US20180237807A1

US20180237807A1 - Method of screening gene for 1,4-bdo production

Info

Publication number: US20180237807A1
Application number: US15/958,548
Authority: US
Inventors: Hwayoung Cho; Jinhwan PARK; Yukyung Jung; Jaechan PARK
Original assignee: Samsung Electronics Co Ltd
Current assignee: Samsung Electronics Co Ltd
Priority date: 2013-08-23
Filing date: 2018-04-20
Publication date: 2018-08-23
Also published as: US20160208292A1; WO2015026195A1; US9963721B2; KR102113254B1; KR20150023175A

Abstract

Provided is a screening method of discovering genes capable of increasing 1,4-BDO production on the basis of proteomics data. Over-expression of proteins screened by the method, NCgl0630 (citrate synthase) and NCgl2145 (hyperthetical protein), increase 1,4-BDO productivity. The method may lead to screening of a protein associated with 1,4-BDO productivity, thereby increasing 1,4-BDO productivity, and thus, the method may be recognized as being industrially applicable.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2013-0100567, filed on Aug. 23, 2013, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
BACKGROUND

1. Field

One aspect relates to a method of screening a protein involved in efficiently producing 1,4-BDO. Another aspect relates to a microorganism the nucleic acid encoding the protein screened by the method. Another aspect also relates to a method of producing 1,4-BDO at a high efficiency using the microorganism.

2. Description of the Related Art

1,4-butandiol (1,4-BDO) is used not only as a solvent for manufacturing plastics and fiber but also as a raw material for producing fiber such as spandex. About 1.3 million tons of 1,4-BDO is produced in a year worldwide from petroleum-based materials such as acetylene, butane, propylene, and butadiene. In addition, about 6% of consumption increase is anticipated each year. 1,4-butandiol is important as it is used throughout the entire chemical industry for the production of various chemicals such as polymers, solvents, and fine chemistry intermediates. Most of the chemicals having a carbon number of four are currently synthesized by being derived from 1,4-butandiol or maleic anhydride, but the chemical production process needs to be improved or replaced by a newly developed process as production costs are increasing due to rising oil prices. Thus, biological processes using microorganisms are suggested as the alternative processes.
Different from the method of producing 1,4-BDO chemically, Genomatica Inc. established in 2011 a biosynthetic pathway of producing 1,4-BDO using succinyl-CoA synthetase (Cat1), succinate semialdehyde dehydrogenase (SucCD), NAD-dependent 4-hydroxybutyrate dehydrogenase (4Hbd), 4-hydroxybutyryl CoA:acetyl-CoA transferase (Cat2), and alcohol dehydrogenase (AdhE2) genes in an Escherichia coli. However, there has been an attempt to establish a new biosynthetic pathway by altering the biological pathways which have already been shown in an Escherichia coli in order to produce 1,4-BDO more efficiently. The attempt is much focused on discovering enzymes of high efficiency by inducing various genetic mutations in enzyme genes.
However, such an approach alone is limited in effectively discovering a protein or a gene related to producing of 1,4-BDO. A novel approach was tried to solve the problem, and the genes screened by the approach were verified to enable a significant increase of 1,4-BDO production.

SUMMARY

An aspect provides a screening method of a protein involved in efficient production of 1,4-BDO. Another aspect provides a microorganism including a nucleic acid encoding a protein screened in the method. Another aspect provides a method of producing 1,4-BDO at a high efficiency using the microorganism.

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. 1 compares growth curves of a wild type CGL strain and a CGL strain capable of producing 1,4-BDO in the cases where wild type CGL strain and the CGL strain capable of producing 1,4-BDO were treated with 1,4-BDO of different concentrations. WT+

BDO

25, 50, and 100 denote that 1,4-BDO was added to the wild type CGL at the concentrations of 25 g/L, 50 g/L, and 100 g/L, respectively. adhE2+

BDO

25, 50, and 100 denote that 1,4-BDO was added to the CGL transformed to produce 1,4-BDO at the concentrations of 25 g/L, 50 g/L, and 100 g/L, respectively;

FIG. 2 shows the result of 2D-gel electrophoresis was obtained from proteins produced in the CGL strain cultured in a LB culture medium at 30° C.;

FIGS. 3a and 3b respectively show the result of 2D-gel electrophoresis of proteins produced in the wild type CGL and the mutant strain CGL (Δldh 4G adhE2) after each of the CGL strain was cultured in a LB culture medium at 30° C.;

FIG. 4 shows 2D-gel electrophoresis spots of CGL strain wherein intensity was increased. The left shows proteins which were expressed when the wild type CGL was treated with 1,4-BDO of a concentration of 100 g/L. The right shows proteins which were expressed when the mutant strain CGL (Δldh 4G adhE2) producing 1,4-BDO was treated with 1,4-BDO of a concentration of 100 g/L; and

FIG. 5 compares the 1,4-BDO productivity of CGL wherein an identified gene is over-expressed. pEKEx1 denotes a null vector as a control group. pEk0827 denotes a vector expressing NCgl0827. pEk2145 denotes a vector expressing NCgl2145. pEk0630 denotes a vector expressing NCgl0630. pEk2826 denotes a vector expressing NCgl2826.

DETAILED DESCRIPTION

An aspect of the present invention provides a method of screening a protein involved in efficient production of 1,4-BDO.
An embodiment of the present invention provides a method of screening a protein positively involved in 1,4-BDO production, including culturing a microorganism producing 1,4-BDO in a culture medium either including 1,4-BDO or not including 1,4-BDO; screening a protein showing an increased expression according to increase of 1,4-BDO concentration from culture solution; and selecting the screened protein as a protein positively involved in 1,4-BDO production.
The protein screening method is described in detail below.
First, the protein screening method includes culturing a microorganism producing 1,4-BDO in a culture medium either including 1,4-BDO or not including 1,4-BDO. The microorganism, which is a microorganism producing 1,4-BDO, may be a wild type microorganism or a transformed mutant microorganism. The microorganism may be a microorganism capable of producing 1,4-BDO. The microorganism may be a wild type microorganism capable of producing 1,4-BDO. Also, the microorganism may be a microorganism where introduction of genes associated with 1,4-BDO biosynthesis makes the microorganism be capable of producing 1,4-BDO. The microorganism may be a microorganism of a Corynebacterium genus. The microorganism of Corynebacterium genus may be an Corynebacterium glutamicum.
The microorganism capable of producing 1,4-BDO may include an enzyme converting succinyl CoA to succinyl semialdehyde, an enzyme converting succinyl semialdehyde to 4-hydroxybutyrate, an enzyme converting 4-hydroxybutyrate to 4-hydroxybutyrate-CoA, an enzyme converting 4-hydroxybutyrate-CoA to 1,4-BDO, or the combination thereof.
The enzyme converting succinyl CoA to succinyl semialdehyde may be CoA-dependent succinate semialdehyde dehydrogenase. The enzyme may be an enzyme classified as EC.1.2.1.76. An example of the enzyme may be SucD. The enzyme converting succinyl semialdehyde to 4-hydroxybutyrate may be 4-hydroxybutyrate dehydrogenase. The enzyme may be an enzyme classified as EC.1.1.1.61. The enzyme may be 4Hbd. In addition, the enzyme converting 4-hydroxybutyrate to 4-hydroxybutyrate-CoA may be 4-hydroxybutyryl CoA:acetyl-CoA transferase. The enzyme may be an enzyme classified as EC.2.8.3.-. An example of the enzyme may be Cat2. The enzyme converting 4-hydroxybutyrate-CoA to 1,4-BDO may be alcohol dehydrogenase. The alcohol dehydrogenase may be an enzyme classified as EC.1.1.1.-. The enzyme may be AdhE or AdhE2. As an example, the microorganism producing 1,4-BDO may be an microorganism expressing the SucD protein, the 4Hbd protein, the Cat2 protein, and the AdhE protein.
The term “protein expression” herein means that a protein or an enzyme exists and has activity in a microorganism. The protein or enzyme may exist through a transcription and a translation where a polynucleotide encoding the protein, existing in the microorganism, is transcribed to an mRNA which is in turn translated into the protein. The polynucleotide encoding the protein may exist either by being inserted in a chromosome of a microorganism or by being inserted in a plasmid vector.
The CoA-dependent succinate semialdehyde dehydrogenase may be a protein derived from an Escherichia genus, a Corynebacterium genus or a Porphyromonas genus. The SucD protein may have an amino acid sequence of SEQ ID NO:10. The polynucleotide encoding the SucD may have a nucleotide sequence of SEQ ID NO:15.
The 4-hydroxybutyrate dehydrogenase may be a protein derived from an Escherichia genus, a Corynebacterium genus or a Porphyromonas genus. The 4Hbd protein may have an amino acid sequence of SEQ ID NO:7. The polynucleotide encoding the 4HbD may have a nucleotide sequence of SEQ ID NO:12.
The 4-hydroxybutyryl CoA:acetyl-CoA transferase may be a protein derived from an Escherichia genus, a Corynebacterium genus or a Porphyromonas genus. The Cat2 protein may have an amino acid sequence of SEQ ID NO:8. The polynucleotide encoding the Cat2 may have a nucleotide sequence of SEQ ID NO:13.
The alcohol dehydrogenase may be a protein derived from Clostridium acetobutylicum. The AdhE protein may have an amino acid sequence of SEQ ID NO:9. The polynucleotide encoding the AdhE may have a nucleotide sequence of SEQ ID NO:14.
The microorganism may additionally include succinyl CoA:coenzyme A transferase. The succinyl CoA:coenzyme A transferase may have an activity to catalyze a reaction converting succinate to succinyl CoA. The enzyme may be an enzyme classified as EC.2.8.3.-. As an example, the enzyme may be Cat1. The Cat1 may have an amino acid sequence of SEQ ID NO:11. The polynucleotide encoding the Cat1 may have a nucleotide sequence of SEQ ID NO:16.
The microorganism may be a microorganism wherein a pathway synthesizing lactate from pyruvate is inactivated or decreased. The microorganism may have the eliminated or decreased activity of lactate dehydrogenase (Ldh). The Ldh may have an activity catalyzing a reaction converting pyruvate to lactate. The Ldh may be an enzyme classified as EC.1.1.1.27.The microorganism may have the inactivated or attenuated gene encoding lactate dehydrogenase.
The term “inactivation” herein may mean that a gene which is not expressed or which is expressed but produces the enzyme or the protein without activity, is produced. The term “attenuation” may mean that a gene of which expression is decreased to a level lower than an expression level of wild type strain, a strain which is not genetically engineered or a parent strain or a gene which is expressed but produces the enzyme or the protein with a decreased activity, is produced. A decreased Ldh activity in the microorganism may be lower than 30%, 20% or 10% of the Ldh activity of wild type microorganism. The Ldh activity in the microorganism may be completely eliminated. The inactivation or the attenuation may be caused by homologous recombination. The inactivation or attenuation may be performed by introducing a vector including a part of the sequence of the genes into a cell, culturing the cell so that homologous recombination between the sequence and an endogenous gene of the cell may occur, and then selecting a cell wherein homologous recombination has occurred using a selection marker. The microorganism may be a microorganism wherein activity of an enzyme encoded by the gene may be eliminated or decreased by inactivation or attenuation of the gene. The term “decrease” may relatively represent the activity of the genetically engineered microorganism in comparison to the activity of a microorganism which is not genetically engineered.
Activity of the lactate dehydrogenase may be inactivated or attenuated in the microorganism by a mutation of gene encoding the lactate dehydrogenase. The mutation may be performed by substitution, partial or total deletion, or addition of a nucleotide. Activity of the lactate dehydrogenase in the microorganism may be decreased by eliminating endogenous lactate dehydrogenase gene. The elimination includes not only physical elimination of the gene but also prevention of functional expression of the gene. The elimination may be performed by homologous recombination.
The term “transformation” herein refers to introducing a gene to a microorganism so that the gene may be expressed in the microorganism. The introduced gene, if the gene is expressed in the microorganism, may be inserted into a chromosome of the microorganism or exists outside a chromosome. The gene may be a polynucleotide capable of encoding a polypeptide, which may be DNA or RNA. The introduction of the gene may be any type of introduction, only if the gene may be introduced into and expressed in the microorganism. For example, the gene may be introduced into a microorganism in the form of an expression cassette, a polynucleotide construct including all elements necessary to be expressed by itself. The expression cassette usually includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked with the gene. The expression cassette may be an expression vector capable of self-replication. The gene may be introduced as itself or in the form of a polynucleotide construct to a host cell and be operably linked with a sequence required for an expression in the microorganism.
The term “sequence identity” of a nucleic acid or a polypeptide herein means the degree of identity with reference to base-to-base or amino acid-to-amino acid comparison or with reference to function or structure in a whole window of comparison. Therefore, “percentage of sequence identity” may be calculated, for example, by comparing two optimally aligned sequences in a whole window of comparison, determining the number of positions wherein the same base or the same amino acid is located in both sequences and obtaining the number of matched positions, and by dividing the number of the matched positions with the total number of positions (i.e., window size) and then multiplying 100 with the resulting value. The percent sequence identity may be determined by using known sequence comparing software such as BLASTn (NCBI) and MEGALIGN™ (DNASTAR Inc). Various levels of sequence identity may be used to identify many polypeptides or genes having an identical or similar function or activity. For example, a percent sequence identity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% may be used.
1,4-BDO may be added to culture medium in any phase in the growth curve of a microorganism. 1,4-BDO may be added to culture medium in exponential phase wherein a microorganism grows most actively. In addition, concentration of the added 1,4-BDO may be from about 0 to about 500 g/L. For example, the concentration of the added 1,4-BDO may be selected from the range from about 0 to about 400 g/L, from about 0 to about 300 g/L, from about 0 to about 200 g/L, or from about 0 to about 100 g/L. Two or n different concentrations of 1,4-BDO may be treated (n is a integer equal to or greater than 2.). When 1,4-BDO is added, at least two different concentrations, for example, three or more, four or more, or five or more different concentrations of 1,4-BDO may be added to culture medium.
Microorganism culture conditions may be dependent on the microorganism. The term “culture conditions” refers to conditions to culture a microorganism. The culture condition may be, for example, carbon source, nitrogen source or oxygen conditions. Carbon sources which may be used by a microorganism include monosaccharide, disaccharide or polysaccharide. Specifically, glucose, fructose, mannose, or galactose etc. may be used. Nitrogen sources which may be used by a microorganism include organic nitrogen compounds and inorganic nitrogen compounds. Specifically, amino acids, amides, amines, nitrates or ammonium salts etc. may be used.
The protein screening method also includes screening a protein showing an increased expression according to increase of 1,4-BDO concentration from culture solution. A protein produced by a microorganism refers to all proteins produced by a microorganism, and may be proteins existing in or secreted by a microorganism.
The protein screening method may additionally include a step of collecting cultured microorganism and a step of extracting a protein from the microorganism in order to compare the protein quantity. Methods including SDS-PAGE or Western blot may be used to compare the protein quantity. In addition, the protein quantity may be verified through two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS).
A protein expressed to a higher level at a higher 1,4-BDO concentration may be screened in the steps. After treating with 1, 4-BDO of two different concentrations, a protein expressed to a higher level under a higher 1,4-BDO concentration may be screened. In addition, when 1,4-BDO of two or n different concentrations is treated, a protein commonly expressed to a higher level in comparison to an expression level at the lowest 1,4-BDO concentration may be screened (n is a integer equal to or greater than 2.). In addition, a protein of which expression level is increased as the 1,4-BDO concentration is increased may be screened.
The protein screening method also includes selecting the screened protein as a protein positively involved in 1,4-BDO production. The selected protein in the above steps may be a protein involved in 1,4-BDO production. The protein may be directly or indirectly involved in 1,4-BDO production by a microorganism. The protein with an increased expression may be expressed in a cell.
The culturing may include culturing a wild type microorganism incapable of producing 1,4-BDO and a mutant microorganism thereof capable of producing 1,4-BDO. The screening may additionally include screening of a protein of which expression is higher in the mutant microorganism capable of producing 1,4-BDO than that in the wild type microorganism incapable of producing 1,4-BDO.
The method may include culturing a wild type microorganism incapable of producing 1,4-BDO and a mutant thereof capable of producing 1,4-BDO, analyzing a protein produced by the microorganisms, and screening a protein of which expression is higher in a mutant capable of producing 1,4-BDO than that in a wild type microorganism.
A wild type microorganism incapable of producing 1,4-BDO and a mutant microorganism capable of producing 1,4-BDO may be the same type as the microorganism producing 1,4-BDO used in the method. In addition, the wild type microorganism refers to a microorganism which is not yet mutated to produce 1,4-BDO. The culture conditions may be the same as the culture conditions used in the screening method. A wild type microorganism and a mutant thereof capable of producing 1,4-BDO may be cultured at the same 1,4-BDO concentration or in the absence of 1,4-BDO. The protein of which expression is increased may be expressed in a cell.
The protein screening method also includes comparing proteins expressed at a high level in the culturing and selecting a protein commonly expressed in the screening.
When 1,4-BDO of different concentrations is added, a protein expressed at a higher level in a microorganism to which 1,4-BDO of a higher concentration is added may be screened. In addition, a protein expressed at a higher level in a microorganism to which 1,4-BDO is added than that in a microorganism to which 1,4-BDO is not added may be screened. When 1,4-BDO of a higher concentration is added, a protein expressed at a higher level may be screened. In addition, a protein commonly expressed in all microorganisms to which 1,4-BDO of different concentrations is added may be screened. In addition, a protein of which expression level is increased as 1,4-BDO is increased may be screened.
An over-expressed protein may be verified by the screening. For example, the protein may be citrate synthase NCgl0630 or NCgl2145. Production of 1,4-BDO may be increased by introducing a nucleic acid sequence encoding an over-expressed protein such as NCgl0630 or NCgl2145 into a microorganism capable of producing 1,4-BDO. In an Example of the present invention, 1,4-BDO productivity of a strain wherein NCgl0630 was introduced was 49% higher than that of the control group. In addition, 1,4-BDO productivity of a strain wherein NCgl2145 was introduced was 18% higher than that of the control group. These results verified that the a protein involved in 1,4-BDO production may be effectively screened by the screening method.
An aspect relates to a microorganism including a nucleic acid encoding a protein screened by the method. An Example of the present invention provides a microorganism, which is capable of producing 1,4-BDO, wherein activity of citrate synthase is increased. The activity of citrate synthase may be increased in comparison with that of a wild type of the microorganism. In addition, the microorganism is capable of producing 1,4-BDO at a high level.
The microorganism, which is a microorganism producing 1,4-BDO, may be a wild type microorganism or a transformed microorganism. The transformed microorganism may be prepared by an addition, deletion, or substitution of a gene to produce 1,4-BDO in a wild type microorganism. In addition, the transformation may be performed by mutating one or more genes. The microorganism may be Corynebacterium glutamicum (CGL). Activity of Ldh in a wild type CGL may be eliminated for producing 1,4-BDO. In addition, cat1, sucD, 4hbD, cat2, and adhE genes may be introduced into a wild type CGL for producing 1,4-BDO.
The citrate synthase may be a gene derived from a CGL. In amino acids constituting the citrate synthase, part of the amino acid may be substituted, altered or deleted, as long as the sequence retains original activity of citrate synthase. In addition, the citrate synthase may include an amino acid sequence having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO:1. For example, the citrate synthase may include an amino acid sequence of SEQ ID NO:1.
A nucleic acid sequence encoding the citrate synthase may include a nucleic acid sequence encoding an amino acid sequence having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO:1. For example, the nucleic acid may have a nucleic acid sequence of SEQ ID NO:2. Part of the nucleic acid sequence may be substituted, altered, or deleted, as long as the protein encoded by the nucleic acid sequence retains original activity of citrate synthase. The nucleic acid may be a nucleic acid sequence having 80% or higher, 90% or higher, or 95% or higher sequence identity with the nucleic acid sequence, as long as the protein encoded by the nucleic acid sequence retains original activity of citrate synthase.
Activity of citrate synthase may be increased by over-expressing a nucleic acid encoding citrate synthase in a cell. The nucleic acid may be introduced to a microorganism as itself or as it is inserted to a vector. The nucleic acid may be expressed within a vector or over-expressed as it is inserted into a chromosome of a microorganism.
The term “vector” herein refers to a DNA product including a DNA sequence operably linked to an appropriate regulatory sequence capable of expressing DNA in an appropriate host. The vector may be a plasmid vector, a bacteriophage vector, or a cosmid vector.
To operate as an expression vector, a vector may include a replication origin, a promoter, a multi-cloning site (MCS), and a selection marker. A replication origin gives a function to a plasmid to replicate itself independently of host cell chromosome. A promoter operates in transcription process of an inserted foreign gene. An MCS enables a foreign gene to be inserted through various restriction enzyme sites. A selection marker verifies whether a vector has been properly introduced to a host cell or not. A selection includes an antibiotic-resistant gene generally used in the art. For example, a selection marker may include a gene resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, or tetracycline. Considering the cost, ampicillin or gentamycin-resistant gene may be used.
When a vector of an aspect of the present invention uses a prokaryotic cell as host cell, a strong promoter, for example, lamda-PL promoter, trp promoter, lac promoter, T7 promoter, or tac promoter is included in the vector. If a vector uses a eukaryotic cell as host cell, the vector may include a promoter derived from genome of a mammal (metallothionin promoter, e.g.) or a promoter derived from a mammal virus (adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter or tk promoter of HSV promoter, e.g.). The promoter may be a lamda-PL promoter, trp promoter, lac promoter, T7 promoter, or tac promoter. In this manner, a promoter is operably linked with a sequence encoding a gene.
The term “operably linked” herein may mean a functional linkage between a nucleic acid expression regulatory sequence (promoter, signal sequence, or a sequence at transcription regulation factor binding site) and another nucleic acid sequence. Through the functional linkage, the regulatory sequence may control transcription and/or translation of a nucleic acid encoding the gene.
A microorganism in another Example of the present invention may include NCgl2145 protein which is not included in a wild type microorganism. The microorganism provides a microorganism, which is capable of producing 1,4-BDO, including NCgl2145 protein. NCgl2145 protein may include an amino acid sequence having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO:3. For example, NCgl2145 protein may include an amino acid sequence of SEQ ID NO:3.
A nucleic acid sequence encoding the NCgl2145 protein may include a nucleic acid sequence encoding an amino acid sequence having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO:3. For example, the nucleic acid encoding the NCgl2145 protein may have a nucleic acid sequence of SEQ ID NO:4. The nucleic acid encoding amino acid sequence of SEQ ID NO:3 may be a nucleic acid sequence having 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence identity with a nucleic acid sequence of SEQ ID NO:4 or a fragment thereof. For example, the amino acid sequence of SEQ ID NO:3 may be encoded by the sequence of SEQ ID NO:4. In addition, the microorganism provides a microorganism, which is capable of producing 1,4-BDO, wherein a nucleic acid sequence encoding an amino acid sequence having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO:3 is introduced. The microorganism may additionally include a nucleic acid encoding an amino acid sequence having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO:3, in comparison with a wild type microorganism. The microorganism is capable of producing 1,4-BDO at a high level.
The microorganism may be CGL. Ldh activity may be eliminated in a wild type CGL for producing 1,4-BDO. For example, cat1, sucD, 4hbD, cat2, and adhE genes may be introduced into a wild type CGL for producing 1,4-BDO.
Another example of the present invention provides a microorganism wherein nucleic acids encoding citrate synthase and NCgl2145 (hypothetical protein) are introduced. The microorganism overexpressing the two enzymes at the same time is capable of producing 1,4-BDO at a high level.
The microorganism may be CGL. The citrate synthase may have an amino acid sequence of SEQ ID NO:1. The amino acid sequence of SEQ ID NO:1 may be encoded by a nucleic acid sequence of SEQ ID NO:2. In addition, the citrate synthase may be derived from CGL. The NCgl2145 may have an amino acid sequence of SEQ ID NO:3. In addition, the amino acid sequence of SEQ ID NO:3 may be encoded by a nucleic acid sequence of SEQ ID NO:4. The microorganism may be CGL. Ldh activity may be eliminated in a wild type CGL for producing 1,4-BDO. Nucleic acids encoding Cat1, SucD, 4HbD, Cat2, and AdhE may be introduced into a wild type CGL for producing 1,4-BDO.
Another aspect provides a method of producing a microorganism having an increased capability of producing 1,4-BDO, the method including introduction of a nucleic acid encoding a screened protein positively involved in 1,4-BDO production to a microorganism capable of producing 1,4-BDO.
The protein positively involved in 1,4-BDO production may be citrate synthase or a protein having an amino acid sequence of SEQ ID NO:3. The citrate synthase may have an amino acid sequence of SEQ ID NO:1. The citrate synthase may be encoded by a nucleic acid sequence of SEQ ID NO:2. In addition, the amino acid sequence of SEQ ID NO:3 may be encoded by a nucleic acid sequence of SEQ ID NO:4. The microorganism may be CGL. Ldh activity may be eliminated in a wild type CGL for producing 1,4-BDO. A nucleic acid encoding one protein selected from the group consisting of Cat1, SucD, 4HbD, Cat2 , and AdhE may be introduced into a wild type CGL for producing 1,4-BDO. In addition, the microorganism may include Cat1, SucD, 4HbD, Cat2 , and AdhE all together.
Another aspect provides a method of producing 1,4-BDO including culturing a microorganism wherein a nucleic acid encoding a protein positively involved in 1,4-BDO production is introduced; and obtaining 1,4-BDO from culture medium.
The microorganism may be CGL. Ldh activity may be eliminated in a wild type CGL for producing 1,4-BDO. sucD, 4hbD, cat2, and adhE genes may be introduced into a wild type CGL for producing 1,4-BDO. In addition, the microorganism may additionally include a nucleic acid encoding Cat1 or nucleic acid encoding SucCD.
To over-expressing in a cell a nucleic acid encoding a protein screened by the screening method, a nucleic acid may be introduced to a microorganism as itself or as it is inserted to a vector. The nucleic acid may be expressed within a vector or over-expressed as it is inserted into a chromosome of a microorganism. A vector for expression may include a replication origin, a promoter, an MCS, and a selection marker.
Another example of the present invention provides a method of producing 1,4-BDO at a high yield including culturing a microorganism wherein citrate synthase is over-expressed, Ncgl2145 is introduced, or a nucleic acid encoding a screened protein is introduced; and obtaining 1,4-BDO from culture medium.
The culturing may be performed under an appropriate culture medium and culture conditions known in this art. The culture medium and culture conditions may be conveniently adjusted according to the selected microorganism. The culturing method may include batch culturing, continuous culturing, fed-batch culturing or a combination thereof.
The culture medium may include various carbon sources, nitrogen sources, and trace elements.
The carbon source may include a carbohydrate such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, a lipid such as soybean oil, sunflower oil, castor oil, and coconut oil, a fatty acid such as palmitic acid, stearic acid, and linoleic acid, an organic acid such as acetic acid or a combination thereof. The culturing may be performed by using glucose as a carbon source. The nitrogen source may include an organic nitrogen source such as peptone, yeast extract, meat extract, malt extract, corn steep liquid, and soybean, an inorganic nitrogen source such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate or a combination thereof. The culture medium may include as a phosphorous source, for example, potassium dihydrogen phosphate, dipotassium phosphate, a sodium-containing salt corresponding to potassium dihydrogen phosphate, and dipotassium phosphate, and a metal salt such as magnesium sulfate and iron sulfate. The culture medium or an individual component may be added to the culture in a batch mode or a continuous mode. The culture medium or an individual component may be added to the culture solution in a batch mode or a continuous mode.
In addition, pH of the culture may be adjusted during the culturing by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid or sulfuric acid to the culture in an appropriate mode. In addition, bubble formation may be repressed by using an endoplasmic reticulum such as fatty acid polyglycol ester.
The culturing may be performed under anaerobic conditions. The term “anaerobic conditions” herein refers to a state wherein oxygen content is lower than that of normal atmospheric state. Anaerobic conditions may be formed, for example, by supplying carbon dioxide or nitrogen at a flow rate range from about 0.1 vvm (Volume per Volume per Minute) to about 0.4 vvm, from about 0.2 vvm to about 0.3 vvm or at a flow rate of 0.25 vvm. In addition, anaerobic conditions may be formed by setting an aeration rate in the range from about 0 vvm and to 0.4 vvm, from about 0.1 vvm to about 0.3 vvm or from 0.15 vvm to about 0.25 vvm.
The method of producing 1,4-BDO includes recovering of the produced 1,4-BDO from the culture medium. For example, the recovery of 1,4-BDO may be performed by using known separation and purification methods. The recovery may be performed by centrifugation, ion exchange chromatography, filtration, precipitation or a combination thereof.
As described above, according to a screening method of one Example of the present invention, a gene for producing 1,4-BDO at a high efficiency may be effectively screened. In addition, a microorganism over-expressing NCgl0630 gene encoding citrate synthase and NCgl2145 gene screened by the screening method is capable of producing 1,4-BDO effectively. 1,4-BDO may be effectively produced by the method and with the genes.
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.

Example 1. Preparation of Corynebacterium Microorganism Wherein Endogenous Lactate Dehydrogenase Gene is Deleted

A decrease in intracellular acetyl-CoA concentration was found when culturing Corynebacterium glutamicum ATCC13032 under anaerobic conditions. Therefore, it was assumed that decrease in TCA cycle activity may be caused by the decrease in the acetyl-CoA concentration. In addition, an experiment was designed in search of a method to resolve the problem. For this, a Δldh Corynebacterium microorganism ATCC13032 wherein endogenous lactate dehydrogenase gene is deleted (“basic strain” hereinafter) was prepared by deleting the endogenous lactate dehydrogenase gene so that the Pdh enzyme activity might be conveniently measured in the natural Corynebacterium glutamicum.
1.1 Preparation of replacement vector
The L-lactate dehydrogenase gene of Corynebacterium glutamicum (CGL) ATCC13032 was inactivated by homologous recombination using a pK19 mobsacB (ATCC87098) vector. The two homologous regions for the elimination of the ldhA gene were obtained by PCR amplification using the genome DNA of CGL ATCC13032. Two homologous regions for the elimination of the ldh gene were located upstream and downstream from the gene and obtained by PCR amplification using a primer set including ldhA_5′_HindIII (SEQ ID NO:17) and ldhA_up_3′_Xhol (SEQ ID NO:18) and a primer set including ldhA_dn_5′_Xhol (SEQ ID NO:19) and ldhA_3′_EcoRI (SEQ ID NO:20). The PCR amplification was performed by repeating, 30 times, a cycle including a denaturation step at 95° C. for 30 seconds, an annealing step at 55° C. for 30 seconds, and an extension step at 72° C. for 30 seconds. All the PCR amplifications hereinafter were performed under the same conditions. A pK19_ΔldhA vector was prepared by cloning the obtained amplification product to the HindIII and EcoRI restriction enzyme positions of pK19 mobsacB vector.
1.2 Preparation of CGL (ΔldhA) strain
The pK19_ΔldhA vector was introduced to CGL ATCC13032 by electroporation. The strain wherein the pK19_ΔldhA vector was introduced was cultured at 30° C. by streaking the strain on a lactobacillus selection (LBHIS) culture medium including kanamycin 25 μg/ml. The LBHIS culture medium included brain-heart infusion broth 18.5 g/L, 0.5 M sorbitol, 5 g/L bacto-tryptone, 2.5 g/L bacto-yeast extract, 5 g/L NaCl, and 18 g/L bacto-agar. Hereinafter, the composition of the LBHIS culture medium is the same. Colonies on the culture medium were streaked on an LB-sucrose culture medium and cultured at 30° C., and then only the colonies wherein double crossing-over occurred were selected. After separating genomic DNA from the selected colonies, deletion of the ldhA gene was verified by PCR using a primer set including ldhA up (SEQ ID NO:21) and ldhA down (SEQ ID NO:22). CGL (ΔldhA) strain (B005) was obtained as a result.

Example 2. Introduction of Genes for 1,4-BDO Production 2.1 Preparation of pK19 gapA::4G vector

A CGL strain capable of producing 1,4-BDO was prepared on the basis of the strain prepared above. To insert four genes of cat1, sucD, 4hbD, and cat2 into a chromosome of the strain, pK19 gapA::4G vector for the insertion of cat1, sucD 4hbD, and cat2 genes was prepared on the basis of pK19 mobsacB. The pK19 gapA::4G vector was prepared by synthesizing whole 4G gene having a nucleotide sequence of SEQ ID NO:23 and cloning the 4G gene into the Nhel and Xbal restriction enzyme sites of the pK19 mobsacB vector.
2.2 Preparation of CGL (ΔldhA) strain
The pK19 gapA::4G vector was introduced to CGL (Δldh) by electroporation. The strain wherein the pK19 gapA::4G vector was introduced was cultured at 30° C. by streaking the strain on LBHIS culture medium including kanamycin 25 μg/ml. The colony was streaked on LB-sucrose culture medium and cultured at 30° C. Then, only the colonies wherein double crossing-over occurred were selected. The genome DNA was separated from the selected colonies, and introduction of the 4G genes was verified through PCR by using primer sets 0049-1 for (SEQ ID NO:24) and 0049-2 rev (SEQ ID NO:25). CGL (Δldh 4G) strain was obtained as a result.

Example 3. Preparation of Strain Wherein adhE2 is Introduced

3.1 Preparation of pK19 gapA::adhE2 vector
To insert the adhE2 gene to the chromosome, the pK19 gapA::adhE2 vector for insertion of adhE2 gene was prepared on the basis of pK19 mobsacB. The pK19 gapA::adhE2 was prepared by synthesizing whole adhE2 gene having a nucleotide sequence of SEQ ID NO:26 and the cloning the adhE2 gene into the Smal restriction enzyme site of the pK19 mobsacB vector.
3.2 Preparation of CGL (ΔldhA 4G adhE2) strain
The pK19 gapA::adhE2 vector was introduced to CGL (Δldh 4G) by electroporation. The strain wherein the pK19 gapA::adhE2 vector was introduced was cultured at 30 ° C. by streaking the strain on LBHIS culture medium including kanamycin 25 μ/ml. The colony was streaked on LB-sucrose culture medium and cultured at 30° C. Then, only the colonies wherein double crossing over occurred were selected. The genome DNA was separated from the selected colonies, and introduction of the adhE2 gene was verified through PCR by using primer sets AdhE2_1_F for (SEQ ID NO:27) and AdhE2_2260_R (SEQ ID NO:28). CGL (ΔldhA 4G adhE2) strain capable of producing 1,4-BDO was obtained as a result.

Example 4. Screening of a Protein Related to Producing of 1,4-BDO

A wild type CGL and the mutant capable of producing 1,4-BDO, which was prepared above, were cultured in LB culture medium at 30° C. In an exponential phase of the wild type CGL and the mutant capable of producing 1,4-BDO (ΔldhA, cat1, sucD 4hbD, cat2, and adhE), 1,4-BDO of a concentration of 0, 25, 50, and 100 g/L was added to the culture medium at the time when the value of OD₆₀₀was in the range from about 1.5 to about 2.5. Samples were taken at the time lapse of 0, 1, 3, and 5 hours, and expression of the total proteins was compared by 2D-gel electrophoresis (FIG. 3). Seven spots wherein electrophoresis expression intensity was increased in redundancy were selected (FIG. 4). Proteins of which electrophoresis expression level was increased in redundancy were identified as NCgl0827, NCgl2145, NCgl0630, and NCgl2826 by MALDI/MS.

Example 5. Preparation of a Strain, which is Capable of Producing 1,4-BDO, Over-Expressing a Screened Protein

To verify whether or not a screened protein actually affects 1,4-BDO production, a microorganism wherein a screened protein is introduced was prepared. For this, a sequence encoding a screened protein was introduced into a vector, and the vector was in turn introduced into a CGL. The CGL was a strain which was transformed to be capable of producing 1,4-BDO. NCgl0630 gene (SEQ ID NO:2), NCgl2145 gene (SEQ ID NO:4), NCgl0827 gene (SEQ ID NO:5), and NCgl2826 gene (SEQ ID NO:6) were inserted into a MCS of pEKEx1 vector (a family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression and promoter probing. Gene, 102 (1991) 93-98) and then expressed by tac promoter. NCgl0630 gene was inserted by using the restriction enzymes EcoRI and SalI recognition sequences. NCgl2145, NCgl0827, and NCgl2826 genes were inserted by using the restriction enzymes BamHI and PstI recognition sites; restriction enzyme EcoRI (single) recognition site; and restriction enzymes EcoRI and BamHI recognition sites, respectively. A total of five strains were prepared by introducing expression vectors wherein each of the genes was introduced (pEK0630, pEK2145, pEK0827, and pEK2826) and a pEKEx1null vector (Ref) as a control group to a mutant strain CO24 (ΔldhA, cat1 sucD 4hbD cat2 adhE2) capable of producing 1,4-BDO.

Example 6. Comparison Of 1,4-BDO Productivity of CGL Over-Expressing an Identified Gene

1,4-BDO productivity was compared after fermenting a total of five CGL mutants prepared above, including the control group. Firstly, to provide equal growth conditions, the strains were fermented under aerobic conditions. Fermentation was performed by changing the conditions into anaerobic conditions by reducing oxygen in culture medium after a predetermined time passed.
Colonies of the five mutants were injected respectively inoculated to LB culture medium 3 mL including kanamycin 25 μg/ml and cultured at 30° C. at a stirring rate of 220 rpm for 12 hours. The culture solutions were respectively inoculated to LB culture medium 50 mL including kanamycin 25 μg/ml and isopropyl β-D-1-thiogalactopyranoside (IPTG), which is a substance to induce expression of tac promoter, and cultured in 250 mL flasks at 30° C. at a stirring rate of 220 rpm for 24 hours. To change the culture conditions into anaerobic conditions, the culture solutions were respectively transported to 125 mL flasks and the flasks were sealed with film so that air might not be supplied. Then, the culture solutions were cultured at 30° C. at a stirring rate of 90 rpm for 72 hours. Cells of the strains were separated from the final culture solutions by centrifugation, and 1,4-BDO was quantified by analyzing supernatants by HPLC. 1,4-BDO productivity of the strains wherein NCgl0630 was expressed was 49% higher than that of the control group. 1,4-BDO productivity of the strains wherein NCgl2145 was expressed was 18% higher than that of the control group.

Claims

1. A method of screening for a protein positively involved in 1,4-BDO production, comprising:

culturing a microorganism that produces 1,4-BDO in a culture medium comprising 1,4-BDO and in a culture medium not comprising 1,4-BDO;

analyzing protein expression in the microorganisms; and

selecting a protein showing increased expression in the microorganism cultured with 1,4-BDO as compared to the microorganism cultured without 1,4-BDO

as a protein positively involved in 1,4-BDO production.

2. The method of claim 1, wherein the microorganism that produces 1,4-BDO is a mutant microorganism, and the method further comprises culturing a wild type microorganism incapable of producing 1,4-BDO in a culture medium comprising 1,4-BDO and in a culture medium not comprising 1,4-BDO a protein showing (a) increased expression in the microorganism cultured with 1,4-BDO as compared to the microorganism cultured without 1,4-BDO, and (b) increased expression in the mutant microorganism capable of producing 1,4-BDO than in the wild type microorganism.

3. The method of claim 1, wherein the microorganism is Corynebacterium glutamicum.

4. The method of claim 1, wherein the microorganism does not have lactate dehydrogenase activity.

5. The method of claim 1, wherein the microorganism comprises genes encoding coenzyme A-dependent succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl CoA:acetyl-CoA transferase, and alcohol dehydrogenase.

6. The method of claim 1, wherein the 1,4-BDO is added to the culture medium during an exponential growth phase of the microorganism.

7-18. (canceled)

19. The method of claim 2, wherein the mutant microorganism is Corynebacterium glutamicum modified to produce 1,4-BDO, and the wild-type microorganism is Corynebacterium glutamicum.