KR20200080106A - 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 D-xylonic acid detection method using the same. The promoter represented by nucleotide sequences of SEQ ID NO: 1 according to the present invention has high reactivity 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 a final product through the pH control by adjusting the accumulation of D-xylonic acid.

Description

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 genetic circuit for detecting D-xylonic acid containing 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 in the isomerization step, releasing D-xylulose. This intermediate is further metabolized through the pentose phosphate pathway (PPP). Next, the oxo-reductive pathway (ORP) produces D-xylulose as an intermediate, but begins with a pathway that reduces D-xylose and oxidizes xylitol. The third pathway, called the Xylose Oxidation Path (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 glycolaldehyde. It is another dehydration reaction to form an aldolase reaction or 2-ketoglutarate semialdehyde. The former is called the Dahms pathway, and the latter is called the Weimberg pathway.

Xylose oxidation pathway (XOP) is 1,2,4-butanetriol (1,2,4-butanetriol), xylonic acid (xylonic acid), ethylene glycol (ethylene glycol), glycolic acid (glycolic acid), gamma It has been used in the development of recombinant strains capable of producing various valuable compounds such as amino-butyric 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 aldol cleavage step results in low carbon yields for all target products, and secondly, the intermediate chain D-xylonic acid accumulates in the medium, resulting in a decrease in pH. Previous studies have attempted to address this problem by identifying the optimal genes for the pathway and the 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 biological tools. It uses genetic elements or components to build synthetic circuits or networks that allow some degree of predictability for mutant traits. The parts used to make these synthetic genetic circuits are promoters, operators, regulators or inducing molecules and reporter proteins. The basic goal is to create conditions in which the presence of an inducing molecule or external force causes a reaction by the host microorganism. These reactions range from simple biosensing to more complex gene regulation. XOP, especially the Dahms pathway of Escherichia coli, currently has limited research on synthetic genetic tools.

Thus, the present inventors have 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 it is applied to a synthetic circuit for detecting D-xylonic acid in E. coli because of its high reactivity, it is confirmed that the production yield of the final product can be increased through pH adjustment by controlling the accumulation of D-xylonic acid, and the present invention is 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 an expression vector comprising the D-xylonic acid reactive promoter, a host cell transformed with the expression vector, and a biosensor for detecting D-xylonic acid containing the host cell.

Another object of the present invention is to provide a redesigned genetic circuit for detecting D-xylonic acid containing the D-xylonic acid reactive promoter and a recombinant microorganism for detecting D-xylonic acid containing the genetic 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 a non-toxic nucleic acid sequence upstream of the coding region, which includes a binding site for a polymerase and has transcription initiation activity of a promoter subgene 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 The base sequence of the D-xylonic acid-reactive promoter was confirmed by confirming the expression.

The sequence encoding the promoter of the present invention can be modified to a certain extent. Those skilled in the art that the nucleotide sequence that maintains 70% or more homology 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 gene expression desired in the present invention. Will be easy to understand.

In the present invention, the term "homology" refers to the same degree as the wild type nucleic acid sequence, and the comparison of homology is the percentage of homology between two or more sequences using a comparison program that is easy to purchase 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 preferably comprises at least 70%, more preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical nucleic acid sequence do.

In addition, the promoter of the present invention includes a variant having a promoter nucleic acid sequence modified by substitution, deletion, insertion or a combination of one or more nucleic acid bases as long as it retains promoter activity. Promoter nucleic acid sequences can induce sequence variations by deletion, insertion, non-conservative or conservative substitutions, or combinations thereof. Through the sequence variation, the same activity as the natural promoter may be exhibited, but preferably, the function of the promoter can be improved to suit the purpose, such as a promoter having increased activity and a promoter having increased specificity for an inducer.

Nucleic acid molecules encoding all the categories of promoters are provided as promoter components of expression vectors that induce expression of target genes, and modifications of various vectors using the promoters 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" means a gene construct containing essential regulatory elements such as a promoter so that a target gene can be expressed in a suitable host cell. The expression vector related to the present invention is a vector in which the promoter is a D-xylonic acid-reactive promoter, and the promoter is operably linked to induce 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 (operably linked)" refers to a functional link between the nucleotide sequence encoding the gene of interest and the D-xylonic acid reactive expression control sequence to perform a general function. Operational linkage with recombinant vectors can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage uses enzymes, etc., 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 an expression control sequence capable of affecting the expression of a protein, for example, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer , Signal sequence for membrane targeting or secretion.

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 transformable with the vector can be used as long as it is known as a host cell producing a recombinant protein. As a host cell, bacteria, yeast, fungi, and the like are possible, but are not limited thereto, and are preferably characterized by Escherichia coli .

According to another aspect of the present invention, the present invention provides a biosensor for detecting xylonic acid containing 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. to provide.

According to another aspect of the present invention, the present invention exposes the biosensor to D-xylonic acid, and then measures the degree of optical, electrochemical or biochemical expression of the fluorescent protein by the operation of the xylonic acid reactive promoter to determine D- It provides a method for detecting xylonic acid.

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

According to another aspect of the invention, the invention is i) a reporter gene coating a red fluorescent protein; ii) a promoter that regulates the expression of downstream red fluorescent protein; iii) lacI gene coating lactose protein; iv) reporter gene coating green fluorescent protein; And iv) a promoter represented by the nucleotide sequence of SEQ ID NO: 1 that detects D-xylonic acid and induces expression of a downstream lacI gene and a reporter gene coating the downstream green fluorescent protein. Design genetic circuits are provided.

The redesigned gene circuit for D-xylonic acid detection of the present invention includes a lacI gene coating lactose protein and a reporter gene coating green fluorescent protein and a reporter gene detecting D-xylonic acid coating the lacI gene and green fluorescent protein It consists of a promoter represented by the nucleotide sequence of SEQ ID NO: 1 that induces the expression, and the promoter reacted with this in proportion to the concentration of D-xylonic acid induces the expression of the lacI gene and the gene encoding the green fluorescent protein. , It can be said to be a redesigned genetic circuit capable of regulating D-xylonic acid accumulation. As shown in Figs. 9A to 9C, quantitative fluorescence analysis can be performed by the D-xylonic acid detection genetic circuit 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 the D-xylonic acid present in a certain concentration or more and controlling the accumulation of D-xylonic acid (Example 3) And, FIGS. 9A to 9C).

In the redesigned genetic 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, the genetic circuit suppresses sfGFP and lacI when D-xylonic acid is not detected and mCherry is continuously expressed to generate red fluorescence, and D-xyl When ronic acid is detected, sfGFP and lacI are expressed to produce green fluorescence. That is, the genetic circuit according to the present invention can confirm the production 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 comprises: i) an xdh gene encoding a xylose dehydrogenase protein; ii) a promoter that regulates the expression of downstream xdh gene according to the expression of lactose protein; iii) lacI gene coating 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 D-xylonic acid detection of the present invention consists of a lacI gene coating 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 downstream lacI gene. Produced in response to the concentration of D-xylonic acid, the promoter reacted with this 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 that LacI can inhibit. xdh produces D-xylonic acid in D-xylose, LacI is expressed at a specific concentration of accumulated D-xylonic acid, effectively inhibiting Xdh production, temporarily stopping D-xylonic acid accumulation, and simultaneously D- The downstream gene using xylonic acid is activated. This suggests that the genetic circuit of the present invention can increase the production yield of the final product through pH adjustment by detecting the D-xylonic acid present in a certain concentration or more and controlling the accumulation of D-xylonic acid (Example 4 and, see FIG. 10).

In the redesigned genetic 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 xylon acid is at least 5 mM, and is detectable even in the above concentration range It is characterized by.

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 to stop the production of xylose dehydrogenase, thereby temporarily stopping the accumulation of D- xylonic acid. It is characterized by.

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, the microorganism can be used as long as it is known as a microorganism that produces a recombinant protein. As a microorganism, bacteria, yeast, fungi, and the like are possible, but are not limited thereto, and are preferably E. coli.

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

1 is a schematic diagram showing a metabolic exaggeration of D-xylonic acid accumulation in E. coli W3110.
2 is a result of confirming the relative gene expression of yag and yjh genes in E. coli W3110 ((2a)M9 medium, (2b)D-xylose M9 medium).
Figure 3 is a quantitative PCR analysis of the yag and yjh genes of E. coli W3110.
4 is a plasmid map including a D-xylogen-reactive promoter as a genetic element.
FIG. 5 is a result showing promoter responses to D-xylonic acid (FIG. 5A), D-xylose (FIG. 5B) and D-glucose (FIG. 5C).
6 is a result of analyzing the promoter using BPROM to predict the bacterial promoter.
7 is a result of analyzing the promoter using NNPP.
8 is a result of analyzing the properties of P _yjhI ((8a) as a result of predicting two crp binding sites, (8b) as a result of confirming mCherry expression in a form of differently reducing the base of _yjhI , (8c) P for D- _xylonic acid dose response of _yjhI ).
FIG. 9 shows a D- _xylonic acid-reactive gene circuit (9a) using P _yjhI as a promoter genetic element, and a _result of the D- _xylonic acid reaction result ((9b) D-xylonic acid medium, (9c) ) D-xylonic acid).
Figure 10 shows the genetic circuit using the D- _xylonic acid response promoter P _yjhI 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 only for illustrating the present invention, it should not be construed that the scope of the present invention is limited by these examples.

In order to construct a redesigned genetic circuit for detecting D-xylonic acid, the present invention identifies structural genes and promoters that respond to D-xylonic acid, and constructs a new genetic circuit for detecting D-xylonic acid. The detailed experimental 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 Gibson isothermal assembly strategies were used for DNA assembly (Gibson et al. 2009). The list of recombinant plasmid and E. coli strains used in the present invention are shown in Table 1. The plasmid sfGFP-pBAD is Michael Davidson and Geoffrey Waldo (Addgene plasmid # 54519) (P

delacq et al. 2006), and was used as a material for the sfGFP gene. The plasmid pETmCherryLIC was donated by 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 gene 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 GGCATGGG 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 TCACATGGTCGAGACTCGGA 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 AGGAAGCTGA GTTGGCTGCT GCCACCGCTG AGCAATAACT AGCATAACCC CTTGGGGCCT CTAAACGGGT CTTGAGGGGT TTTTTGCTGA AAGGAGGAAC TATATCCGGA T 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

Culture of plasmid

Plasmid propagation and maintenance was performed using E. coli DH5α grown in LB medium or agar medium supplemented with appropriate antibiotics (50 μg/mL kanamycin or 100 μg/mL ampicillin). Modified M9 (MM9) containing 33.7 mM Na ₂ HPO ₄ , 22.0 mM KH ₂ PO ₄ , 8.55 mM NaCl, 9.35 mM NH ₄ Cl, 1.0 mM MgSO ₄ , 0.5 g/L yeast extract and 1.0 g/L peptone Medium was used throughout the experiment. M9 medium was used by modifying the selected substrate to an appropriate concentration as required by the test conditions. For real-time PCR studies, 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 in the range of 0 to 100 mM, and 20 mM D-xylonic acid was used at the optimum concentration to induce an optimal response. For the gene switch experiment, 20 mM D-xylonic acid was used as the inducer concentration.

Quantitative real-time PCR

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 appropriate substrate(s) and incubated at 37° C. for 3 hours. Strains grown only in MM9 medium were selected as controls. Test conditions include strains growing on D-xylose (X), D-glucose (G), D-xylonic acid (XA) or any combination of these substrates. Total mRNA of growth 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 at room temperature for 5 minutes before adding RLT buffer (included in 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 of Part 1). The isolated RNA was confirmed using a NanoDrop 1000 spectrophotometer from Thermo Scientific (Waltham, MA, USA) and agarose gel electrophoresis. RNA was converted to cDNA using a QuantiTect Reverse Transcription kit (Qiagen, Germany) according to a protocol comprising removing genomic DNA. qPCR reaction was performed using QuantiFast® SYBR® Green PCR Kit (Qiagen, Germany). Each reaction included a suitable qPCR primer (Table 4) with a final concentration of 1 μM. The expression levels of the target gene were appropriately normalized using the reference genes rrsB, mdoG and cysG. The qPCR reaction was annealed and extended for 10 cycles at 62°C for 40 cycles of denaturation at 95°C using Rotor-Gene Q (Qiagen, Germany). qPCR raw data was processed through RG mode in 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

Cells were incubated overnight in LB medium, 1 ml of each sample was centrifuged at 21206xg for 2 minutes and the supernatant was discarded. The pellets were resuspended in 500 μl of M9 minimal medium and then 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 samples were incubated at 37° C. vigorously, until 0.3 to 0.4 absorbance was reached. Samples were taken from each medium before replenishing the sugar substrate. The amount collected in each sample depends on the OD600 absorbance. After collection, each medium was supplemented with 20 mM 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 for 2 minutes at 21206xg, the supernatant was discarded and 1 ml of water was added to each sample. Cells were normalized based on absorbance at OD600. Cells were subjected to fluorescence analysis using a fluorescence spectrometer FluoroMate FS-2 (Scinco, Korea). The fluorescence spectrometer was set to 587 nm excitation, 610 nm emission for sfGFP, and 485 excitation, 507 emission for mCherry.

Bioinformatics analysis

Gene and protein sequences of enzymes related to E. coli yag and yjh operons 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 the 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 NNPP (Neural Network Promoter Prediction) (Reese 2001).

Example 1: Screening of D-xylonic-reactive gene elements

Gene expression analysis was performed through real-time PCR to obtain information on the expression profiles of structural genes and regulatory genes. Transporter-coding genes were excluded from the analysis. Real-time PCR was performed on 8 genes (yagA, yagE, yagF, yagI, yjhG, yjhH, yjhI, yjhU) under MM9 medium without a substrate (control) and MM9 with at least one substrate (example) conditions. The results are shown in FIGS. 2 and 3.

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

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

In addition, FIG. 3 is a yag (FIGS. 3A and 3B) of E. coli W3110 supplemented with D-xylonic acid, D-xylose, D-glucose (FIGS. 3A and 3C) or a combination (FIGS. 3B and 3D ), and As a result of quantitative PCR analysis of the yjh (FIGS. 3C and 3D) gene, strains grown on modified M9 medium (M9 salt with 1 g/L peptone and 0.5 g/L yeast extract) were used as a control. In Figure 3, D-xylose, D-xylonic acid and D-glucose are denoted by X, XA and G, respectively. Statistical analysis of two-sided T-tests is marked with a value with a significant difference compared to the control group (p <0.05, n≥3).

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

Through the RT-PCR results, it was confirmed that the expression levels of the structural genes yagE, yagF, yjhH and yjhG and the regulatory genes yjhI were increased when exposed to D-xylonic acid. In addition, as can be seen in Table 3 and Figure 1b, it can be confirmed 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 marked with red fluorescence. Then, the prepared plasmid was introduced into host E.coli W3110 and treated with three different saccharides, D-xylose, D-glucose, and D-xylonic acid, and the results are shown in FIG. 5.

FIG. 5 is a result showing promoter responses to D-xylonic acid (FIG. 5A), D-xylose (FIG. 5B) and D-glucose (FIG. 5C). When OD600 reached 0.4 absorbance units, 20 mM of the 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 control strains using the IPTG inducible T7 promoter. On the other hand, no reaction was seen when exposed to D-xylose and D-glucose. These results indicate that P _yjhI has low basal expression and excellent response to D- _xylonic acid. Based on these results, the following characteristic analysis was further conducted.

Example 2: Characterization of promoter elements

Partial characterization of the DNA sequence of P _yjhI was performed using the BPROM and NNPP online databases (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 were identified for binding of RNA polymerase and transcription start site through BPROM analysis (cutoff is set to 0.80) (FIG. 6 ). , Through a second analysis through NNPP, one possible site for -10 and -35 that matches one site predicted by BPROM was identified (FIG. 7).

In addition, two crp binding sites were predicted based on bioinformatics research and database (FIG. 8A). Referring to FIG. 8, FIG. 8A shows essential parts of P _yjhI based on bioinformatics research and databases, and FIG. 8B shows mCherry expression in a form of differently reducing the base of P _yjhI . MCherry was used as a positive control (T7 + IPTG) under the control of the T7 promoter. Total and total (+XA) represent the total length of mCherry's P _yjhI upstream. 8B shows that (+XA) was induced by 20 mM D-xylonic acid. The expression of mCherry under the control of a differently reduced form of P _yjhI (Δ symbol and number of bases removed) shows that it was induced by 20 mM D-xylonic acid. Figure 8c is a result of the dose response of P _yjhI to D- _xylonic acid, the sample was collected after 24 hours of culture.

Specifically, based on the bioinformatics analysis prediction (FIG. 8A), a promoter sequence with reduced base was located upstream of the mCherry ORF. About 50 bp was removed step by step and mCherry production induced by D-xylonic acid was monitored (FIG. 8B). As a result, when the nucleotide sequence of 200bp was removed from the promoter sequence, it was confirmed that the fluorescence was very 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, when the dose response was tested as the concentration of the D-xylonic acid salt was increased using the entire 302 bp base sequence of P _yjhI (FIG. 8C), when the D-xylonic acid salt concentration was low (0 to 20 mM) It was confirmed that the degree of fluorescence increase rapidly. On the other hand, the increase in fluorescence was low at a concentration of 30 mM or more. This suggests that P _yjhI is sensitive to D- _xylonic acid, so it can exhibit a good detection reaction even when the concentration of the D- _xylonic acid salt is low.

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

The D- _xylonic acid-responsive gene circuit was designed as shown in FIG. 9A using P _yjhI as a promoter genetic element. Specifically, the red fluorescent protein mCherry is constitutively expressed downstream of P _T7-lacO (host strain lacks the LacI gene) while sfGFP and lacI are _located downstream of P _yjhI . Without D-xylonic acid, mCherry is continuously expressed while sfGFP and lacI are inhibited. Once D-xylonic acid is introduced, lacI and sfGFP are generated to produce green fluorescence. The LacI protein binds lacO, which stops the production of mCherry protein and reduces red fluorescence. The genetic elements of the synthetic circuit are made from the pRSET-A vector to make the plasmid pRSET-XAS (Figure 4). The plasmid pRESET-XAS is then introduced into the target host and cultured in two different conditions of medium; (1) MM9 medium containing glucose to support growth (Figure 9b) and (2) MM9 medium containing D-xylonic acid to induce glucose and sfGFP expression to support growth (Figure 9c). As a result, red fluorescence was related in both conditions before adding the D-xylonic acid salt. After 12 hours, increased green fluorescence was observed and red fluorescence was significantly reduced under the conditions shown in FIG. 9C. On the other hand, although sfGFP did not increase, 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 a synthetic circuit to control the xylose oxidation pathway

The promoter P _yjhI is integrated into the autonomic 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 LacI can inhibit. xdh produces D-xylonic acid in D-xylose, and LacI is expressed at a specific concentration of accumulated D-xylonic acid salt, effectively inhibiting Xdh production, temporarily stopping D-xylonic acid accumulation, and simultaneously D- The downstream gene using xylonic acid is activated.

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

D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.
An 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 comprising a D-xylonic acid reactive promoter represented by the nucleotide sequence of SEQ ID NO: 1.
According to claim 3,
The host cell is Escherichia coli , characterized in that the host cell.
Biosensor for detecting D-xylonic acid containing 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.
A method of detecting D-xylonic acid by exposing the biosensor according to claim 5 to D-xylonic acid, and then measuring the degree of optical, electrochemical or biochemical expression of the protein by the action of a xylonic acid reactive promoter.
Redesigned genetic circuit for D-xylonic acid detection, including:
i) a reporter gene coating a red fluorescent protein;
ii) a promoter that regulates the expression of downstream red fluorescent protein;
iii) lacI gene coating lactose protein;
iv) reporter gene coating 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 the downstream lacI gene and the reporter gene that coats the downstream green fluorescent protein.
The method of claim 7,
The red fluorescent protein is mCheery, the green fluorescent protein is redesigned genetic circuit for D-xylonic acid detection, characterized in that sfGFP.
The method of claim 7,
In the gene circuit, 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 D-xylonic acid detection, characterized in that.
Redesigned genetic circuit for D-xylonic acid detection, including:
i) xdh gene encoding xylose dehydrogenase protein; And
ii) a promoter that regulates the expression of downstream xdh gene according to the expression of lactose protein;
iii) lacI gene coating 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 downstream lacI gene.
The method of claim 10,
The promoter of iv) is a redesigned gene circuit for D-xylonic acid detection, characterized in that inducing the expression of the lacI gene when the concentration of xylon acid is at least 5 mM.
The method of claim 10,
The redesign gene circuit for D-xylonic acid detection is characterized in that the expression of the lacI gene temporarily stops the accumulation of xylose dehydrogenase by inhibiting the expression of the xdh gene, thereby temporarily stopping the accumulation of D-xylonic acid.
A 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 it is E. coli.

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