CN114540396A - Construction method of glucose metabolic pathway in Shewanella strain - Google Patents

Abstract

The invention relates to a method for constructing a glucose metabolic pathway in a Shewanella strain; through a heterologous expression technology, combining means such as genetic engineering and the like, a glucose transmembrane transport module is combined with an ED module to synthesize key genes for enhancing glucose metabolism (zwf, pgl, edd, eda, pfkA and pykA), recombinant plasmids are constructed through Biobrick and are introduced into wild Shewanella MR-1, and a metabolic pathway of Shewanella is reconstructed, so that the Shewanella can use glucose as a unique carbon source for growth and metabolism, and the usable substrate spectrum of Shewanella is widened. 13 engineering strains capable of carrying out glucose growth and metabolism are obtained. Provides a practical basis for the design of substrate co-utilization of the microbial fuel cell, and is beneficial to improving the extracellular electron transfer efficiency of the strain.

Description

Construction method of glucose metabolism pathway in Shewanella strains

technical field

The invention belongs to the technical field of genetic engineering and biological metabolism, in particular to a method for constructing a strain that enables Shewanella to use glucose as the sole carbon source for growth and metabolism.

Background technique

Genetic engineering, also known as gene splicing technology and DNA recombination technology, is based on molecular genetics and uses modern methods of molecular biology and microbiology as means to combine genes from different sources according to pre-designed blueprints. The operation of constructing hybrid DNA molecules in vitro, and then introducing them into living cells, so that foreign genes can be replicated, transcribed, translated and expressed in recipient cells, so as to change the original genetic characteristics of organisms, obtain new varieties, and produce new products.

Glucose (chemical formula C ₆ H ₁₂ O ₆ ), abbreviated as glucose. Chemical name: 2,3,4,5,6-pentahydroxyhexanal, it is the most widely distributed and most important monosaccharide in nature, and it is a polyhydroxyaldehyde. Pure glucose is colorless crystal, easily soluble in water, slightly soluble in ethanol, insoluble in ether. Glucose plays an important role in the field of biology. Plants can produce glucose through photosynthesis, which is the energy source and metabolic intermediate product of living cells, that is, the main energy supply material for organisms. Glucose enters red blood cells to assist in the diffusion into cells, and the process of entering other cells is active transport, which requires the participation of carrier proteins. Glucose entering the cell is completely oxidized to carbon dioxide and water through the EMP pathway → tricarboxylic acid cycle → respiratory chain, and energy is generated from it.

Shewanella oneidensis is a facultative anaerobe with a clear genetic background and easy manipulation. The model electrogenic microorganism, Shewanella oneidensis MR-1 (MR-1 for short), is the most extensively studied strain of Shewanella in terms of genome sequence annotation and genetic characterization. The strain is able to transfer electrons to the anode in a microbial fuel cell without the addition of an exogenous medium, making it one of the model organisms to study how microorganisms generate electrical current in MFCs. Wild-type Shewanella generally uses lactic acid as a substrate for growth and metabolism, and cannot use sugars such as glucose and xylose, which makes the spectrum of available substrates too narrow and the extracellular electron transfer efficiency is low.

The EMP pathway (Embden-Meyerhof pathway) refers to the process in which glucose is decomposed into pyruvate and a small amount of ATP is released under anaerobic conditions. It can be roughly divided into two stages: the first stage only generates two molecules of the main intermediate metabolite 3-phosphate-glyceraldehyde. In the second stage, a redox reaction occurs, releasing ATP for energy synthesis and simultaneously forming two molecules of pyruvate. Through the EMP pathway, two molecules of ATP are obtained for each molecule of glucose oxidized, which provides energy and material preparation for biological life activities. The ED pathway is also known as the 2-keto-3-deoxy-6-phosphogluconate (KDPG) pathway. This is an alternative glucose-degrading pathway that exists in some strictly aerobic bacteria that lack an intact EMP pathway and is unique to microorganisms. The characteristic is that glucose can quickly obtain pyruvate which can only be formed by 10 steps of EMP pathway after only 4 steps of reaction. The key to this pathway is the cleavage of 2-keto-3-deoxy-6-phosphogluconate, i.e., into glyceraldehyde-3-phosphate and pyruvate. Glyceraldehyde 3-phosphate can be converted into pyruvate by entering the EMP pathway. (Characteristic enzyme: KDPG aldolase)

The invention discloses a method for constructing a strain that enables Shewanella to use glucose as the sole carbon source for growth and metabolism, aiming to construct 13 available recombinant plasmids through heterologous expression technology combined with genetic engineering and other means, and utilize glucose The key genes of metabolism were introduced into MR-1, and the metabolic pathway of Shewanella strains was reconstructed from two modules: "synthetic biology modification and optimization of glucose transmembrane transport module" and "glucose transport module combined with ED module to enhance glucose metabolism". The engineered Shewanella can grow and metabolize in a modified M9 liquid medium with glucose as the sole carbon source, thereby broadening the available substrate spectrum of Shewanella.

Invention content:

The present invention aims to combine "synthetic biology modification and optimization of glucose transmembrane transport module" (Ompc, galp, glf, glk) and "glucose transport module combined with ED module to enhance glucose metabolism through heterologous expression technology combined with genetic engineering and other means. "(zwf, pgl, edd, eda, pfkA, pykA) The key genes of the two modules were integrated through Biobrick's construction strategy, the recombinant plasmid was constructed, and imported into wild-type Shewanella oneidensis MR-1 to reconstruct Shewanella The metabolic pathway enables it to use glucose as the sole carbon source for growth metabolism, thereby broadening the available substrate spectrum of Shewanella.

The technical scheme of the present invention is summarized as follows:

A method for constructing a glucose metabolism pathway in Shewanella strains; the transmembrane transmembrane transport module is synthesized into biologically modified and optimized genes Ompc, galp, glf or/and glk; the glucose transport module combined with the ED module enhances the glucose metabolism genes zwf, pgl, edd, eda, pfkA or/and pykA; the key genes of the two modules were integrated through Biobrick 's construction strategy to construct a recombinant plasmid and imported into wild-type Shewanella oneidensis MR-1 to reconstruct the metabolic pathway of Shewanella , using glucose as the sole carbon source for growth metabolism.

The construction method described; the key genes of the two modules are connected to the vector plasmid pYYDT to form a recombinant plasmid, and then introduced into S. oneidensis MR-1 to obtain 13 kinds of engineering Sheva's containing the recombinant plasmid and capable of glucose metabolism and utilization. strains.

The construction method; the optimized nucleotide sequence of the gene ompC is shown in SEQ ID NO.1; the optimized nucleotide sequence of the gene galp is shown in SEQ ID NO.2; the optimized nucleotide sequence of the gene glf The sequence is shown in SEQ ID NO.3; the optimized nucleotide sequence of gene glk is shown in SEQ ID NO.4; the optimized nucleotide sequence of gene zwf is shown in SEQ ID NO.5; the optimized nucleotide sequence of gene pgl is shown in SEQ ID NO.5; The nucleotide sequence is shown in SEQ ID NO.6; the optimized nucleotide sequence of gene edd is shown in SEQ ID NO.7; the optimized nucleotide sequence of gene eda is shown in SEQ ID NO.8; The optimized nucleotide sequence of pfkA is shown in SEQ ID NO.9; the optimized nucleotide sequence of gene pykA is shown in SEQ ID NO.10.

The construction method; the nucleotide sequence of the vector plasmid pYYDT is shown in SEQ ID NO.11.

The described construction method; the codon-optimized gene is expressed through the Ptac promoter and T1 terminator, and the construction strategy of Biobrick is adopted, using SpeI and XbaI enzymes to be homozygous enzymes, and leaving the same sticky ends after processing. , under the action of T4 ligase, the required foreign genes are connected to the base plasmid one by one.

The present invention constructs 13 engineered Shewanella strains containing recombinant plasmids and capable of glucose metabolism and utilization as follows: Module 1: Synthetic biology transformation and optimization of glucose transmembrane transport module

①pSH1(pYYDT-glf-glk);

②pSH2(pYYDT-galp-glk);

③pSH3(pYYDT-ompC-galp-glk);

④pSH4(pYYDT-ompC-glf-glk);

Module 2: Glucose transport module combined with ED module to enhance glucose metabolism

⑤pSH5(pYYDT-ompC-glf-glk-zwf);

⑥pSH6(pYYDT-ompC-glf-glk-pgl);

⑦pSH7(pYYDT-ompC-glf-glk-edd);

⑧pSH8(pYYDT-ompC-glf-glk-eda);

⑨pSH9(pYYDT-ompC-glf-glk-pfkA);

⑩pSH10(pYYDT-ompC-glf-glk-zwf-pgl);

pSH11(pYYDT-ompC-glf-glk-zwf-pgl-edd)；

pSH11(pYYDT-ompC-glf-glk-zwf-pgl-edd);

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda)；

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda);

pSH13(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA)。

pSH13 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA).

The specific construction method is described as follows:

Using the Biobrick method, construct the recombinant plasmid:

To obtain the optimized sequence, add XbaI enzyme (known) cutting site at the 5' end of the target gene fragment respectively, and add SpeI and SbfI enzyme (known) cutting site successively at the 3' end; The pYYDT plasmid of the restriction site (the plasmid map is shown in Figure 1(1), the nucleotide sequence is shown in SEQ ID NO.11, and the reference EnhancingBidirectional Electron Transfer of Shewanella oneidensis by a Synthetic FlavinPathway) was digested; because XbaI It is a homozygous enzyme with SpeI enzyme, and the same sticky end will be generated after enzyme digestion, and then ligated by T4 ligase to construct a recombinant plasmid containing pYYDT as a vector and containing the target gene.

Module 1: Synthetic biology modification and optimization of glucose transmembrane transport module

①Construction of pSH1 (pYYDT-glf-glk) plasmid.

Use XbaI and SbfI enzymes to digest the target gene glf (nucleotide sequence is SEQ ID NO.3); use SpeI and SbfI enzymes to digest the pYYDT plasmid; then connect by T4 ligase to construct a gene glf containing Recombinant plasmid pYYDT-glf.

The gene glk was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO.4), and the newly constructed recombinant plasmid pYYDT-glf containing gene glf was digested with SpeI and SbfI enzymes, and passed through T4 ligase. Finally, a recombinant plasmid containing two target genes of glf and glk was obtained: pSH1 (pYYDT-glf-glk).

②Construction of pSH2 (pYYDT-galp-glk) plasmid.

Use XbaI and SbfI enzymes to digest the target gene galp (nucleotide sequence is SEQ ID NO. 2); use SpeI and SbfI enzymes to digest the pYYDT plasmid; then connect by T4 ligase to construct a gene containing galp. Recombinant plasmid pYYDT-galp.

The gene glk was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO. 4), and the newly constructed recombinant plasmid pYYDT-galp containing gene galp was digested with SpeI and SbfI enzymes, and passed through T4 ligase. Finally, a recombinant plasmid containing two target genes of galp and glk was finally obtained: pSH2 (pYYDT-galp-glk).

③pSH3(pYYDT-ompC-galp-glk),

The target gene ompC was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO.1); the pYYDT plasmid was digested with SpeI and SbfI enzymes; Recombinant plasmid pYYDT-ompC.

The gene galp was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO. 2), and the newly constructed recombinant plasmid pYYDT-ompC containing the gene ompC was digested with SpeI and SbfI enzymes. T4 ligase Afterwards, a recombinant plasmid pYYDT-ompC-galp containing the genes ompC and galp was constructed.

The gene glk is digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO.4), and the recombinant plasmid pYYDT-ompC-galp containing the gene ompC and galp just constructed is digested with SpeI and SbfI enzymes, After T4 ligase, a recombinant plasmid containing three target genes of ompC, galp and glk was finally obtained: pSH3 (pYYDT-ompC-galp-glk).

④pSH4(pYYDT-ompC-glf-glk);

The target gene ompC was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO.1); the pYYDT plasmid was digested with SpeI and SbfI enzymes; Recombinant plasmid pYYDT-ompC.

The gene glf was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO. 3), and the newly constructed recombinant plasmid pYYDT-ompC containing gene ompC was digested with SpeI and SbfI enzymes, and passed through T4 ligase. Then, the recombinant plasmid pYYDT-ompC-glf containing the gene ompC,glf was constructed.

The gene glk was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO.4), and the recombinant plasmid pYYDT-ompC-glf containing gene ompC, glf was just constructed with SpeI and SbfI enzymes. After T4 ligase, a recombinant plasmid containing three target genes of ompC, glf and glk was finally obtained: pSH4 (pYYDT-ompC-glf-glk),

Similarly, according to the construction method of the recombinant plasmid in the above-mentioned module 1, the construction of the recombinant plasmid for module 2: the glucose transport module combined with the ED module to enhance the glucose metabolism is completed:

⑤pSH5(pYYDT-ompC-glf-glk-zwf);

The recombinant plasmid pYYDT-ompC-glf-glk constructed in module one was digested with SpeI and SbfI enzymes with XbaI and SbfI enzymes; the target gene zwf was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO. .5), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing four target genes of ompC, glf, glk and zwf: pSH5 (pYYDT-ompC-glf-glk-zwf).

⑥pSH6 (pYYDT-ompC-glf-glk-pgl)

The recombinant plasmid pYYDT-ompC-glf-glk constructed in module one was digested with SpeI and SbfI enzymes with XbaI and SbfI enzymes; the target gene pgl was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO. .6), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing four target genes of ompC, glf, glk and pgl: pSH6 (pYYDT-ompC-glf-glk-pgl).

⑦pSH7(pYYDT-ompC-glf-glk-edd)

The recombinant plasmid pYYDT-ompC-glf-glk constructed in module one was digested with SpeI and SbfI enzymes with XbaI and SbfI enzymes; the target gene edd was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO. .7), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing four target genes of ompC, glf, glk and edd: pSH7 (pYYDT-ompC-glf-glk-edd).

⑧pSH8(pYYDT-ompC-glf-glk-eda)

The recombinant plasmid pYYDT-ompC-glf-glk constructed in module one was digested with SpeI and SbfI enzymes with XbaI and SbfI enzymes; the target gene eda was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO. .8), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing four target genes of ompC, glf, glk and eda: pSH8 (pYYDT-ompC-glf-glk-eda).

⑨pSH9(pYYDT-ompC-glf-glk-pfkA)

The recombinant plasmid pYYDT-ompC-glf-glk constructed in module 1 was digested with SpeI and SbfI enzymes with XbaI and SbfI enzymes; the target gene pfkA was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO. 9), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing four target genes of ompC, glf, glk, pfkA: pSH9 (pYYDT-ompC-glf-glk-pfkA).

⑩pSH10(pYYDT-ompC-glf-glk-zwf-pgl)

Use XbaI and SbfI enzymes to digest the recombinant plasmid pYYDT-ompC-glf-glk-zwf constructed in module two with SpeI and SbfI enzymes; use XbaI and SbfI enzymes to digest the target gene pgl (the nucleotide sequence is SEQ ID NO.6), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing five target genes of ompC, glf, glk, zwf and pgl: pSH10 (pYYDT-ompC-glf-glk-zwf-pgl).

pSH11(pYYDT-ompC-glf-glk-zwf-pgl-edd)

pSH11(pYYDT-ompC-glf-glk-zwf-pgl-edd)

Use XbaI and SbfI enzymes to digest the recombinant plasmid pYYDT-ompC-glf-glk-zwf-pgl constructed in module two with SpeI and SbfI enzymes; use XbaI and SbfI enzymes to digest the target gene edd (nucleotides) The sequence is SEQID NO.7), and then connected by T4 ligase to finally obtain a recombinant plasmid containing six target genes of ompC, glf, glk, zwf, pgl, edd: pSH11 (pYYDT-ompC-glf-glk-zwf- pgl-edd).

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda)

pSH12 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda)

The recombinant plasmid pYYDT-ompC-glf-glk-zwf-pgl-edd constructed in module two was digested with SpeI and SbfI enzymes with XbaI and SbfI enzymes; the target gene eda was digested with XbaI and SbfI enzymes (nucleolysis) The nucleotide sequence is SEQ ID NO.8), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing seven target genes of ompC, glf, glk, zwf, pgl, edd, eda: pSH12 (pYYDT-ompC-glf -glk-zwf-pgl-edd-eda).

pSH13(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA)

pSH13 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA)

The recombinant plasmid pYYDT-ompC-glf-glk-zwf-pgl-edd-eda constructed in module two was digested with XbaI and SbfI enzymes with SpeI and SbfI enzymes; the target gene pykA was digested with XbaI and SbfI enzymes (the nucleotide sequence is SEQ ID NO.10), and then ligated by T4 ligase to finally obtain a recombinant plasmid containing eight target genes of ompC, glf, glk, zwf, pgl, edd, eda, pykA: pSH13 (pYYDT -ompC-glf-glk-zwf-pgl-edd-eda-pykA).

The corresponding plasmid maps are shown in Figures 1(2)-(14). According to the above steps and methods, the construction and connection of plasmids were completed, and 13 recombinant plasmids were finally obtained.

The metabolically recombinant Shewanella strain obtained by the method of the present invention is used for aerobic fermentation in the improved M9 liquid medium with glucose as the sole carbon source, and the biomass and sugar metabolism in the fermentation broth are determined.

The metabolically recombinant Shewanella strain obtained by the method of the present invention is used for anaerobic fermentation in the improved M9 liquid medium with glucose as the sole carbon source, and the biomass and sugar metabolism in the fermentation broth are determined.

Overcoming the deficiencies of the prior art, a method for constructing a strain that enables Shewanella to use glucose as the sole carbon source for growth and metabolism is provided. The main technical scheme is to use heterologous expression technology, combined with genetic engineering and other means to construct recombinant plasmids, which will promote glucose uptake into cells (glucose transmembrane transport module: ompC, galp, glf, glk) to promote intracellular metabolism (optimized ED pathway). : zwf, pgl, edd, eda, pfkA, pykA) the key genes of the two modules were introduced into Shewanella oneidensis MR-1 to reconstruct the metabolic pathway of S. oneidensis MR-1, so that the engineered Shewanella could use glucose as the Growth metabolism in modified M9 liquid medium with a sole carbon source, thereby broadening the available substrate spectrum for Shewanella. The transformed strain was fermented aerobic and anaerobic, and its growth curve and residual sugar content in the modified M9 liquid medium with 3 g/L glucose as the sole carbon source were determined.

In module one, the data showed that the control strain WT, which was introduced with an empty plasmid, hardly took up metabolic glucose, whether it was aerobic or anaerobic fermentation; the constructed engineered strain could take up a certain amount of glucose, strain PSH3: pYYDT-ompC-glf- The aerobic and anaerobic fermentation biomass of glk was relatively higher than that of the control strain WT, and more glucose was ingested and utilized. After 12h aerobic fermentation of PSH3, the growth plateau was reached, the biomass (OD ₆₀₀ =0.51333) was 2.4 times that of WT (OD ₆₀₀ =0.21467), and the glucose in the medium was almost exhausted after 8h; after 8h of anaerobic fermentation of PSH3 The growth plateau was reached, the biomass ( _OD600 =0.19967) was about 1.6 times that of WT ( _OD600 =0.12833), and about 45% of the glucose was finally consumed in the medium. Therefore, we obtained the optimized engineered strain PSH3: pYYDT-ompC-glf-glk after "synthetic biology modification of glucose transmembrane transport module".

In module 2, the data show that the engineered strain PSH3: pYYDT-ompC-glf-glk after the optimization of module 1 continues to be modified, whether it is aerobic or anaerobic fermentation, the control strains WT and The engineering strain PSH9 hardly takes up and metabolizes glucose; the other 8 engineered strains PSH5, PSH6, PSH7, PSH8, PSH10, PSH11, PSH12, and PSH13 can take up a certain amount of glucose, among which strain PSH11: pYYDT-ompC-glf-glk The biomass of aerobic and anaerobic fermentation of -zwf-pgl-edd in the modified M9 liquid medium with 3g/L glucose as the only carbon source was relatively high, and the amount of glucose uptake and utilization was the most. PSH11 reached the growth plateau after 24 hours of aerobic fermentation, the biomass (OD ₆₀₀ =0.58267) was 3 times that of WT (OD ₆₀₀ =0.19133), and the glucose in the medium was almost exhausted after 16 hours; after 48 hours of anaerobic fermentation of PSH11 The growth plateau was reached, the biomass ( _OD600 =0.22467) was about 1.7 times that of WT ( _OD600 =0.13300), and about 59% of the glucose was consumed in the medium.

The effect of the present invention is as follows:

1. The present invention provides a strain construction method that enables Shewanella to use glucose as the sole carbon source for growth and metabolism. Using heterologous expression technology, combined with genetic engineering and other means, from the construction of recombinant plasmids, the key genes of the two modules of "synthetic biology modification and optimization of glucose transmembrane transport module" and "glucose transport module combined with ED module to enhance glucose metabolism" were introduced into In MR-1, 13 engineered strains capable of glucose growth and metabolism were obtained.

2. In the constructed engineering strain, PSH11 reached the growth plateau after 24 hours of aerobic fermentation, the biomass (OD ₆₀₀ =0.58267) was 3 times that of WT (OD ₆₀₀ =0.19133), and the glucose in the medium was almost consumed after 16 hours After 48 h of anaerobic fermentation of PSH11, the growth plateau was reached, the biomass (OD ₆₀₀ =0.22467) was about 1.7 times that of WT (OD ₆₀₀ =0.13300), and about 59% of the glucose was consumed in the medium.

3. The present invention broadens the available substrate spectrum of the model electrogenic bacteria MR-1, provides a realistic basis for the design of the co-utilization of microbial fuel cell substrates, and is beneficial to improve the extracellular electron transfer efficiency of the strain.

Description of drawings

Figure 1: Maps and nucleotide sequences of pYYDT and recombinant plasmids constructed for

Fig. 2: Schematic diagram for the design of the gene fragment bio-brick of the present invention

Fig. 3: the engineering Shewanella glucose metabolism design schematic diagram constructed for the present invention

Fig. 4: for the engineering strain of the present invention under aerobic conditions, the growth and sugar consumption situation in the improved M9 medium with glucose as the sole carbon source

Fig. 5: for the engineering strain of the present invention under anaerobic conditions, the growth and sugar consumption situation in the improved M9 medium with glucose as the sole carbon source

Detailed ways

The plasmid constructed in the present invention is designed in this laboratory. In the process of total synthesis, each exogenous gene required by the present invention needs to be connected to the plasmid vector and stored. Below in conjunction with embodiment, the present invention is further elaborated.

First, query the required target gene sequence information on KEGG. The target genes include key genes from two modules: Ompc, galp, glf, glk, zwf, pgl, edd, eda, pfkA, pykA (as shown in SEQ ID NO. 1-10); connected to the vector plasmid pYYDT (the plasmid map is shown in Figure 1(1), and the sequence information is as shown in SEQ ID NO. 11), and finally obtained 13 recombinant plasmids connected with the target gene on pYYDT, named respectively:

Module 1: Synthetic biology modification and optimization of glucose transmembrane transport module

①pSH1 (pYYDT-glf-glk); the map of the recombinant plasmid pSH1 is shown in Figure 1(2);

②pSH2 (pYYDT-galp-glk); the map of the recombinant plasmid pSH2 is shown in Figure 1(3);

③pSH3 (pYYDT-ompC-galp-glk); the map of the recombinant plasmid pSH3 is shown in Figure 1(4);

④pSH4 (pYYDT-ompC-glf-glk); the map of the recombinant plasmid pSH3 is shown in Figure 1(5).

Module 2: Glucose transport module combined with ED module to enhance glucose metabolism

⑤pSH5 (pYYDT-ompC-glf-glk-zwf); the map of the recombinant plasmid pSH3 is shown in Figure 1(6);

⑥pSH6 (pYYDT-ompC-glf-glk-pgl); the map of the recombinant plasmid pSH3 is shown in Figure 1(7);

⑦ pSH7 (pYYDT-ompC-glf-glk-edd); the map of the recombinant plasmid pSH3 is shown in Figure 1(8);

⑧pSH8 (pYYDT-ompC-glf-glk-eda); the map of the recombinant plasmid pSH3 is shown in Figure 1(9);

⑨pSH9 (pYYDT-ompC-glf-glk-pfkA); the map of the recombinant plasmid pSH3 is shown in Figure 1(10);

⑩pSH10 (pYYDT-ompC-glf-glk-zwf-pgl); the map of the recombinant plasmid pSH3 is shown in Figure 1(11);

pSH11(pYYDT-ompC-glf-glk-zwf-pgl-edd)；重组质粒pSH3图谱如图1(12)所示；

pSH11 (pYYDT-ompC-glf-glk-zwf-pgl-edd); the map of the recombinant plasmid pSH3 is shown in Figure 1 (12);

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda)；重组质粒pSH3图谱如图1(13)所示；

pSH12 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda); the map of the recombinant plasmid pSH3 is shown in Figure 1 (13);

pSH13(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA)；重组质粒pSH3图谱如图1(14) 所示。

pSH13 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA); the map of the recombinant plasmid pSH3 is shown in Figure 1 (14).

glk: the glucokinase gene from Escherichia coli K-12

galp: from the galactose transporter gene in Escherichia coli K-12

glf: from the UDP-galactopyranosyl mutase gene in Escherichia coli K-12

ompC: an outer membrane porin gene from Escherichia coli K-12 that enhances cell membrane permeability

zwf: from the glucose-6-phosphate dehydrogenase gene in Escherichia coli K-12

pgl: from the 6-phosphogluconolactonase gene in Escherichia coli K-12

pfkA: from the 6-phosphofructokinase gene in Escherichia coli K-12

edd: from the phosphogluconate dehydratase gene in Sheva's MR-1

eda: from the 2-dehydro-3-deoxyphosphoglucuronide aldolase gene in Sheva's MR-1

pykA: from the pyruvate kinase gene in Sheva's MR-1

In the present invention, the above-mentioned codon-optimized gene is expressed through the Ptac promoter and T1 terminator, adopts the construction strategy of Biobrick, utilizes the SpeI and XbaI enzymes to be homologous enzymes, and leaves the same sticky end after processing. Under the action of ligase, the required foreign genes are connected to the basic plasmid one by one, and the connected foreign genes are not affected by subsequent endonucleases, so as to construct a recombinant plasmid to exert different functions.

The engineering bacteria amplified by the selected recombinant plasmids is the auxotrophic strain E.coil WM3064, and the final host engineering bacteria is S. oneidensis MR-1. The transformation method of WM3064 can adopt conventional methods in the art, such as physical transformation method. Then the conjugation was transferred to MR-1 bacteria to obtain the constructed engineering bacteria. The wild-type strain introduced with the pYYDT empty plasmid was named WT, and the engineered strains introduced with other recombinant plasmids were named:

In the above construction method, the technical effect achieved by the expression of the recombinant plasmid in the present invention proposes that the Shewanella strain can use glucose as the sole carbon source to carry out aerobic and anaerobic growth and metabolism, thereby broadening the available base of Shewanella Spectrum.

Example 1: Construction of recombinant plasmids

(1) Acquisition and sequence optimization of the target gene:

First, query the required target gene sequence information on KEGG (http://www.kegg.jp/kegg/genes.html), the target gene includes the promotion of glucose uptake into cells (glucose transmembrane transport module: ompC, galp, glf, glk) to promote intracellular metabolism (optimized ED pathway: zwf, pgl, edd, eda, pfkA, pykA) key genes of two modules; use Jcat (http://www.jcat.de/) to code the coding sequence Sub-optimization and site-directed mutagenesis of the bio-brick restriction sites in the sequence were selected for optimization in Shewanella oneidensis MR-1, and the restriction sites EcoRI, XbaI, SpeI and PstI needed for future experiments were avoided.

(2) Using the Biobrick method, construct the recombinant plasmid:

To obtain the optimized sequence, add an XbaI restriction site at the 5' end of each target gene fragment, and add SpeI and SbfI restriction sites in sequence at the 3' end, and synthesize them. Using the Biobrick method, the above genes were connected by enzyme digestion to construct a recombinant plasmid ①pSH1 (pYYDT-glf-glk).

First, use XbaI and SbfI enzymes (well known) to digest the gene glf (nucleotide sequence is SEQ ID NO. 3), and use SpeI and SbfI enzymes (well known) to digest the pYYDT plasmid with the restriction site (plasmid map As shown in Figure 1(1), the nucleotide sequence is shown in SEQ ID NO. 11, and the reference is Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway) for enzyme digestion. Since XbaI and SpeI enzymes are homologous enzymes, the same sticky ends will be generated after enzyme digestion, and then ligated by T4 ligase to construct a recombinant plasmid containing the gene glf.

The gene glk was digested with XbaI and SbfI enzymes (nucleotide sequence is SEQ ID NO. 4), and the newly constructed recombinant plasmid containing gene glf was digested with SpeI and SbfI enzymes. After T4 ligase, the final A recombinant plasmid containing two target genes of glf and glk was obtained: ① pSH1 (pYYDT-glf-glk), the plasmid map is shown in Figure 1(2).

Also according to the method in the above step (2), the construction and connection of other plasmids were completed, and finally 13 kinds of recombinant plasmid modules were obtained. One: synthetic biology transformation and optimization of glucose transmembrane transport module

①pSH1(pYYDT-glf-glk), ②pSH2(pYYDT-galp-glk), ③pSH3(pYYDT-ompC-galp-glk), ④pSH4(pYYDT-ompC-glf-glk);

Module 2: Glucose transport module combined with ED module to enhance glucose metabolism

pSH11 (pYYDT-ompC-glf-glk-zwf-pgl-edd)，

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda)，

pSH13(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA)。⑤pSH5(pYYDT-ompC-glf-glk-zwf), ⑥pSH6(pYYDT-ompC-glf-glk-pgl), ⑦pSH7(pYYDT-ompC-glf-glk-edd), ⑧pSH8(pYYDT-ompC-glf-glk-eda) ), ⑨pSH9 (pYYDT-ompC-glf-glk-pfkA), ⑩pSH10 (pYYDT-ompC-glf-glk-zwf-pgl),

pSH11 (pYYDT-ompC-glf-glk-zwf-pgl-edd),

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda),

pSH13 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA).

The plasmid maps are shown in Figures 1(2)-(14), respectively.

The construction of the recombinant plasmid was completed in turn according to the above steps. The design principle of the bio-brick for constructing the plasmid is shown in Figure 2. Since pYYDT as the basic plasmid vector contains the kanamycin resistance gene sequence, it can be used for the directional screening of later engineering strains.

Example 2: Construction of recombinant Shewanella strains

①Transformation: The recombinant plasmid obtained above was introduced into E.coliWM3064 by physical transformation method for the construction of the subsequent engineering Shewanella.

Remove 50 μL of E.coliWM3064 competent cells from the -80°C refrigerator, thaw naturally in an ice box, add 33 μL of recombinant plasmid pSH, let stand on ice for 30 min, heat shock at 42°C for 90 s, and let stand on ice for 2-3 min. Add 1mL LB+DAP liquid medium (5g/L yeast extract, 10g/L tryptone, 10g/LNaCl, 0.059g/L 2,6-diaminopimelic acid) to the tube, and place at 37°C, 220rpm. Resuscitate in a shaker for 1 h. After centrifugation, it was spread on LB+DAP+kana solid plate (5g/L yeast extract, 10g/L tryptone, 10g/LNaCl, 0.059g/L DAP, 50mg/mL kanamycin (kana, 1:1000). ), 15g/L agar powder) and finally put it upside down in a constant temperature incubator at 37°C for overnight culture, pick out positive clones, and store the strains, take 500 μL of the overnight cultured positive clones and 500 μL of 50% glycerol, and add them to the storage tube. stored at -80°C.

②Conjugation transfer: The recombinant plasmid in the above-obtained positive cloned bacteria was transferred to the model electrogenic bacteria S.oneidensis MR-1 by conjugation to obtain an engineered strain capable of glucose uptake and utilization.

The transformed E. coli was inoculated into 3mL LB+DPA+kana liquid medium (5g/L yeast extract, 10g/L tryptone, 10g/LNaCl, 0.059g/L DAP, 50mg/mL kanamycin (kanamycin). , 1:1000)), 37°C, 200rpm for 10-12h; wild-type strain MR-1 was inoculated into 3mL LB liquid medium (5g/L yeast extract, 10g/L tryptone, 10g/L NaCl) Medium culture, 30°C, 200rpm for 10-12h. 500 μL of the obtained E. coli seed solution and MR-1 seed solution were mixed into a 1.5 mL sterile EP tube, mixed evenly, centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. Resuspend with 1 mL of LB+DAP liquid medium and let stand at 30 °C for 2 h. After standing, mix well and inoculate 50 μL of bacterial liquid on the solid plate of LB+kana (5g/L yeast extract, 10g/L tryptone, 10g/L NaCl, 15g/L agar powder, 50mg/mL kanamycin (kana, 1:1000)) was placed in a 30° C. incubator for more than 12 hours to obtain Shewanella engineered bacteria.

Colony PCR for verification: After the bacteria conjugated to the transfer coating plate grows an obvious single colony (about 0.5-1 mm in diameter), prepare bacteria P to verify whether the plasmid is successfully transferred. You need to prepare the LB+kana plate and draw a grid to make a mark. The PCR system is prepared and divided into 96-well plates, and 2-5 more systems can be prepared than the actual need to prevent the loss caused by the solution of the pipette tip. Prepare enough sterilized toothpicks. In the ultra-clean bench, a toothpick picks a single colony and puts it into a well of a system; after picking, each toothpick is streaked in the corresponding grid of the new plate, and the toothpick is discarded. After all the scratches are completed, the 96-well plate is sealed tightly with a sealing pad, placed in the PCR machine, the parameters are set, and the operation is started. Put the plate into a 30°C constant temperature incubator (growth not less than 10h, depending on the growth of the colony, when it grows to a certain extent, it can be wrapped in plastic wrap and placed at 4°C). Single PCR reaction system configuration (trans fast Taq): ddH ₂ 0 11.55 μL; upstream and downstream primers 0.3 μL each; 10x buffer 1.5 μL; dNTPS, 1.2 μL; Taq enzyme 0.15 μL; a total of 15 μL in one reaction system. The PCR reaction conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 30s, 53°C annealing for 30s, 72°C extension for Xs (1 kb/30s, only sufficient time but not enough), 30 cycles, and finally 72°C for 7min extension, 4°C incubation .

Agarose gel electrophoresis: make gel first, 100mL of 1XTAE buffer, 1.0g of agarose, heat to dissolve, add 5μL of nucleic acid dye after proper cooling, shake well and pour into the glue tank where the transparent pad and comb are placed, and wait for solidification.

Gel running: After PCR, add DNA Loading buffer, spot samples, 10 μL per well, and electrophoresis for 8-12 min. Observe whether there is a target band and record the corresponding number.

Bacteria preservation: The engineering Shewanella bacteria that have been verified successfully will be preserved for future use. Take 500 μL of Shewanella engineering bacteria cultured overnight and 500 μL of 50% glycerol, add them to a bacterial preservation tube, and store at -80°C.

The wild-type Shewanella strain introduced with the empty plasmid (pYYDT) was named WT, and the engineered strains introduced with other recombinant plasmids were named:

Module 1: Synthetic biology modification and optimization of glucose transmembrane transport module

①PSH1(pYYDT-glf-glk);②PSH2(pYYDT-galp-glk);③PSH3(pYYDT-ompC-galp-glk);④PSH4(pYYDT-ompC-glf-glk);

Module 2: Glucose transport module combined with ED module to enhance glucose metabolism

PSH11 (pYYDT-ompC-glf-glk-zwf-pgl-edd)；

PSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda)；

PSH13(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA)。⑤PSH5(pYYDT-ompC-glf-glk-zwf);⑥PSH6(pYYDT-ompC-glf-glk-pgl);⑦PSH7(pYYDT-ompC-glf-glk-edd);⑧PSH8(pYYDT-ompC-glf-glk-eda) ); ⑨PSH9(pYYDT-ompC-glf-glk-pfkA);⑩PSH10(pYYDT-ompC-glf-glk-zwf-pgl);

PSH11 (pYYDT-ompC-glf-glk-zwf-pgl-edd);

PSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda);

PSH13 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA).

As shown in Figure 3, it is the schematic diagram of the engineering Shewanella glucose metabolism design constructed by the present invention, which is designed from the source, including the synthetic biology transformation and optimization of the glucose transmembrane transport module that promotes glucose uptake into cells (ompC, galp, glf, glk) to the promotion of intracellular metabolism (optimized ED pathway: zwf, pgl, edd, eda, pfkA, pykA) to obtain an engineered Shewanella that can metabolize glucose.

Example 3: Shewanella engineering bacteria WT, PSH1-13 were fermented and cultured in a modified M9 medium containing a concentration of 3 g/L glucose solution (including aerobic and anaerobic)

First inoculate (1:100) each engineering strain into the liquid medium of LB+Kana (5g/L yeast extract, 10g/L tryptone, 10g/LNaCl, 50mg/mL kanamycin (kana, 1:1000) )), and then further aerobic and anaerobic fermentation was carried out in the fermentation flask, and an appropriate amount of IPTG (1M, 1:2000) was added to induce the expression of exogenous genes. Every 2h, take an appropriate amount of fermentation broth in the ultra-clean workbench to detect the OD ₆₀₀ value of each strain and record it; use a liquid chromatograph to detect the residual sugar content in each sampled fermentation broth. The wild-type strain WT introduced with the empty plasmid (pYYDT) was selected as the blank control group, and three groups of parallel experiments were performed for each strain.

Glucose metabolism growth curve of engineering Shewanella and determination of residual sugar:

1. Bacterial activation: In the morning, take several 10mL EP tubes sterilized by high pressure steam, add 3mL LB+kana liquid medium to the ultra-clean workbench, inoculate the bacteria, and cultivate at 30°C, 200rpm/min for 12h.

2. First-class seeds: inoculate each activated bacterial solution at 1% into a 250-mL conical flask containing 100 mL of LB+kana liquid medium, and cultivate at 30° C., 200 rpm/min for 12 hours.

3. Secondary seeds, fermentation: measure the OD ₆₀₀ of the primary seed liquid, adjust each strain to the same growth state, and inoculate it into a 250 mL conical flask containing 90 mL M9+10 mL Glucose+100 μL kana+50 μL IPTG liquid medium at 2% medium, 30°C, 200rpm/min for aerobic fermentation culture, and samples were periodically sampled and stored at 4°C for later use.

At the same time, prepare a batch of sugar-eating experimental groups of strains in anaerobic environment: take several 10mL EP tubes sterilized by high pressure steam (white tubes have poor dissolved oxygen effect), add 7885μL M9+1mL Glucose+10μLkana+5μL IPTG+ 100 μL of bacterial liquid + 1 mL of fumaric acid (electron acceptor), put it in a 30°C incubator and let it stand for anaerobic fermentation culture, and periodically sample and store at 4°C for later use.

4. Measure the OD ₆₀₀ of the fermentation broth for 0-24 h, record the data, and reserve 1 mL of the fermentation sample liquid for each group to centrifuge and take the supernatant through the liquid phase to measure the residual sugar content.

In this experiment, a sufficient amount of 30g/L glucose solution was prepared, passed through a 0.25 μm filter membrane in an ultra-clean workbench, and stored at 4°C for later use; a sufficient amount of 5×M9 solution was prepared, and dH ₂ O was added to dilute to 1×M9 solution. Sterilized by high pressure steam, and stored at 4°C after cooling.

in,

5×M9 mother liquor (1 L): 2.5 g NaCl, 5 g NH ₄ Cl, 15 g KH ₂ PO ₄ , 30 g Na ₂ HPO ₄ , sterilized.

Kanamycin stock solution (50mg/mL): 0.5g kana powder, dilute to 10mL with ddH ₂ O, sterilize with 0.22μm filter, divide into 1mL/tube, and store at -20°C.

IPTG stock solution (1M): 1.9064g IPTG, ddH ₂ O, dilute the volume to 8mL, sterilize with 0.22μm filter membrane, aliquot into 1mL/tube, and store at -20°C.

The growth and sugar consumption of the engineered strains in the modified M9 medium with glucose as the sole carbon source under aerobic (anaerobic) conditions are shown in Figure 4 (Figure 5): The data show that in module one, whether it is Aerobic or anaerobic fermentation, the control strain WT into which the empty plasmid was introduced hardly took up metabolic glucose; the constructed engineering strain could take up a certain amount of glucose, the aerobic and anaerobic fermentation biomass of strain PSH3: pYYDT-ompC-glf-glk Compared with the control strain WT were relatively higher, and the amount of glucose uptake and utilization was more. After 12h aerobic fermentation of PSH3, the growth plateau was reached, the biomass (OD ₆₀₀ =0.51333) was 2.4 times that of WT (OD ₆₀₀ =0.21467), and the glucose in the medium was almost exhausted after 8h; after 8h of anaerobic fermentation of PSH3 The growth plateau was reached, the biomass ( _OD600 =0.19967) was about 1.6 times that of WT ( _OD600 =0.12833), and about 45% of the glucose was finally consumed in the medium. Therefore, we obtained the optimized engineered strain PSH3: pYYDT-ompC-glf-glk after "synthetic biology modification of glucose transmembrane transport module". In module 2, the engineered strain PSH3 after module 1 optimization: pYYDT-ompC-glf-glk is a strain that continues to be transformed. Regardless of aerobic or anaerobic fermentation, the control strain WT and engineered strain PSH9 into which empty plasmids were introduced barely took up metabolic glucose; the other 8 engineered strains PSH5, PSH6, PSH7, PSH8, PSH10, PSH11, PSH12, and PSH13 were all able to A certain amount of glucose was ingested, wherein the biomass of strain PSH11: pYYDT-ompC-glf-glk-zwf-pgl-edd in the modified M9 liquid medium with 3 g/L glucose as the sole carbon source were both relative relative aerobic and anaerobic fermentation biomass higher, and the highest amount of glucose is ingested and utilized. PSH11 reached the growth plateau after 24 hours of aerobic fermentation, the biomass (OD ₆₀₀ =0.58267) was 3 times that of WT (OD ₆₀₀ =0.19133), and the glucose in the medium was almost exhausted after 16 hours; after 48 hours of anaerobic fermentation of PSH11 The growth plateau was reached, the biomass ( _OD600 =0.22467) was about 1.7 times that of WT ( _OD600 =0.13300), and about 59% of the glucose was consumed in the medium.

The engineering bacteria amplified by the selected recombinant plasmids is auxotrophic strain E.coilWM3064 (commercially available, ColiGenetic Stock Center http://cgsc.biology.yale.edu/), and the final host engineering bacteria is S.oneidensis MR- 1 (commercially available, ATCC 700550); the restriction enzymes, DNA ligases and molecular biology reagents used above were purchased from Thermo (http://www.thermoscientificbio.com/fermentas); other biochemical reagents used were from Purchased from Sangon Bioengineering (Shanghai) Co., Ltd. (http://www.sangon.com/).

The technical solutions disclosed and proposed in the present invention can be realized by those skilled in the art by referring to the content of this article and appropriately changing the conditions, routes and other links. The methods and technical routes described herein can be modified or recombined without departing from the content, spirit and scope of the present invention to achieve the final preparation technology. It should be particularly pointed out that all similar substitutions and modifications apparent to those skilled in the art are deemed to be included in the spirit, scope and content of the present invention. Matters not covered by the present invention belong to the known technology.

sequence listing

<110> Tianjin University

<120> Construction method of glucose metabolism pathway in Shewanella strains

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1144

<212> DNA

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gctctagata ctagagaaag aggagaaaat gatgaaacgt aacatcttag ctgttatcgt 60

tccagcgtta ttagttgctg gcactgctaa cgctgctgaa atctacaaca aagatggtaa 120

caaagttgat ttatacggta aagctgttgg tttacactac ttctctaaag gtaacggtga 180

aaactcttac ggtggtaacg gtgatatgac ttacgctcgt ttaggtttca aaggtgaaac 240

tcaaatcaac tctgatttaa ctggttacgg tcaatgggaa tacaacttcc aaggtaacaa 300

ctctgaaggt gctgatgctc aaactggtaa caaaactcgt ttagctttcg ctggtttaaa 360

atacgctgat gttggttctt tcgattacgg tcgtaactac ggtgttgttt acgatgcttt 420

aggttacact gatatgttac cagaattcgg tggtgatact gcttactctg atgatttctt 480

cgttggtcgt gttggtggtg ttgctactta ccgtaactct aacttcttcg gtttagttga 540

tggtttaaac ttcgctgttc aatacttagg taaaaacgaa cgtgatactg ctcgtcgttc 600

taacggtgat ggtgttggtg gttctatctc ttacgaatac gaaggtttcg gtatcgttgg 660

tgcttacggt gctgctgatc gtactaactt acaagaagct caaccattag gtaacggtaa 720

aaaagctgaa caatgggcta ctggtttaaa atacgatgct aacaacatct acttagctgc 780

taactacggt gaaactcgta acgctactcc aatcactaac aaattcacta acacttctgg 840

tttcgctaac aaaactcaag atgttttatt agttgctcaa taccaattcg atttcggttt 900

acgtccatct atcgcttaca ctaaatctaa agctaaagat gttgaaggta tcggtgatgt 960

tgatttagtt aactacttcg aagttggtgc tacttactac ttcaacaaaa acatgtctac 1020

ttacgttgat tacatcatca accaaatcga ttctgataac aaattaggtg ttggttctga 1080

tgatactgtt gctgttggta tcgtttacca attctaatct ccccaaacta gtccctgcag 1140

ggct 1144

<210> 2

<211> 1458

<212> DNA

<213> Artificial Sequence

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gctctagata ctagagaaag aggagaaata ctagagatgc cagatgctaa aaaacaaggt 60

cgttctaaca aagctatgac tttcttcgtt tgtttcttag ctgctctcgc gggcttactc 120

ttcggtttag atatcggtgt tatcgctggt gctttaccat tcatcgctga tgaattccaa 180

atcacttctc acactcaaga atgggttgtt tcttctatga tgttcggtgc tgctgttggt 240

gctgttggtt ctggttggtt atctttcaaa ttaggtcgta aaaaatcttt aatgatcggt 300

gctatcttat tcgttgctgg ttctttattc tctgctgctg ctcccaacgt tgaagtacta 360

atcttatctc gtgttttatt aggtttagct gttggtgttg cttcttacac tgctccatta 420

tacttatctg aaatcgctcc agaaaaaatc cgtggttcta tgatctctat gtaccaatta 480

atgatcacta tcggtatctt aggtgcttac ttatctgata ctgctttctc ttacactggt 540

gcttggcgtt ggatgttagg tgttatcatc atcccagcta tcttattatt aatcggtgtt 600

ttcttcttac cagattctcc acgttggttc gctgctaaac gtcgtttcgt tgatgctgag 660

cgtgtcctct tacgtttacg tgatacttct gctgaagcta aacgtgaatt agatgaaatc 720

cgtgaatctt tacaagttaa acaatctggt tgggctttat tcaaagaaaa ctctaacttc 780

cgtcgtgctg ttttcttagg tgttttatta caagttatgc aacaattcac tggtatgaac 840

gttatcatgt actacgctcc aaaaatcttc gaattagctg gttacactaa cactactgaa 900

caaatgtggg gtactgttat agttggttta actaacgtgc tggctacttt catcgctatc 960

ggtttagttg atcgttgggg tcgtaaacca actttaactt taggtttctt agttatggct 1020

gctggtatgg gtgttttagg tactatgatg cacatcggta tccactctcc atctgctcaa 1080

tacttcgcta tcgctatgtt attaatgttc atcgttggtt tcgctatgtc tgctggtcca 1140

ttaatctggg ttttatgttc tgaaatccaa ccattaaaag gtcgtgattt cggtatcact 1200

tgttctactg ctactaactg gatcgctaac atgatcgttg gtgctacttt cttaactatg 1260

ttaaacactt taggtaacgc taacactttc tgggtttacg ctgctttaaa cgttttattc 1320

atcttattaa ctttatggtt agttccagaa acaaagcacg taagcttaga acacatcgaa 1380

cgtaacttaa tgaaaggtcg taaattacgt gaaatcggtg ctcacgatta atctccccaa 1440

actagtccct gcagggct 1458

<210> 3

<211> 1485

<212> DNA

<213> Artificial Sequence

<400> 3

gctctagata ctagagaaag aggagaaata ctagagatgt cttctgaatc ttctcaaggt 60

ttagttactc gtttagcttt aatcgctgct atcggtggtt tattattcgg ttacgattct 120

gctgttatcg ctgctatcgg tactccagtt gatatccact tcatcgctcc acgtcactta 180

tctgctactg ctgctgcttc tttatctggt atggttgttg ttgctgtttt agttggttgt 240

gttactggtt ctttattatc tggttggata ggcatcaggt tcggtcgtcg tggcggttta 300

ttaatgtctt ctatctgttt cgttgctgct ggtttcggtg ctgctttaac tgaaaaatta 360

ttcggtactg gtggttctgc tttacaaatc ttctgtttct tccgtttctt agctggttta 420

ggtatcggtg ttgtttctac tttaactcca acttacatcg ctgaaatcgc tccaccagat 480

aaacgtggtc aaatggtttc tggtcaacaa atggctatcg ttactggtgc tttaactggt 540

tacatcttca cttggttatt agctcacttc ggttctatcg attgggttaa cgcttctggt 600

tggtgttggt ctccagcgtc ggaaggtctc atcggtatcg cgttcttatt attattatta 660

actgctccag atactccaca ctggttagtt atgaaaggtc gtcactctga agcttctaaa 720

atactcgctc gtctcgaacc acaagcggat ccaaacttaa ctatccaaaa aatcaaagct 780

ggtttcgata aagctatgga taaatcttct gctggtttat tcgctttcgg catcactgtt 840

gtgttcgcgg gtgtttctgt tgctgctttc caacaattag ttggcataaa cgctgtgctc 900

tactacgctc cacaaatgtt ccaaaactta ggtttcggtg ccgatacagc gttattacaa 960

actatatcta tcggtgttgt taacttcatc ttcactatga tcgcttctcg tgttgttgat 1020

cgtttcggtc gtaaaccatt attaatctgg ggtgctttag gtatggctgc gatgatggct 1080

gtgctcggtt gttgtttctg gttcaaagtt ggtggtgttt taccactcgc gtctgtacta 1140

ttatacatcg ctgttttcgg tatgtcttgg ggtccagttt gttgggttgt tttatctgaa 1200

atgttcccat cttctatcaa aggtgctgct atgccaatcg ctgttactgg tcaatggtta 1260

gctaacatct tagttaactt cttattcaaa gttgctgatg gttcgcccgc tctcaaccaa 1320

acttttaacc acggtttctc ttacttagtt ttcgctgctt tatctatctt aggtggttta 1380

atcgttgctc gtttcgttcc agaaactaaa ggtcgttctt tagatgaaat cgaagaaatg 1440

tggcgttctc aaaaataatc tccccaaact agtccctgca gggct 1485

<210> 4

<211> 1038

<212> DNA

<213> Artificial Sequence

<400> 4

gctctagata ctagagaaag aggagaaata ctagagatgg aaatcgttgc tatcgatatc 60

ggtggtactc acgctcgttt ctctatcgct gaagtttcta acggtcgtgt tttatcttta 120

ggtgaagaaa ctactttcaa aactgctgaa cacgcttctt tacaattagc ttgggaacgt 180

ttcggtgaaa aattaggtcg tccattacca cgtgctgctg ctatcgcttg ggctggtcca 240

gttcacggtg aagttttaaa attaactaac aacccatggg ttttacgtcc agctacttta 300

aacgaaaaat tagatatcga tactcacgta ttaataaacg acttcggtgc tgttgcgcac 360

gctgttgctc acatggattc ttcttactta gatcacatct gtggtccaga cgaagctctc 420

ccatctgatg gcgttatcac tatcttaggt ccgggtacag gtctcggtgt tgctcattta 480

ttacgtactg aaggtcgtta cttcgttatc gaaactgaag gtggtcacat cgatttcgct 540

ccattagatc gtttagaaga taaaatctta gctcgtttac gtgagaggtt ccgtcgtgtt 600

tcgatcgaac gtatcatctc tggtccaggt ttaggtaaca tctacgaagc tttagctgct 660

atcgaaggtg ttccattctc tttattagat gatatcaaat tatggcaaat ggctttagaa 720

ggtaaagata acttagctga agctgcgctc gataggttct gtttaagttt aggtgctatc 780

gctggtgatt tagctttagc tcaaggtgct acttctgttg ttatcggtgg tggtgttggt 840

ttacgtatcg cttctcactt accagagagt ggcttccgtc aacgttttgt ttctaaaggt 900

cgtttcgaac gtgttatgtc taaaatccca gttaaattaa tcacttaccc acaaccaggt 960

ttattaggtg ctgctgctgc ttacgctaac aaatactctg aagttgaata atctccccaa 1020

actagtccct gcagggct 1038

<210> 5

<211> 1574

<212> DNA

<213> Artificial Sequence

<400> 5

gctctagatt gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca 60

atactagaga aagaggagaa atactagaga tgactaacac tgtttctact atgatcttat 120

tcggctctac tggtgactta tctcaacgta tgttattacc atctttatac ggtttagatg 180

ctgatggttt attagctgat gatttacgta tcgtttgtac ttctcgttct gaatacgata 240

ctgatggttt ccgtgatttc gctgaaaaag ctttagatcg tttcgttgct tctgatcgtt 300

taaacgatga tgctaaagct aaattcttaa acaaattatt ctacgctact gttgatatca 360

ctgatccaac tcaattcggt aaattagctg atttatgtgg tccagttgaa aaaggtatcg 420

ctatctactt atctactgct ccatctttat tcgaaggtgc tatcgctggt ttaaagcagg 480

ctggtctcgc tggtccaact tctcgtttag ctttagaaaa accattaggt caagatttag 540

cttcttctga tcacatcaat gacgctgtac tcaaagtttt ctctgaaaaa caagtttacc 600

gtatcgatca ctacttaggt aaagaaactg ttcaaaactt attaacttta aggttcggca 660

acgcgttatt cgaaccactc tggaactcta aaggtatcga tcacgttcaa atctctgttg 720

ctgaaactgt tggtttagaa ggtcgtatcg gttacttcga tggttctggt tctttacgtg 780

atatggttca atctcacatc ttacaattag ttgctttagt tgctatggaa ccaccagctc 840

acatggaagc taacgctgtt cgtgatgaaa aagttaaagt tttccgtgct ttacgtccaa 900

tcaacaacga tactgttttc actcacactg ttactggtca atacggtgct ggtgtttctg 960

gtggtaaaga agttgctggt tacatcgatg aattaggtca accatctgat actgaaactt 1020

tcgttgctat caaagctcac gttgataact ggcgttggca aggtgttcca ttctacatcc 1080

gtactggtaa acgtttacca gctcgtcgtt ctgaaatcgt tgttcaattc aaaccagttc 1140

cacactctat cttctcttct tctggtggta tcttacaacc aaacaaatta cgtatcgttt 1200

tacaaccaga tgaaactatc caaatctcta tgatggttaa agagccaggt ctcgatcgta 1260

acggcgctca catgcgtgaa gtttggttag atttatcttt aactgatgtt ttcaaagatc 1320

gtaaacgtcg tatcgcgtac gaaaggttaa tgttagatct catcgaaggg gacgctacat 1380

tattcgttcg tcgtgatgaa gttgaagctc aatgggtttg gatcgatggt atccgtgaag 1440

gttggaaagc taactctatg aaaccaaaaa cttacgtttc tggtacttgg ggtccatcta 1500

ctgctatcgc tttagctgaa cgtgatggtg ttacttggta cgattaatct ccccaaacta 1560

gtccctgcag ggct 1574

<210> 6

<211> 771

<212> DNA

<213> Artificial Sequence

<400> 6

gctctagata ctagagaaag aggagaaata ctagagatga ctgaagctga atggtgggaa 60

ttcgaaaacg ttgaagctat ggctaaacaa atcgctgatg atatcgaatt catcatcaaa 120

caagctatcg aaaaaaaagg tcgtgcttta atcatcgttc caggtggttc tactccaaaa 180

ttagttttcc caactttagc tgctcgtgat ttagattggt ctaaagttac tttaatgtta 240

actgatgatc gtttagttgc taaagataac ccattatcta acttcggttt attaactaaa 300

cacttcggtt cttctggtgc tgaattagtt tctttaatcg atgaaaacta cttagatgat 360

cgtgctgctg ctggtcgtgc tgctgatcaa aaattagctt cttacaaatg gccagctgat 420

ttagtttggt taggtatggg taacgatggt cacactgctt ctatcttccc aggtccaaac 480

ttcgatgaag ctgttaacgg tccaagggaa cgtcgtgcgt taggtttatt accagttcca 540

ttaccaccag aagctccagt tgctcgtgtt actttatctt tatctacttt agcttctgct 600

cacactgtta tggttgttat cactggtgat cacaaacgta ctgttttaac tgatgcttta 660

aaagaaggtg cttcgtcgcg tctcccagtt ggtcgtgttt taggtgaaac tgaagcttct 720

atcgatgttt actggtctaa ataatctccc caaactagtc cctgcagggc t 771

<210> 7

<211> 1887

<212> DNA

<213> Artificial Sequence

<400> 7

gctctagata ctagagaaag aggagaaata ctagagatga ctgatttaca ctctactgtt 60

gaaaaagtta ctgctcgtgt tatcgaacgt tctcgtgaaa ctcgtaaagc ttacttagat 120

ttaatccaat acgaacgtga aaaaggtgtt gatcgtccaa acttatcttg ttctaactta 180

gctcacggtt tcgctgctat gaacggtgat aaaccagctt tacgtgattt caaccgtatg 240

aacatcggtg ttgttacttc ttacaacgat atgttatctg ctcacgaacc atactaccgt 300

tacccagaac aaatgaaagt tttcgctcgt gaagttggtg ctactgttca agttgctggt 360

ggtgttccag ctatgtgtga tggtgttact caaggtcaac caggtatgga agaatcttta 420

ttctctcgtg atgttatagc tctcgctact tctgtttctt tatctcacgg tatgttcgaa 480

ggtgctgctt tattaggtat ctgtgataaa atcgttccag gtttattaat gggtgcttta 540

cgtttcggtc acttaccaac tatcttagtt ccatctggtc caatgactac tggtatccca 600

aacaaagaaa aaatccgtat ccgtcaatta tacgctcaag gtaaaatcgg tcaaaaagaa 660

ttattagata tggaagctgc ttgttaccac gctgaaggta cttgtacttt ctacggtact 720

gctaacacta accaaatggt tatggaagtg ctaggtttac acatgccagg ttctgctttc 780

gttactccag gtactccatt acgtcaagct ttaactcgtg ctgctgttca ccgtgttgct 840

gaattaggtt ggaaaggtga tgattaccgt ccattaggta aaatcatcga tgaaaaatct 900

atcgttaacg ctatcgttgg tttattagct actggtggtt ctactaacca cactatgcac 960

atcccagcta tcgctcgtgc tgctggtgtt atcgtaaact ggaacgactt ccacgacctg 1020

tctgaagttg ttccattaat cgctcgtatc tacccaaacg gtccacgtga tatcaacgaa 1080

ttccaaaacg ctggtggtat ggcttacgtt atcaaagaat tattatctgc taacttatta 1140

aaccgtgatg ttactactat cgctaaaggt ggtatcgaag aatacgctaa agctccagct 1200

ttaaacgatg ctggtgaatt agtttggaaa ccagctggtg aaccaggtga tgatactatc 1260

ttacgtccag tttctaaccc attcgctaaa gatggtggtt tacgtttatt agaaggtaac 1320

ttaggtcgtg ctatgtacaa agcttctgct gttgatccaa aattctggac tatcgaagct 1380

ccagttcgtg ttttctctga tcaagatgat gttcaaaaag ctttcaaagc tggtgaatta 1440

aacaaagatg ttatcgttgt tgttcgtttc caaggtccac gtgctaacgg tatgccagaa 1500

ttacacaaat taactccagc tttaggtgtt ttacaagata acggttacaa agttgcttta 1560

gttactgatg gtcgtatgtc tggtgctaca ggtaaagttc cagttgctct ccacgtttct 1620

ccagaggctc tcggtggtgg tgcgatcggt aaattacgtg atggtgatat cgttcgtatc 1680

tctgttgaag aaggtaaatt agaagcttta gttccagctg atgaatggaa cgctcgtcca 1740

cacgctgaaa agcccgcgtt ccgtccaggt actggtcgtg aattattcga tatcttccgt 1800

caaaacgctg ctaaagctga agatggtgct gttgctatct acgctggtgc tggtatctaa 1860

tctccccaaa ctagtccctg cagggct 1887

<210> 8

<211> 743

<212> DNA

<213> Artificial Sequence

<400> 8

gctctagatt gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca 60

atactagaga aagaggagaa atactagaga tgcgtgatat cgattctgtt atgcgtttag 120

ctccagttat gccagtttta gttatcgaag atatcgctga tgctaaacca atcgctgaag 180

ctttagttgc tggtggttta aacgttttag aagttacttt acgtactcca tgtgctttag 240

aagctatcaa aatcatgaaa gaagttccag gtgctgttgt tggtgctggt actgttttaa 300

acgctaaaat gttagatcaa gctcaagaag ctggttgtga attcttcgtt tctccaggtt 360

taactgctga tttaggtaaa cacgctgttg ctcaaaaagc tgctttatta ccaggtgttg 420

ctaacgctgc tgatgttatg ttaggtttag atttaggcct cgataggttc aaattcttcc 480

cagctgaaaa catcggtggt ctcccagctc tcaaatctat ggcgtctgtt ttccgtcaag 540

ttcgtttctg tccaactggt ggtatcactc caacttctgc tccaaaatac ttagaaaacc 600

catctatctt atgtgttggt ggttcttggg ttgttccagc tggtaaacca gatgttgcta 660

aaatcactgc tttagctaaa gaagcttctg ctttcaaacg tgctgctgtt gcttaatctc 720

cccaaactag tccctgcagg gct 743

<210> 9

<211> 1071

<212> DNA

<213> Artificial Sequence

<400> 9

gctctagatt gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca 60

atactagaga aagaggagaa aatgatcaaa aaaatcggtg ttttaacttc tggtggtgat 120

gctccaggta tgaacgctgc tatccgtggt gttgttcgtt ctgctttaac tgaaggttta 180

gaagttatgg gtatctacga tggttactta ggtttatacg aagatcgtat ggttcaatta 240

gatcgttact ctgtttctga tatgatcaac cgtggtggta ctttcttagg ttctgctcgt 300

ttcccagaat tccgtgatga aaacatccgt gctgttgcta tcgaaaactt aaaaaaacgt 360

ggtatcgatg ctttagttgt tatcggtggt gatggttctt atatgggcgc tatgcgtctc 420

actgaaatgg gcttcccatg tatcggttta ccaggtacta tcgataacga tatcaaaggt 480

actgattaca ctatcggttt cttcactgct ttatctactg ttgttgaagc tatcgatcgt 540

ttacgtgata cttcttcttc tcaccaacgt atctctgttg ttgaagttat gggtcgttac 600

tgtggtgatt taactttagc tgctgctatc gctggtggtt gtgaattcgt tgttgttcca 660

gaagttgaat tctctcgtga agatttagtt aacgaaatca aagctggtat cgctaaaggt 720

aaaaaacacg ctatcgttgc tatcactgaa cacatgtgtg atgttgatga attagctcac 780

ttcatcgaaa aagaaactgg tcgtgaaact cgtgctactg ttttaggtca catccaacgt 840

ggtggttctc cagttccata cgatcgtatc ttagcttctc gtatgggtgc ttacgctatc 900

gatttattat tagctggtta cggtggtcgt tgtgttggta tccaaaacga acaattagtt 960

caccacgata tcatcgatgc tatcgaaaac atgaaacgtc cattcaaagg tgattggtta 1020

gattgtgcta aaaaattata ctaatctccc caaactagtc cctgcagggc t 1071

<210> 10

<211> 1503

<212> DNA

<213> Artificial Sequence

<400> 10

gctctagata ctagagaaag aggagaaata ctagagatgt tccgtcgtac taaaatcgtt 60

actactttag gtccagctac tgatcgtgat gataacttac gtcgtatcat cgctgctggt 120

gctaacgttg ttcgtttaaa cttctctcac ggttctccag aagatcactt aaaacgtgct 180

actcaagctc gtgaaatcgc taaagaatta ggtgttcacg ttgctatctt aggtgattta 240

caaggtccaa aaatccgtgt ttctactttc aaagataaca aaaaaatcca attaaaatta 300

ggtcaaactt acatcttaga tgctgaatta gctaaaggtg aaggtgatga aaaccaagtt 360

ggtatcgatt acaaacaatt accagatgat gttaacgttg gtgatatctt aatgttagat 420

gatggtcgtg ttcaattacg tgttgaacgt gttgaaggtc gtaaagttca cactactgtt 480

actgttgctg gtccattatc taacaacaaa ggtatcaaca aacaaggtgg tggtttatct 540

gctgctgctt taactgaaaa agataaagct gatatcttaa ctgctgctat gatccaagtt 600

gattacttag ctgtttcttt cccacgttct ggtgctgatt tagaatacgc tcgttcttta 660

gctcaacaag ctggttctaa cgctttaatc gttgctaaag ttgaacgtgc tgaagctgtt 720

gcttctgatg aagctatgga tgatgttatc ttagcttctg atgttgttat ggttgctcgt 780

ggtgatttag gtgttgaaat cggtgatgct gctttagttg ctgttcaaaa aaaattaatc 840

gctcgttctc gtcaattaaa caaaatcgtt atcactgcta ctcaaatgat ggaatctatg 900

atctcttctc caatgccaac tcgtgctgaa gttatggatg ttgctaacgc tgttttagat 960

ggtactgatg ctgttatgtt atctgctgaa actgctgctg gtgatttccc agaagaaact 1020

gttaaagcta tggctaacgt ttgtgttggt gctgaatctc acccatctgt taaagtttct 1080

aaacaccgtc tcgacgctag gttcacttct gttgaggaaa ctatcgcttt atctactatg 1140

tacgctgcta accacttaga aggtgttaaa gctatcatcg ctttaactga atctggtgct 1200

actccaaaat taatgtctcg tatctcttct tctttaccaa tcttaggttt atctcgtcac 1260

gatactactt tagcgaagat ggcgttatac cgtggtgttt taccaatcta cttcgattct 1320

actatctacc cagctgatga attagctcaa aaagctttag aatctttaac taaagctggt 1380

tacttacact ctggtgattt agttttaatg actaaaggtg atgctatgga aactatcggt 1440

ggtactaaca cttgtaaagt tttaatcgtt gcttaatctc cccaaactag tccctgcagg 1500

gct 1503

<210> 11

<211> 5904

<212> DNA

<213> Artificial Sequence

<400> 11

cacctcgcta acggattcac cgtttttatc aggctctggg aggcagaata aatgatcata 60

tcgtcaatta ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag 120

cacacggtca cactgcttcc ggtagtcaat aaaccggtca gaatttcaga taaaaaaaat 180

ccttagcttt cgctaaggat gatttctgtg gtacctcgga tcccggggag ctagcacgaa 240

ttcgcggccg cttctagacc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat 300

gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg 360

atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca 420

gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca 480

ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca 540

cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg 600

atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta 660

aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc 720

tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc 780

ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc 840

gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc 900

cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca 960

atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac 1020

aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc 1080

agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata 1140

tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca 1200

ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 1260

ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa 1320

ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 1380

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta 1440

agttagctca ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacggt 1500

gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt 1560

aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgtttttttgc 1620

gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg 1680

gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagccagtcc 1740

gtttaggtgt tttcacgagc acttcaccaa caaggaccat agcatatgcc actagtagcg 1800

gccgcctgca ggtggtcgac cactcgaggc caggcatcaa ataaaacgaa aggctcagtc 1860

gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca 1920

cactggctca ccttcgggtg ggcctttctg cgtttataac cggtaaacca gcaatagaca 1980

taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaac 2040

cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat 2100

cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt 2160

cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc 2220

cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat 2280

cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca 2340

gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 2400

cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 2460

tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 2520

caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 2580

cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca 2640

gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct 2700

tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 2760

cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 2820

ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag 2880

atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc 2940

agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 3000

ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 3060

ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc ccaggtggca 3120

cttttcgggg aaatgtgcgc gcccgcgttc ctgctggcgc tgggcctgtt tctggcgctg 3180

gacttcccgc tgttccgtca gcagcttttc gcccacggcc ttgatgatcg cggcggcctt 3240

ggcctgcata tcccgattca acggccccag ggcgtccaga acgggcttca ggcgctcccg 3300

aaggtctcgg gccgtctctt gggcttgatc ggccttcttg cgcatctcac gcgctcctgc 3360

ggcggcctgt agggcaggct catacccctg ccgaaccgct tttgtcagcc ggtcggccac 3420

ggcttccggc gtctcaacgc gctttgagat tcccagcttt tcggccaatc cctgcggtgc 3480

ataggcgcgt ggctcgaccg cttgcgggct gatggtgacg tggcccactg gtggccgctc 3540

cagggcctcg tagaacgcct gaatgcgcgt gtgacgtgcc ttgctgccct cgatgccccg 3600

ttgcagccct agatcggcca cagcggccgc aaacgtggtc tggtcgcggg tcatctgcgc 3660

tttgttgccg atgaactcct tggccgacag cctgccgtcc tgcgtcagcg gcaccacgaa 3720

cgcggtcatg tgcgggctgg tttcgtcacg gtggatgctg gccgtcacga tgcgatccgc 3780

cccgtacttg tccgccagcc acttgtgcgc cttctcgaag aacgccgcct gctgttcttg 3840

gctggccgac ttccaccatt ccgggctggc cgtcatgacg tactcgaccg ccaacacagc 3900

gtccttgcgc cgcttctctg gcagcaactc gcgcagtcgg cccatcgctt catcggtgct 3960

gctggccgcc cagtgctcgt tctctggcgt cctgctggcg tcagcgttgg gcgtctcgcg 4020

ctcgcggtag gcgtgcttga gactggccgc cacgttgccc attttcgcca gcttcttgca 4080

tcgcatgatc gcgtatgccg ccatgcctgc ccctcccttt tggtgtccaa ccggctcgac 4140

gggggcagcg caaggcggtg cctccggcgg gccactcaat gcttgagtat actcactaga 4200

ctttgcttcg caaagtcgtg accgcctacg gcggctgcgg cgccctacgg gcttgctctc 4260

cgggcttcgc cctgcgcggt cgctgcgctc ccttgccagc ccgtggatat gtggacgatg 4320

gccgcgagcg gccaccggct ggctcgcttc gctcggcccg tggacaaccc tgctggacaa 4380

gctgatggac aggctgcgcc tgcccacgag cttgaccaca gggattgccc accggctacc 4440

cagccttcga ccacataccc accggctcca actgcgcggc ctgcggcctt gccccatcaa 4500

ttttttttaat tttctctggg gaaaagcctc cggcctgcgg cctgcgcgct tcgcttgccg 4560

gttggacacc aagtggaagg cgggtcaagg ctcgcgcagc gaccgcgcag cggcttggcc 4620

ttgacgcgcc tggaacgacc caagcctatg cgagtggggg cagtcgaagg cgaagcccgc 4680

ccgcctgccc cccgagcctc acggcggcga gtgcgggggt tccaaggggg cagcgccacc 4740

ttgggcaagg ccgaaggccg cgcagtcgat caacaagccc cggaggggcc actttttgcc 4800

ggagggggag ccgcgccgaa ggcgtggggg aaccccgcag gggtgccctt ctttgggcac 4860

caaagaacta gatatagggc gaaatgcgaa agacttaaaa atcaacaact taaaaaaggg 4920

gggtacgcaa cagctcattg cggcaccccc cgcaatagct cattgcgtag gttaaagaaa 4980

atctgtaatt gactgccact tttacgcaac gcataattgt tgtcgcgctg ccgaaaagtt 5040

gcagctgatt gcgcatggtg ccgcaaccgt gcggcaccct accgcatgga gataagcatg 5100

gccacgcagt ccagagaaat cggcattcaa gccaagaaca agcccggtca ctgggtgcaa 5160

acggaacgca aagcgcatga ggcgtgggcc gggcttattg cgaggaaacc cacggcggca 5220

atgctgctgc atcacctcgt ggcgcagatg ggccaccaga acgccgtggt ggtcagccag 5280

aagacacttt ccaagctcat cggacgttct ttgcggacgg tccaatacgc agtcaaggac 5340

ttggtggccg agcgctggat ctccgtcgtg aagctcaacg gccccggcac cgtgtcggcc 5400

tacgtggtca atgaccgcgt ggcgtggggc cagccccgcg accagttgcg cctgtcggtg 5460

ttcagtgccg ccgtggtggt tgatcacgac gaccaggacg aatcgctgtt ggggcatggc 5520

gacctgcgcc gcatcccgac cctgtatccg ggcgagcagc aactaccgac cggccccggc 5580

gaggagccgc ccagccagcc cggcattccg ggcatggaac cagacctgcc agccttgacc 5640

gaaacggagg aatgggaacg gcgcgggcag cagcgcctgc cgatgcccga tgagccgtgt 5700

tttctggacg atggcgagcc gttggagccg ccgacacggg tcacgctgcc gcgccggtag 5760

cacttgggtt gcgcagcaac ccgtaagtgc gctgttccag actatcggct gtagccgcct 5820

cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac 5880

ctcgacctga atggaagccg gcgg 5904

Claims (6)

1. the construction method of glucose metabolism pathway in Shewanella strain; it is characterized in that, the gene Ompc, galp, glf or/and glk of synthetic biology modification and optimization of glucose transmembrane transport module; glucose transport module combined with ED module to enhance glucose Metabolic genes zwf, pgl, edd, eda, pfkA or/and pykA; the key genes of the two modules were integrated through Biobrick 's construction strategy to construct a recombinant plasmid, and introduced into wild-type Shewanella oneidensis MR-1 to reconstitute the The metabolic pathway of var. var. uses glucose as the sole carbon source for growth and metabolism. 2. construction method as claimed in claim 1; It is characterized in that, the key gene of two modules is connected on carrier plasmid pYYDT to form recombinant plasmid, is introduced in S.oneidensis MR-1 again, obtains 13 kinds of containing recombinant plasmid and can Engineered Shewanella strains for glucose metabolism and utilization. 3. construction method as claimed in claim 1; It is characterized in that, the nucleotide sequence after gene ompC optimization is as shown in SEQ ID NO.1; The nucleotide sequence after gene galp optimization is as shown in SEQ ID NO.2 The optimized nucleotide sequence of gene glf is as shown in SEQ ID NO.3; the optimized nucleotide sequence of gene glk is as shown in SEQ ID NO.4; the optimized nucleotide sequence of gene zwf is as shown in SEQ ID NO. .5; the optimized nucleotide sequence of gene pgl is shown in SEQ ID NO.6; the optimized nucleotide sequence of gene edd is shown in SEQ ID NO.7; the optimized nucleotide sequence of gene eda As shown in SEQ ID NO.8; the optimized nucleotide sequence of the gene pfkA is shown in SEQ ID NO.9; the optimized nucleotide sequence of the gene pykA is shown as SEQ ID NO.10. 4. The construction method according to claim 2; characterized in that, the nucleotide sequence of the vector plasmid pYYDT is shown in SEQ ID NO.11. 5. construction method as claimed in claim 1; It is characterized in that, the gene after codon optimization will complete expression by Ptac promoter and T1 terminator, adopt the construction strategy of Biobrick, utilize SpeI and XbaI enzyme to be the same tail enzyme , after treatment, the same sticky ends are left, and the required foreign genes are connected to the base plasmid one by one under the action of T4 ligase. 6. construction method as claimed in claim 5; It is characterized in that, construct 13 kinds of engineering Shewanella strains that contain recombinant plasmid and can carry out glucose metabolism and utilization as follows: Module 1: Synthetic biology modification and optimization of glucose transmembrane transport module ①pSH1(pYYDT-glf-glk); ②pSH2(pYYDT-galp-glk); ③pSH3(pYYDT-ompC-galp-glk); ④pSH4(pYYDT-ompC-glf-glk); Module 2: Glucose transport module combined with ED module to enhance glucose metabolism ⑤pSH5(pYYDT-ompC-glf-glk-zwf); ⑥pSH6(pYYDT-ompC-glf-glk-pgl); ⑦pSH7(pYYDT-ompC-glf-glk-edd); ⑧pSH8(pYYDT-ompC-glf-glk-eda); ⑨pSH9(pYYDT-ompC-glf-glk-pfkA); ⑩pSH10(pYYDT-ompC-glf-glk-zwf-pgl);

pSH11(pYYDT-ompC-glf-glk-zwf-pgl-edd);

pSH12(pYYDT-ompC-glf-glk-zwf-pgl-edd-eda);

pSH13 (pYYDT-ompC-glf-glk-zwf-pgl-edd-eda-pykA).

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