate semialdehyde, a polynucleotide encoding 4-hydroxybutyrate dehydrogenase (4Hbd) that converts succinate semialdehyde into 4-hydroxybutyrate, and a polynucleotide encoding 4-hydroxybutyryl CoA:acetyl-CoA transferase (Cat2) that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA. In one embodiment, the microorganism may be E. coli (see, e.g., Yim et al., Nat. Chem. Biol., 7(7): 445-452 (2011).
The enzyme that converts succinate into succinyl-CoA may be succinyl-CoA:coenzyme A transferase (e.g., cat1) belonging to EC 2.8.3.-. The succinyl-CoA:coenzyme A transferase may be Cat1 having the amino acid sequence of SEQ ID NO:10. The gene encoding succinyl-CoA:coenzyme A transferase Cat1 may have the nucleotide sequence of SEQ ID NO: 11.
The enzyme that converts succinyl-CoA into succinate semialdehyde may be CoA-dependent succinate semialdehyde dehydrogenase, and may be classified as an enzyme belonging to EC 1.2.1., such as EC 1.2.1.76. For example, the enzyme may be succinate semialdehyde dehydrogenase (SucD). In addition, the CoA-dependent succinate semialdehyde dehydrogenase may be a gene derived from E. coli, Clostridium, or Porphyromonas. For example, a sucD protein may have the amino acid sequence of SEQ ID NO: 12. The polynucleotide encoding the SucD may have the nucleotide sequence of SEQ ID NO: 13.
The enzyme that converts succinate semialdehyde into 4-hydroxybutyrate may be 4-hydroxybutyrate dehydrogenase, and may be classified as an enzyme belonging to EC 1.1.1. For example, the enzyme may be NAD-dependent 4-hydroxybutyrate dehydrogenase (4Hbd). Herein, a 4HB dehydrogenase may be a protein derived from E. coli, Clostridium, or Porphyromonas. For example, a 4Hbd protein may have the amino acid sequence of SEQ ID NO: 14. The polynucleotide encoding the 4HbD may have the nucleotide sequence of SEQ ID NO: 15.
The enzyme that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA may be 4-hydroxybutyryl-CoA:acetyl-CoA transferase, and may be classified as an enzyme belonging to EC 2.8.3. For example, the enzyme may be 4-hydroxybutyryl-CoA:acetyl-CoA transferase (Cat2). Herein, a 4-hydroxybutyryl-CoA transferase may be a protein derived from E. coli, Clostridium, or Porphyromonas. For example, a Cat2 protein may have the amino acid sequence of SEQ ID NO: 16. The polynucleotide encoding the Cat2 may have the nucleotide sequence of SEQ ID NO: 17.
In one embodiment, the recombinant microorganism capable of producing 1,4-BDO may be a recombinant microorganism capable of expressing the sucD protein, the 4Hbd protein, the Cat2 protein, and the Cat1 protein. The recombinant microorganism may be E. coli. 
The term “protein expression” as used herein denotes that a protein or an enzyme is present in a recombinant microorganism and has an activity. In addition, the protein or the enzyme may be present in a recombinant microorganism by which a polynucleotide encoding a protein in a recombinant microorganism is transcribed to mRNA, and the mRNA is translated to a protein. Herein, the polynucleotide encoding a protein may be inserted into a chromosome in a recombinant microorganism, or may be present within a plasmid vector.
A recombinant microorganism producing a 1,4-BDO compound may be a recombinant microorganism in which a synthesis pathway of lactate from pyruvate is inactivated or reduced. That is, an activity of L-lactate dehydrogenase (Ldh) may be removed or reduced in the recombinant microorganism. The Ldh may have a catalytic activity for converting pyruvate into lactate, and may be classified as an enzyme belonging to EC 1.1.1.27. In addition, an activity of a gene encoding the Ldh may be inactivated or attenuated in the recombinant microorganism.
The term “inactivation” used herein refers to a gene, which is not expressed at all or a gene, which is expressed/generated, but does not have a functional activity, for example, enzymatic activity. The term “attenuation” used herein refers that gene expression is reduced to a level lower than that of a wild strain, a strain that is not engineered, or a parent strain. Alternatively, the term refers that a gene is expressed, but the enzymatic activity thereof is reduced. In the recombinant microorganism, the activity of the Ldh may be reduced to a level less than 30%, 20%, or 10% than that of the Ldh in a wild type recombinant microorganism. In addition, the activity of the Ldh in the recombinant microorganism may be completely removed in the recombinant microorganism. The inactivation or attenuation of genes may be caused by homologous recombination. That is, the inactivation or attenuation of genes may be caused, by which vectors including a partial sequence of the gene sequence are transformed into cells, and the cells are cultured so that the partial sequence may be subjected to homologous recombination with endogenous cellular genes, thereby selecting the recombinant cells by the using selectable markers. The inactivation or attenuation or genes in the recombinant microorganism may result in removal or reduction of the activity of the enzyme encoded by the genes. The term “reduction” used herein refers to a relative activity of a recombinant microorganism that is engineered, compared to an activity of a recombinant microorganism that is not engineered.
The inactivation or attenuation of the lactate dehydrogenase activity in the recombinant microorganism may be caused by an Ldh-coding gene mutant. Such a mutant may be caused by a substitution, a partial or total deletion, or an addition of a nucleotide. In addition, the reduction of the lactate dehydrogenase activity in the recombinant microorganism may be caused by removal of intrinsic genes of the lactate dehydrogenase. Such removal includes not only physical removal of the genes, but also inhibition of functional expression of the genes. In addition, such removal may be caused by homologous recombination.
The term “transformation” used in the specification refers to introduction of genes into a recombinant microorganism for gene expression therein. As long as the transformed genes are expressed in the recombinant microorganism, the transformed genes may be inserted into a chromosome in the recombinant microorganism, or may be present in any location other than the chromosome. In addition, the transformed genes may include polynucleotides such as DNA and RNA, which may encode polypeptides. The transformed genes may be introduced into the recombinant microorganism in any form, so long as they are introduced and expressed therein. For example, the transformed genes may be introduced into the recombinant microorganism in a form of an expression cassette, which is a polynucleotide structure including all the elements necessary for autonomous gene expression. Such an expression cassette typically includes a promoter that is operably linked to the genes, a transcription termination signal, a ribosome-binding site, and a translation termination signal. The expression cassette may be in a form of a vector to enable self-replication. In addition, the transformed genes may be introduced into a host in a form of the gene itself, or in a form of a polynucleotide structure, so as to be operably connected with sequences required for expression in the recombinant microorganism.
Also, a recombinant vector (e.g., expression vector) comprising a polynucleotide that encodes the recombinant Bld is provided.
The term “vector” refers to a DNA composite including DNA sequences operably connected with appropriate regulatory sequences that are capable of expressing DNA within an appropriate host. The vector may be a plasmid vector, a bacteriophage vector, a cosmid vector, a viral vector, or the like.
The vector (e.g., expression vector) can comprise a replication origin, a promoter, a multiple cloning site (MCS), and/or a selection marker. A replication origin enables a plasmid to replicate separately from a chromosome of a host. A promoter functions in the process of transcription of an inserted foreign gene. A MCS enables a foreign gene to be inserted via various restriction enzyme sites, and a selection marker confirms that a vector is properly inserted in a host cell. A selection marker includes antibiotic resistance genes that are commonly used in the art. Examples of the resistance genes are genes that are resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. For example, in consideration of costs, the resistance genes may be genes that are resistant to ampicillin or gentamicin.
When the vector according to the present invention has a prokaryotic cell as a host, the vector may include a strong promoter, such as lambda PL promoter, trp promoter, lac promoter, T7 promoter, or the like. Meanwhile, when the vector has a eukaryotic cell as a host, the vector may include a promoter derived from the genome of mammalian cells (e.g., metallothionein) or a promoter derived from mammalian viruses (e.g., adenovirus late promoter, Vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV). Exemplary promoters include the lambda PL promoter, trp promoter, lac promoter, or T7 promoter. Such promoters preferably are operably connected (i.e., linked) with foreign polynucleotide sequences (genes or cDNA) that encode a proteins of interest (e.g., Bld or mutants thereof).
The term “operably connected” refers to a functional connection between nucleic acid expression regulatory sequences (e.g., a promoter, a signal sequence, or an array on a transcription regulatory factor-binding site) and other nucleic acid sequences. Due to the operable connection, the regulatory sequences regulate a transcription and/or a translation of the nucleic acid sequences that encode the proteins of interest (e.g., Bld or mutants thereof).
According to another aspect of the present invention, a method of producing 4-hydroxybutyaldehyde includes contacting 4-hydroxybutyryl-CoA with butyraldehyde dehydrogenase or a butyraldehyde dehydrogenase mutant. The butyraldehyde dehydrogenase or butyraldehyde dehydrogenase mutant may comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, respectively.
According to another aspect of the present invention, a method of producing an 1,4-butanediol includes contacting a 4-hydroxybutyaldehyde with butanol dehydrogenase, thereby producing 1,4-butanediol. The Bdh may be encoded by the nucleotide sequence of SEQ ID NO: 9.
According to another aspect of the present invention, a method of producing 1,4-BDO includes contacting 4-hydroxybutyryl-CoA with Bld or a Bld mutant; and contacting the resultant reaction product with a bdh, thereby producing 1,4-BDO.
According to another aspect of the present invention, a method of producing 1,4-BDO includes introducing butyraldehyde dehydrogenase or a butyraldehyde dehydrogenase mutant, and a bdh to a microorganism; incubating the microorganism; and separating 1,4-BDO from the incubation product. The step of incubating may be culturing the microorganism in a medium to produce 1,4-BDO into the microorganism or into the extracellular medium. The medium any be a liquid or solid medium containing nutrients and/or minerals. The nutrients may include a carbon source, nitrogen source, etc.
In each of the above-described methods, the butyraldehyde dehydrogenase, butyraldehyde dehydrogenase mutant, and/or bdh may be introduced as a polynucleotide (e.g., cDNA or vector) or polypeptide.
Available carbon sources that the microorganism may be monosaccharide, disaccharide, polysaccharide, or the like. For example, glucose, fructose, mannose, galactose, or the like may be used. Also, available nitrogen sources that the microorganism may be organic nitrogen compounds, inorganic nitrogen compounds, or the like. For example, amino acids, amides, amines, nitrates, ammonium salts, or the like may be used. An oxygen condition for incubating the microorganism may be an aerobic condition of normal oxygen partial pressure, a hypoxic condition containing from larger than 0 to 10%, for example, 0.1 ̃10% oxygen of the saturation concentration, or an oxygen-free anaerobic condition.
The term “microaerobic condition” used herein refers to a condition having less oxygen than oxygen concentration obtained when the culture medium is in contact with a normal atmosphere. The microaerobic condition or anaerobic condition may be formed by, for example, supplying carbon dioxide or nitrogen at a flow rate in a range of about 0.1 to about 0.4 volume per volume per minute (vvm), for example, about 0.2 to about 0.3 vvm, or about 0.25 vvm. In addition, the microaerobic condition or the anaerobic condition may have a flow rate in a range of about 0 to about 0.4 vvm, about 0.1 to about 0.3 vvm, about 0.15 to about 0.25 vvm.
The introduction steps of the above-described methods may include introducing a polynucleotide (e.g., gene or cDNA) encoding sucCD or Cat1 that converts succinate into succinyl CoA, a polynucleotide (e.g., gene or cDNA) encoding sucD that converts succinyl-CoA into succinate semialdehyde, a polynucleotide (e.g., gene or cDNA) encoding 4hbd that converts succinate semialdehyde into 4-hydroxybutyrate, and a polynucleotide (e.g., gene or cDNA) encoding cat2 that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA.
According to another aspect of the present invention, a method of confirming the yield of 1,4-BDO comprises introducing Bld or a Bld mutant to a microorganism; contacting the microorganism with Schiff's reagent; and measuring absorbance. Herein, the confirmation of the yield of 1,4-BDO yield is performed by measurement of the yield of 4-hydroxybutyraldehyde.
EXAMPLES
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description.
Example 1
Host Cell for Transformation and Manufactured Expression Vector for Transformation
Recombinant microorganisms used to efficiently produce 1,4-BDO and expression vectors used to transform the microorganisms are shown in Table 1 below.









TABLE 1





Strains and plasmids
Relevant properties
Source or reference







Strains




Escherichia coli XL1-Blue
F′::Tn10 proA+B+ lacIq Δ(lacZ)M15/recA1
Stratagene



endA1 gyrA96 (NaIr)



thihsdR17 (rK−mK+) glnV44 relA1 lac


E. Coli TOP10
F- mcrA Δ(mrr-hsdRMS-mcrBC)
Invitrogen



φ80lacZΔM15 ΔlacX74 nupG recA1



araD139 Δ(ara-leu)7697 galE15 galK16



rpsL(StrR) endA1


E. Coli BL21 (DE3)
F- ompT gal [dcm] [lon] hsdSB (rB− mB−; an
NEB



E. coli B strain) with DE3, a λ prophage



carrying the T7 RNA polymerase gene


Clostridium
Source for bld and bdh
KCTCa


saccharoperbutylacetonicum


KCTC 5577


Clostridium
Source for adh1, adhE1, adhE2, bdhA, and
KCTC


acetobutylicum KTCT 1790
bdhB


Plasmids


pUCM
Cloning vector modified from pUC19;
(Kim et al., 2010)



constitutive lac promoter, Apr


pUCM-bld
Constitutively expressed bld of
This study



C. saccharoperbutylacetonicum


pUCM-adh1
Constitutively expressed adh1 of
This study



C. acetobutylicum


pUCM-adhE1
Constitutively expressed adhE1 of
This study



C. acetobutylicum


pUCM-adhE2
Constitutively expressed adhE2 of
This study



C. acetobutylicum


pUCM-bdhA
Constitutively expressed bdhA of
This study



C. acetobutylicum


pUCM-bdhB
Constitutively expressed bdhB of
This study



C. acetobutylicum


pUCM-bdh
Constitutively expressed bdh of
This study



C. saccharoperbutylacetonicum


pUCM-bld-M1-5 series
Constitutively expressed bld mutant 1-5
This study



generated by random mutagenesis


pUCM-bld-S1-6 series
Constitutively expressed Bld mutant A176T,
This study



L273I, K279R, M371R, N409T, or A467S.


pUCM-bld-L273X series
Constitutively expressed 18 Bld mutants
This study



having different amino acids at the position



of Leu273 except for L273I


pBBR1MCS2
Broad-host-range plasmid, Kmr
(Kovach et al., 1995)


pBBR-bdh
Constitutively expressed bdh of
This study



C. saccharoperbutylacetonicum, Kmr


pBBR-bdhA
Constitutively expressed bdhA of
This study



C. acetobutylicum


pBBR-bdhB
Constitutively expressed bdhB of
This study



C. acetobutylicum


pSTV28
Plasmid with a replication origin of
Takara



pACYC184, Cmr


pSTV-cs4c
Constitutively expressed cat1, sucD, 4hbd,
This study



and cat2 together


pET21a
f1 origin, T7 promoter, C-terminal His-tag
Novagen



sequence, Apr


pET-bld_WT
Inducible expression of Hig6-tagged wild-
This study



type Bld on pET21a


pET-bld_L273I
Inducible expression of Hig6-tagged Bld
This study



L273I on pET21a


pET-bld_L273T
Inducible expression of Hig6-tagged Bld
This study



L273T on pET21a


pET-AdhE2
Inducible expression of Hig6-tagged AdhE2
This study



on pET21a


pET-Bdh
Inducible expression of Hig6-tagged Bdh on
This study



pET21a









    1. Kim, S. H., Y. H. Park, C. Schmidt-Dannert, and P. C. Lee. 2010. Redesign, reconstruction, and directed extension of the brevibacterium linens C40 carotenoid pathway in Escherichia coli. Applied and Environmental Microbiology 76:5199-5206.
    2. Kovach M E. Et al., 1995. Four new derivatives of the broad-host-range cloning vector pBBR1 MCS, carrying different antibiotic-resistance cassettes. Gene 166:175-176.


Example 2
Modularization of Biosynthetic Pathway Genes
cat1-sucD-4hbd-cat2 genes synthesized in a pGEM vector were cloned at positions of Xba I and Not I of a pUCM vector with a promoter. Then, subcloning was performed at positions of Sac I and BamH I of a pSTV 28 vector to obtain pSTV28-cs4C (SEQ ID NO:98).
AdhE2 was amplified from Clostridium acetobutylicum's chromosomal DNA by PCR, and then, cloning was performed at positions of Xba I and Not I of pUCM vector. PCR was performed by using DNA engine thermal cycler (Bio-Rad), for 4 minutes at the temperature of 95° C., following by 1 minute at the temperature of 94° C., 40 seconds at the temperature of 50° C., and 1 minute at the temperature of 72° C., and the latter three processes were repeatedly performed 32 times. Finally the PCR was further performed at the temperature of 72° C. for 7 minutes.
DNA sequences for each primer are shown in Table 2 below.










TABLE 2








Enzyme


Gene
Sequence
SEQ ID NO
site


















bdh
F; 5′-GCTCTAGAAGGAGGATTACAAAATGGAGAATTT
SEQ ID NO: 18
Xba I



TAGATTTAATG





R; 5′-TTCCCTTGCGGCCGCTTAAAGGGACATTTCTAA
SEQ ID NO: 19
Not I


 


bld
F; 5′-GCCCCGGGAGGAGGATTACAAAATGATTAAAGA
SEQ ID NO: 20
Xma I



CACGCTAGTTTC





R; 5′-TTCCCTTGCGGCCGCTTAACCGGCGAGTA
SEQ ID NO: 21




CACATC




 


cs4c
F; 5′-GCTCTAGAAGGAGGATTACAAAATGAGTAAAGG
SEQ ID NO: 22
Xba I



GATTAAGAAC





R; 5′-TTCCCTTGCGGCCGCTTAACCAAAACGTTT
SEQ ID NO: 23
Not I



GCG




 


Sub_BamH
R; 5′-CGGGATCCCGGTGTGAAATACCG
SEQ ID NO: 24
BamH I


I_R





 


Sub_EcoR
R; 5′-GAATTCCGGTGTGAAATACCG
SEQ ID NO: 25
EcoR I


I_R





 


Sub_Sacl_F
F; 5′-GAGCTCCCGACTGGAAAGCG
SEQ ID NO: 26
Sac I


 


Sub_Sall_F
F; 5′-ACGCGTCGACCCGACTGGAAAGCG
SEQ ID NO: 27
Sall


adhE2





 



F; 5′-GCTCTAGAAGGAGGATTACAAAATGATTTTGCA
SEQ ID NO: 28
Xbal



TCTGCTG





R; 5′-TTCCCTTGCGGCCGCTTAAAACGACTTGAT
SEQ ID NO: 29
Notl



GTAGAT




 


adh1
F; 5′-GCTCTAGAAGGAGGATTACAAAATGATGAGATT
SEQ ID NO: 30
Xbal



TACATTACCAAG





R; 5′-TTCCCTTGCGGCCGCTTAAAAATCAACTTC
SEQ ID NO: 31
Notl



TGTACC




 


adhE1
F; 5′-GCTCTAGAAGGAGGATTACAAAATGAAAGTCAC
SEQ ID NO: 32
Xbal



AACAGTAAAG





R; 5′-TTCCCTTGCGGCCGCTTAAGGTTGTTTTTT
SEQ ID NO: 33
Notl



AAAAC




 


adhE2
F; 5′-GCTCTAGAAGGAGGATTACAAAATGATTTTGCA
SEQ ID NO: 34
Xbal



TCTGCTG





R; 5′-TTCCCTTGCGGCCGCTTAAAACGACTTGAT
SEQ ID NO: 35
Notl



GTAGAT




 


bdhA
F; 5′-GCTCTAGAAGGAGGATTACAAAATGCTAAGTTT
SEQ ID NO: 36
Xbal



TGATTATTCA





R; 5′-TTCCCTTGCGGCCGCTTATAAGATTTTTTA
SEQ ID NO: 37
Notl



AATATCTC




 


bdhB
F;5′-GCCCCGGGAGGAGGATTACAAAATGGTTGATTTC
SEQ ID NO: 38
Xmal



GAATATTCAATAC





R; 5′-TTCCCTTGCGGCCGCTTACACAGATTTTTTGAA
SEQ ID NO: 39
Notl



TATTTG




 


bld
F; 5′-GCGAATTCATGATTAAAGACACGCT
SEQ ID NO: 40
EcoRl


(pET21a)
AGTTTC





R; 5′-AAAACTCGAGACCGGCGAGTACACATCT
SEQ ID NO: 41
Xhol


 


adhE2
F; 5′-GCGGATCCATGATTTTGCATCTGCTGCGA
SEQ ID NO: 42
BamHI


(pET21a)






R; 5′-AAAACTCGAGAAACGACTTGATGTAG
SEQ ID NO: 43
Xhol



ATATCC




 


bdh
F; 5′-GCGAATTCATGGAGAATTTTAGATTTAAT
SEQ ID NO: 44
EcoRl


(pET21a)





 
R; 5′-AAAACTCGAGAAGGGACATTTCTAAAATTT
SEQ ID NO: 45
Xhol



TATA









Example 3
Genetic Screening of Bld and Bdh Genes
In order to produce 4-hydroxybutyryl-CoA in E. coli, a vector (pSTV-cs4C) was manufactured. The synthesized CS4C was used to express cat1 genes, sucD genes, 4hbd genes, and cat2 genes. In the case of expression of the CS4C module, 1,4-BDO is manufactured in a small amount (about up to 2.0 mg/L). In order to examine the production of 1,4-BDO, Bld, Adh1, and AdhE2 were analyzed. As a result, it was found that the Bld was an enzyme converting 4-hydroxybutyryl-CoA into 4-hydroxybutyraldehyde. Regarding the production of 1,4-BDO with respect to the three candidates above, i.e., Bld, Adh1, and AdhE2, it was confirmed that 29 mg/L of 1,4-BDO was produced with respect to AdhE2, 10 mg/L of 1,4-BDO was produced with respect to bld, and 1.8 mg/L of 1,4-BDO was produced with respect to Adh1.
Thereafter, the production of 1,4-BDO was confirmed with respect to the CS4C module, bld, and three candidates, i.e., Bdh, BdhA, and BdhB. It was confirmed that Bdh has a catalytic activity for converting 4-hydroxybutyraldehyde into 1,4-BDO. As a result of the expression, with respect to the CS4C module, bld, and each of 3 enzymes, 1,4-BDO was produced as the following. 19 mg/L of 1,4-BDO was produced with respect to Bdh, 16 mg/L of 1,4-BDO was produced with respect to BdhB, and 15 mg/L of 1,4-BDO was produced with respect to BdhA.
Example 4
Genetic Improvement and Screening
<4-1> Manufacture of Bld Mutants
A bld gene was transformed by directed evolution to increase the production of 1,4-BDO. Sequences of the bld gene were changed by error prone PCR. In this regard, 2.5 mM MgCl2 and a subcloning primer were used. By using G-rich dNTP (T:A:C:G=1:1:1:4) and T-rich dNTP (T:A:C:G=4:1:1:1) separately, a variety of errors were increased. These bld mutants were inserted at positions of Xma I and Not I of a pUCM vector to produce pUCM-bld.
<4-2> Screening of Bld mutants for highly efficient production of 1,4-BDO
pUCM-bld was introduced to TOP10 to which the pSTV 28-sucCD-sucD-4hbd-cat2 (pSTV-cs4c) vector was introduced.
Schiff's reagent was used to search for a bld mutant that is capable of increasing the yield among libraries. Schiff's reagent was a solution including 30 mg/ml sodium bisulfate (in water), 0.5 M KCl (in water), and 2 mg/ml pararosaniline (in ethanol) which were mixed at a ratio of 2:1:2, and the solution was added to 0.8% agar (in water) for reaction on a plate with colonies floating thereon. After mixing the two solutions, the mixture was poured onto a plate and a reaction was performed at 37° C. for 3 hours. Then, red colonies were selected and incubated on 2 ml LB culture under the conditions of 37° C., 250 rpm, and 12 hours. 200 μl of the supernatant (obtained by centrifuging 1 ml of the culture medium at 13,000 rpm for 10 minutes) and 100 μl of Schiff's reagent were mixed together and reacted at 37° C. for about 1 to 5 hours. Absorbance was measured at 540 nm. Colonies showing high absorbance were introduced to E. Coli TOP 10 along with pSTV-cs4c and pBBR-bdh and incubated (see FIG. 3).
Example 5
E. coli Incubation and 1,4-BDO Production
E. coli strain TOP10 was used to produce 1,4-BDO by the cloning and expression of the gene modules.
Recombinant E. coli including 3 plasmids (pSTV-cs4c, pBBR-bdh, and pUCM-bld) were incubated using a serum bottle under anaerobic conditions of 30° C., 250 rpm, and 48 hours. The medium composition was 100 ml of LB containing 0.6% calcium carbonate and 2% glucose, and 50 μg/ml chloramphenicol, 100 μg/ml ampicillin, and 50 μg/ml kanamycin were all added thereto.
An incubation condition was prepared as an anaerobic condition by injecting nitrogen and the incubation was performed at 30° C., 250 rpm, and 18 hours. The medium composition was 1 L of LB medium including 2% glucose, and 50 μg/ml chloramphenicol, 100 μg/ml ampicillin, and 50 μg/ml kanamycin were all added thereto.
When the modulated genes that were associated with biosynthesis of 1,4-BDO were transformed within E. coli by the method mentioned above, the recombinant E. coli produced 1,4-BDO. However, less 1,4-BDO was produced since 4-hydroxybutyrate accumulated first. Therefore, experiments were designed in a way that 4-hydroxybutyraldehydes were produced in great quantities to make a biosynthetic pathway towards 1,4-BDO.
Example 6
Analysis of 1,4-BDO
1 ml of 100 ml culture obtained in Example 4 was extracted and centrifuged at 13000 rpm for 30 minutes, and the supernatant was centrifuged again under the same condition. Then 800 μl was filtered through a 0.45 um filter to prepare a sample. 10 μl of the sample was used for HPLC analysis. HPLC was performed by using Agilent 1100 device equipped with Refractive index detector (RID). 4 mM H2SO4 solution was used as a mobile phase and BIO-RAD Aminex HPX-87H Column was used as a stationary phase wherein the flow rate is 0.7 ml/min. Temperature of the column and detector was both 50° C.
The yield of 1,4-BDO was analyzed and results show that more 1,4-BDO was produced when the mutant bld gene was introduced and incubated than when the existing Bld gene was expressed with cs4c and bdh genes within E. coli TOP10. The Bld-M2 sample produced about 0.04 g/L concentration of 1,4-BDO, more than twice compared to others (see FIG. 4). Bld-M1, Bld-M3, Bld-M4, and Bld-M5 samples also showed higher 1,4-BDO productivity than the control (Bld-WT) (see FIG. 4). As a result of analyzing nucleotide sequences of the Bld mutants, the sequences were identified as shown in Table 3.
From the results above, it was confirmed that when the butyraldehyde dehydrogenase has high activity, more 4-hydroxybutyraldehyde was produced, and the hydroxybutyraldehyde bound to Schiff's reagent to produce color, which is useful for screening.











TABLE 3







Mutant
Nucleotide Mutation
Amino Acid Mutation









Bld-M1
AAC → ACC
N409T




AGG → AGT
R361S




GCC → TCC
A467S



Bld-M2
AGG → AGT
R361S



Bld-M3
AGG → AGT
R361S




GCC → TCC
A467S



Bld-M4
AGG → AGT
R361S




ATG → AGG
M371R




GCC → TCC
A467S



Bld-M5
GCT → ACT
A176T




TTA → ATA
L273I




AAA → AGA
K279R




AGG → AGT
R361S




GCC → TCC
A467S



Bld-S1
GCT → ACT
A176T



Bld-S2
TTA → ATA
L273I



Bld-S3
AAA → AGA
K279R



Bld-S4
ATG → AGG
M371R



Bld-S5
AAC → ACC
N409T



Bld-S6
GCC → TCC
A467S










Example 7
Screening the Most Effective Bld Mutant
As shown in Table 3 above, the Bld-M1 to Bld-M5 mutants were confirmed to have from 1 to as many as 5 mutated amino acids. Herein, in order to find out which mutant was the most effective, yields of 1,4-BDO of a total 6 mutants were measured in the same manner as Examples 4 and 5. As shown in FIG. 4, a microorganism producing the Bld-S2 (L273I) mutant was confirmed to have the highest yield of 1,4-BDO (0.08 g/L). Other mutants (Bld-S5 and Bld-S6) also showed a slight improvement. Notably, Bld-S2 having the L273I mutation showed more than three times greater effects than adhE2, which is known to have the highest performance among others of this kind.
Example 8
Confirmation of Bld Mutant Activity Via a Mutant 273
Among the bld mutants, L273I had influences on the production of 1,4-BDO. In addition, as a result of the 3D structure analysis of the bld, it was confirmed that amino acid residue 273 was a catalytic site including a binding site to NAD(P)H. Thus, in order to manufacture a mutant to make bld produce 4-hydroxybutyraldehyde in great quantities from 4-hydroxybutyryl-CoA, the amino acid residue 273 was substituted with 18 different amino acids. In comparison with a case of L273I, L273T showed high activity at the level of 15-18% (see FIG. 8). In addition, it was confirmed that 4 bld mutants, i.e., L273C, L273M, L273S, and L273V, had an activity between the activity of the wild-type bld and the activity of the L273I bld mutant. Also, other 13 mutants had reduced activities compared to the activity of the wild-type bld as shown in Tables 4 and 5 below. Table 4 shows a nucleotide sequence of a primer set used for the production of the mutant bld gene and Table 5 shows the nucleotide and amino acid change for each bld mutant.









TABLE 4







SEQ


Gene
Sequence
ID NO:







A176T
F; 5′-GCTAAAAAATGTGTTACCTTTGCTGTCGAA/
46



R; 5′-TTCGACAGCAAAGGTAACACATTTTTTAGC
47


 


L273I
F; 5′-TCTTTTGATAATAATATACCTTGTATTGCA/
48



R; 5′-TGCAATACAAGGTATATTATTATCAAAAGA
49


 


K279R
F; 5′-CCTTGTATTGCAGAAAGAGAAGTATTTGTT/
50



R; 5′-AACAAATACTTCTCTTTCTGCAATACAAGG
51


 


M371R
F; 5′-TATGACAGAACTCATGAGGCCAATATTACC/
52



R; 5′-GGTAATATTGGCCTCATGAGTTCTGTCATA
53


 


N409T
F; 5′-TCAAAAAATATAGACACCCTAAATAGGTTTG/
54



R; 5′-CAAACCTATTTAGGGTGTCTATATTTTTTGA
55


 


A467S
F; 5′-AGAAGATGTGTACTCTCCGGTTAAGCGGCC/
56



R; 5′-GGCCGCTTAACCGGAGAGTACACATCTTCT
57


 


L273A
F; 5′-TCTTTTGATAATAATGCGCCTTGTATTGCA/
58



R; 5′-TGCAATACAAGGGCGATTATTATCAAAAGA
59


 


L273C
F; 5′-TCTTTTGATAATAATTGCCCTTGTATTGCA/
60



R; 5′-TGCAATACAAGGGCAATTATTATCAAAAGA
61


 


L273D
F; 5′-TCTTTTGATAATAATGATCCTTGTATTGCA/
62



R; 5′-TGCAATACAAGGATCATTATTATCAAAAGA
63


 


L273E
F; 5′-TCTTTTGATAATAATGAACCTTGTATTGCA/
64



R; 5′-TGCAATACAAGGTTCATTATTATCAAAAGA
65


 


L273F
F; 5′-TCTTTTGATAATAATTTTCCTTGTATTGCA/
66



R; 5′-TGCAATACAAGGAAAATTATTATCAAAAGA
67


 


L273G
F; 5′-TCTTTTGATAATAATGGCCCTTGTATTGCA/
68



R; 5′-TGCAATACAAGGGCCATTATTATCAAAAGA
69


 


L273H
F; 5′-TCTTTTGATAATAATCATCCTTGTATTGCA/
70



R; 5′-TGCAATACAAGGATGATTATTATCAAAAGA
71


 


L273K
F; 5′-TCTTTTGATAATAATAAACCTTGTATTGCA/
72



R; 5′-TGCAATACAAGGTTTATTATTATCAAAAGA
73


 


L273M
F; 5′-TCTTTTGATAATAATATGCCTTGTATTGCA/
74



R; 5′-TGCAATACAAGGCATATTATTATCAAAAGA
75


 


L273N
F; 5′-TCTTTTGATAATAATAACCCTTGTATTGCA/
76



R; 5′-TGCAATACAAGGGTTATTATTATCAAAAGA
77


 


L273P
F; 5′-TCTTTTGATAATAATCCGCCTTGTATTGCA/
78



R; 5′-TGCAATACAAGGCGGATTATTATCAAAAGA
79


 


L273Q
F; 5′-TCTTTTGATAATAATCAGCCTTGTATTGCA/
80



R; 5′-TGCAATACAAGGCTGATTATTATCAAAAGA
81


 


L273S
F; 5′-TCTTTTGATAATAATAGCCCTTGTATTGCA/
82



R; 5′-TGCAATACAAGGGCTATTATTATCAAAAGA
83


 


L273T
F; 5′-TCTTTTGATAATAATACCCCTTGTATTGCA/
84



R; 5′-TGCAATACAAGGGGTATTATTATCAAAAGA
85


 


L273V
F; 5′-TCTTTTGATAATAATGTGCCTTGTATTGCA/
86



R; 5′-TGCAATACAAGGCACATTATTATCAAAAGA
87


 


L273W
F; 5′-TCTTTTGATAATAATTGGCCTTGTATTGCA/
88



R; 5′-TGCAATACAAGGCCAATTATTATCAAAAGA
89


 


L273Y
F; 5′-TCTTTTGATAATAATTATCCTTGTATTGCA/
90



R; 5′-TGCAATACAAGGATAATTATTATCAAAAGA
91




















TABLE 5







Bld Mutant
Nucleotide change
Amino acid change









L273I
TTA−>ATA
L273I



L273A
TTA−>GCG
L273A



L273C
TTA−>TGC
L273C



L273D
TTA−>GAT
L273D



L273E
TTA−>GAA
L273E



L273F
TTA−>TTT
L273F



L273G
TTA−>GGC
L273G



L273H
TTA−>CAT
L273H



L273K
TTA−>AAA
L273K



L273M
TTA−>ATG
L273M



L273N
TTA−>AAC
L273N



L273P
TTA−>CCG
L273P



L273Q
TTA−>CAG
L273Q



L273R
TTA−>CGT
L273R



L273S
TTA−>AGC
L273S



L273T
TTA−>ACC
L273T



L273V
TTA−>GTG
L273V



L273W
TTA−>TGG
L273W



L273Y
TTA−>TAT
L273Y










Example 9
Measurement of Enzymatic Activity of Wild-Type Bld, L273I, and L273T
In order to confirm the correlation between the bld mutants and the increased 1,4-BDO productivity, butyryl-CoA was used as a substrate as to measure specific activities of purified His6-tagged wild-type bld, a L273I mutant, a L273T mutant, and AdhE2 (D'mbrosio et al., 2006). As a result, the L273I mutant and the L273T mutant each had specific activity of 2.9±0.60 and 3.1±0.30 pmol·mg−1·min−1. As expected, the specific activities of the mutants were 25-30% higher than that of the wild-type bld (2.3±0.46 pmol·mg−1·min−1) (see FIG. 9A).
As an alternative for AdhE2 having two functions, a purified butanol dehydrogenase was added to an enzyme assay of the wild-type bld, the L273I mutant, and the L273T mutant, to evaluate butyraldehyde dehydrogenase and butanol dehydrogenase. In such a condition including butanol dehydrogenase, the specific activities of the wild-type bld, the L273I mutant, and the L273T mutant were measured 2.0±0.48, 2.5±0.44, and 3.0±0.47 pmol·mg−1·min−1, respectively. These specific activity measurements were about 2 to 3 times higher than the specific activity of AdhE2 (0.9±0.16 pmol·mg−1·min−1) (see FIG. 9B). As a result, it was confirmed that bld and bdh may replace AdhE2 in the production pathway of 1,4-BDO.
Example 10
Homology Modeling of Bld
Revealing the effect of mutants on an activity of an enzyme requires identification of a three-dimensional structure of the enzyme. However, the three-dimensional structure of the Bld enzyme was not identified yet. Therefore, the three-dimensional structure of the Bld was newly created by using a method of homology modeling. First, a protein structure having similar sequences with the Bld enzyme was searched for, and as a result, two proteins (Protein Data Bank ID: 3K9D, 3MY7) having the highest similarity were identified. By using the sequences of these two proteins as a template, the sequences of the Bld enzyme were arranged in this template (FIG. 6). Finally, the template-based three dimensional structure of Bld was created (FIGS. 7A and 7B). All the modeling methods used Discovery Studio 3.1 software.
According to the reaction mechanism of aldehyde dehydrogenase, a substrate that reacts with amino acids exists, and this is well preserved as cysteine amino acid in various aldehyde dehydrogenases (see, e.g., J. Mol. Biol (2007) 366, 481-493; Nat. Struct. Mol. Biol. (1997) 4, 317-326). Through the sequence alignment results, the cysteine amino acid was confirmed to be preserved in the Bld enzyme as well, which is the 275th amino acid (Cys275) (FIG. 6). When analyzing the mutants that have an improved activity of the Bld based on the three-dimensional structure, the activity of the enzyme was shown to be increased when the mutation occurs near Cys275 or near the coenzyme binding site (FIGS. 7A and 7B). FIG. 7A shows a three-dimensional structure of the Bld enzyme which is produced by homology modeling. Cys275 and Leu273 amino acids were illustrated as yellow stick models and the coenzyme was illustrated as a pink stick model. FIG. 7B is a close-up view of the catalytic site, and the coenzyme is not shown to reveal the location of the two amino acids described above better.
From these results, possibilities for the improvement of the Bld enzyme were confirmed by mutating amino acids near Cys275. Namely, it was confirmed that the transformation of amino acids near the catalytic site that reacts with a substrate contributes to the improvement of the activity of the corresponding enzyme.
When a new enzyme according to an embodiment of the present invention is used, 1,4-BDO productivity is increased. Accordingly, when the activity of the Bld enzyme is enhanced by directed evolution, this may be very usefully utilized in industry.
According to an embodiment of the present invention, it was confirmed that E. coli having bld gene and bdh gene within the biosynthetic pathway of 1,4-BDO produces 1,4-BDO with high productivity. In addition, a Bld mutant protein enabling high-efficiency production of 1,4-BDO is obtained, and a recombinant microorganism having a gene encoding the mutant protein is obtained whose 1,4-BDO production concentration is improved more than twice than a parent cell. When the recombinant microorganism is used, 1,4-BDO may be efficiently produced.
It should be understood that the exemplary embodiments described therein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A butyraldehyde dehydrogenase (bld) mutant polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7′ and wherein the mutant polypeptide has butyraldehyde dehydrogenase activity“.

2. A polynucleotide that encodes the bld mutant of claim 1.

3. A microorganism that produces 1,4-butanediol, the microorganism comprising the bld mutant polypeptide of claim 1.

4. The microorganism of claim 3, wherein the microorganism comprises an exogenous polynucleotide encoding the bld mutant polypeptide of claim 1.

5. The microorganism of claim 3, further comprising a butanol dehydrogenase enzyme that catalyzes the conversion of 4-hydroxybutyraldehyde into 1,4-butanediol.

6. The microorganism of claim 5, wherein the microorganism comprises a gene encoding the butanol dehydrogenase enzyme.

7. The microorganism of claim 6, wherein the butanol dehydrogenase enzyme is from Clostridium saccharoperbutylacetonicum.

8. The microorganism of claim 3, further comprising
a gene encoding succinyl-CoA:coenzyme A transferase that converts succinate into succinyl-CoA,
a gene encoding CoA-dependent succinate semialdehyde dehydrogenase that converts succinyl-CoA into succinate semialdehyde,
a gene encoding 4-hydroxybutyrate dehydrogenase that converts succinate semialdehyde into 4-hydroxybutyrate, and
a gene encoding 4-hydroxybutyryl-CoA:acetyl-CoA transferase that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA.

9. The microorganism of claim 3, wherein the microorganism is Escherichia coli.

10. A method of producing 4-hydroxybutyaldehyde, the method comprising:
contacting 4-hydroxybutyryl-CoA with the bld mutant polypeptide of claim 1, whereby 4-hydroxybutyaldehyde is produced.

11. A method of producing 1,4-BDO, the method comprising:
contacting 4-hydroxybutyryl-CoA with the bld mutant polypeptide of claim 1; and
contacting the obtained reaction product with butanol dehydrogenase, whereby 1,4-BDO is produced.

12. A method of producing 1,4-BDO, the method comprising:
incubating a microorganism comprising a polynucleotide encoding the bld mutant of claim 1 and a polynucleotide encoding butanol dehydrogenase (bdh) with a carbon source, whereby the microorganism produces 1,4-BDO; and
separating 1,4-BDO from the incubation product.

13. The method of claim 12, further comprising introducing the polynucleotide encoding the bld mutant of claim 1 and the polynucleotide encoding butanol dehydrogenase into the microorganism prior to incubating the microorganism with a carbon source.

14. The method of claim 12, wherein the microorganism further comprises
a gene encoding succinyl-CoA:coenzyme A transferase that converts succinate into succinyl-CoA,
a gene encoding CoA-dependent succinate semialdehyde dehydrogenate that converts succinyl-CoA into succinate semialdehyde,
a gene encoding 4-hydroxybutyrate dehydrogenase that converts succinate semialdehyde into 4-hydroxybutyrate, and
a gene encoding 4-hydroxybutyryl-CoA:acetyl-CoA transferase that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA.

15. The mutant polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

16. The mutant polypeptide of claim 1, wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 3.

17. The mutant polypeptide of claim 1, wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 4.

18. The mutant polypeptide of claim 1, wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 5.

19. The mutant polypeptide of claim 1, wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 6.

20. The mutant polypeptide of claim 1, wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 7.