KR102241682B1 - D-xylonate-responsive promoter, artificial genetic circuits comprising d-xylonate-responsive promoter and method for detection of d-xylonate using artificial genetic circuit - Google Patents

Abstract

The present invention relates to a D-xylonic acid reactive promoter, a redesigned gene circuit for detecting D-xylonic acid including the same, and a method for detecting D-xylonic acid using the same, which is represented by the nucleotide sequence of SEQ ID NO: 1 according to the present invention. Promoter is highly reactive to D-xylonic acid, so when applied to a synthetic circuit for detecting D-xylonic acid in E. coli, it is possible to increase the production yield of the final product through pH control by controlling the accumulation of D-xylonic acid. .

Description

D-xylonate-reactive promoter, a redesigned gene circuit for detecting D-xylonic acid, and a method for detecting D-xylonic acid using the same {D-XYLONATE-RESPONSIVE PROMOTER, ARTIFICIAL GENETIC CIRCUITS COMPRISING D-XYLONATE-RESPONSIVE PROMOTER AND METHOD FOR DETECTION OF D-XYLONATE USING ARTIFICIAL GENETIC CIRCUIT}

The present invention relates to a D-xylonic acid reactive promoter, a redesigned gene circuit for detecting D-xylonic acid including the same, and a method for detecting D-xylonic acid using the same.

There are three pathways for the metabolism of D-xylose in microorganisms. First, the Xylose isomerase pathway (XIP) begins at the isomerization step that releases D-xylulose. This intermediate is further metabolized through the pentose phosphate pathway (PPP). Next, the oxo-reductive pathway (ORP) begins with a pathway that produces D-xylulose as an intermediate, but reduces D-xylose and oxidizes xylitol. The third pathway, called the xylose oxidation pathway (XOP), is initiated by the oxidation of D-xylose to form D-xylonic acid. This intermediate is dehydrated to form 2-keto 3-deoxy D-pentonate, which is divided into two pathways, which form pyruvate and glycol aldehyde. It is another dehydration reaction that forms an aldolase reaction or 2-ketoglutarate semialdehyde. The former is called the Dahms path, and the latter is called the Weimberg path.

The xylose oxidation pathway (XOP) is 1,2,4-butanetriol, xylonic acid, ethylene glycol, glycolic acid, and gamma. -It has been used to develop recombinant strains capable of producing various valuable compounds such as gama-aminobutyric acid and 3,4-dihydroxybutyric acid (Liu et al. 2012; Liu et al. 2013; Valdehuesa et al. 2014; Cabulong et al. 2017; Wang et al. 2017; Zhao et al. 2017; Cabulong et al. 2018). However, the use of XOP in these strains presents two drawbacks. First, when the Dahms pathway is used, the carbon yield for all target products is low due to the aldol cleavage step, and second, the intermediate D-xylonic acid accumulates in the medium, resulting in a decrease in pH. Previous studies have attempted to solve this problem by identifying the optimal genes for the pathway and their respective expression intensity (Cabulong et al. 2017; Cabulong et al. 2018).

The current trend in the development of recombinant strains involves the integration of synthetic biology tools. It uses genetic elements or components to build synthetic circuits or networks that allow some predictability of the mutant trait. The parts used to make these synthetic genetic circuits are promoters, operators, regulators or inducers and reporter proteins. The basic goal is to create conditions under which the presence of an inducing molecule or an external force triggers a reaction by the host microorganism. This response can range from simple biosensing to more complex gene regulation. In the case of XOP, particularly the Dahms pathway of Escherichia coli, research on synthetic genetic tools is currently limited.

Thus, the present inventors overcome the problems of the prior art, and as a result of confirming a new promoter element involved in the D-xylonic acid reaction, the promoter represented by the nucleotide sequence of SEQ ID NO: 1 according to the present invention is for D-xylonic acid. When applied to a synthetic circuit for detecting D-xylonic acid in Escherichia coli because of its high reactivity, it was confirmed that the production yield of the final product can be increased through pH control by controlling the accumulation of D-xylonic acid, and the present invention was completed. Was done.

The main object of the present invention is to provide a D-xylonic acid-reactive promoter having reactivity to D-xylonic acid.

Another object of the present invention is to provide a biosensor for detecting an expression vector comprising the D-xylonic acid reactive promoter, a host cell transformed with the expression vector, and D-xylonic acid containing the host cell.

Another object of the present invention is to provide a redesigned gene circuit for detecting D-xylonic acid including the D-xylonic acid-reactive promoter and a recombinant microorganism for detecting D-xylonic acid containing the gene circuit.

According to one aspect of the present invention, the present invention provides a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the term "promoter" refers to an untranslated nucleic acid sequence upstream of the coding region, which includes a binding site for a polymerase and has an activity to initiate transcription of a lower gene of a promoter into mRNA. Promoter with respect to the present invention is mCherry in check for one site (site) as possible to the binding of RNA polymerase and transcription start site, based on the genomic information of E. coli, and reduced otherwise the required portion and the base of the P _yjhI of P _yjhI form By confirming the expression, the nucleotide sequence of the D-xylonic acid reactive promoter was confirmed.

The sequence encoding the promoter of the present invention can be modified to a certain degree. Those skilled in the art know that the nucleotide sequence in which 70% or more homology is maintained by such artificial modification is equivalent to that derived from the nucleotide sequence of the present invention, as long as it retains the promoter activity for expression of the gene of interest in the present invention. It will be easy to understand.

In the present invention, the term "homology" refers to the degree of homology with a wild type nucleic acid sequence, and the comparison of homology is a percentage of homology between two or more sequences using a comparison program that is easy to purchase or with the naked eye. It can be calculated as (%). The nucleic acid sequence encoding the promoter region of SEQ ID NO: 1 of the present invention is preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the same nucleic acid sequence. do.

In addition, the promoter of the present invention includes variants having a promoter nucleic acid sequence in which one or more nucleic acid bases are mutated by substitution, deletion, insertion, or a combination thereof, as long as it retains promoter activity. The promoter nucleic acid sequence can induce a variation in sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Although the same activity as the natural promoter may be exhibited through such sequence variation, the function of the promoter can be improved suitably for a purpose, such as a promoter with increased activity and a promoter with increased specificity for an inducing agent.

Nucleic acid molecules encoding all of the above categories of promoters are provided as a promoter component of an expression vector that induces expression of a gene of interest, and modifications of various vectors using the promoter are included in the scope of the present invention.

According to another aspect of the present invention, the present invention provides an expression vector comprising a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the term "expression vector" refers to a gene construct including essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell. The expression vector related to the present invention is a vector in which the promoter is a D-xylonic acid reactive promoter, the promoter is operably linked to induce the expression of a target gene, and the vector may be a form integrated into the genome of a host cell.

In the present invention, "operably linked" refers to a functional link between a D-xylonic acid reactive expression control sequence and a nucleotide sequence encoding a gene of interest to perform a general function. The operative linkage with the recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.

The expression vector of the present invention essentially contains a D-xylonic acid reactive promoter as a regulatory element, and expression control sequences that can affect the expression of the protein, for example, start codon, stop codon, polyadenylation signal, enhancer , A signal sequence for membrane targeting or secretion, and the like.

According to another aspect of the present invention, the present invention provides a host cell transformed with an expression vector comprising a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.

In the host cell transformed with the expression vector of the present invention, any host cell capable of transforming with the vector may be used as long as it is known as a host cell producing a recombinant protein. The host cell may include bacteria, yeast, fungi, etc., but is not limited thereto, and is preferably characterized in that it is Escherichia coli.

According to another aspect of the present invention, the present invention provides a biosensor for detecting xylonic acid containing host cells transformed with an expression vector containing a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1. to provide.

According to another aspect of the present invention, after exposing the biosensor to D-xylonic acid, the D- Provides a way to detect xylonic acid.

The method of detecting D-xylonic acid using the biosensor according to the present invention can be appropriately selected and performed by a person of ordinary skill in the art. For example, in the case of using E. coli transformed with a promoter having a nucleic acid molecule sequence of SEQ ID NO: 1, a device that contacts a biosensor cell and a device that measures the light output of the cell is used to detect D-xylonic acid. can do.

According to another aspect of the present invention, the present invention provides i) a reporter gene encoding a red fluorescent protein; ii) a promoter that regulates the expression of the downstream red fluorescent protein; iii) the lacI gene encoding the lactose protein; iv) a reporter gene encoding a green fluorescent protein; And iv) D-xylonic acid detection material comprising a promoter represented by the nucleotide sequence of SEQ ID NO: 1 that detects D-xylonic acid and induces the expression of the downstream lacI gene and the reporter gene encoding the downstream green fluorescent protein. Provides a design genetic circuit.

The redesigned gene circuit for detecting D-xylonic acid of the present invention detects the lacI gene encoding the lactose protein, the reporter gene encoding the green fluorescent protein, and the D-xylonic acid to detect the lacI gene and the reporter gene encoding the green fluorescent protein. It consists of a promoter represented by the nucleotide sequence of SEQ ID NO: 1, which induces the expression of, and the promoter reacting with it in proportion to the concentration of D-xylonic acid induces the expression of the lacI gene and the expression of the gene encoding the green fluorescent protein. It can be said that it is a redesigned gene circuit capable of regulating the accumulation of D-xylonic acid. As shown in Figs. 9A to 9C, quantitative fluorescence analysis can be performed on D-xylonic acid by the genetic circuit for detecting D-xylonic acid prepared in the present invention. This suggests that the genetic circuit of the present invention can increase the production yield of the final product through pH control by detecting D-xylonic acid present at a specific concentration or higher and controlling the accumulation of D-xylonic acid (Example 3 And, see FIGS. 9A to 9C).

In the redesigned gene circuit for detecting D-xylonic acid of the present invention, the red fluorescent protein is mCheery, and the green fluorescent protein is sfGFP.

In the redesigned genetic circuit for detecting D-xylonic acid of the present invention, in the genetic circuit, when D-xylonic acid is not detected, sfGFP and lacI are inhibited and mCherry is continuously expressed to generate red fluorescence, and D-xyl It is characterized in that sfGFP and lacI are expressed when ronic acid is detected to generate green fluorescence. That is, the gene circuit according to the present invention can confirm the generation of D-xylonic acid according to the expression level of the green fluorescent protein.

According to another aspect of the present invention, the present invention provides i) xdh gene encoding a xylose dehydrogenase protein; ii) a promoter that regulates the expression of the xdh gene downstream according to the expression of the lactose protein; iii) the lacI gene encoding the lactose protein; And iv) a promoter represented by the nucleotide sequence of SEQ ID NO: 1 that detects D-xylonic acid and induces the expression of the downstream lacI gene.

The redesigned gene circuit for detecting D-xylonic acid of the present invention consists of a lacI gene encoding a lactose protein and a promoter represented by the nucleotide sequence of SEQ ID NO: 1 that detects D-xylonic acid and induces the expression of the downstream lacI gene. In proportion to the concentration of D-xylonic acid, the promoter reacting with it induces the expression of the lacI gene, thereby inhibiting the production of xylose dehydrogenase (xdh), which forms D-xylose into D-xylonic acid, It can be said to be a redesigned genetic circuit capable of regulating D-xylonic acid accumulation. As shown in FIG. 10, xylose dehydrogenase (xdh) is located downstream _{of P T7-locO, a promoter capable of inhibiting LacI.} xdh produces D-xylonic acid from D-xylose, and LacI is expressed at a specific concentration of accumulated D-xylonic acid, effectively inhibiting Xdh production, thereby temporarily stopping D-xylonic acid accumulation and at the same time D- Downstream genes using xylonic acid are activated. This suggests that the genetic circuit of the present invention can increase the production yield of the final product through pH control by controlling the accumulation of D-xylonic acid by detecting D-xylonic acid present at a specific concentration or higher (Example 4 and, see Fig. 10).

In the redesigned gene circuit for detecting D-xylonic acid of the present invention, the promoter of iv) can induce the expression of the lacI gene when the concentration of xylonic acid is at least 5 mM, and detectable even in the above concentration range. It is characterized.

In the redesigned gene circuit for detecting D- xylonic acid of the present invention, the expression of the lacI gene inhibits the expression of the xdh gene and stops the production of xylose dehydrogenase, thereby temporarily stopping the accumulation of D-xylonic acid. It is characterized.

According to another aspect of the present invention, the present invention provides a recombinant microorganism for detecting D-xylonic acid containing the genetic circuit.

In the recombinant microorganism for detecting D-xylonic acid of the present invention, any microorganism may be used as long as it is known as a microorganism that produces a recombinant protein. Microorganisms may include bacteria, yeast, fungi, etc., but are not limited thereto, and are preferably E. coli.

As described above, when the promoter represented by the nucleotide sequence of SEQ ID NO: 1 according to the present invention has high reactivity to D-xylonic acid, when applied to a synthetic circuit for detecting D-xylonic acid in E. coli, D-xylonic acid By controlling the accumulation, it is possible to increase the production yield of the final product through pH control and consequently.

1 is a schematic diagram showing an exaggeration of D-xylonic acid accumulation and metabolism in E. coli W3110.
Figure 2 is a result of confirming the relative gene expression of the yag and yjh genes in E. coli W3110 ((2a) M9 medium, (2b) M9 medium with D-xylose).
3 is a result of quantitative PCR analysis of yag and yjh genes of Escherichia coli W3110.
4 is a plasmid map including a D-xylon line reactive promoter as a genetic element.
Figure 5 is a result showing the promoter response to D-xylonic acid (Figure 5a), D-xylose (Figure 5b) and D-glucose (Figure 5c).
6 is a result of analyzing the promoter using BPROM in order to predict the bacterial promoter.
7 is a result of analyzing the promoter using NNPP.
Figure 8 is the _{result of the characteristic analysis of P yjhI} ((8a) two crp binding site prediction results, (8b) the result of confirming mCherry expression in the form of differently reduced bases of _{yjhI, (8c) P for D-xylonic acid. yjhI} dose response results).
_{9 is a D-xylonic} acid-reactive gene circuit (9a) using P yjhI as a promoter genetic element and the results of the D-xylonic acid reaction of the gene circuit ((9b) a medium containing no D-xylonic acid, (9c) ) D-xylonic acid-containing medium).
FIG. 10 shows a genetic circuit to _{which P yjhI} , a D-xylonic acid reaction promoter, is applied in the autonomous regulation of D-xylose metabolism through the xylose oxidation pathway (XOP).

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

In the present invention, in order to construct a redesigned gene circuit for detecting D-xylonic acid, structural genes and promoters reacting to D-xylonic acid were identified, and a new gene circuit for detecting D-xylonic acid was constructed. The specific test method and design method are as follows.

[Experiment method]

DNA manipulation

delacq et al. 2006)로부터 기증받았으며, sfGFP 유전자를 위한 재료로 이용하였다. 플라스미드 pETmCherryLIC은 Scott Gradia(Addgene plasmid # 29769)로부터 기증받아, mCherry 유전자의 공급원으로 사용되었다. 모든 PCR 반응은 표 2에 기재된 적절한 올리고 뉴클레오타이드를 사용하여 Phusion High-fidelity DNA 중합 효소를 사용하여 수행되었다. 본 발명에서 사용된 유전자 요소와 유전자의 전체 목록은 표 3에 나타내었다. 1 단계 TSS(형질 전환 및 저장 용액) 프로토콜을 사용하여 플라스미드 형질 전환을 수행 하였다.Standard techniques were used for all genetic manipulation experiments (Green and Sambrook 2012), and the Gibson isothermal assembly strategy was used for DNA assembly (Gibson et al. 2009). A list of recombinant plasmids and E. coli strains used in the present invention is shown in Table 1. Plasmid sfGFP-pBAD is derived from Michael Davidson and Geoffrey Waldo (Addgene plasmid # 54519) (P

delacq et al. 2006), and used as a material for the sfGFP gene. Plasmid pETmCherryLIC was donated from Scott Gradia (Addgene plasmid # 29769) and used as a source of mCherry gene. All PCR reactions were performed using Phusion High-fidelity DNA Polymerase using the appropriate oligonucleotides listed in Table 2. The entire list of genetic elements and genes used in the present invention is shown in Table 3. Plasmid transformation was performed using a one-step TSS (transformation and stock solution) protocol.

Names Relevant Characteristics Source Plasmids pET28A pBR322 ori , P _T7 , Kan ^R Invitrogen pRSET-A pUC ori , P _T7 ,Amp ^R Invitrogen sfGFP-pBAD P _pBAD - sfGFP (Addgene #54519) * pET mCherry LIC P _T7 - mCherry , (Addgene #29769) ** pET28A-T7 pET28A derivative; P _T7 - mCherry This work pET28-PyagE pET28A derivative; P _yagE - mcherry This work pET28-PyjhI pET28A derivative; P _yjhI - mcherry This work pRSET-A-XAS P _T7- mcherry, P _{yjhI -} lacI, P _{yjhI -} sfGFP This work Strains MG1655 E. coli MG1655 (DE3) This work MGMC0 MG1655 pET28A This work MGMC1 MG1655 pET28A-T7 This work MGMC2 MG1655 pET28-PyagE This work MGMC3 MG1655 pET28-PyjhI This work MGMC4 MG1655 pRSET-A-XAS This work

* Plasmid sfGFP-pBAD was donated by Michael Davidson and Geoffrey Waldo (Addgene plasmid # 54519).

** pET mCherry LIC cloning vector (u-mCherry) was donated by Scott Gradia (Addgene plasmid # 29769).

order
number name Sequence (5'→3')* 8 T7MC-F CTTTAAGAAGGAGATATAC ATGGCCATCATCAAGGAGTTC 9 T7MC-R CAGTGGTGGTGGTGGTGGTGC CTACTTGTACAGCTCGTCCATG 10 PyagEMC1-F CGTCCGGCGTAGAGGATCGA AAACGGGTTCTTATGCCTTAGTTG 11 PyagEMC1-R TGATGGCCAT GAGATCTCCTTGCTGAATCATTTTGTTC 12 PyagEMC2-F AGGAGATCTC ATGGCCATCATCAAGGAGTTC 13 PyagEMC2-R AGTGGTGGTGGTGGTGGTGC CTACTTGTACAGCTCGTCCATG 14 PyjhIMC1-F CGTCCGGCGTAGAGGATCGA TAAGTAAGTTCATTCGAGAGGGATTTCAAG 15 PyjhIMC1-R TGATGGCCAT AATGGCTCCTCCTTGCTCATG 16 PyjhIMC2-F AGGAGCCATT ATGGCCATCATCAAGGAGTTC 17 PyjhIMC2-R AGTGGTGGTGGTGGTGGTGC CTACTTGTACAGCTCGTCCATG 18 T7SF-F ACTTTAAGAAGGAGATATAC ATGGTGAGCAAGGGCGAGGAG 19 T7SF-R AGTGGTGGTGGTGGTGGTGC CTTGTACAGCTCGTCCATGCC 20 PyagESF1-F CGTCCGGCGTAGAGGATCGA AAACGGGTTCTTATGCCTTAGTTG 21 PyagESF1-R TGCTCACCAT GAGATCTCCTTGCTGAATCATTTTGTTC 22 PyagESF2-F AGGAGATCTC ATGGTGAGCAAGGGCGAGGAG 23 PyagESF2-R AGTGGTGGTGGTGGTGGTGC CTTGTACAGCTCGTCCATGCC 24 PyjhISF1-F GTCCGGCGTAGAGGATCGA TAAGTAAGTTCATTCGAGAGGGATTTCAAG 25 PyjhISF1-R TGCTCACCAT AATGGCTCCTCCTTGCTCATG 26 PyjhISF2-F AGGAGCCATT ATGGTGAGCAAGGGCGAGGAG 27 PyjhISF2-R AGTGGTGGTGGTGGTGGTGC CTTGTACAGCTCGTCCATGCC 28 pRSETXAS-F CCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCC 29 pRSETXAS-R AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCGGG 30 lacO-mCherry-T7T F TAATACGACTCACTATAGGG GGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTG 31 lacO-mCherry-T7T R GAACCCGTTT ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAG 32 XAS yjhIp F ATATCCGGAT AAACGGGTTCTTATGCCTTAGTTGTAAGTGTCTACCATGTC 33 XAS yjhIp R CTGGTTTCAC GAGATCTCCTTGCTGAATCATTTTGTTCTACATTATAGAACAG 34 lacI F AGGAGATCTC GTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCG 35 lacI R AACTTACTTA CTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAG 36 yjhIp-sfGFP-T7T F TGAAATCTAG TAAGTAAGTTCATTCGAGAGGGATTTCAAGCAAAAATAATCAATGG 37 yjhIp-sfGFP-T7T R GAGGGAAACCGTTGTGGTCT ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAG

order
number name Sequence (5’→3’) One
P _yjhI TAAGTAAGTT CATTCGAGAG GGATTTCAAG CAAAAATAAT CAATGGCACC CAATAGAAAA TATTGGCGAT GCGCTCGAAC GAATAAAGAA GCTCTAGGCG CAATCCACAC ACTACGATGT TGCAACAACA CGCCATCTAC TTTTTATTCT CATTCACTAA ATGTGGCTGT TCTGGATTCA TTATTCAAAG TGTGTACAAG ATCACATTTA ATCACATCAT TACGGTTCAG CATGCTGAAC AAAGCATATT TTCCACTATG TAATGCCGAT ACCATTTATT CCATGAGCAA GGAGGAGCCA TT 2
P _yagE AAACGGGTTC TTATGCCTTA GTTGTAAGTG TCTACCATGT CCCCGAACAA GTGTTCACTA TGTCCCCGGA CCGTACACCC CAAAGGGGAG AGGGGACTGC ACCGAGCCAT CTTTTCCCCC TCGCCCCTTT GGGGAGAGGG CCGGGGTGAG GGGCAATATG TGATCCAGCT TAAATTTCCC GCACTCCCTC TTCCCTTCCG ATTTACCTCT CCTTGTTCTG CGTCATAGTA TGATCGTTAA ATAAACGAAC GCTGTTCTAT AATGTAGAAC AAAATGATTC AGCAAGGAGA TCTC 3
mCherry ATGGCCATCA TCAAGGAGTT CATGCGCTTC AAGGTGCACA TGGAGGGCTC CGTGAACGGC CACGAGTTCG AGATCGAGGG CGAGGGCGAG GGCCGCCCCT ACGAGGGCAC CCAGACCGCC AAGCTGAAGG TGACCAAGGG TGGCCCCCTG CCCTTCGCCT GGGACATCCT GTCCCCTCAG TTCATGTACG GCTCCAAGGC CTACGTGAAG CACCCCGCCG ACATCCCCGA CTACTTGAAG CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG CGCGTGATGA ACTTCGAGGA CGGCGGCGTG GTGACCGTGA CCCAGGACTC CTCCCTCCAG GACGGCGAGT TCATCTACAA GGTGAAGCTG CGCGGCACCA ACTTCCCCTC CGACGGCCCC GTAATGCAGA AGAAGACCAT GGGCTGGGAG GCCTCCTCCG AGCGGATGTA CCCCGAGGAC GGCGCCCTGA AGGGCGAGAT CAAGCAGAGG CTGAAGCTGA AGGACGGCGG CCACTACGAC GCTGAGGTCA AGACCACCTA CAAGGCCAAG AAGCCCGTGC AGCTGCCCGG CGCCTACAAC GTCAACATCA AGTTGGACAT CACCTCCCAC AACGAGGACT ACACCATCGT GGAACAGTAC GAACGCGCCG AGGGCCGCCA CTCCACCGGC GGCATGGACG AGCTGTACAA 4
sfGFP ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC CCATCCTGGT CGAGCTGGAC GGCGACGTAA ACGGCCACAA GTTCAGCGTG CGCGGCGAGG GCGAGGGCGA TGCCACCAAC GGCAAGCTGA CCCTGAAGTT CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTCGTGACCA CCCTGACCTA CGGCGTGCAG TGCTTCAGCC GCTACCCCGA CCACATGAAG CGCCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG CACCATCAGC TTCAAGGACG ACGGCACCTA CAAGACCCGC GCCGAGGTGA AGTTCGAGGG CGACACCCTG GTGAACCGCA TCGAGCTGAA GGGCATCGAC TTCAAGGAGG ACGGCAACAT CCTGGGGCAC AAGCTGGAGT ACAACTTCAA CAGCCACAAC GTCTATATCA CCGCCGACAA GCAGAAGAAC GGCATCAAGG CCAACTTCAA GATCCGCCAC AACGTGGAGG ACGGCAGCGT GCAGCTCGCC GACCACTACC AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC CGACAACCAC TACCTGAGCA CCCAGTCCGT GCTGAGCAAA GACCCCAACG AGAAGCGCGA TCACATGGTCTC GACGC GCCGGTCGAGTGAGC GGCGCGCGAGT 5




P _yjhI -sfGFP-T _T7 TAAGTAAGTT CATTCGAGAG GGATTTCAAG CAAAAATAAT CAATGGCACC CAATAGAAAA TATTGGCGAT GCGCTCGAAC GAATAAAGAA GCTCTAGGCG CAATCCACAC ACTACGATGT TGCAACAACA CGCCATCTAC TTTTTATTCT CATTCACTAA ATGTGGCTGT TCTGGATTCA TTATTCAAAG TGTGTACAAG ATCACATTTA ATCACATCAT TACGGTTCAG CATGCTGAAC AAAGCATATT TTCCACTATG TAATGCCGAT ACCATTTATT CCATGAGCAA GGAGGAGCCA TTATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT GCCCATCCTG GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGCGCGGCGA GGGCGAGGGC GATGCCACCA ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCCGTG CCCTGGCCCA CCCTCGTGAC CACCCTGACC TACGGCGTGC AGTGCTTCAG CCGCTACCCC GACCACATGA AGCGCCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA CGTCCAGGAG CGCACCATCA GCTTCAAGGA CGACGGCACC TACAAGACCC GCGCCGAGGT GAAGTTCGAG GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA GGACGGCAAC ATCCTGGGGC ACAAGCTGGA GTACAACTTC AACAGCCACA ACGTCTATAT CACCGCCGAC AAGCAGAAGA ACGGCATCAA GGCCAACTTC AAGATCCGCC ACAACGTGGA GGACGGCAGC GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC CGTGCTGCTG CCCGACAACC ACTACCTGAG CACCCAGTCC GTGCTGAGCA AAGACCCCAA CGAGAAGCGC GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCACGG CATGGACGAG CTGTACAAGT CGAGCACCAC CACCACCACC ACTGAGATCC GGCTGCTAAC AAAGCCCGAA AGGAAGCCCC GGCTGCTAAC AAAGCCCGAA AGGAAGCCCGC GATGCTGACTGTGACTGACTGACTGGCTGCTAGGCTGACTGGCTGCTAGGCTGACTGGCTGCTAGGCTGACTGGCTG 6 P _T7-lacO -mCherry -T _T7 GGAATTGTGA GCGGATAACA ATTCCCCTCT AGAAATAATT TTGTTTAACT TTAAGAAGGA GATATACATG GCCATCATCA AGGAGTTCAT GCGCTTCAAG GTGCACATGG AGGGCTCCGT GAACGGCCAC GAGTTCGAGA TCGAGGGCGA GGGCGAGGGC CGCCCCTACG AGGGCACCCA GACCGCCAAG CTGAAGGTGA CCAAGGGTGG CCCCCTGCCC TTCGCCTGGG ACATCCTGTC CCCTCAGTTC ATGTACGGCT CCAAGGCCTA CGTGAAGCAC CCCGCCGACA TCCCCGACTA CTTGAAGCTG TCCTTCCCCG AGGGCTTCAA GTGGGAGCGC GTGATGAACT TCGAGGACGG CGGCGTGGTG ACCGTGACCC AGGACTCCTC CCTCCAGGAC GGCGAGTTCA TCTACAAGGT GAAGCTGCGC GGCACCAACT TCCCCTCCGA CGGCCCCGTA ATGCAGAAGA AGACCATGGG CTGGGAGGCC TCCTCCGAGC GGATGTACCC CGAGGACGGC GCCCTGAAGG GCGAGATCAA GCAGAGGCTG AAGCTGAAGG ACGGCGGCCA CTACGACGCT GAGGTCAAGA CCACCTACAA GGCCAAGAAG CCCGTGCAGC TGCCCGGCGC CTACAACGTC AACATCAAGT TGGACATCAC CTCCCACAAC GAGGACTACA CCATCGTGGA ACAGTACGAA CGCGCCGAGG GCCGCCACTC CACCGGCGGC ATGGACGAGC TGTACAAGTA GTCGAGCACC ACCACCACCA CCACTGAGAT CCGGCTGCTA ACAAAGCCCG AAAGGAAGCT GAGTTGGCTG CTGCCACCGC TGAGCAATAA CTAGCATAAC CCCTTGGGGC CTCTAAACGG GTCTTGAGGG GTTTTTTGCT GAAAGGAGGA ACTATATCCG GAT 7
lacI GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAA CCGCGTGGCA CAACAACTGG CGGGCAAACA GTCGTTGCTG ATTGGCGTTG CCACCTCCAG TCTGGCCCTG CACGCGCCGT CGCAAATTGT CGCGGCGATT AAATCTCGCG CCGATCAACT GGGTGCCAGC GTGGTGGTGT CGATGGTAGA ACGAAGCGGC GTCGAAGCCT GTAAAGCGGC GGTGCACAAT CTTCTCGCGC AACGCGTCAG TGGGCTGATC ATTAACTATC CGCTGGATGA CCAGGATGCC ATTGCTGTGG AAGCTGCCTG CACTAATGTT CCGGCGTTAT TTCTTGATGT CTCTGACCAG ACACCCATCA ACAGTATTAT TTTCTCCCAT GAAGACGGTA CGCGACTGGG CGTGGAGCAT CTGGTCGCAT TGGGTCACCA GCAAATCGCG CTGTTAGCGG GCCCATTAAG TTCTGTCTCG GCGCGTCTGC GTCTGGCTGG CTGGCATAAA TATCTCACTC GCAATCAAAT TCAGCCGATA GCGGAACGGG AAGGCGACTG GAGTGCCATG TCCGGTTTTC AACAAACCAT GCAAATGCTG AATGAGGGCA TCGTTCCCAC TGCGATGCTG GTTGCCAACG ATCAGATGGC GCTGGGCGCA ATGCGCGCCA TTACCGAGTC CGGGCTGCGC GTTGGTGCGG ACATCTCGGT AGTGGGATAC GACGATACCG AAGACAGCTC ATGTTATATC CCGCCGTTAA CCACCATCAA ACAGGATTTT CGCCTGCTGG GGCAAACCAG CGTGGACCGC TTGCTGCAAC TCTCTCAGGG CCAGGCGGTG AAGGGCAATC AGCTGTTGCC CGTCTCACTG GTGAAAAGAA AAACCACCCT GGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTAAGTTAGC TCACTCATTA GGCACCGGGA TCTCGACCGA TGCCCTTGAG AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC ATGACTAACA TGAGAATTAC AACTTATATC GTATGGGGCT GACTTCAGGT GCTACATTTG AAGAGATAAA TTGCACTGAA ATCTAG

Plasmid cultivation

Plasmid proliferation and maintenance was performed using E. coli DH5α grown on LB medium or agar medium supplemented with appropriate antibiotics (50 μg/mL kanamycin or 100 μg/mL ampicillin). 33.7mM Na ₂ HPO ₄ , 22.0mM KH ₂ PO ₄ , 8.55mM NaCl, 9.35mM NH ₄ Cl _{, 1.0mM MgSO 4} , modified M9 (MM9) containing 0.5 g/L yeast extract and 1.0 g/L peptone The medium was used throughout the experiment. M9 medium was used by modifying the selected substrate to an appropriate concentration as required in the test conditions. For the real-time PCR study, 10 mM D-xylose, D-glucose, D-xylonic acid, or a combination thereof was used. For the dose response test, mCherry expression was induced using D-xylonic acid ranging from 0 to 100mM, and 20mM D-xylonic acid was used at an optimal concentration to induce an optimal response. For the gene switch experiment, 20mM D-xylonic acid was used as an inducing agent concentration.

Quantitative real-time PCR

The wild-type E. coli W3110 strain was selected for qRT-PCR analysis. Incubation was performed in a 50 mL tube containing 5 mL MM9 with an appropriate substrate(s), and incubated at 37° C. for 3 hours. A strain grown only in MM9 medium was selected as a control. Test conditions include strains growing on D-xylose (X), D-glucose (G), D-xylonic acid (XA) or any combination of these substrates. The total mRNA of the growing cells was purified by modifying the protocol provided by the manufacturer using the RNeasy Mini Kit (Qiagen, Germany). Specifically, cells from the culture sample were harvested and resuspended in Tris-EDTA buffer containing lysozyme. The mixture was incubated for 5 minutes at room temperature before adding RLT buffer (included in the kit) and ethanol. The remaining steps for RNA purification were performed according to the protocol provided by the manufacturer (Quick-Start Protocol RNeasy Mini Kit, starting from step 3 in Part 1). The isolated RNA was confirmed using a NanoDrop 1000 spectrophotometer of Thermo Scientific (Waltham, MA, USA) and agarose gel electrophoresis. RNA was converted to cDNA using the QuantiTect Reverse Transcription kit (Qiagen, Germany) according to a protocol comprising the step of removing genomic DNA. The qPCR reaction was performed using QuantiFast® SYBR® Green PCR Kit (Qiagen, Germany). Each reaction contains an appropriate qPCR primer (Table 4) with a final concentration of 1 μM. The expression levels of the target genes were properly normalized using the reference genes rrsB, mdoG and cysG. The qPCR reaction was subjected to denaturation at 95° C. for 40 cycles and annealing and extension at 62° C. for 10 seconds using Rotor-Gene Q (Qiagen, Germany). qPCR raw data was processed through the RG mode of REST 2009 software.

Sequence number name Sequence (5’→3’) Target gene 38 RTyagA-F GTCGCTTCGGCATTTCAC yagA 39 RTyagA-R CAGGTTATGGACGGTGCTG yagA 40 RTyagE-F AAGTGTCGGAAGCGAACC yagE 41 RTyagE-R CGATGGTGTCTTTGATGCC yagE 42 RTyagF-F GACCTGCGATAAAGGGCT yagF 43 RTyagF-R AGGGAGAGTTCGTGGTTGG yagF 44 RTyagI-F TCGCCTATTCACGCATCG yagI 45 RTyagI-R CTCGTTCTCTTCGCTGTCC yagI 46 RTyjhU-F TGAGAATGGTGATGTGCTGG yjhU 47 RTyjhU-R TAGGCGATTTGCGATGAGG yjhU 48 RTyjhG-F CCTTATTCGCTCTCTGCCC yjhG 49 RTyjhG-R GCCGTTGTCTTCTCCATCC yjhG 50 RTyjhH-F CTACTACTGGAAAGTCGCACC yjhH 51 RTyjhH-R TACGCAAGTGACCAACGC yjhH 52 RTyjhI-F GGAAGACACGAGAAGAACTG yjhI 53 RTyjhI-R TCGCCAGAAAGTGAGATTG yjhI 54 RTrrsB-F TACCCGCAGAAGAAGCACC rrsB* 55 RTrrsB-R CGCATTTCACCGCTACACC rrsB* 56 RTcysG-F GCGTTTATTCCACAGTTCACC cysG* 57 RTcysG-R GTTACAGAAGATGCGACGAG cysG* 58 mdoG-F ATACACCATCACCTTCAGCC mdoG* 59 mdoG-R CGTGCTTTCAACTATCTCACC mdoG*

Fluorescence analysis

The cells were cultured in LB medium overnight, and 1 ml of each sample was centrifuged at 21206xg for 2 minutes, and the supernatant was discarded. The pellets were resuspended with 500 μl of M9 minimal medium and then they ^{were transferred to M9 medium with 1 gL -1} peptone and 0.5 gL -1 yeast extract and placed in a 100 ml volumetric flask. The sample was incubated with vigorous shaking at 37°C until it reached an absorbance of 0.3 to 0.4. Samples were taken from each medium prior to supplementation with the sugar substrate. The amount collected in each sample depends on the OD600 absorbance. After collection, each medium was supplemented with 20 mM of a sugar substrate, i.e. glucose for the first medium, xylose for the second medium, and xylonic acid for the final medium. For the positive control, 0.1 mM IPTG was added. The collected samples were centrifuged at 21206xg for 2 minutes, the supernatant was discarded, and 1 ml of water was added to each sample. Cells were normalized based on absorbance at OD600. Cells were analyzed for fluorescence using a fluorescence spectrometer FluoroMate FS-2 (Scinco, Korea). The fluorescence spectrometer was set to 587 nm excitation and 610 nm emission for sfGFP, and 485 excitation and 507 emission for mCherry.

Bioinformatics analysis

Genes and protein sequences of enzymes related to the yag and yjh operons of Escherichia coli were analyzed using Clustal O online databas (Sievers et al. 2011). Blastp analysis was performed using the blast function of UNIPROT, and the phylogram was generated using CLC Sequence Viewer (Qiagen). Promoter sequences were analyzed using the online tool BPROM prediction of bacterial promoters (Solovyev and Salamov 2011; Umarov and Solovyev 2017) and Neural Network Promoter Prediction (NNPP) (Reese 2001).

Example 1: Screening of D-xylonic acid-reactive genetic elements

Gene expression analysis was performed through real-time PCR to obtain information on the expression profiles of structural genes and regulatory genes. The transporter-encoding gene was excluded from the analysis. Real-time PCR was performed on 8 genes (yagA, yagE, yagF, yagI, yjhG, yjhH, yjhI, yjhU) under the conditions of MM9 medium without substrate (control) and MM9 with one or more substrates (Example). The results are shown in FIGS. 2 and 3.

2 is a result of confirming the relative gene expression of the yag and yjh genes in E. coli W3110, as compared with cells grown only in M9 medium (FIG. 2A) or in M9 medium with D-xylose (FIG. 2B ). When xylonic acid was added, the fold conversion value was measured. The fold conversion value was calculated using comparative CT.

As a result, as can be seen in Figure 2, the expression of structural genes yjhG and yjhH increased more than twice when treated with D-xylonic acid compared to the control (Figure 2a), compared with the strain grown in D-xylose. It also appeared the same when (Fig. 2b).

In addition, Figure 3 is a yag (Figure 3a and Figure 3b) of E. coli W3110 supplemented with D-xylonic acid, D-xylose, D-glucose (Figures 3a and 3c) or a combination (Figures 3b and 3d) and As a result of quantitative PCR analysis of the yjh (Fig. 3c and Fig. 3d) gene, a strain grown on a modified M9 medium (M9 salt with 1 g/L peptone and 0.5 g/L yeast extract) was used as a control. In FIG. 3, D-xylose, D-xylonic acid, and D-glucose are denoted by X, XA, and G, respectively. For statistical analysis of both T-tests, values with significant differences compared to those of the control group were marked with * (p <0.05, n≥3).

As a result, as can be seen in Fig. 3, the expression level of the yjh operon and the yagE and yagF gene genes increased only when exposed to D-xylonic acid, whereas mRNA when treated with D-xylose or D-glucose No significant difference was observed in the level (see FIGS. 3A to 3D).

Through the RT-PCR results, it was confirmed that the structural genes yagE, yagF, yjhH and yjhG and the regulatory genes yjhI increased their expression levels when exposed to D-xylonic acid. In addition, as can be seen in Table 3 and Fig. 1b, it can be seen that the yagE and yagF genes have P _yagE as a common promoter, and yjhH, yjhG and yjhI have P _yjhI as a common promoter.

Therefore, in order to confirm whether this common promoter (genetic element) can elicit a response to D-xylonic acid, the reporter gene mCherry was cloned upstream as shown in FIG. 4 and displayed in red fluorescence. Then, the prepared plasmid was introduced into a host E. coli W3110 and treated with three different saccharides of D-xylose, D-glucose and D-xylonic acid, and the results are shown in FIG. 5.

Figure 5 is a result showing the promoter response to D-xylonic acid (Figure 5a), D-xylose (Figure 5b) and D-glucose (Figure 5c). When OD600 reached 0.4 absorbance units, 20 mM of inducing molecule (or sugar substrate) was added to the culture.

As a result, as can be seen in Figure 5, P _yagE showed a high expression level for mCherry when treated with D-xylose, D-glucose and D-xylonic acid. In contrast, _{treatment with D-xylonic} acid in P yjh increased mCherry production similar to the control strain using the IPTG inducible T7 promoter. On the other hand, no reaction was observed when exposed to D-xylose and D-glucose. These results suggest that P _yjhI has low basal expression and excellent response to D-xylonic acid. Based on these results, the following characteristic analysis was additionally conducted.

Example 2: Characterization of promoter elements

Partial characterization of the DNA sequence of P _yjhI was performed using BPROM and NNPP online database (FIGS. 6 and 7). 6 is a result of analyzing a promoter using BPROM to predict a bacterial promoter, and FIG. 7 is a result of analyzing a promoter using NNPP.

As a result, as can be seen in FIGS. 6 and 7, five possible essential sites for the binding of RNA polymerase and transcription start site were identified through BPROM analysis (cutoff is set to 0.80) (FIG. 6) , Through a second analysis through NNPP, one possible site for -10 and -35, which is consistent with one site predicted by BPROM, was identified (FIG. 7).

In addition, two crp binding sites were predicted based on bioinformatics studies and databases (Fig. 8A). Referring to FIG. 8, FIG. 8A shows _{an essential part of P yjhI} based on bioinformatics research and a database, and FIG. 8B shows mCherry expression in the form of differently reduced bases _{of P yjhI.} Under the control of the T7 promoter, mCherry was used as a positive control (T7 + IPTG). All and all (+XA) represent the total length _{of P yjhI upstream of mCherry.} In Figure 8b (+XA) shows that it was induced by 20 mM D-xylonic acid. Expression of mCherry under the control of the differently shortened form of P _yjhI (Δ sign and number of removed bases) was induced by 20 mM D-xylonic acid. 8C is a _{result of the dose response of P yjhI} to D-xylonic acid, and samples were taken after 24 hours of incubation.

Specifically, based on the bioinformatics analysis prediction (FIG. 8A), the promoter sequence of the reduced base was located upstream of the mCherry ORF. About 50 bp were removed stepwise and mCherry production induced by D-xylonic acid was monitored (Fig. 8b). As a result, it was confirmed that when the 200bp nucleotide sequence was removed from the promoter sequence, the fluorescence was greatly reduced. This truncated version of the promoter effectively removes the predicted -10 and -35 sequences, indicating that this region is essential and a potential binding site for RNA polymerase. In addition, as a result of testing the dose response according to the increase in the concentration of D-xylonate using the total 302bp nucleotide sequence of _{P yjhI (Fig. 8c), when the concentration of D-xylonate is low (0 to 20 mM)} It was confirmed that the degree of fluorescence increased rapidly. On the other hand, at a concentration of 30 mM or higher, the degree of increase in fluorescence was low. _{This suggests} that since P yjhI is sensitive to D-xylonic acid, a good detection reaction can be exhibited even when the concentration of D-xylonic acid salt is low.

Example 3: Design of a novel D-xylonic acid reactive synthetic dielectric circuit

_{Using P yjhI} as a promoter genetic element, a D-xylonic acid-reactive gene circuit was designed as shown in FIG. 9A. Specifically, mCherry, a red fluorescent protein, is _{constitutively expressed downstream of P T7-lacO} (the host strain lacks the LacI gene), whereas sfGFP and lacI _{are located} downstream of P yjhI. In the absence of D-xylonic acid, mCherry is continuously expressed while sfGFP and lacI are inhibited. Once D-xylonic acid is introduced, lacI and sfGFP are produced, producing green fluorescence. The LacI protein binds to lacO, which stops the production of mCherry protein and reduces red fluorescence. The genetic elements of the synthetic circuit were made from the pRSET-A vector to create the plasmid pRSET-XAS (Fig. 4). Then, the plasmid pRESET-XAS is introduced into a target host and cultured in a medium under two different conditions; (1) MM9 medium containing glucose to support growth (Fig. 9b) and (2) MM9 medium containing glucose to support growth and D-xylonic acid to induce sfGFP expression (Fig. 9c). As a result, before the addition of D-xylonate salt, red fluorescence was related in both conditions. After 12 hours, under the conditions as shown in FIG. 9C, increased green fluorescence was observed, and red fluorescence was remarkably decreased. On the other hand, sfGFP did not increase, but red fluorescence increased further under the conditions shown in FIG. 9B. This suggests that the synthetic genetic circuit for the presence of D-xylonic acid works well.

Example 4: Application of synthetic circuit for regulation of xylose oxidation pathway

The promoter P _yjhI is integrated into the autonomous regulation of the xylose oxidation pathway in recombinant bacteria (FIG. 10 ). The first enzyme in the pathway, xylose dehydrogenase (xdh), is located downstream _{of P T7-lacO, a promoter that can be inhibited by LacI.} xdh produces D-xylonic acid from D-xylose, and LacI is expressed at a specific concentration of accumulated D-xylonate, effectively inhibiting Xdh production, thereby temporarily stopping D-xylonic acid accumulation and at the same time D- Downstream genes using xylonic acid are activated.

Another application of P _yjhI application is to place all XOP genes under constitutive expression allowing the host recombinant strain to grow in D-xylose. Activation of LacI at specific concentrations of accumulated D-xylonate inhibits total XOP and activates conversion to the production of target compounds such as ethylene glycol, glycolic acid, 1,2,4-butanetriol or 1,4-butanediol. I can make it.

<110> MYONGJI UNIVERSITY INDUSTRY AND ACADEMIA COOPERATION FOUNDATION <120> D-XYLONATE-RESPONSIVE PROMOTER, ARTIFICIAL GENETIC CIRCUITS COMPRISING D-XYLONATE-RESPONSIVE PROMOTER AND METHOD FOR DETECTION OF D-XYLONATE USING ARTIFICIAL GENETIC CIRCUIT <130> P19-0013/MJU <150> KR 2018/0169381 <151> 2018-12-26 <160> 59 <170> KoPatentIn 3.0 <210> 1 <211> 302 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of Promoter yjhI <400> 1 taagtaagtt cattcgagag ggatttcaag caaaaataat caatggcacc caatagaaaa 60 tattggcgat gcgctcgaac gaataaagaa gctctaggcg caatccacac actacgatgt 120 tgcaacaaca cgccatctac tttttattct cattcactaa atgtggctgt tctggattca 180 ttattcaaag tgtgtacaag atcacattta atcacatcat tacggttcag catgctgaac 240 aaagcatatt ttccactatg taatgccgat accatttatt ccatgagcaa ggaggagcca 300 tt 302 <210> 2 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of Promoter yagE <400> 2 aaacgggttc ttatgcctta gttgtaagtg tctaccatgt ccccgaacaa gtgttcacta 60 tgtccccgga ccgtacaccc caaaggggag aggggactgc accgagccat cttttccccc 120 tcgccccttt ggggagaggg ccggggtgag gggcaatatg tgatccagct taaatttccc 180 gcactccctc ttcccttccg atttacctct ccttgttctg cgtcatagta tgatcgttaa 240 ataaacgaac gctgttctat aatgtagaac aaaatgattc agcaaggaga tctc 294 <210> 3 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of reporter gene mCherry <400> 3 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc 60 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc 120 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag 180 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag 240 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg 300 gtgaccgtga cccaggactc ctccctccag gacggcgagt tcatctacaa ggtgaagctg 360 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag 420 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg 480 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag 540 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac 600 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc 660 ggcatggacg agctgtacaa g 681 <210> 4 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of reporter gene sfGFP <400> 4 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cgccacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcagc 300 ttcaaggacg acggcaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaacttcaa cagccacaac gtctatatca ccgccgacaa gcagaagaac 480 ggcatcaagg ccaacttcaa gatccgccac aacgtggagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgt gctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actcacggca tggacgagct gtacaag 717 <210> 5 <211> 1181 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of promoter yjhI, reporter gene sfGFP and TT7 <400> 5 taagtaagtt cattcgagag ggatttcaag caaaaataat caatggcacc caatagaaaa 60 tattggcgat gcgctcgaac gaataaagaa gctctaggcg caatccacac actacgatgt 120 tgcaacaaca cgccatctac tttttattct cattcactaa atgtggctgt tctggattca 180 ttattcaaag tgtgtacaag atcacattta atcacatcat tacggttcag catgctgaac 240 aaagcatatt ttccactatg taatgccgat accatttatt ccatgagcaa ggaggagcca 300 ttatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 360 acggcgacgt aaacggccac aagttcagcg tgcgcggcga gggcgagggc gatgccacca 420 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 480 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 540 agcgccacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatca 600 gcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 660 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 720 acaagctgga gtacaacttc aacagccaca acgtctatat caccgccgac aagcagaaga 780 acggcatcaa ggccaacttc aagatccgcc acaacgtgga ggacggcagc gtgcagctcg 840 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 900 actacctgag cacccagtcc gtgctgagca aagaccccaa cgagaagcgc gatcacatgg 960 tcctgctgga gttcgtgacc gccgccggga tcactcacgg catggacgag ctgtacaagt 1020 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga 1080 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 1140 cttgaggggt tttttgctga aaggaggaac tatatccgga t 1181 <210> 6 <211> 913 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of promoter T7-lacOI, reporter gene mCherry and TT7 <400> 6 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga 60 gatatacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt 120 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca 180 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc 240 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta 300 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg 360 cggcgtggtg accgtgaccc aggactcctc cctccaggac ggcgagttca tctacaaggt 420 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg 480 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa 540 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa 600 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac 660 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc 720 caccggcggc atggacgagc tgtacaagta gtcgagcacc accaccacca ccactgagat 780 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa 840 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga 900 actatatccg gat 913 <210> 7 <211> 1275 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of lacI <400> 7 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 60 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 120 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 180 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 240 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 300 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 360 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 420 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 480 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 540 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 600 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 660 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 720 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 780 gggctgcgcg ttggtgcgga catctcggta gtgggatacg acgataccga agacagctca 840 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 900 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 960 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 1020 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 1080 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat 1140 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactaacat 1200 gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat 1260 tgcactgaaa tctag 1275 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> T7MC (forward primer) <400> 8 ctttaagaag gagatataca tggccatcat caaggagttc 40 <210> 9 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> T7MC (reverse primer) <400> 9 cagtggtggt ggtggtggtg cctacttgta cagctcgtcc atg 43 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> PyagEMC1 (forward primer) <400> 10 cgtccggcgt agaggatcga aaacgggttc ttatgcctta gttg 44 <210> 11 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PyagEMC1 (reverse primer) <400> 11 tgatggccat gagatctcct tgctgaatca ttttgttc 38 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PyagEMC2 (forward primer) <400> 12 aggagatctc atggccatca tcaaggagtt c 31 <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> PyagEMC2 (reverse primer) <400> 13 agtggtggtg gtggtggtgc ctacttgtac agctcgtcca tg 42 <210> 14 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> PyjhIMC1 (forward primer) <400> 14 cgtccggcgt agaggatcga taagtaagtt cattcgagag ggatttcaag 50 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PyjhIMC1 (reverse primer) <400> 15 tgatggccat aatggctcct ccttgctcat g 31 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PyjhIMC2(forward primer) <400> 16 aggagccatt atggccatca tcaaggagtt c 31 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> PyjhIMC2(reverse primer) <400> 17 agtggtggtg gtggtggtgc ctacttgtac agctcgtcca tg 42 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> T7SF (forward primer) <400> 18 actttaagaa ggagatatac atggtgagca agggcgagga g 41 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> T7SF (reverse primer) <400> 19 agtggtggtg gtggtggtgc cttgtacagc tcgtccatgc c 41 <210> 20 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> PyagESF1 (forward primer) <400> 20 cgtccggcgt agaggatcga aaacgggttc ttatgcctta gttg 44 <210> 21 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PyagESF1 (reverse primer) <400> 21 tgctcaccat gagatctcct tgctgaatca ttttgttc 38 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PyagESF2 (forward primer) <400> 22 aggagatctc atggtgagca agggcgagga g 31 <210> 23 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PyagESF2 (reverse primer) <400> 23 agtggtggtg gtggtggtgc cttgtacagc tcgtccatgc c 41 <210> 24 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> PyjhISF1 (forward primer) <400> 24 gtccggcgta gaggatcgat aagtaagttc attcgagagg gatttcaag 49 <210> 25 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PyjhISF1 (reverse primer) <400> 25 tgctcaccat aatggctcct ccttgctcat g 31 <210> 26 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PyjhISF2 (forward primer) <400> 26 aggagccatt atggtgagca agggcgagga g 31 <210> 27 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PyjhISF2 (reverse primer) <400> 27 agtggtggtg gtggtggtgc cttgtacagc tcgtccatgc c 41 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRSETXAS (forward primer) <400> 28 ccctatagtg agtcgtatta atttcgcggg atcgagatcc 40 <210> 29 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> pRSETXAS (reverse primer) <400> 29 agaccacaac ggtttccctc tagaaataat tttgtttaac tttaagaagg agatatacat 60 atgcggg 67 <210> 30 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> lacO-mCherry-T7T (forward primer) <400> 30 taatacgact cactataggg ggaattgtga gcggataaca attcccctct agaaataatt 60 ttg 63 <210> 31 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> lacO-mCherry-T7T (reverse primer) <400> 31 gaacccgttt atccggatat agttcctcct ttcagcaaaa aacccctcaa g 51 <210> 32 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> XAS yjhIp (forward primer) <400> 32 atatccggat aaacgggttc ttatgcctta gttgtaagtg tctaccatgt c 51 <210> 33 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> XAS yjhIp (reverse primer) <400> 33 ctggtttcac gagatctcct tgctgaatca ttttgttcta cattatagaa cag 53 <210> 34 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> lacI (forward primer) <400> 34 aggagatctc gtgaaaccag taacgttata cgatgtcgca gagtatgccg 50 <210> 35 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> lacI (reverse primer) <400> 35 aacttactta ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaag 54 <210> 36 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> yjhIp-sfGFP-T7T (forward primer) <400> 36 tgaaatctag taagtaagtt cattcgagag ggatttcaag caaaaataat caatgg 56 <210> 37 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> yjhIp-sfGFP-T7T (reverse primer) <400> 37 gagggaaacc gttgtggtct atccggatat agttcctcct ttcagcaaaa aacccctcaa 60 g 61 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RTyagA (forward primer) <400> 38 gtcgcttcgg catttcac 18 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyagA (reverse primer) <400> 39 caggttatgg acggtgctg 19 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RTyagE (forward primer) <400> 40 aagtgtcgga agcgaacc 18 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyagE (reverse primer) <400> 41 cgatggtgtc tttgatgcc 19 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RTyagF (forward primer) <400> 42 gacctgcgat aaagggct 18 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyagF (reverse primer) <400> 43 agggagagtt cgtggttgg 19 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RTyagI (forward primer) <400> 44 tcgcctattc acgcatcg 18 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyagI (reverse primer) <400> 45 ctcgttctct tcgctgtcc 19 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RTyjhU (forward primer) <400> 46 tgagaatggt gatgtgctgg 20 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyjhU (reverse primer) <400> 47 taggcgattt gcgatgagg 19 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyjhG (forward primer) <400> 48 ccttattcgc tctctgccc 19 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyjhG (reverse primer) <400> 49 gccgttgtct tctccatcc 19 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RTyjhH (forward primer) <400> 50 ctactactgg aaagtcgcac c 21 <210> 51 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RTyjhH (reverse primer) <400> 51 tacgcaagtg accaacgc 18 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RTyjhI (forward primer) <400> 52 ggaagacacg agaagaactg 20 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTyjhI (reverse primer) <400> 53 tcgccagaaa gtgagattg 19 <210> 54 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTrrsB (forward primer) <400> 54 tacccgcaga agaagcacc 19 <210> 55 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RTrrsB (reverse primer) <400> 55 cgcatttcac cgctacacc 19 <210> 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RTcysG (forward primer) <400> 56 gcgtttattc cacagttcac c 21 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RTcysG (reverse primer) <400> 57 gttacagaag atgcgacgag 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdoG (forward primer) <400> 58 atacaccatc accttcagcc 20 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mdoG (reverse primer) <400> 59 cgtgctttca actatctcac c 21

Claims (14)

delete Expression vector comprising a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.
A host cell transformed with an expression vector containing a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.
The method of claim 3,
The host cell is E. coli ( Escherichia coli ), characterized in that the host cell.
A biosensor for detecting D-xylonic acid containing host cells transformed with an expression vector containing a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.
After exposing the biosensor according to claim 5 to D-xylonic acid, a method of detecting D-xylonic acid by measuring the degree of optical, electrochemical or biochemical expression of the protein by the operation of the xylonic acid-reactive promoter.
Redesigned genetic circuit for detection of D-xylonic acid, including:
i) a reporter gene encoding a red fluorescent protein;
ii) a promoter that regulates the expression of the downstream red fluorescent protein;
iii) the lacI gene encoding the lactose protein;
iv) a reporter gene encoding a green fluorescent protein; And
iv) a promoter represented by the nucleotide sequence of SEQ ID NO: 1 that detects D-xylonic acid and induces the expression of a downstream lacI gene and a reporter gene encoding a downstream green fluorescent protein.
The method of claim 7,
The red fluorescent protein is mCheery, the green fluorescent protein is a redesigned gene circuit for detecting D-xylonic acid, characterized in that sfGFP.
The method of claim 7,
When D-xylonic acid is not detected, sfGFP and lacI are suppressed and mCherry is continuously expressed to generate red fluorescence, and when D-xylonic acid is detected, sfGFP and lacI are expressed to generate green fluorescence. Redesigned genetic circuit for detecting D-xylonic acid, characterized in that.
Redesigned genetic circuit for detection of D-xylonic acid, including:
i) xdh gene encoding xylose dehydrogenase protein; And
ii) a promoter that regulates the expression of the xdh gene downstream according to the expression of the lactose protein;
iii) the lacI gene encoding the lactose protein; And
iv) A promoter represented by the nucleotide sequence of SEQ ID NO: 1 that detects D-xylonic acid and induces the expression of the downstream lacI gene.
The method of claim 10,
The promoter of iv) is a redesigned gene circuit for detecting D-xylonic acid, characterized in that when the concentration of xylonic acid is at least 5 mM, it induces the expression of the lacI gene.
The method of claim 10,
The expression of the lacI gene is a redesigned gene circuit for detecting D-xylonic acid, characterized in that by stopping the production of xylose dehydrogenase by inhibiting the expression of the xdh gene, D-xylonic acid accumulation is temporarily stopped.
Recombinant microorganism for detecting D-xylonic acid containing the genetic circuit of claim 10.
The method of claim 13,
The microorganism is a recombinant microorganism for detecting D-xylonic acid, characterized in that E. coli.

Images