WO2018117773A2 - Recombinant strain having modified sugar metabolic pathway and method for screening sugar isomerase using same - Google Patents

Recombinant strain having modified sugar metabolic pathway and method for screening sugar isomerase using same Download PDF

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WO2018117773A2
WO2018117773A2 PCT/KR2017/015435 KR2017015435W WO2018117773A2 WO 2018117773 A2 WO2018117773 A2 WO 2018117773A2 KR 2017015435 W KR2017015435 W KR 2017015435W WO 2018117773 A2 WO2018117773 A2 WO 2018117773A2
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sugar
strain
gene
galactose
metabolism
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WO2018117773A3 (en
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이동우
신선미
주윤혜
성재윤
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경북대학교 산학협력단
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Priority claimed from KR1020170044740A external-priority patent/KR101979213B1/en
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Priority to EP17882917.2A priority Critical patent/EP3578651A4/en
Priority to US16/603,116 priority patent/US20200087647A1/en
Publication of WO2018117773A2 publication Critical patent/WO2018117773A2/en
Publication of WO2018117773A3 publication Critical patent/WO2018117773A3/en

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    • C12Y503/01004—L-Arabinose isomerase (5.3.1.4)
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    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells

Definitions

  • the present invention relates to a recombinant strain having a modified sugar metabolic pathway by introducing enzymes derived from other strains into a strain, and to obtain a useful substance or to screen various variants and mutant enzymes capable of producing a useful substance using ultrafast screening. It is about how it can be.
  • L-arabinose isomerase (EC 5.3.1.5) is an enzyme that converts L-arabinose to L-ribulose in vivo , but in vitro In vitro , D-galactose, a structurally similar substrate, is converted to D-tagatose. Therefore, when L-arabinose isomerase having high affinity and high activity for D-galactose is used, D-tagatose can be produced by an efficient process.
  • D-tagatose is an isomer of D-galactose and is a natural sugar present in fruits, milk, cheese, and the like. D-tagatose has a variety of health functional properties and sweetness very similar to sugar, so it is used as an alternative sweetener to satisfy both health and taste at the same time.
  • molecular evolution (directed evolution) technology is generally used as an improved technology for converting the properties of the enzyme to meet the desired purpose, such as to enhance the activity and structural stability of the enzyme or to impart activity on a new substrate.
  • the most common method used to prepare the mutant strain library for performing this technique is the error-prone PCR method, which introduces random mutations by controlling the error rate of DNA polymerase during PCR. After expressing the protein using the mutants thus made, by selecting the mutants with good activity, an improved enzyme having excellent activity is obtained.
  • Developing an efficient screening technology suitable for the characteristics and purposes of the desired enzyme is the core technology of molecular evolution. can do.
  • the inventors of the present invention while developing a new metabolic pathway for the production of useful substances and the rapid screening method of the new glycoconverting enzyme, D-tagatose-1,6-bisphosphate aldolase (D-tagatose)
  • D-tagatose D-tagatose-1,6-bisphosphate aldolase
  • a gene encoding 1, 6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced into a D-galactose non-metabolizing strain, a strain having a new sugar metabolic pathway can be obtained.
  • the present invention was completed by confirming that a novel sugar isomerase can be screened quickly by constructing a library inducing random displacement to a gene encoding arabinose isomerase and measuring strain growth using the same.
  • an object of the present invention is to provide a method for rapidly screening strains having new sugar isomerization ability and sugar metabolism ability by using newly constructed sugar metabolism pathway, and through this, to screen novel variant sugar isomerase at high speed.
  • the present invention comprises the steps of: 1) selecting an enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism;
  • a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); And 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and checking the growth and growth rate of the bacterium. It includes, wherein the first sugar and the second sugar is mutual isomers, it provides an ultra-fast screening method of variants having a second sugar isomerization activity and a first sugar metabolic ability.
  • the present invention comprises the steps of: 1) selecting the enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and selecting strains having high growth capacity; And 6) identifying the mutant enzyme introduced into the strain selected in step 5); To include, wherein the first sugar and the second sugar is mutual isomers, it provides an ultra-fast screening method of mutant glycoisomerase having a second sugar isomerization activity
  • the present invention also provides a recombinant vector comprising a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase.
  • the present invention also provides a recombinant strain having D-galactose metabolism, wherein a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced. do.
  • the present invention comprises the steps of introducing a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase into a D-galactose non-metabolic strain; It provides a method for producing a recombinant strain having a D-galactose metabolism comprising a.
  • the present invention provides a L- arabinose isomerase mutase having a sugar isomerization ability, represented by one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4.
  • recombinant vector and strain according to the present invention not only can a new sugar metabolic pathway be constructed in the strain to effectively obtain D-tagatose from D-galactose, but also randomly modified sugar isomerization After the introduction of the enzyme, by performing a cell growth-based ultra-fast screening method, there is an advantage that can quickly screen useful glycosylation mutagens.
  • FIG. 1 is a schematic diagram showing a new sugar metabolic pathway introduced into the strain to build a "cell growth-associated screening system (cell growth-associated screening system)" of the present invention.
  • FIG. 2 is a diagram showing E. coli cell growth curves in complex (LB) medium and restriction (M9) medium in which glucose (glc) and D-galactose (gal) were added as the only carbon sources.
  • Figure 3 is a view showing the comparison of the gene group of the microorganisms with D- tagatose metabolism and microorganisms lacking D- tagatose metabolism.
  • FIG. 4 is a schematic diagram of expression vector of D-tagatose metabolic capacity related gatY gene recombinant cloning.
  • FIG. 5 is a diagram showing the results of confirming the expression of the protein by SDS-PAGE by introducing a foreign gene by transforming the gatY gene in E. coli. (Lane1: E. coli without the gatY gene introduced, Lane2: E. coli with the gatY gene introduced)
  • Figure 6 is a diagram showing the results confirmed by the growth curve of the D- Tagawa Sat agarose Metabolism strain in the gatY Gene E. coli.
  • FIG. 7 is a diagram showing the results of the growth of the strain according to the D- tagatose metabolic activity in the E. coli gatY gene introduced in the restriction solid medium to which tagatose is added as the only carbon source.
  • FIG. 8 is a schematic diagram of the pET-22b (+)- araA recombinant cloning expression vector.
  • FIG. 9 is a diagram showing the results of confirming the expression of the gatY gene (31 kDa) and the araA gene (56 kDa) encoding L-arabinose isomerase through SDS-PAGE.
  • FIG. 10 shows a recombinant strain into which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are cultured in a glucose or D-galactose containing medium. Shows the result of confirming cell growth.
  • FIG. 11 is a diagram showing cell growth curves of glycoisomerase library variants using cell growth-based ultrafast screening.
  • Figure 12 is a diagram showing the results of comparing the activity of D-galactose of glycoisomerase mutase selected using the cell growth-based ultrafast screening method with wild type.
  • the present invention comprises the steps of: 1) selecting an enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); And 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and checking the growth and growth rate of the bacterium.
  • the first sugar and the second sugar are mutual isomers, it provides an ultra-fast screening method of variants having a second sugar isomerization activity and a first sugar metabolism.
  • the variant obtained through the screening method of the present invention is a variant in which the sugar metabolism pathway capable of the second sugar isomerization and the first sugar metabolism is newly established, and the second sugar is isomerized to the first sugar by the newly constructed sugar metabolism pathway. It is possible to effectively metabolize the first sugar produced through isomerization. Therefore, when the mutant of the present invention is cultured in a medium containing a second sugar as the only carbon source, it may exhibit higher growth compared to wild-type strains without altering sugar metabolic pathways, and induce random gene mutation through comparison of growth capacity.
  • One variant of the second sugar isomerization enzyme can quickly select a strain containing an enzyme capable of effectively isomerizing the second sugar.
  • the first sugar and the second sugar in the interisomer relationship used in the present invention may include without limitation all of the common sugar isomers known in the art, but preferably the first sugar is tagatose and the second sugar A first combination which is galactose; A second combination wherein the first sugar is fructose and the second sugar is glucose; A third combination wherein the first sugar is tagatose and the second sugar is fructose; A fourth combination wherein the first sugar is mannose and the second sugar is glucose; A fifth combination wherein the first sugar is glucose and the second sugar is galactose; And a sixth combination wherein the first sugar is fructose and the second sugar is galactose; It may be characterized in that the combination selected from the group consisting of.
  • one skilled in the art can select and use the first and second sugars in a mutual isomeric relationship as appropriate to establish the sugar metabolism pathways required to obtain the desired variant glycosylation enzymes.
  • one embodiment of the present invention discloses a method for obtaining new variant isomerases capable of converting D-galactose (second sugar) to D-tagatose (first sugar).
  • D-tagatose-1,6-bisphosphate aldolase an enzyme capable of conferring D-tagatose metabolism as a first sugar
  • was selected and the gene gatY encoding the enzyme, gatY , lacks D-galactose metabolism. Introduced.
  • a random mutation-induced L-arabinose isomerase coding gene library was constructed and the gene of the library was added to the taga gene into which the gatY gene was introduced. It was introduced into a recombinant strain with toss metabolism.
  • the recombinant strain is cultured in a medium having only D-galactose as the only carbon source, only strains capable of producing D-tagatose by effectively converting D-galactose can be grown.
  • Strains with novel D-galactose mutant isomerase, ie L-arabinose mutant isomerase, having D-galactose converting ability can be selected.
  • a person skilled in the art performs the step of selecting the enzymes essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain of the first step and the strain lacking the first sugar metabolic ability. Comparison and genome analysis of strains necessary to derive the essential enzymes related to the metabolic capacity of monosaccharide can be carried out without limitation through known methods known in the art.
  • Step 3) is a step of obtaining a genetic mutation library by inducing a random genetic mutation in the gene encoding a second sugar isomerase, such as L-arabinose isomerase, random mutation is a mutation-induced PCT
  • the error-prone PCR method is used to induce any mutation in the normal sequence as a template.
  • the genetic variation library refers to a population and a set of variation genes obtained through random displacement.
  • Step 4) refers to a step of introducing the mutation gene of the gene mutation library into the first sugar metabolic recombinant strain, such as D-tagatose metabolic strain of step 2) to obtain a strain library, D-tagatose metabolic This means that the strain additionally expresses the mutant gene, thereby obtaining a library of strains that have established new sugar metabolism pathways.
  • Individual mutation genes of the gene mutation library can be introduced and expressed in D-tagatose metabolic strains of step 2), and through this process, a strain library in which various mutation genes are newly introduced can be obtained.
  • Step 5) is a step of culturing the strain of the strain library in a medium containing a second sugar as the only carbon source to check the growth of the bacteria, by confirming the growth of the bacteria whether the strain obtained the second sugar conversion ability You can quickly check whether or not.
  • strains showing excellent growth of cells can be selected as strains obtained with the second sugar conversion ability, and the second sugar conversion ability is given by identifying the mutation gene of the gene mutation library of step 3) introduced into the strain. It is possible to derive a variant having a second sugar isomerization ability and a variant enzyme introduced thereto.
  • the present invention comprises the steps of: 1) selecting an enzyme essential for first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and selecting strains having high growth capacity; And 6) identifying the mutant enzyme introduced into the strain selected in step 5); It includes, The first sugar and the second sugar provides an ultra-fast screening method of mutant glycoisomerase having a second sugar isomerization activity, characterized in that the mutual isomers.
  • Each step of the method can be carried out according to the description of the method for fast screening of variants having a second sugar isomerization capacity and a first sugar metabolism capacity described above.
  • the high-speed screening method of L-arabinose isomerase mutase and D-galactose glycoisomer variant having glycisomerization ability disclosed in one embodiment of the present invention is based on the "cell growth based ultrafast screening method”. It is done.
  • Cell growth-based ultrafast screening method of the present invention means that by introducing a variety of candidate mutation genes to the platform strain, and confirming the cell growth for this, it is possible to quickly select and search for the mutant gene showing activity based on this. More specifically, the present invention forms a library of various mutant L-arabinose isomerase coding genes, introduces it into a strain lacking D-galactose isomerization, and cultures the strain in a restriction medium containing D-galactose, thereby promoting growth of the strain.
  • a specific mutant gene capable of effectively assigning D-galactose isomerization among a number of mutant L-arabinose isomerase coding genes can be derived and used as a D-galactose glycosylation mutant.
  • the present invention also provides a recombinant vector comprising a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase.
  • the present invention also provides a recombinant strain having D-galactose isomerization, in which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced. do.
  • the gene encoding the D-tagatose-1,6-bisphosphate aldolase may be a gene derived from a microorganism having tagatose metabolism, and preferably, Bacillus licheniformis GatY gene from SEQ ID NO: 14580 (SEQ ID NO: 5). The amino acid sequence encoded therefrom is shown in SEQ ID NO: 7.
  • the gene encoding the L-arabinose isomerase of the present invention may be a gene derived from a microorganism having galactose metabolism, preferably an araA gene derived from Escherichia coli (SEQ ID NO: 6). The amino acid sequence encoded therefrom is shown in SEQ ID NO: 8.
  • D-tagatose-1 A recombinant strain having D-galactose metabolism, into which a gene encoding a 6-bisphosphate aldolase and a gene encoding L-arabinose isomerase was introduced, the recombinant strain introduced a foreign gene.
  • a new modified sugar metabolic pathway is established, it is possible to newly have a metabolism capable of producing a useful substance D-tagatose using a sugar, preferably D-galactose, which has not been conventionally available.
  • the new sugar metabolism pathway constructed by such gene introduction is shown in the schematic diagram in FIG. 1.
  • sucgar isomerization and “sugar conversion” may be used interchangeably.
  • the "vector" of the present invention refers to a carrier for introducing a foreign gene into a strain, and may preferably be a plasmid
  • the plasmid may be used without limitation, which is commonly used in the art, and specifically, in FIG. introducing gatY gene using the plasmid having the sequence shown map, and can be introduced into the araA gene by using the plasmid having the sequence map shown in Fig.
  • Recombinant strains of the present invention by the introduction of the gene encoding the foreign gene D-tagatose-1,6-bisphosphate aldolase and the gene encoding the L- arabinose isomerase D-galactose metabolism ability It is a new strain, in particular, a strain in which a gene is introduced into a strain that does not have D-galactose metabolism.
  • Strains lacking D-galactose metabolism do not have any particular limitation, but Agrobacterium, Azospirillum Bacillus, Burkholderia, Clostridium, Enterotrope Enterobacter, Enterococcus, Escherichia, Flavoobacterium, Klebsiella, Lactobacillus, Rhodobacter, Geobacillus E. coli belonging to the genus of one species selected from the group consisting of) and Salmonella, in particular, Enterobacter most commonly used for recombination ( E. coli ).
  • Recombinant strain having D-galactose metabolism wherein the gene encoding the D-tagatose-1,6-bisphosphate aldolase and the gene encoding the L-arabinose isomerase were introduced into D-galactose It is a recombinant strain that can be converted to D-tagatose and used for glycolysis, and may be characterized as a strain having both D-galactose and D-tagatose metabolic or magnetizing ability.
  • the present invention comprises the steps of introducing a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase into a D-galactose non-metabolic strain; It provides a method for producing a recombinant strain having a D-galactose isomerization ability comprising a.
  • the method of introducing the two kinds of genes into the host gene is not particularly limited, but by inserting the gene into a vector, the recombinant microorganism may be incorporated into a recombinant microorganism using an electrophoresis and a heat shock transformation method.
  • the recombinant microorganism may be incorporated into a recombinant microorganism using an electrophoresis and a heat shock transformation method.
  • the introduction of the two genes may be performed simultaneously or sequentially. If used sequentially, the order is not limited.
  • the present invention also provides a recombinant strain having D-galactose metabolism, in which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced. Culturing in a galactose containing medium; It provides a D- tagatose production method comprising a.
  • the gene encoding D-tagatose-1,6-bisphosphate aldolase and the gene encoding L-arabinose isomerase are introduced.
  • D-tagatose-1,6-bisphosphate aldolase and the gene encoding L-arabinose isomerase are introduced.
  • the D-galactose containing medium may be a medium containing a component corresponding to the growth of a recombinant strain, and may be a restriction medium containing only D-galactose and / or glucose as a carbon source.
  • the present invention provides an L-arabinose isomerase mutase having glycoisomerization ability, represented by one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4.
  • the mutant enzyme is a mutant amino acid sequence of glycoisomerase variants screened by cell growth-based ultrafast screening method, randomly mutated arabinose isomerase and introduced into the strain, followed by culturing the recombinant strain in D-galactose containing medium. It is an enzyme derived by checking growth.
  • L-Arabinose isomerase mutase having a glycisomerizing ability of the present invention is 90 to 99%, respectively, preferably with the enzymes represented by SEQ ID NOs: 2, 3 and 4, as long as the function of sugar isomerization is equivalent.
  • Example 1 D-galactose Metabolism Seizure of E. coli missing
  • Escherichia coli DH5 ⁇ can use glucose and D-galactose as a carbon source, while E. coli BL21 (DE3) was confirmed through experiments that cell growth does not occur in D-galactose addition medium.
  • D- tagatose microorganisms metabolic capability Bacillus licheniformis 14580 and Microorganism E. coli Without D-tagatose Metabolism Genome information of the BL21 (DE3) strain was directly compared and analyzed, and the results are schematically shown in FIG. 3.
  • Bacillus licheniformis 14580 a microorganism having D-tagatose metabolism, is E. coli without D-tagatose metabolism. Compared with BL21 (DE3), it was confirmed that the gatY gene encoding D-tagatose 1 and 6-bisphosphate aldolase was selected as an essential enzyme for D-tagatose metabolism.
  • Example 2 an expression vector was prepared using the gatY gene expected to be essential for D-tagatose metabolism.
  • a forward primer (Bli03552 gatY Nde I F 5-CATATGCTGACAAATACGAAAAAAATGC-3) and a reverse primer (Bli03552 gatY Xho IR 5-CTCGAGTTAGTATCTGTCATTGCTCATGC-3) containing respective restriction enzyme sequences ( Nde I or Xho I)
  • PCR was performed using genomic DNA of Bacillus licheniformis cells as a template for gene amplification.
  • primestar HS DNA polymerase from Takara was used for 30 seconds at 98 ° C, 5 seconds at 57 ° C, and 30 minutes at 72 ° C for 1 minute. After performing the cycle, it was carried out at 72 °C 10 minutes.
  • the amplified PCR product was purified by gel extraction kit (Qiagen) after electrophoresis and cloned using pTOP blunt V2 cloning kit (Enzynomics), followed by DNA sequencing (Solgent). Confirmed.
  • the gene expression vector prepared after checking for the absence of the mutation in a gene obtained through PCR was processed the pET-28 (+) (Novagen ) vector with restriction enzymes (Nde I, Xho I), it is cloned into the pTOP Blunt V2 vector
  • the restriction enzymes corresponding to the expression vector were processed and isolated and purified using electrophoresis and gel extraction kit (Qiagen). Restriction enzyme-treated vectors and recovered genes were ligated using DNA ligation kit (promega) to prepare expression vector pET-28a (+)- gatY .
  • the gene map of the expression vector is shown in FIG.
  • E in order to prepare E.
  • the expression vector of Figure 4 was introduced into E. coli BL21 (DE3) confirmed D-galactose metabolism.
  • the transforming bacteria were plated in LB solid medium containing kanamycin, an antibiotic suitable for the antibiotic resistance gene included in the expression vector, at a final concentration of 50 ⁇ g / ml and incubated at 37 ° C. To form a single colony.
  • Recombinant overexpression vector pET-28a (+) gatY obtained in 3.1 was transformed into Escherichia coli BL21 (DE3) strain, and then precultured to 1% precultured LB to kanamycin (50 ⁇ g / ml) in the flask When the absorbance (600 nm) was 0.4-0.6 by inoculation , the expression of gatY gene was induced at 37 ° C. for 6 hours using IPTG (Isopropyl -D-1-thiogalactopyranoside) at a final concentration of 1 mM.
  • IPTG Isopropyl -D-1-thiogalactopyranoside
  • E. coli BL21 (DE3) E. coli BL21 (DE3) host cells obtained D-tagatose metabolism according to the expression of the recombinant gene
  • transformed novel E. coli host cells pET-28a (+) ) -gatY / E. coli BL21 (DE3) was cultured in a restriction (M9) medium containing 5 g / L D-tagatose as the only carbon source and cell growth was confirmed.
  • M9 medium containing 5 g / L D-tagatose as the only carbon source and cell growth was confirmed. The results are shown in FIGS. 6 and 7.
  • the transgenic bacteria introduced with the foreign gene gatY exhibited excellent growth and specific growth rate similar to that of the glucose-containing medium even in the restriction medium containing D-tagatose.
  • E. coli BL21 (DE3) host cells overexpressing the gatY exogenous gene encoding D-tagatose 1 and 6-bisphosphate aldolase were found to have a favorable cell growth according to foreign protein expression. However, this did not show cell growth in a restriction medium containing 0.5% D-galactose as the only carbon source. Therefore, it was confirmed that an additional modification of the sugar metabolism pathway is required to impart D-galactose metabolism.
  • an expression vector of L-arabinose isomerase protein was prepared.
  • a forward primer (ECAI Nde I F, 5'-CATATGACGATTTTTGATAATTATGAAGTGTG-3 ') and a reverse primer (ECAI Hin dIII R, 5'- AAGCTTTTAGCGACGAAACCCGTAATAC), each containing a restriction enzyme sequence ( Nde I or Hin dIII), were first prepared.
  • -3 ' was used to amplify the gene, and genomic DNA of Escherichia coli cells was used as a template for gene amplification by PCR method.
  • the check after pET-22b (+) (Novagen ) vector for the gene expression vector prepared restriction enzyme were treated with (Nde I, Hin dIII), the expression vector for the to the number of genes that are cloned in pTOP Blunt V2 vector that The restriction enzymes were treated and isolated and purified using electrophoresis and gel extraction kit (Qiagen) .
  • the vector treated with restriction enzyme and recovered genes were ligated using DNA ligation kit (promega) to express the expression vector pET-. 22b (+)-araA was prepared A gene map of the expression vector thus prepared is shown in FIG.
  • E. coli transformants were prepared for expression of the genes as follows.
  • the recombinant expression vector shown in FIG. 8 was introduced into the E. coli pET-28a (+)-gatY / BL21 (DE3) strain in which the D-galactose hypertrophy was confirmed and the D-tagatose metabolism was loaded.
  • an antibiotic, ampicillin which is suitable for the antibiotic resistance gene included in the expression vector, is plated in an LB solid medium containing a final concentration of 100 ⁇ g / ml and cultured at 37 ° C. to form a single colony. Was formed.
  • transformed Escherichia coli expressed the gatY gene (31 kDa) encoding the D-tagatose 1, 6-bisphosphate aldolase enzyme and the araA gene (56 kDa) encoding the L-arabinose isomerase enzyme.
  • E. coli BL21 (DE3) transformed with the two plasmids, pET-22b (+)- araA and pET-28 (+)- gatY , to confirm the activity comparison and cell growth of the isomerase of the prepared cell growth-based screening method ), 0.05 g / L fructose and 0.45 g / L D-galactose, final concentration 0.2 mM IPTG, and empicillin (50 ⁇ g / ml) and kanamycin (25 ⁇ g / ml) suitable for the two plasmids. Cultured in the restriction (M9) medium to confirm the cell growth curve by the D-galactose isomerization of glycoisomerase, the results are shown in FIG.
  • the gatY gene encoding the D-tagatose 1 and 6-bisphosphate aldolase enzymes and the araA encoding the L-arabinose isomerase enzymes in E. coli BL21 (DE3) strains that do not grow in a medium containing D-galactose.
  • the strain into which the gene was introduced had cell growth, and it was confirmed that D-galactose could be used as the only carbon source due to the activity of isomerase.
  • Example 5 araA Through mutation-induced genes, Sugar isomerization enzyme Variant Selection
  • mutation PCR was performed with a PCR randomization mutation kit (Clontech, USA). 50 ng of the mutation-induced PCR library DNA was transformed into Escherichia coli BL21 (DE3) containing plasmid pET-28a (+)-gatY, with a restriction containing 0.05 g / L of fructose and 0.45% of D-galactose ( Culture was carried out in M9) medium. Thereafter, colonies formed were collected and plasmids were extracted using the plasmid purification kit. Among them, 8 nucleotide sequences of some plasmids were analyzed, and the gene diversity of the library was confirmed.
  • Plasmids were isolated from glycoisomerase variants and wild type selected by the cell growth based screening assay of 5.2 above: pET-22b (+)-ECAI H17R / R159S / V168A, pET-22b (+)-ECAI E22D / M95L / H157L, and pET-22b (+)-ECAI-V368A / E493D.
  • the mutated amino acid sequence of the variants selected by the screening search method through the sequencing derived from the isolated plasmid was analyzed, which are shown in SEQ ID NOs: 2 to 4, respectively.
  • the amino acid sequence of wild type ECAI is shown in SEQ ID NO: 1.
  • Ni2 + column chromatography was performed to purely separate the mutant enzymes and to compare the activity with the ECAI wild type enzymes.
  • the host strain (Host), ECAI wild-type and glycoisomerase variants were measured and compared the activity against D-galactose, shown in Figure 12.

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Abstract

The present invention relates to a recombinant strain having a modified sugar metabolic pathway resulting from the introduction of an enzyme derived from a different strain to a strain, and a high throughput screening method for various variant strains and mutant enzymes by which useful materials can be obtained or which can produce useful materials. The use of a recombinant vector and a strain according to the present invention not only can construct a new metabolic pathway in a strain to effectively obtain D-tagatose from D-galactose, but also can introduce randomly modified sugar isomerases and then allow D-galactose isomerase mutants to be rapidly screened by conducting a cell growth-based high-throughput screening method.

Description

변형된 당 대사 경로를 갖는 재조합 균주 및 이를 이용한 당이성화 효소의 스크리닝 방법 Recombinant strain having modified sugar metabolism pathway and screening method of glycosylation enzyme using same
본 발명은 균주에 타 균주 유래의 효소를 도입하여 변형된 당 대사 경로를 갖는 재조합 균주에 관한 것이며, 이를 이용하여 유용물질을 얻거나, 유용물질을 생산할 수 있는 다양한 변이체 및 변이 효소를 초고속 스크리닝 할 수 있는 방법에 관한 것이다.The present invention relates to a recombinant strain having a modified sugar metabolic pathway by introducing enzymes derived from other strains into a strain, and to obtain a useful substance or to screen various variants and mutant enzymes capable of producing a useful substance using ultrafast screening. It is about how it can be.
L-아라비노오스 이성화효소(L-arabinose isomerase; EC 5.3.1.5)는 생체내(in vivo)에서 L-아라비노오스를 L-리불로오스(L-ribulose)로 전환시키는 효소이나, 시험관내(in vitro)에서는 구조적으로 유사한 기질인 D-갈락토오스(D-galactose)를 D-타가토오스(D-tagatose)로 전환한다. 따라서, D-갈락토오스에 대해 고친화도 및 고활성의 L-아라비노오스 이성화효소를 이용할 경우, 효율적인 공정으로 D-타가토오스를 생산할 수 있다. L-arabinose isomerase (EC 5.3.1.5) is an enzyme that converts L-arabinose to L-ribulose in vivo , but in vitro In vitro , D-galactose, a structurally similar substrate, is converted to D-tagatose. Therefore, when L-arabinose isomerase having high affinity and high activity for D-galactose is used, D-tagatose can be produced by an efficient process.
D-타가토오스는 D-갈락토오스의 이성질체이며 과일, 우유, 치즈 등에 존재하는 천연 당류이다. D-타가토오스는 다양한 건강 기능적 특성과 설탕과 매우 유사한 단맛을 가지고 있기 때문에 여러 제품 적용 시 건강과 맛을 동시에 만족시킬 수 있는 대체 감미료로 사용된다. D-tagatose is an isomer of D-galactose and is a natural sugar present in fruits, milk, cheese, and the like. D-tagatose has a variety of health functional properties and sweetness very similar to sugar, so it is used as an alternative sweetener to satisfy both health and taste at the same time.
한편, 일반적으로 효소의 활성 및 구조적 안정성을 증진시키거나 새로운 기질에 대한 활성을 부여하는 등 원하는 목적에 부합하도록 효소의 특성을 변환시키는 개량기술로 분자진화 (directed evolution) 기술이 사용되고 있다. 이러한 기술을 수행하기 위한 변이주 라이브러리를 제조하기 위해 가장 일반적인 방법으로 많이 사용되는 것은 error-prone PCR 방법으로 PCR 수행시 DNA 중합효소의 에러발생율을 조절하여 무작위적으로 돌연변이를 도입하는 방법이다. 이렇게 만들어진 변이주들을 이용하여 단백질을 발현시킨 후, 활성이 좋은 변이주를 선별함으로써 우수한 활성을 갖는 개량 효소를 수득하게 되는데 원하는 효소의 특징과 목적에 맞는 효율적인 스크리닝 기술을 개발하는 것이 분자진화의 핵심기술이라 할 수 있다.On the other hand, molecular evolution (directed evolution) technology is generally used as an improved technology for converting the properties of the enzyme to meet the desired purpose, such as to enhance the activity and structural stability of the enzyme or to impart activity on a new substrate. The most common method used to prepare the mutant strain library for performing this technique is the error-prone PCR method, which introduces random mutations by controlling the error rate of DNA polymerase during PCR. After expressing the protein using the mutants thus made, by selecting the mutants with good activity, an improved enzyme having excellent activity is obtained. Developing an efficient screening technology suitable for the characteristics and purposes of the desired enzyme is the core technology of molecular evolution. can do.
효소개량 라이브러리 구축을 위하여 무작위 돌연변이법 및 포인트 돌연변이법 등 다양한 변이주 제작을 위한 시도가 있었으나, 이러한 방법은 개량된 효소의 초고속 탐색방법의 부재로 107 이상의 변이체를 스크리닝하는 분자진화기술을 적용하기엔 한계가 있다. 즉, 당전환 효소의 단백질 공학기술 적용을 위해서 초고속 탐색이 불가한 기존의 방법을 대체할 수 있는 신규한 효소 탐색법 연구가 필요한 실정이다.Attempts have been made to produce a variety of mutant strains, including random mutations and point mutations, for the construction of enzyme-enhanced libraries.However, these methods are limited to applying molecular evolution techniques to screen more than 10 7 variants due to the lack of an ultrafast search method for improved enzymes. There is. In other words, there is a need for a new enzyme search method that can replace the existing methods that cannot be searched for high speed in order to apply the protein engineering technology of the glycoconversion enzyme.
본 발명자들은 유용물질을 생산하기 위한 새로운 대사경로 구축과, 새로운 당전환 효소의 빠른 스크리닝법에 관한 연구를 수행하던 중, D-타가토오스-1,6-비스포스페이트 알돌라아제(D-tagatose 1, 6-bisphosphate aldolase)를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자를 D-갈락토오스 비대사성 균주에 도입하는 경우, 새로운 당 대사 경로를 갖는 균주를 수득할 수 있으며, L-아라비노오스 이성화효소를 코딩하는 유전자에 무작위 변위를 유발한 라이브러리 구축과 이를 이용한 균주 성장 측정을 통해 신규한 당 이성화 효소를 빠르게 스크리닝 할 수 있음을 확인하고 본 발명을 완성하였다. The inventors of the present invention, while developing a new metabolic pathway for the production of useful substances and the rapid screening method of the new glycoconverting enzyme, D-tagatose-1,6-bisphosphate aldolase (D-tagatose) When a gene encoding 1, 6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced into a D-galactose non-metabolizing strain, a strain having a new sugar metabolic pathway can be obtained. The present invention was completed by confirming that a novel sugar isomerase can be screened quickly by constructing a library inducing random displacement to a gene encoding arabinose isomerase and measuring strain growth using the same.
따라서 본 발명의 목적은 새롭게 구축된 당 대사 경로를 이용하여 새로운 당 이성화능 및 당 대사능을 갖는 균주를 신속하게 스크리닝 하고, 이를 통해 신규한 변이 당 이성화 효소를 초고속으로 스크리닝 하는 방법을 제공하는 것이다. Accordingly, an object of the present invention is to provide a method for rapidly screening strains having new sugar isomerization ability and sugar metabolism ability by using newly constructed sugar metabolism pathway, and through this, to screen novel variant sugar isomerase at high speed. .
상기와 같은 목적을 달성하기 위하여 본 발명은 1) 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계; In order to achieve the above object, the present invention comprises the steps of: 1) selecting an enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism;
2) 제1당 대사능 및 제2당 이성화능이 결여된 균주에 상기 1) 단계에서 선별된 효소를 암호화하는 유전자를 도입하여 제1당 대사성 재조합 균주를 제조하는 단계; 3) 제2당 이성화 효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계; 4) 상기 3) 단계에서 수득된 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계에서 제조된 제1당 대사성 재조합 균주에 도입하여 균주 라이브러리를 얻는 단계; 및 5) 제2당을 유일 탄소원으로 포함하는 제한 배지에서 상기 4) 단계에서 수득된 라이브러리의 균주를 배양하고 균의 성장 및 생장속도를 확인하는 단계; 를 포함하며, 상기 제1당과 제2당은 상호 이성질체인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 및 제1당 대사능을 갖는 변이체의 초고속 스크리닝 방법을 제공한다. 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); And 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and checking the growth and growth rate of the bacterium. It includes, wherein the first sugar and the second sugar is mutual isomers, it provides an ultra-fast screening method of variants having a second sugar isomerization activity and a first sugar metabolic ability.
또한 본 발명은 1) 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계; 2) 제1당 대사능 및 제2당 이성화능이 결여된 균주에 상기 1) 단계에서 선별된 효소를 암호화하는 유전자를 도입하여 제1당 대사성 재조합 균주를 제조하는 단계; 3) 제2당 이성화 효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계; 4) 상기 3) 단계에서 수득된 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계에서 제조된 제1당 대사성 재조합 균주에 도입하여 균주 라이브러리를 얻는 단계; 5) 제2당을 유일 탄소원으로 포함하는 제한 배지에서 상기 4) 단계에서 수득된 라이브러리의 균주를 배양하고 높은 성장능을 갖는 균주를 선별하는 단계; 및 6) 상기 5) 단계에서 선별된 균주에 도입된 변이 효소를 확인하는 단계; 를 포함하며, 상기 제1당과 제2당은 상호 이성질체인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 을 갖는 변이 당이성화 효소의 초고속 스크리닝 방법을 제공한다. In another aspect, the present invention comprises the steps of: 1) selecting the enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and selecting strains having high growth capacity; And 6) identifying the mutant enzyme introduced into the strain selected in step 5); To include, wherein the first sugar and the second sugar is mutual isomers, it provides an ultra-fast screening method of mutant glycoisomerase having a second sugar isomerization activity (isomerization activity).
또한 본 발명은 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자를 포함하는, 재조합 벡터를 제공한다. The present invention also provides a recombinant vector comprising a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase.
또한 본 발명은 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자가 도입된, D-갈락토오스 대사능을 갖는 재조합 균주를 제공한다. The present invention also provides a recombinant strain having D-galactose metabolism, wherein a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced. do.
또한 본 발명은 D-갈락토오스 비대사성 균주에 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자를 도입하는 단계; 를 포함하는 D-갈락토오스 대사능을 갖는 재조합 균주의 제조방법을 제공한다. In another aspect, the present invention comprises the steps of introducing a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase into a D-galactose non-metabolic strain; It provides a method for producing a recombinant strain having a D-galactose metabolism comprising a.
또한 본 발명은 서열번호 2 내지 4로 이루어진 군에서 선택된 1종의 아미노산 서열로 표시되는, 당 이성화능을 갖는 L-아라비노오스 이성화효소 변이효소를 제공한다. In another aspect, the present invention provides a L- arabinose isomerase mutase having a sugar isomerization ability, represented by one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4.
본 발명에 따른 초고속 스크리닝 방법, 재조합 벡터 및 균주를 이용하면, 새로운 당 대사경로를 균주에 구축하여 D-갈락토오스로부터 D-타가토오스를 효과적으로 수득할 수 있을 뿐만 아니라, 무작위적으로 변형된 당 이성화 효소를 도입한 후, 세포 성장 기반 초고속 탐색법을 수행함으로써, 유용한 당이성화 변이효소를 빠르게 스크리닝할 수 있는 장점이 있다. Using the ultra-fast screening method, recombinant vector and strain according to the present invention, not only can a new sugar metabolic pathway be constructed in the strain to effectively obtain D-tagatose from D-galactose, but also randomly modified sugar isomerization After the introduction of the enzyme, by performing a cell growth-based ultra-fast screening method, there is an advantage that can quickly screen useful glycosylation mutagens.
도 1은 본 발명의 “세포성장 기반 초고속 탐색법” (cell growth-associated screening system)을 구축하기 위하여 균주에 도입되는 새로운 당 대사경로를 나타낸 모식도이다. 1 is a schematic diagram showing a new sugar metabolic pathway introduced into the strain to build a "cell growth-associated screening system (cell growth-associated screening system)" of the present invention.
도 2는 복합 (LB) 배지와 포도당(glc) 및 D-갈락토오스(gal) 가 유일 탄소원으로 첨가된 제한 (M9) 배지에서의 대장균 균체 성장곡선을 나타낸 도이다. FIG. 2 is a diagram showing E. coli cell growth curves in complex (LB) medium and restriction (M9) medium in which glucose (glc) and D-galactose (gal) were added as the only carbon sources.
도 3은 D-타가토오스 대사능이 있는 미생물과 D-타가토오스 대사능이 결여된 미생물의 유전자군 비교 결과를 나타낸 도이다.Figure 3 is a view showing the comparison of the gene group of the microorganisms with D- tagatose metabolism and microorganisms lacking D- tagatose metabolism.
도 4은 D-타가토오스 대사능 관련 gatY 유전자 재조합 클로닝의 발현 벡터 모식도이다.4 is a schematic diagram of expression vector of D-tagatose metabolic capacity related gatY gene recombinant cloning.
도 5는 gatY 유전자를 대장균 내에 형질전환하여 외래 유전자 도입에 의한 단백질의 발현을 SDS-PAGE를 통해 확인한 결과를 나타낸 도이다. (Lane1: gatY 유전자가 도입되지 않은 대장균, Lane2: gatY 유전자가 도입된 대장균) 5 is a diagram showing the results of confirming the expression of the protein by SDS-PAGE by introducing a foreign gene by transforming the gatY gene in E. coli. (Lane1: E. coli without the gatY gene introduced, Lane2: E. coli with the gatY gene introduced)
도 6는 gatY 유전자가 도입된 대장균에서 D-타가토오스 대사능을 균주의 성장곡선을 통해 확인한 결과를 나타낸 도이다. Figure 6 is a diagram showing the results confirmed by the growth curve of the D- Tagawa Sat agarose Metabolism strain in the gatY Gene E. coli.
도 7은 gatY 유전자가 도입된 대장균에서 D-타가토오스 대사능에 따른 균주의 성장을 타가토스가 유일 탄소원으로 첨가된 제한 고체배지에서 확인한 결과를 나타낸 도이다.7 is a diagram showing the results of the growth of the strain according to the D- tagatose metabolic activity in the E. coli gatY gene introduced in the restriction solid medium to which tagatose is added as the only carbon source.
도 8은 pET-22b(+)-araA 재조합 클로닝 발현 벡터의 모식도이다.8 is a schematic diagram of the pET-22b (+)- araA recombinant cloning expression vector.
도 9는 gatY 유전자 (31 kDa) 및 L-아라비노오스 이성화효소를 암호화하는 araA 유전자 (56 kDa) 의 발현을 SDS-PAGE를 통해 확인한 결과를 나타낸 도이다. 9 is a diagram showing the results of confirming the expression of the gatY gene (31 kDa) and the araA gene (56 kDa) encoding L-arabinose isomerase through SDS-PAGE.
도 10은 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자가 도입된 재조합 균주를 글루코오스 또는 D-갈락토오스 포함 배지에서 배양하고, 세포성장을 확인한 결과를 나타낸 도이다. FIG. 10 shows a recombinant strain into which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are cultured in a glucose or D-galactose containing medium. Shows the result of confirming cell growth.
도 11은 세포성장 기반 초고속 탐색법을 이용하여 당이성화 효소 라이브러리 변이체의 균체 성장곡선을 나타낸 도이다.FIG. 11 is a diagram showing cell growth curves of glycoisomerase library variants using cell growth-based ultrafast screening.
도 12은 세포성장 기반 초고속 탐색법을 이용하여 선별된 당이성화효소 변이효소의 D-galactose 에 대한 활성을 야생형과 비교한 결과를 나타낸 도이다.Figure 12 is a diagram showing the results of comparing the activity of D-galactose of glycoisomerase mutase selected using the cell growth-based ultrafast screening method with wild type.
본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Terms not defined otherwise in this specification are intended to have a meaning commonly used in the art to which the present invention pertains.
본 발명은 1) 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계; 2) 제1당 대사능 및 제2당 이성화능이 결여된 균주에 상기 1) 단계에서 선별된 효소를 암호화하는 유전자를 도입하여 제1당 대사성 재조합 균주를 제조하는 단계; 3) 제2당 이성화 효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계; 4) 상기 3) 단계에서 수득된 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계에서 제조된 제1당 대사성 재조합 균주에 도입하여 균주 라이브러리를 얻는 단계; 및 5) 제2당을 유일 탄소원으로 포함하는 제한 배지에서 상기 4) 단계에서 수득된 라이브러리의 균주를 배양하고 균의 성장 및 생장속도를 확인하는 단계; 를 포함하며, The present invention comprises the steps of: 1) selecting an enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); And 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and checking the growth and growth rate of the bacterium. Including;
상기 제1당과 제2당은 상호 이성질체인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 및 제1당 대사능을 갖는 변이체의 초고속 스크리닝 방법을 제공한다. The first sugar and the second sugar are mutual isomers, it provides an ultra-fast screening method of variants having a second sugar isomerization activity and a first sugar metabolism.
본 발명의 스크리닝 방법을 통해 수득되는 변이체는 제2당 이성화능 및 제1당 대사능이 가능한 당 대사 경로가 새롭게 구축된 변이체이며, 새롭게 구축된 당 대사 경로에 의하여 제2당을 제1당으로 이성화시킬 수 있고, 이성화를 통해 생산된 제1당을 효과적으로 대사할 수 있다. 따라서 본 발명의 변이체는 제2당을 유일 탄소원으로 포함하는 배지에서 배양되는 경우, 당 대사 경로가 변경되지 않은 야생형 균주와 비교하여 높은 생장을 나타낼 수 있으며, 생장능 비교를 통해 무작위 유전자 변이를 유발한 변이 제2당 이성화 효소 중 제2당을 효과적으로 이성화시킬 수 있는 효소를 포함하는 균주를 빠르게 선별해 낼 수 있다. The variant obtained through the screening method of the present invention is a variant in which the sugar metabolism pathway capable of the second sugar isomerization and the first sugar metabolism is newly established, and the second sugar is isomerized to the first sugar by the newly constructed sugar metabolism pathway. It is possible to effectively metabolize the first sugar produced through isomerization. Therefore, when the mutant of the present invention is cultured in a medium containing a second sugar as the only carbon source, it may exhibit higher growth compared to wild-type strains without altering sugar metabolic pathways, and induce random gene mutation through comparison of growth capacity. One variant of the second sugar isomerization enzyme can quickly select a strain containing an enzyme capable of effectively isomerizing the second sugar.
본 발명에 사용되는 상호 이성질체 관계에 있는 제1당 및 제2당은 당 분야에 알려진 통상의 당 이성질체를 모두 제한없이 포함할 수 있으나, 바람직하게는 제1당은 타가토오스 및 제2당은 갈락토오스인 제1조합; 제1당은 프럭토오스 및 제2당은 글루코오스인 제2조합; 제1당은 타가토오스 및 제2당은 프럭토오스인 제3조합; 제1당은 만노오스 및 제2당은 글루코오스인 제4조합; 제1당은 글루코오스 및 제2당은 갈락토오스인 제5조합; 및 제1당은 프럭토오스 및 제2당은 갈락토오스인 제6조합; 으로 이루어진 군에서 선택된 조합인 것을 특징으로 할 수 있다. The first sugar and the second sugar in the interisomer relationship used in the present invention may include without limitation all of the common sugar isomers known in the art, but preferably the first sugar is tagatose and the second sugar A first combination which is galactose; A second combination wherein the first sugar is fructose and the second sugar is glucose; A third combination wherein the first sugar is tagatose and the second sugar is fructose; A fourth combination wherein the first sugar is mannose and the second sugar is glucose; A fifth combination wherein the first sugar is glucose and the second sugar is galactose; And a sixth combination wherein the first sugar is fructose and the second sugar is galactose; It may be characterized in that the combination selected from the group consisting of.
당 분야의 통상의 기술자는 목적으로 하는 변이 당이성화 효소를 수득하기 위하여 필요한 당 대사 경로를 구축하기에 적절한 상호 이성질체 관계의 제1당 및 제2당을 선택하여 사용할 수 있다. 예컨대 본원 발명의 일 실시예에서는 D-갈락토오스 (제2당) 를 D-타가토오스 (제1당)로 전환시킬 수 있는 새로운 변이 이성화 효소를 수득하기 위한 방법을 개시한다. 먼저 제1당인 D-타가토오스 대사능을 부여할 수 있는 효소인 D-타가토오스-1,6-비스포스페이트 알돌라아제를 선별하여 이를 암호화하는 유전자 gatY 를 D-갈락토오스 대사능이 결여된 균주에 도입하였다. 이 후, 제2당인 D-갈락토오스를 이성화 시킬 수 있는 변이 효소를 고속 스크리닝하기 위하여, 무작위 돌연변이가 유도된 L-아라비노오스 이성화효소 암호화 유전자 라이브러리를 구축하고 라이브러리의 유전자를 gatY 유전자가 도입된 타가토오스 대사능을 갖는 재조합 균주에 도입하였다. 재조합된 균주를 D-갈락토오스만을 유일 탄소원으로 하는 배지에서 배양하면, D-갈락토오스를 효과적으로 전환하여 D-타가토오스를 생산할 수 있는 균주만 생장이 가능하므로, 균주의 생장을 비교하는 방법을 통해 빠르게, D-갈락토오스 전환능을 갖는 신규한 D-갈락토오스 변이 이성화 효소, 즉, L-아라비노오스 변이 이성화효소를 갖는 균주를 선별해 낼 수 있다. One skilled in the art can select and use the first and second sugars in a mutual isomeric relationship as appropriate to establish the sugar metabolism pathways required to obtain the desired variant glycosylation enzymes. For example, one embodiment of the present invention discloses a method for obtaining new variant isomerases capable of converting D-galactose (second sugar) to D-tagatose (first sugar). First, D-tagatose-1,6-bisphosphate aldolase, an enzyme capable of conferring D-tagatose metabolism as a first sugar , was selected, and the gene gatY encoding the enzyme, gatY , lacks D-galactose metabolism. Introduced. Subsequently, in order to rapidly screen the mutant enzyme capable of isomerizing the second sugar D-galactose, a random mutation-induced L-arabinose isomerase coding gene library was constructed and the gene of the library was added to the taga gene into which the gatY gene was introduced. It was introduced into a recombinant strain with toss metabolism. When the recombinant strain is cultured in a medium having only D-galactose as the only carbon source, only strains capable of producing D-tagatose by effectively converting D-galactose can be grown. , Strains with novel D-galactose mutant isomerase, ie L-arabinose mutant isomerase, having D-galactose converting ability can be selected.
당 분야의 통상의 기술자는 제 1단계의 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계를 수행함에 있어서, 목적으로 하는 제1당의 대사능과 관련된 필수적인 효소를 도출하기 위해 필요한 균주의 비교 및 유전체 분석을 당 분야에 알려진 공지의 방법을 통해 제한없이 수행할 수 있다. A person skilled in the art performs the step of selecting the enzymes essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain of the first step and the strain lacking the first sugar metabolic ability. Comparison and genome analysis of strains necessary to derive the essential enzymes related to the metabolic capacity of monosaccharide can be carried out without limitation through known methods known in the art.
상기 1) 및 2) 단계와 같이 제1당 대사에 필수적인 효소, 예컨대 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자가 도입되는 경우, 종래 D-타가토오스만이 탄소원으로 첨가된 배지에서 균체 성장이 일어나지 않았던 D-타가토오스 대사능이 결여된 균주에 새로운 당 대사 경로가 구축됨으로써, D-타가토오스 첨가 배지에서 균체 성장이 일어날 수 있게 된다. When a gene encoding an enzyme essential for first sugar metabolism, such as D-tagatose-1,6-bisphosphate aldolase, is introduced as in steps 1) and 2), only conventional D-tagatose is a carbon source. As a new sugar metabolism pathway is established in a strain lacking D-tagatose metabolism in which no cell growth has occurred in the medium added thereto, cell growth may occur in the medium containing D-tagatose.
상기 3) 단계는 제2당 이성화 효소를 코딩하는 효소, 예컨대 L-아라비노오스 이성화효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계이며, 무작위 유전자 변이란 변이유발 PCT인 error-prone PCR 법을 통해 정상 서열을 주형으로 임의의 변이를 유발하는 것을 말한다. 상기 유전자 변이 라이브러리는 무작위 변위를 통해 수득된 변이 유전자의 집단, 집합을 말한다. Step 3) is a step of obtaining a genetic mutation library by inducing a random genetic mutation in the gene encoding a second sugar isomerase, such as L-arabinose isomerase, random mutation is a mutation-induced PCT The error-prone PCR method is used to induce any mutation in the normal sequence as a template. The genetic variation library refers to a population and a set of variation genes obtained through random displacement.
상기 4) 단계는 상기 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계의 제1당 대사성 재조합 균주, 예컨대 D-타가토오스 대사성 균주에 도입하여 균주 라이브러리를 얻는 단계를 말하며, D-타가토오스 대사성 균주가 변이 유전자를 추가적으로 발현하고, 이를 통해 새로운 당 대사 경로를 구축한 균주의 라이브러리를 얻는 것을 의미한다. Step 4) refers to a step of introducing the mutation gene of the gene mutation library into the first sugar metabolic recombinant strain, such as D-tagatose metabolic strain of step 2) to obtain a strain library, D-tagatose metabolic This means that the strain additionally expresses the mutant gene, thereby obtaining a library of strains that have established new sugar metabolism pathways.
유전자 변이 라이브러리의 개별 변이 유전자가 1종씩 2) 단계의 D-타가토오스 대사성 균주에 도입되어 발현될 수 있으며, 이러한 공정을 통해 다양한 변이 유전자가 새롭게 도입된 균주 라이브러리를 얻을 수 있다. Individual mutation genes of the gene mutation library can be introduced and expressed in D-tagatose metabolic strains of step 2), and through this process, a strain library in which various mutation genes are newly introduced can be obtained.
상기 5) 단계는 상기 균주 라이브러리의 균주를 제2당을 유일 탄소원으로 포함하는 배지에서 배양하여 균의 성장을 확인하는 단계이며, 균의 성장을 확인하여 해당 균주가 제2당 전환능을 획득하였는지 여부를 빠르게 확인할 수 있다. 특히 균체의 성장이 우수하게 나타나는 균주를 제2당 전환능이 획득된 균주로 선별할 수 있으며, 해당 균주에 도입된 3) 단계의 유전자 변이 라이브러리의 변이 유전자를 확인함으로써, 제2당 전환능을 부여하는 제2당 이성화능을 갖는 변이체 및 이에 도입된 변이 효소를 도출해 낼 수 있다. Step 5) is a step of culturing the strain of the strain library in a medium containing a second sugar as the only carbon source to check the growth of the bacteria, by confirming the growth of the bacteria whether the strain obtained the second sugar conversion ability You can quickly check whether or not. In particular, strains showing excellent growth of cells can be selected as strains obtained with the second sugar conversion ability, and the second sugar conversion ability is given by identifying the mutation gene of the gene mutation library of step 3) introduced into the strain. It is possible to derive a variant having a second sugar isomerization ability and a variant enzyme introduced thereto.
따라서 본 발명은 1) 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계; 2) 제1당 대사능 및 제2당 이성화능이 결여된 균주에 상기 1) 단계에서 선별된 효소를 암호화하는 유전자를 도입하여 제1당 대사성 재조합 균주를 제조하는 단계; 3) 제2당 이성화 효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계; 4) 상기 3) 단계에서 수득된 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계에서 제조된 제1당 대사성 재조합 균주에 도입하여 균주 라이브러리를 얻는 단계; 5) 제2당을 유일 탄소원으로 포함하는 제한 배지에서 상기 4) 단계에서 수득된 라이브러리의 균주를 배양하고 높은 성장능을 갖는 균주를 선별하는 단계; 및 6) 상기 5) 단계에서 선별된 균주에 도입된 변이 효소를 확인하는 단계; 를 포함하며,상기 제1당과 제2당은 상호 이성질체인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 을 갖는 변이 당이성화 효소의 초고속 스크리닝 방법을 제공한다. Therefore, the present invention comprises the steps of: 1) selecting an enzyme essential for first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); 5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and selecting strains having high growth capacity; And 6) identifying the mutant enzyme introduced into the strain selected in step 5); It includes, The first sugar and the second sugar provides an ultra-fast screening method of mutant glycoisomerase having a second sugar isomerization activity, characterized in that the mutual isomers.
상기 방법의 각 단계는 앞서 기술된 제2당 이성화능 및 제1당 대사능을 갖는 변이체의 고속 스크리닝 방법에 대한 설명에 따라 수행될 수 있다. Each step of the method can be carried out according to the description of the method for fast screening of variants having a second sugar isomerization capacity and a first sugar metabolism capacity described above.
본 발명에서 일 실시예로 개시한, 당이성화능을 갖는 L-아라비노오스 이성화효소 변이효소 및 D-갈락토오스 당이성화 변이체의 고속 스크리닝 방법은 “세포성장 기반 초고속 탐색법”을 기반으로 하는 것을 특징으로 한다. The high-speed screening method of L-arabinose isomerase mutase and D-galactose glycoisomer variant having glycisomerization ability disclosed in one embodiment of the present invention is based on the "cell growth based ultrafast screening method". It is done.
본 발명의 세포성장 기반 초고속 탐색법은 다양한 후보 변이 유전자들을 플랫폼 균주에 도입하고, 이에 대한 세포 성장을 확인함으로써 이를 기초로 활성을 나타내는 변이 유전자를 빠르게 선별, 탐색할 수 있는 것을 의미한다. 보다 구체적으로 본 발명에서는 다양한 변이 L-아라비노오스 이성화효소 코딩 유전자의 라이브러리를 형성하고, 이를 D-갈락토오스 이성화능이 결여된 균주에 도입하고 D-갈락토오스 포함 제한 배지에서 균주를 배양함으로써 균주의 성장을 확인하는 방법을 통해, 수많은 변이 L-아라비노오스 이성화효소 코딩 유전자 중 D-갈락토오스 이성화능을 효과적으로 부여할 수 있는 특정 변이 유전자를 도출하여 D-갈락토오스 당이성화 변이효소로 이용할 수 있도록 한다. Cell growth-based ultrafast screening method of the present invention means that by introducing a variety of candidate mutation genes to the platform strain, and confirming the cell growth for this, it is possible to quickly select and search for the mutant gene showing activity based on this. More specifically, the present invention forms a library of various mutant L-arabinose isomerase coding genes, introduces it into a strain lacking D-galactose isomerization, and cultures the strain in a restriction medium containing D-galactose, thereby promoting growth of the strain. Through the identification method, a specific mutant gene capable of effectively assigning D-galactose isomerization among a number of mutant L-arabinose isomerase coding genes can be derived and used as a D-galactose glycosylation mutant.
또한 본 발명은 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자를 포함하는, 재조합 벡터를 제공한다. The present invention also provides a recombinant vector comprising a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase.
또한 본 발명은 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자가 도입된, D-갈락토오스 이성화능을 갖는 재조합 균주를 제공한다. The present invention also provides a recombinant strain having D-galactose isomerization, in which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced. do.
상기 D-타가토오스-1,6-비스포스페이트 알돌라아제를 암호화하는 유전자는 타가토스 대사능이 있는 미생물로부터 유래된 유전자일 수 있으며, 바람직하게는 Bacillus licheniformis 14580 유래의 gatY 유전자 (서열번호 5) 일 수 있다. 이로부터 코딩된 아미노산 서열은 서열번호 7로 나타내었다. The gene encoding the D-tagatose-1,6-bisphosphate aldolase may be a gene derived from a microorganism having tagatose metabolism, and preferably, Bacillus licheniformis GatY gene from SEQ ID NO: 14580 (SEQ ID NO: 5). The amino acid sequence encoded therefrom is shown in SEQ ID NO: 7.
또한 본 발명의 L-아라비노오스 이성화효소를 코딩하는 유전자는 갈락토스 대사능이 있는 미생물로부터 유래된 유전자일 수 있으며, 바람직하게는 Escherichia coli 유래의 araA 유전자 (서열번호 6) 일 수 있다. 이로부터 코딩된 아미노산 서열은 서열번호 8로 나타내었다. In addition, the gene encoding the L-arabinose isomerase of the present invention may be a gene derived from a microorganism having galactose metabolism, preferably an araA gene derived from Escherichia coli (SEQ ID NO: 6). The amino acid sequence encoded therefrom is shown in SEQ ID NO: 8.
본 발명의 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자를 포함하는 재조합 벡터를 이용하면, D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자가 도입된, D-갈락토오스 대사능을 갖는 재조합 균주를 수득할 수 있으며, 상기 재조합 균주는 외래 유전자 도입으로 새로운 변형된 당 대사 경로가 구축되므로, 종래 이용할 수 없었던 당, 바람직하게는 D-갈락토오스를 이용하여 유용물질인 D-타가토오스를 생산할 수 있는 대사능을 새롭게 가질 수 있다. 이와 같은 유전자 도입에 따라 구축된 새로운 당 대사 경로를 도 1에 모식도로 나타내었다. Using a recombinant vector comprising a gene encoding D-tagatose-1,6-bisphosphate aldolase of the present invention and a gene encoding L-arabinose isomerase, D-tagatose-1 A recombinant strain having D-galactose metabolism, into which a gene encoding a 6-bisphosphate aldolase and a gene encoding L-arabinose isomerase was introduced, the recombinant strain introduced a foreign gene. As a new modified sugar metabolic pathway is established, it is possible to newly have a metabolism capable of producing a useful substance D-tagatose using a sugar, preferably D-galactose, which has not been conventionally available. The new sugar metabolism pathway constructed by such gene introduction is shown in the schematic diagram in FIG. 1.
본 발명에서 "당 이성화" 및 "당 전환"은 상호 교환적으로 사용될 수 있다. In the present invention, "sugar isomerization" and "sugar conversion" may be used interchangeably.
본 발명의 "벡터”는 외래 유전자를 균주에 도입하기 위한 전달체를 말하는 것으로 바람직하게는 플라스미드일 수 있다. 상기 플라스미드는 당 분야에 통상적으로 널리 이용되는 것을 제한없이 사용할 수 있으나, 구체적으로 도 4에 나타낸 계열지도를 갖는 플라스미드를 이용하여 gatY 유전자를 도입하고, 도 8에 나타낸 계열지도를 갖는 플라스미드를 이용하여 araA 유전자를 도입할 수 있다. The "vector" of the present invention refers to a carrier for introducing a foreign gene into a strain, and may preferably be a plasmid The plasmid may be used without limitation, which is commonly used in the art, and specifically, in FIG. introducing gatY gene using the plasmid having the sequence shown map, and can be introduced into the araA gene by using the plasmid having the sequence map shown in Fig.
본 발명의 재조합 균주는 외래 유전자인 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자의 도입에 의하여 D-갈락토오스 대사능을 새롭게 가지게 된 균주이며, 특히 D-갈락토오스 대사능을 가지고 있지 않은 균주에 유전자가 도입된 균주이다. D-갈락토오스 대사능이 결여된 균주는 그 종류를 특별히 제한하지 않으나, 아그로박터리엄(Agrobacterium), 아조스피릴엄(Azospirillum) 바실러스(Bacillus), 버크홀데리아(Burkholderia), 클로스트리디엄(Clostridium), 엔테로박터(Enterobacter), 엔트로코커스(Enterococcus), 에스케리키아(Escherichia), 플라보박터리엄(Flavobacterium), 클렙시엘라(Klebsiella), 락토바실러스(Lactobacillus), 로도박터(Rhodobacter), 지오바실러스 (Geobacillus) 및 살모넬라(Salmonella)로 이루어진 그룹으로부터 선택된 1종의 속에 속하는 것일 수 있으며, 특히, 재조합에 가장 일반적으로 사용되는 엔테로박터(Enterobacter)에 속하는 대장균(E. coli)이 가장 바람직하다.Recombinant strains of the present invention by the introduction of the gene encoding the foreign gene D-tagatose-1,6-bisphosphate aldolase and the gene encoding the L- arabinose isomerase D-galactose metabolism ability It is a new strain, in particular, a strain in which a gene is introduced into a strain that does not have D-galactose metabolism. Strains lacking D-galactose metabolism do not have any particular limitation, but Agrobacterium, Azospirillum Bacillus, Burkholderia, Clostridium, Enterotrope Enterobacter, Enterococcus, Escherichia, Flavoobacterium, Klebsiella, Lactobacillus, Rhodobacter, Geobacillus E. coli belonging to the genus of one species selected from the group consisting of) and Salmonella, in particular, Enterobacter most commonly used for recombination ( E. coli ).
상기 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자가 도입된, D-갈락토오스 대사능을 갖는 재조합 균주는 D-갈락토오스를 D-타가토오스로 전환시키고, 타카토오스를 glycolysis에 이용할 수 있는 재조합 균주로, D-갈락토오스 및 D-타가토오스 대사능 또는 자화능을 모두 갖는 균주인 것을 특징으로 할 수 있다. Recombinant strain having D-galactose metabolism, wherein the gene encoding the D-tagatose-1,6-bisphosphate aldolase and the gene encoding the L-arabinose isomerase were introduced into D-galactose It is a recombinant strain that can be converted to D-tagatose and used for glycolysis, and may be characterized as a strain having both D-galactose and D-tagatose metabolic or magnetizing ability.
또한 본 발명은 D-갈락토오스 비대사성 균주에 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L-아라비노오스 이성화효소를 코딩하는 유전자를 도입하는 단계; 를 포함하는 D-갈락토오스 이성화능을 갖는 재조합 균주의 제조방법을 제공한다.In another aspect, the present invention comprises the steps of introducing a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase into a D-galactose non-metabolic strain; It provides a method for producing a recombinant strain having a D-galactose isomerization ability comprising a.
본 발명에서 상기 2 종의 유전자를 숙주 유전자에 도입하는 방법으로는 특별히 한정하지는 않으나, 벡터에 상기 유전자를 삽입하여 전기충격(electrophoresis) 및 열 충격 방법(heat shock transformation method) 등을 사용해 재조합 미생물 내에 도입함으로써, 재조합 균주에서 함께 발현될 수 있도록 하는 것이 바람직하고, 이용될 수 있는 벡터의 구체적인 예는 도 4 및 도 8에 나타내었다. In the present invention, the method of introducing the two kinds of genes into the host gene is not particularly limited, but by inserting the gene into a vector, the recombinant microorganism may be incorporated into a recombinant microorganism using an electrophoresis and a heat shock transformation method. By introducing, it is desirable to be able to be expressed together in a recombinant strain, and specific examples of vectors that can be used are shown in FIGS. 4 and 8.
균주에 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자를 도입하는 경우, 상기 2종 유전자의 도입은 동시에 또는 순차적으로 이루어질 수 있으며, 순차적으로 이용하는 경우 그 순서에는 제한이 없다. When introducing a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase into a strain, the introduction of the two genes may be performed simultaneously or sequentially. If used sequentially, the order is not limited.
또한 본 발명은 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자가 도입된, D-갈락토오스 대사능을 갖는 재조합 균주를 D-갈락토오스 포함 배지에서 배양하는 단계; 를 포함하는 D-타가토오스 생산 방법을 제공한다. The present invention also provides a recombinant strain having D-galactose metabolism, in which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced. Culturing in a galactose containing medium; It provides a D- tagatose production method comprising a.
상기 방법에 따르면, 기존에 D-갈락토오스 대사능이 결여된 균주라도 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자를 도입하는 공정을 통해 D-갈락토오스를 대사할 수 있게 함으로써, D-갈락토오스를 기질로 하는 배지에서 유용물질인 D-타가토오스를 생산할 수 있도록 할 수 있다. According to the method, even if the strain previously lacking D-galactose metabolism, the gene encoding D-tagatose-1,6-bisphosphate aldolase and the gene encoding L-arabinose isomerase are introduced. By metabolizing D-galactose through the process, it is possible to produce D-tagatose, a useful substance in a medium based on D-galactose.
상기 D-갈락토오스 포함 배지는 재조합 균주의 생장에 부합하는 성분을 포함하는 배지일 수 있으며, 탄소원으로는 D-갈락토오스 및/또는 포도당만을 포함하고 있는 제한 배지일 수 있다. The D-galactose containing medium may be a medium containing a component corresponding to the growth of a recombinant strain, and may be a restriction medium containing only D-galactose and / or glucose as a carbon source.
또한 본 발명은 서열번호 2 내지 4로 이루어진 군에서 선택된 1종의 아미노산 서열로 표시되는, 당이성화능을 갖는 L-아라비노오스 이성화효소 변이효소를 제공한다. 상기 변이 효소는 세포성장기반 초고속 탐색법으로 스크리닝된 당이성화효소 변이체의 변이 아미노산 서열이며, 아라비노오스 이성화효소를 무작위 변이시키고, 이를 균주에 도입한 후, D-갈락토오스 포함 배지에서 재조합 균주를 배양하고 성장을 확인함으로써 도출된 효소이다. In another aspect, the present invention provides an L-arabinose isomerase mutase having glycoisomerization ability, represented by one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4. The mutant enzyme is a mutant amino acid sequence of glycoisomerase variants screened by cell growth-based ultrafast screening method, randomly mutated arabinose isomerase and introduced into the strain, followed by culturing the recombinant strain in D-galactose containing medium. It is an enzyme derived by checking growth.
본 발명의 당이성화능을 갖는 L-아라비노오스 이성화효소 변이효소는 당 이성화 효소의 기능을 동등하게 나타내는 한, 서열번호 2, 3 및 4로 표시되는 효소들과 각각 90 내지 99%, 바람직하게는 95 내지 97%, 더욱 바람직하게는 97 내지 99%의 상동성을 갖는 서열을 모두 제한없이 포함할 수 있다. L-Arabinose isomerase mutase having a glycisomerizing ability of the present invention is 90 to 99%, respectively, preferably with the enzymes represented by SEQ ID NOs: 2, 3 and 4, as long as the function of sugar isomerization is equivalent. Can include without limitation any sequence having a homology of 95 to 97%, more preferably 97 to 99%.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.
실시예Example 1: D-갈락토오스  1: D-galactose 대사능이Metabolism 결여된 대장균의 확보 Seizure of E. coli missing
기질인 D-갈락토오스에 대해 대사능이 없는 숙주세포를 선별하고 이를 실험적으로 확인하였다. D-갈락토오스에 대사능이 없는 대장균 BL21 (DE3)균주의 유전체 분석을 수행하였으며, 그 결과 해당 균주에서는 탄수화물 대사 경로 중 D-갈락토오스 대사에 관여하는 효소를 암호화하는 galK (EC 2.7.1.6 galactosekinase), galT (EC 2.7.1.12 galactose 1-phosphate uridylyltransferase), 및 galE (EC 5.1.3.2 UDP-galactose 4-epimerase)가 결여되어 있음을 확인하였다. 이러한 정보를 바탕으로, 실험적으로 영양원에 따른 균체 성장을 확인하기 위해 상기 D-갈락토오스 대사 관련 유전자들이 결여된 대장균 BL21 (DE3) 균주와 D-갈락토오스 대사 관련 유전자를 결여되지 않은 대장균 DH5α 를 복합(LB) 배지와 5 g/L 포도당(glc) 또는 5 g/L D-갈락토오스(gal)가 각각 포함된 제한(M9)배지에 배양하여 35시간 동안 균체 성장을 확인하였으며, 그 결과를 도 2에 나타내었다. Host cells without metabolism against the substrate D-galactose were selected and confirmed experimentally. It was performed to analyze genome of E. coli BL21 (DE3) strain with no ability to D- galactose metabolism, resulting in the strain carbohydrate metabolism galK (EC 2.7.1.6 galactosekinase) coding for the enzyme involved in the metabolism of D- galactose path, galT (EC 2.7.1.12 galactose 1-phosphate uridylyltransferase), and galE (EC 5.1.3.2 UDP-galactose 4-epimerase). Based on this information, in order to experimentally confirm cell growth according to nutrient sources, E. coli BL21 (DE3) strain lacking the D-galactose metabolism related gene and E. coli DH5α lacking the D-galactose metabolism related gene were combined (LB ) Cell growth was confirmed for 35 hours by culturing in a medium (M9) medium containing 5 g / L glucose (glc) or 5 g / L D-galactose (gal), respectively, the results are shown in Figure 2 It was.
도 2에 나타낸 바와 같이, 대장균 DH5α는 포도당과 D-갈락토오스를 탄소원으로 사용할 수 있는 반면, 대장균 BL21 (DE3) 는 D-갈락토오스 첨가 배지에서 균체 성장이 일어나지 않는 것을 실험을 통해 확인하였다.As shown in Figure 2, Escherichia coli DH5α can use glucose and D-galactose as a carbon source, while E. coli BL21 (DE3) was confirmed through experiments that cell growth does not occur in D-galactose addition medium.
실시예Example 2: D- 2: D- 타가토오스Tagatose 대사에 필수적인 외래 유전자 탐색 Exploring Foreign Genes Essential for Metabolism
D-타가토오스 대사에 필수적인 효소군 및 유전자를 탐색하기 위하여, D-타가토오스 대사능이 있는 미생물 Bacillus licheniformis 14580 과 D-타가토오스 대사능이 없는 미생물 E. coli BL21 (DE3) 균주의 유전체 정보를 직접 비교 및 분석하였으며, 그 결과를 도 3에 모식화하여 나타내었다. In order to search for an essential enzyme, and the gene D- tagatose metabolism, D- tagatose microorganisms metabolic capability Bacillus licheniformis 14580 and Microorganism E. coli Without D-tagatose Metabolism Genome information of the BL21 (DE3) strain was directly compared and analyzed, and the results are schematically shown in FIG. 3.
도 3에 나타낸 바와 같이, D-타가토오스 대사능이 있는 미생물인 Bacillus licheniformis 14580 은 D-타가토오스 대사능이 없는 E. coli BL21 (DE3) 와 비교하여 D-타카토스 1, 6-bisphosphate aldolase를 암호화하는 gatY 유전자를 가지고 있는 것을 확인하였으며, 이를 D-타가토오스 대사에 필수적인 효소로 선정하였다. As shown in FIG. 3, Bacillus licheniformis 14580, a microorganism having D-tagatose metabolism, is E. coli without D-tagatose metabolism. Compared with BL21 (DE3), it was confirmed that the gatY gene encoding D-tagatose 1 and 6-bisphosphate aldolase was selected as an essential enzyme for D-tagatose metabolism.
실시예Example 3:  3: GatYGatY 도입을 통한 형질전환체의  Of transformants through introduction 대사능Metabolism 획득 확인 Acquisition confirmation
3.1 3.1 GatYGatY 단백질의 발현 벡터 및 대장균  Expression Vectors of Proteins and E. Coli 형질전환균체의Of transformed cells 제조 Produce
상기 실시예 2를 통해 D-타가토오스 대사에 필수적일 것으로 예상된 gatY 유전자를 이용하여 발현벡터를 제조하였다. 발현 벡터를 제조하기 위하여, 먼저 각각의 제한효소 서열 (NdeI 또는 XhoI)이 포함된 정방향 프라이머 (Bli03552 gatY NdeI F 5-CATATGCTGACAAATACGAAAAAAATGC-3) 와 역방향 프라이머(Bli03552 gatY XhoI R 5-CTCGAGTTAGTATCTGTCATTGCTCATGC-3) 를 발현벡터 제조에 맞게끔 쌍으로 PCR에 사용하였다. 유전자 증폭을 위하여 Bacillus licheniformis 균체의 genomic DNA를 주형으로 사용하여 PCR을 수행하였고, 구체적으로 Takara 사의 primeSTAR HS DNA polymerase를 사용하여 98℃에서 30초, 57℃에서 5초 및 72℃에서 1분으로 30 사이클을 수행 후, 72℃에서 10분간 수행하였다. 증폭된 PCR 산물을 전기영동 후 Gel extract kit (Qiagen)를 사용하여 분리 정제하였으며 pTOP blunt V2 cloning kit (Enzynomics)를 사용하여 클로닝한 후, DNA 시퀀싱 (Solgent) 을 통하여 각각의 유전자의 변이삽입 유무를 확인하였다. PCR을 통해 획득한 유전자에 변이가 없는 것을 확인한 후 유전자 발현벡터 제조를 위하여 pET-28(+) (Novagen)벡터를 제한효소 (NdeI, XhoI)로 처리하였으며, pTOP Blunt V2벡터에 클로닝 되어있는 유전자의 회수를 위하여 발현용 벡터에 맞는 제한효소를 처리하여 전기영동 및 Gel extraction kit (Qiagen)을 사용하여 분리, 정제하였다. 제한효소가 처리된 벡터 및 회수된 유전자들을 DNA ligation kit (promega)를 사용하여 라이게이션시켜 발현벡터 pET-28a(+)-gatY를 제조하였다. 발현벡터의 유전자 지도를 도 4 에 나타내었다. 또한 유전자의 발현을 위한 대장균 형질전환체를 제조하기 위하여, 도 4의 발현벡터를 D-갈락토오스 비대사능이 확인된 대장균 BL21 (DE3) 에 도입하였다. 형질전환균을 선택적으로 배양하기 위하여 상기 발현벡터에 포함된 항생제 내성 유전자에 적합한 항생제인 카나마이신 (kanamycin)이 최종농도 50 μg/ml로 포함된 LB 고체배지에 형질전환균을 도말하고 37℃에서 배양하여 단일 콜로니를 형성하였다. In Example 2, an expression vector was prepared using the gatY gene expected to be essential for D-tagatose metabolism. To prepare the expression vector, first, a forward primer (Bli03552 gatY Nde I F 5-CATATGCTGACAAATACGAAAAAAATGC-3) and a reverse primer (Bli03552 gatY Xho IR 5-CTCGAGTTAGTATCTGTCATTGCTCATGC-3) containing respective restriction enzyme sequences ( Nde I or Xho I) Were used in PCR in pairs to suit the expression vector preparation. PCR was performed using genomic DNA of Bacillus licheniformis cells as a template for gene amplification. Specifically, primestar HS DNA polymerase from Takara was used for 30 seconds at 98 ° C, 5 seconds at 57 ° C, and 30 minutes at 72 ° C for 1 minute. After performing the cycle, it was carried out at 72 ℃ 10 minutes. The amplified PCR product was purified by gel extraction kit (Qiagen) after electrophoresis and cloned using pTOP blunt V2 cloning kit (Enzynomics), followed by DNA sequencing (Solgent). Confirmed. For the gene expression vector prepared after checking for the absence of the mutation in a gene obtained through PCR was processed the pET-28 (+) (Novagen ) vector with restriction enzymes (Nde I, Xho I), it is cloned into the pTOP Blunt V2 vector In order to recover the genes, the restriction enzymes corresponding to the expression vector were processed and isolated and purified using electrophoresis and gel extraction kit (Qiagen). Restriction enzyme-treated vectors and recovered genes were ligated using DNA ligation kit (promega) to prepare expression vector pET-28a (+)- gatY . The gene map of the expression vector is shown in FIG. In addition, in order to prepare E. coli transformants for the expression of the gene, the expression vector of Figure 4 was introduced into E. coli BL21 (DE3) confirmed D-galactose metabolism. In order to selectively cultivate the transforming bacteria, the transforming bacteria were plated in LB solid medium containing kanamycin, an antibiotic suitable for the antibiotic resistance gene included in the expression vector, at a final concentration of 50 μg / ml and incubated at 37 ° C. To form a single colony.
3.2 3.2 대장균내의E. coli GatYGatY 단백질의 발현 확인 Confirmation of Protein Expression
상기 3.1에서 수득된 재조합 과발현벡터 pET-28a(+)gatY 를 대장균 BL21 (DE3) 균주에 형질전환하고 전배양 후 플라스크 내에서 카나마이신 (50 μg/ml)을 첨가한 LB에 1%의 전배양균을 접종하고 본배양하여 흡광도 (600 nm)가 0.4-0.6일 때, 최종 농도 1 mM의 IPTG (Isopropyl -D-1-thiogalactopyranoside)를 사용하여 gatY 유전자를 37℃에서 6시간 동안 발현을 유도하였다. Recombinant overexpression vector pET-28a (+) gatY obtained in 3.1 was transformed into Escherichia coli BL21 (DE3) strain, and then precultured to 1% precultured LB to kanamycin (50 μg / ml) in the flask When the absorbance (600 nm) was 0.4-0.6 by inoculation , the expression of gatY gene was induced at 37 ° C. for 6 hours using IPTG (Isopropyl -D-1-thiogalactopyranoside) at a final concentration of 1 mM.
GatY 단백질 발현을 유도시킨 균체의 흡광도 (600 nm) 1 에 해당하는 배양액을 1.5 ml E-tube에 옮긴 후 원심분리 후 배양 균체만을 회수하였으며, 100 μl의 1X SDS sample buffer로 균체를 현탁한 후 끓는 물에서 10분간 처리 후 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 분석을 수행하고 그 결과를 도 5에 나타내었다. After transferring the culture solution corresponding to the absorbance of the GatY protein (600 nm) 1 to 1.5 ml E-tube and centrifugation, only the cultured cells were recovered, and the cells were suspended with 100 μl of 1X SDS sample buffer and boiled. After treatment for 10 minutes in water 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis was performed and the results are shown in FIG.
도 5에 나타낸 바와 같이, GatY 단백질의 발현(30 kDa)이 확인되었으며, gatY 유전자의 도입이 일어났음을 확인하였다. As shown in Figure 5, the expression of GatY protein (30 kDa) was confirmed, it was confirmed that the introduction of the gatY gene.
3.3 D-3.3 D- 타가토오스를Tagatose 이용한  Used 형질전환균의Transgenic 균체성장Cell growth 확인 Confirm
재조합 유전자의 발현에 따라 대장균 BL21(DE3) 숙주 세포가 D-타가토오스 대사능을 획득하였는지 여부를 확인하기 위하여, 상기 3.1 내지 3.2를 통해 수득한 형질전환된 신규 대장균 숙주세포 pET-28a(+)- gatY/E. coli BL21 (DE3)를 유일 탄소원으로 5 g/L D-타가토오스가 포함된 제한(M9)배지에서 배양하고 균체 성장을 확인하였다. 그 결과는 도 6 및 도 7에 나타내었다. In order to confirm whether E. coli BL21 (DE3) host cells obtained D-tagatose metabolism according to the expression of the recombinant gene, transformed novel E. coli host cells pET-28a (+) ) -gatY / E. coli BL21 (DE3) was cultured in a restriction (M9) medium containing 5 g / L D-tagatose as the only carbon source and cell growth was confirmed. The results are shown in FIGS. 6 and 7.
도 6에 나타낸 바와 같이, 외래유전자인 gatY가 도입된 형질전환균은 D-타가토오스 포함 제한배지에서도 글루코스 포함 배지와 유사한 정도로 우수한 성장 및 비증식속도를 나타냈으며, 도 7에 나타낸 바와 같이, D-tagatose 1, 6- bisphosphate aldolase를 암호화하는 gatY 외래유전자가 과발현된 대장균 BL21(DE3) 숙주세포는 외래 단백질 발현에 따라 균체 성장이 원활하게 일어남을 확인하였다. 그러나 이는 0.5% D-갈락토오스를 유일 탄소원으로 하는 제한배지에서는 균체 성장을 나타내지 않았다. 따라서 D-갈락토오스 대사능을 부여하기 위해서는 추가적인 당 대사 경로의 변형이 필요함을 확인하였다. As shown in FIG. 6, the transgenic bacteria introduced with the foreign gene gatY exhibited excellent growth and specific growth rate similar to that of the glucose-containing medium even in the restriction medium containing D-tagatose. As shown in FIG. 7, E. coli BL21 (DE3) host cells overexpressing the gatY exogenous gene encoding D-tagatose 1 and 6-bisphosphate aldolase were found to have a favorable cell growth according to foreign protein expression. However, this did not show cell growth in a restriction medium containing 0.5% D-galactose as the only carbon source. Therefore, it was confirmed that an additional modification of the sugar metabolism pathway is required to impart D-galactose metabolism.
실시예Example 4: D-갈락토오스  4: D-galactose 이성화능을Rationality 갖는 균주의 제조 Preparation of Having Strain
당이성화효소 라이브러리 탐색을 위한 플랫폼 균주 및 스크리닝 시스템 개발을 위해 상기 실시예 3에서 제조된 숙주세포, 즉 D-갈락토오스 대사능이 결여되고, D-타가토오스에 대해서는 대사능을 갖는 균주에 당이성화효소 L-아라비노오스 이성화효소(arabinose isomerase) 단백질 발현이 일어나도록 하였다. Platform strain for screening the glycosylase library and the screening system for the development of the host cell prepared in Example 3, that is, D-galactose metabolism lacking, the strain having metabolism for D-tagatose glycosylase L-arabinose isomerase protein expression was allowed to occur.
이를 위해 L-아라비노오스 이성화효소 단백질의 발현벡터를 제조하였다. 발현벡터를 제조하기 위하여 먼저 각각의 제한효소 서열 (NdeI 또는 HindIII)이 포함된 정방향 프라이머(ECAI NdeI F, 5'- CATATGACGATTTTTGATAATTATGAAGTGTGG-3')와 역방향 프라이머(ECAI HindIII R, 5'- AAGCTTTTAGCGACGAAACCCGTAATAC-3'를 제작하였으며 이를 유전자의 증폭에 사용하였다. PCR 방법을 통한 유전자 증폭을 위하여 Escherichia coli 균체의 genomic DNA를 주형으로 사용하였으며, Takara 사의 primeSTAR HS DNA polymerase를 사용하여 98도에서 30초, 57도에서 5초, 및 72도에서 1분 40초로 30 사이클을 수행 후, 72도에서 10분간 반응을 수행하였다. 증폭된 PCR산물을 전기영동 후 Gel extract kit (Qiagen)를 사용하여 분리 정제하였으며 pTOP blunt V2 cloning kit (Enzynomics)를 사용하여 클로닝한 후, DNA 시퀀싱 (Solgent)을 통하여 각각의 유전자의 변이삽입 유무를 확인하였다. PCR을 통해 획득한 유전자에 변이가 없는 것을 확인한 후 유전자 발현벡터 제조를 위하여 pET-22b(+) (Novagen)벡터를 제한효소 (NdeI, HindIII)로 처리하였으며, pTOP Blunt V2벡터에 클로닝 되어있는 유전자의 회수를 위하여 발현용 벡터에 맞는 제한효소를 처리하여 전기영동 및 Gel extraction kit (Qiagen)을 사용하여 분리, 정제하였다. 제한효소가 처리된 벡터 및 회수된 유전자들을 DNA ligation kit (promega)를 사용하여 라이게이션시켜 발현벡터 pET-22b(+)-araA를 제조하였다. 이와 같이 제조된 발현벡터의 유전자 지도를 도 8에 나타내었다. To this end, an expression vector of L-arabinose isomerase protein was prepared. To prepare an expression vector, a forward primer (ECAI Nde I F, 5'-CATATGACGATTTTTGATAATTATGAAGTGTG-3 ') and a reverse primer (ECAI Hin dIII R, 5'- AAGCTTTTAGCGACGAAACCCGTAATAC), each containing a restriction enzyme sequence ( Nde I or Hin dIII), were first prepared. -3 'was used to amplify the gene, and genomic DNA of Escherichia coli cells was used as a template for gene amplification by PCR method. After 30 cycles of 5 seconds and 1 minute 40 seconds at 72 degrees, the reaction was carried out for 10 minutes at 72 degrees.The amplified PCR product was subjected to electrophoresis and separated and purified using Gel extract kit (Qiagen). After cloning using the blunt V2 cloning kit (Enzynomics), DNA sequencing (Solgent) was used to confirm the presence of mutations in each gene. The check after pET-22b (+) (Novagen ) vector for the gene expression vector prepared restriction enzyme were treated with (Nde I, Hin dIII), the expression vector for the to the number of genes that are cloned in pTOP Blunt V2 vector that The restriction enzymes were treated and isolated and purified using electrophoresis and gel extraction kit (Qiagen) .The vector treated with restriction enzyme and recovered genes were ligated using DNA ligation kit (promega) to express the expression vector pET-. 22b (+)-araA was prepared A gene map of the expression vector thus prepared is shown in FIG.
유전자의 발현을 위해 대장균 형질전환체를 아래와 같이 제조하였다. 상기 D-갈락토오스 비대사능이 확인되고, D-타가토오스 대사능이 탑재된 대장균 pET-28a(+)-gatY/BL21(DE3) 균주에 도 8에 나타낸 재조합 발현 벡터를 도입하였다. 형질전환균을 선택적으로 배양하기 위하여 상기 발현벡터에 포함된 항생제 내성 유전자에 적합한 항생제인 엠피실린 (ampicillin)이 최종농도 100 μg/ml이 포함된 LB 고체배지에 도말하고 37℃에서 배양하여 싱글 콜로니를 형성하였다. 상기 형질전환체를 전배양 후 플라스크 내에서 엠피실린 (100 μg/ml)을 첨가한 LB에 1%의 전배양균을 접종하고 본배양하여 흡광도 (600 nm)가 0.4-0.6일 때, 최종 농도 1 mM의 IPTG (Isopropyl -D-1-thiogalactopyranoside)를 사용하여 araA 유전자의 발현을 37℃에서 6시간 동안 유도하였다. 단백질 발현을 유도시킨 균체의 흡광도 (600 nm) 1에 해당하는 배양액을 1.5 ml E-tube에 옮긴 후 원심분리하고 배양 균체만을 회수하였으며, 100 μl 의 1X SDS sample buffer로 균체를 현탁한 후 끓는 물에서 10분간 처리 후 12% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) 분석을 통해 대장균 내에서의 D-tagatose 1, 6-bisphosphate aldolase 효소를 암호화하는 gatY 유전자의 발현 (31 kDa) 및 L-arabinose isomerase 효소를 암호화하는 araA 유전자의 발현 (56 kDa)여부를 도 9와 같이 확인하였다. E. coli transformants were prepared for expression of the genes as follows. The recombinant expression vector shown in FIG. 8 was introduced into the E. coli pET-28a (+)-gatY / BL21 (DE3) strain in which the D-galactose hypertrophy was confirmed and the D-tagatose metabolism was loaded. In order to selectively cultivate the transformed bacteria, an antibiotic, ampicillin, which is suitable for the antibiotic resistance gene included in the expression vector, is plated in an LB solid medium containing a final concentration of 100 μg / ml and cultured at 37 ° C. to form a single colony. Was formed. After the incubation of the transformant, inoculated with 1% precultured bacteria to LB added with empicillin (100 μg / ml) in the flask, and the main concentration was obtained when the absorbance (600 nm) was 0.4-0.6. 1 mM of IPTG (Isopropyl -D-1-thiogalactopyranoside) was used to induce the expression of the araA gene at 37 ° C for 6 hours. After transferring the culture solution corresponding to the absorbance (600 nm) 1 of the cells that induced protein expression into 1.5 ml E-tube, centrifugation was performed, and only the cultured cells were recovered. The cells were suspended with 100 μl of 1X SDS sample buffer and boiled water. Expression of the gatY gene (31 kDa) and L- encoding the D-tagatose 1 and 6-bisphosphate aldolase enzymes in Escherichia coli via 10% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis Expression of the araA gene encoding the arabinose isomerase enzyme (56 kDa) was confirmed as shown in FIG. 9.
도 9에 나타낸 바와 같이, 형질전환된 대장균에서는 D-tagatose 1, 6-bisphosphate aldolase 효소를 암호화하는 gatY 유전자 (31 kDa) 및 L-arabinose isomerase 효소를 암호화하는 araA 유전자(56 kDa)가 발현하였다. As shown in Figure 9, transformed Escherichia coli expressed the gatY gene (31 kDa) encoding the D-tagatose 1, 6-bisphosphate aldolase enzyme and the araA gene (56 kDa) encoding the L-arabinose isomerase enzyme.
제작된 세포성장기반 스크리닝 탐색법의 이성화 효소의 활성 대비 및 세포성장을 확인하기 위해, 상기 두 플라스미드 pET-22b(+)-araA와 pET-28(+)-gatY가 형질전환된 대장균 BL21(DE3)을 0.05 g/L 프락토오스와 0.45 g/L D-갈락토오스, 최종 농도 0.2 mM IPTG, 및 상기 두 플라스미드에 적합한 항생제인 엠피실린(50 μg/ml)과 카나마이신(25 μg/ml)이 포함된 제한 (M9)배지에서 배양하여 당이성화 효소의 D-갈락토오스 이성화능에 의한 균체 성장곡선을 확인하였으며, 그 결과를 도 10에 나타내었다. E. coli BL21 (DE3) transformed with the two plasmids, pET-22b (+)- araA and pET-28 (+)- gatY , to confirm the activity comparison and cell growth of the isomerase of the prepared cell growth-based screening method ), 0.05 g / L fructose and 0.45 g / L D-galactose, final concentration 0.2 mM IPTG, and empicillin (50 μg / ml) and kanamycin (25 μg / ml) suitable for the two plasmids. Cultured in the restriction (M9) medium to confirm the cell growth curve by the D-galactose isomerization of glycoisomerase, the results are shown in FIG.
도 10에 나타낸 바와 같이, D-갈락토오스를 포함하는 배지에서 성장하지 못하는 대장균 BL21(DE3) 균주에 D-tagatose 1, 6-bisphosphate aldolase 효소를 암호화하는 gatY 유전자 및 L-arabinose isomerase 효소를 암호화하는 araA 유전자가 도입된 균주는 균체 성장이 일어났으며, 이성화 효소의 활성으로 D-갈락토오스를 유일 탄소원으로 이용할 수 있음을 확인하였다.  As shown in FIG. 10, the gatY gene encoding the D-tagatose 1 and 6-bisphosphate aldolase enzymes and the araA encoding the L-arabinose isomerase enzymes in E. coli BL21 (DE3) strains that do not grow in a medium containing D-galactose. The strain into which the gene was introduced had cell growth, and it was confirmed that D-galactose could be used as the only carbon source due to the activity of isomerase.
실시예Example 5:  5: araAaraA 유전자의 변이-유발을 통한,  Through mutation-induced genes, 당이성화Sugar isomerization 효소  enzyme 변이체Variant 선별 Selection
5.1 5.1 araAaraA 유전자의 변이-유발 (error-prone)  Gene-error-prone PCRPCR 및 변이 다양성 확인 And variation diversity
L-아라비노오스 이성화효소 araA 유전자의 변이를 유발하기 위해, PCR 무작이 돌연변이 키트 (Clontech, 미국)로 변이 PCR을 수행하였다. 변이-유발 PCR 라이브러리 DNA 50 ng을 플라스미드 pET-28a(+)-gatY가 포함된 대장균 BL21(DE3)에 형질전환하였으며, 0.05g/L의 프락토스와 0.45%의 D-갈락토오스가 포함된 제한(M9)배지에서 배양을 수행하였다. 그 후, 형성된 콜로니를 모아 플라스미드 정제 키트를 이용하여 플라스미드를 추출하였다. 그 중 일부 플라스미드 8개의 염기서열을 분석하였으며, 라이브러리의 유전자 다양성을 확인하였다. To induce mutations in the L-arabinose isomerase araA gene, mutation PCR was performed with a PCR randomization mutation kit (Clontech, USA). 50 ng of the mutation-induced PCR library DNA was transformed into Escherichia coli BL21 (DE3) containing plasmid pET-28a (+)-gatY, with a restriction containing 0.05 g / L of fructose and 0.45% of D-galactose ( Culture was carried out in M9) medium. Thereafter, colonies formed were collected and plasmids were extracted using the plasmid purification kit. Among them, 8 nucleotide sequences of some plasmids were analyzed, and the gene diversity of the library was confirmed.
5.2 세포성장기반 초고속 탐색법을 이용한 5.2 Using Cell Growth-based Ultrafast Screening 당이성화효소Glycoisomerase 변이체Variant 선발 Selection
상기 다양성이 확인된 당이성화효소 라이브러리 유전자 변이 아라비노오스 이성화효소 유전자 pET-22b(+)-araA library DNA가 형질전환된 pET-22b(+)-araA library DNA 및 변이를 유발하지 않은 pET-28a(+)-gatY/BL21(DE3)를 탄소원으로 0.05 g/L 프락토오스와 0.45 g/L D-갈락토오스, 및 최종 농도 0.2 mM IPTG가 포함된 제한 (M9)배지에서 배양하고 균체성장을 확인하였으며, 그 결과를 도 11에 나타내었다.Glycosylase Library Gene Mutations of the Diversity The arabinos isomerase gene pET-22b (+)-araA library DNA transformed and pET-28a which did not induce mutation Cell growth was confirmed by incubating (+)-gatY / BL21 (DE3) in a restriction (M9) medium containing 0.05 g / L fructose and 0.45 g / L D-galactose as the carbon source and 0.2 mM IPTG at a final concentration. The results are shown in FIG.
도 11에 나타낸 바와 같이, 변이 라이브러리 유전자의 단백질 발현이 다양하게 나타남에 따라 균체 성장의 수준 및 비증식속도는 다르게 나타남을 확인하였으며, 이를 통해 araA 유전자의 도입 및 이의 변이에 따라 D-갈락토오스를 탄소원으로 이용할 수 있는 능력이 각각 상이하게 나타날 수 있음을 확인하였다. As shown in FIG. 11, as the protein expression of the mutant library genes varied, it was confirmed that the level of growth and specific growth rate of the cell growth was different. Through this, D-galactose was used as the carbon source according to the introduction of the araA gene and its variation. As a result, it was confirmed that the ability to be used may appear differently.
5.3. 5.3. 당이성화효소Glycoisomerase 변이효소의 D-갈락토오스  D-galactose of mutant enzyme 이용능Utilization 검증  Verification
상기 5.2의 세포성장기반 스크리닝 탐색법으로 선발된 당이성화효소 변이체 및 야생형으로부터 하기와 같이 플라스미드를 분리하였다: pET-22b(+)-ECAI H17R/R159S/V168A, pET-22b(+)-ECAI E22D/M95L/H157L, 및 pET-22b(+)-ECAI-V368A/E493D. 또한 분리된 플라스미드 유래의 서열분석을 통하여 스크리닝 탐색법으로 선발된 변이체들의 변이 아미노산 서열을 분석하였으며, 이를 각각 서열번호 2 내지 4에 나타내었다. 야생형 ECAI 의 아미노산 서열은 서열번호 1에 나타내었다. Plasmids were isolated from glycoisomerase variants and wild type selected by the cell growth based screening assay of 5.2 above: pET-22b (+)-ECAI H17R / R159S / V168A, pET-22b (+)-ECAI E22D / M95L / H157L, and pET-22b (+)-ECAI-V368A / E493D. In addition, the mutated amino acid sequence of the variants selected by the screening search method through the sequencing derived from the isolated plasmid was analyzed, which are shown in SEQ ID NOs: 2 to 4, respectively. The amino acid sequence of wild type ECAI is shown in SEQ ID NO: 1.
추가적으로 Ni2+ 컬럼 크로마토그래피를 수행하여 변이효소를 순수분리하여 ECAI 야생형 효소와의 활성비교를 수행하였다. 즉, 숙주 균주 (Host), ECAI 야생형 및 당이성화 효소 변이체들의 D-갈락토오스에 대한 활성을 측정, 비교하였으며, 이를 도 12에 나타내었다.In addition, Ni2 + column chromatography was performed to purely separate the mutant enzymes and to compare the activity with the ECAI wild type enzymes. In other words, the host strain (Host), ECAI wild-type and glycoisomerase variants were measured and compared the activity against D-galactose, shown in Figure 12.
도 12에 나타낸 바와 같이, pET-22b(+)-ECAI H17R/R159S/V168A 유래 변이효소 (서열번호 2), pET-22b(+)-ECAI E22D/M95L/H157L 유래 변이효소 (서열번호 3), 및 pET-22b(+)-ECAI-V368A/E493D 유래 변이효소 (서열번호 4) 의 경우 D-갈락토오스에 대한 활성이 최대 80% 배 증가하였음을 확인하였다. As shown in FIG. 12, pET-22b (+)-ECAI H17R / R159S / V168A-derived mutase (SEQ ID NO: 2), pET-22b (+)-ECAI E22D / M95L / H157L-derived mutase (SEQ ID NO: 3) , And pET-22b (+)-ECAI-V368A / E493D-derived mutase (SEQ ID NO: 4) showed up to 80% fold increase in activity against D-galactose.
이와 같은 결과를 통해, L- 아라비노오스 이성화효소의 무작위적인 변이를 유도하고 라이브러리를 형성한 후, 이를 균주에 도입하여 균주 라이브러리를 형성하고 균주 성장을 통한 D-갈락토오스 대사능을 확인하는 경우, D-갈락토오스 대사능을 부여할 수 있는 특정 변이 효소 및 당이성화 효소 변이체를 빠르게 선별할 수 있음을 확인하였다. Through these results, when inducing random variation of the L- arabinose isomerase and forming a library, and then introduced into the strain to form a strain library and confirm the D-galactose metabolism through the growth of the strain, It was confirmed that specific mutation enzymes and glycosylation enzyme variants capable of conferring D-galactose metabolism can be selected quickly.
비록 본 발명이 상기에 언급된 바람직한 실시예로서 설명되었으나, 발명의 요지와 범위로부터 벗어남이 없이 다양한 수정이나 변형을 하는 것이 가능하다. 또한 첨부된 청구 범위는 본 발명의 요지에 속하는 이러한 수정이나 변형을 포함한다.Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the spirit of the invention.

Claims (13)

1) 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계; 1) selecting an enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism ability;
2) 제1당 대사능 및 제2당 이성화능이 결여된 균주에 상기 1) 단계에서 선별된 효소를 암호화하는 유전자를 도입하여 제1당 대사성 재조합 균주를 제조하는 단계; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability;
3) 제2당 이성화 효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library;
4) 상기 3) 단계에서 수득된 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계에서 제조된 제1당 대사성 재조합 균주에 도입하여 균주 라이브러리를 얻는 단계; 및4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2); And
5) 제2당을 유일 탄소원으로 포함하는 제한 배지에서 상기 4) 단계에서 수득된 라이브러리의 균주를 배양하고 균의 성장 및 생장속도를 확인하는 단계; 를 포함하며, 5) culturing the strain of the library obtained in step 4) in a restriction medium containing a second sugar as the only carbon source and checking the growth and growth rate of the bacteria; Including;
상기 제1당과 제2당은 상호 이성질체인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 및 제1당 대사능을 갖는 변이체의 초고속 스크리닝 방법. The first sugar and the second sugar are mutual isomers, characterized in that the ultra-fast screening method of variants having a second sugar isomerization activity (first sugar metabolism) and the first sugar metabolism.
제1항에 있어서, 제1당 및 제2당은 The method of claim 1, wherein the first sugar and the second sugar
제1당은 타가토오스 및 제2당은 갈락토오스인 제1조합; A first combination wherein the first sugar is tagatose and the second sugar is galactose;
제1당은 프럭토오스 및 제2당은 글루코오스인 제2조합; A second combination wherein the first sugar is fructose and the second sugar is glucose;
제1당은 타가토오스 및 제2당은 프럭토오스인 제3조합; A third combination wherein the first sugar is tagatose and the second sugar is fructose;
제1당은 만노오스 및 제2당은 글루코오스인 제4조합;A fourth combination wherein the first sugar is mannose and the second sugar is glucose;
제1당은 글루코오스 및 제2당은 갈락토오스인 제5조합; 및 A fifth combination wherein the first sugar is glucose and the second sugar is galactose; And
제1당은 프럭토오스 및 제2당은 갈락토오스인 제6조합; 으로 이루어진 군에서 선택된 조합인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 및 제1당 대사능을 갖는 변이체의 초고속 스크리닝 방법.A sixth combination wherein the first sugar is fructose and the second sugar is galactose; Ultra fast screening method of a variant having a second sugar isomerization activity (first oligomer) activity and the first sugar metabolism capacity, characterized in that the combination selected from the group consisting of.
제2항에 있어서, 제1당은 타가토오스 및 제2당은 갈락토오스이며, 제1단계에서 선별된 효소는 D-타가토오스-1,6-비스포스페이트 알돌라아제, 제3단계의 이성화 효소는 L- 아라비노오스 이성화효소인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 및 제1당 대사능을 갖는 변이체의 초고속 스크리닝 방법.The method of claim 2, wherein the first sugar is tagatose and the second sugar is galactose, and the enzyme selected in the first step is D-tagatose-1,6-bisphosphate aldolase, the third step of isomerization. The enzyme is characterized in that the L- arabinose isomerase, ultra-fast screening method of variants having a second sugar isomerization activity (first sugar metabolism) and the first sugar metabolism.
제1항에 있어서, 상기 3) 단계의 무작위 유전자 변이는 변이-유발 (error-prone) PCR을 통해 수행되는 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 및 제1당 대사능을 갖는 변이체의 초고속 스크리닝 방법.The method of claim 1, wherein the random genetic variation of step 3) is characterized in that it is carried out through error-prone (error-prone) PCR, having a second sugar isomerization activity (first sugar metabolism activity) and the first sugar metabolic capacity Ultra-fast screening method of variants.
1) 제1당 대사 균주와 제1당 대사능이 결여된 균주의 유전체 분석을 통해 제1당 대사에 필수적인 효소를 선별하는 단계; 1) selecting an enzyme essential for the first sugar metabolism through genome analysis of the first sugar metabolism strain and the strain lacking the first sugar metabolism ability;
2) 제1당 대사능 및 제2당 이성화능이 결여된 균주에 상기 1) 단계에서 선별된 효소를 암호화하는 유전자를 도입하여 제1당 대사성 재조합 균주를 제조하는 단계; 2) preparing a first sugar metabolic recombinant strain by introducing a gene encoding the enzyme selected in step 1) into a strain lacking the first sugar metabolism and the second sugar isomerization ability;
3) 제2당 이성화 효소를 코딩하는 유전자에 무작위 유전자 변이를 유발하여 유전자 변이 라이브러리를 수득하는 단계; 3) inducing random gene mutation in the gene encoding the second sugar isomerase to obtain a gene mutation library;
4) 상기 3) 단계에서 수득된 유전자 변이 라이브러리의 변이 유전자를 상기 2) 단계에서 제조된 제1당 대사성 재조합 균주에 도입하여 균주 라이브러리를 얻는 단계; 4) obtaining a strain library by introducing the mutation gene of the gene mutation library obtained in step 3) into the first sugar metabolic recombinant strain prepared in step 2);
5) 제2당을 유일 탄소원으로 포함하는 제한 배지에서 상기 4) 단계에서 수득된 라이브러리의 균주를 배양하고 높은 성장능을 갖는 균주를 선별하는 단계; 및5) culturing the strain of the library obtained in step 4) in a restriction medium containing the second sugar as the only carbon source and selecting strains having high growth capacity; And
6) 상기 5) 단계에서 선별된 균주에 도입된 변이 효소를 확인하는 단계; 를 포함하며,6) identifying the mutant enzyme introduced into the strain selected in step 5); Including;
상기 제1당과 제2당은 상호 이성질체인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 을 갖는 변이 당이성화 효소의 초고속 스크리닝 방법. The first sugar and the second sugar are mutual isomers, characterized in that the ultra-fast screening method of mutant glycoisomerase having a second sugar isomerization activity (isomerization activity).
제5항에 있어서, 제1당 및 제2당은 The method of claim 5, wherein the first sugar and the second sugar
제1당은 타가토오스 및 제2당은 갈락토오스인 제1조합; A first combination wherein the first sugar is tagatose and the second sugar is galactose;
제1당은 프럭토오스 및 제2당은 글루코오스인 제2조합; A second combination wherein the first sugar is fructose and the second sugar is glucose;
제1당은 타가토오스 및 제2당은 프럭토오스인 제3조합; A third combination wherein the first sugar is tagatose and the second sugar is fructose;
제1당은 만노오스 및 제2당은 글루코오스인 제4조합;A fourth combination wherein the first sugar is mannose and the second sugar is glucose;
제1당은 글루코오스 및 제2당은 갈락토오스인 제5조합; 및 A fifth combination wherein the first sugar is glucose and the second sugar is galactose; And
제1당은 프럭토오스 및 제2당은 갈락토오스인 제6조합; 으로 이루어진 군에서 선택된 조합인 것을 특징으로 하는, 제2당 이성화능 (isomerization activity) 을 갖는 변이 당이성화 효소의 초고속 스크리닝 방법.A sixth combination wherein the first sugar is fructose and the second sugar is galactose; Ultra fast screening method of mutated glycosylation enzyme having a second sugar isomerization activity, characterized in that the combination selected from the group consisting of.
D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자를 포함하는, 재조합 벡터. A recombinant vector comprising a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase.
제7항에 있어서, 상기 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자는 gatY 유전자인 것을 특징으로 하는, 재조합 벡터. 8. The gene of claim 7, wherein the gene encoding D-tagatose-1,6-bisphosphate aldolase is gatY. Recombinant vector, characterized in that the gene.
제7항에 있어서, 상기 L- 아라비노오스 이성화효소를 코딩하는 유전자는 araA 유전자인 것을, 특징으로 하는, 재조합 벡터.The recombinant vector according to claim 7, wherein the gene encoding the L-arabinose isomerase is an araA gene.
D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자가 도입된, D-갈락토오스 이성화능을 갖는 재조합 균주. A recombinant strain having D-galactose isomerization, into which a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase are introduced.
D-갈락토오스 비대사성 균주에 D-타가토오스-1,6-비스포스페이트 알돌라아제를 코딩하는 유전자 및 L- 아라비노오스 이성화효소를 코딩하는 유전자를 도입하는 단계; 를 포함하는 D-갈락토오스 이성화능을 갖는 재조합 균주의 제조방법. Introducing a gene encoding D-tagatose-1,6-bisphosphate aldolase and a gene encoding L-arabinose isomerase into a D-galactose non-metabolizing strain; Method for producing a recombinant strain having a D-galactose isomerization ability comprising a.
제10항의 재조합 균주를 D-갈락토오스 포함배지에서 배양하는 단계; 를 포함하는 D-타가토오스 생산 방법. Culturing the recombinant strain of claim 10 in a D-galactose containing medium; D- tagatose production method comprising a.
서열번호 2 내지 4로 이루어진 군에서 선택된 1종의 아미노산 서열로 표시되는, D-갈락토오스 이성화능을 갖는 L-아라비노오스 이성화효소 변이효소. L-arabinose isomerase mutase having D-galactose isomerization ability, represented by one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4.
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