A heterologous expression in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 and its application

Abstract

The present invention belongs to the field of cellulose synthesis, and specifically relates to a heterologous expression Escherichia coli biosynthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 and its application, including a heterologous expression vector of cellulose synthase subunit PtCesA1 or a heterologous expression vector of cellulose synthase subunit PtCesA8; and a heterologous cell containing the above vector. PtCesA1 and PtCesA8 are expressed in heterologous cells respectively to form cellulose synthase, which is used to catalyze the synthesis of cellulose microfibrils. The present invention realizes the biosynthesis of cellulose in vivo, and directly synthesizes a cellulose product with high purity similar to plants and free of lignin and hemicellulose from the bacteria by heterologously expressing plant cellulose synthase subunits in Escherichia coli.

Description

Technical Field

[0001] The invention belongs to the field of cellulose synthesis, and specifically relates to a heterologous expression Escherichia coli in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 and an application thereof. Background Art

[0002] Cellulose is a natural organic polymer compound. It is the most widely distributed and abundant renewable resource in nature. It plays an important role and has great value in the fields of papermaking, textiles, bioenergy, etc. With the rapid development of social economy, people have an increasingly high demand for the application of plant cellulose. However, traditional cellulose production methods consume a lot of energy and cause serious environmental pollution. Artificial biosynthesis of cellulose, as a green and environmentally friendly synthesis method, is of great significance to the biomass materials industry. However, due to the unclear understanding of the cellulose synthesis mechanism in plants and the structure and action mechanism of the cellulose synthase complex, the output and quality of artificial biosynthesis products of cellulose have not yet met the application needs.

[0003] The chemical structure of cellulose is composed of D-pyranose glucose rings connected by β-1,4-glycosidic bonds. 4 C1 chair conformation. In plants, cellulose is synthesized by cellulose synthase complex (CSCs) on the cytoplasmic membrane. In primary and secondary cell walls, cellulose synthesis is controlled by CSCs containing different combinations of types. Cellulose synthase gene (CesA) is a gene encoding the catalytic subunit of cellulose synthase complex. Most plants express multiple different types of CesA genes, and the functions of different gene types are different. The number of members of the CesA gene family in different plants also varies to a certain extent.

[0004] In the prior art, cellulose microfibrils can be synthesized in vitro by cellulose synthase, but the synthesized cellulose microfibrils are limited and are more disordered than the cellulose microfibrils synthesized by in situ expression in plants.

[0005] In the prior art, it is not disclosed that the subunits of cellulose synthase from Populus trichocarpa can be used to synthesize cellulose. In the prior art, it is difficult to obtain a cellulose product with an orderly structure, integrity and high yield through in vivo biosynthesis of a single cellulose synthase subunit. Summary of the invention

[0006] The object of the present invention is to provide a heterologous expression in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 and application thereof.

[0007] The first aspect of the present invention is to provide a heterologous expression in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8, including a heterologous expression vector of cellulose synthase subunit PtCesA1 or a heterologous expression vector of cellulose synthase subunit PtCesA8; and a heterologous cell containing the above vector.

[0008] In a preferred embodiment, the heterologous expression vector for cellulose synthase subunit PtCesA1 comprises the PtCesA1 gene at 174-3428 in SEQ ID NO:1, and the heterologous expression vector for cellulose synthase subunit PtCesA8 comprises the PtCesA8 gene at 171-3125 in SEQ ID NO:2.

[0009] Furthermore, the heterologous cells include but are not limited to Escherichia coli, and PtCesA1 and PtCesA8 are respectively expressed and recombined to form cellulose synthase in Escherichia coli. In the presence of isopropyl-β-D-thiogalactoside (IPTG), the cellulose synthase catalyzes the in situ synthesis of cellulose microfibrils.

[0010] The second aspect of the present invention discloses heterologous expression vectors of cellulose synthase subunits PtCesA1 and PtCesA8, including a heterologous expression vector of cellulose synthase subunit PtCesA1 or a heterologous expression vector of cellulose synthase subunit PtCesA8; the heterologous expression vector of cellulose synthase subunit PtCesA1 comprises the PtCesA1 gene at 174-3428 in SEQ ID NO:1, and the heterologous expression vector of cellulose synthase subunit PtCesA8 comprises the PtCesA8 gene at 171-3125 in SEQ ID NO:2.

[0011] In a preferred embodiment, the sequence of the heterologous expression vector of the cellulose synthase subunit PtCesA1 is shown as SEQ ID NO:1, and the sequence of the heterologous expression vector of the cellulose synthase subunit PtCesA8 is shown as SEQ ID NO:2.

[0012] The third aspect of the present invention is to provide a heterologous expression in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 or a method for constructing the aforementioned heterologous expression vector.

[0013] The fourth aspect of the present invention discloses a method for biosynthesis of cellulose microfibrils, which uses the aforementioned heterologous expression in vivo synthesis system of the cellulose synthase subunits PtCesA1 and PtCesA8 to synthesize cellulose microfibrils.

[0014] In a preferred embodiment, Escherichia coli cells containing heterologous expression vectors of cellulose synthase subunits PtCesA1 and PtCesA8 are cultured, and isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.3-0.5 mM / L isopropyl-β-D-thiogalactoside (IPTG) is used to induce the PtCesA1 gene and PtCesA8 gene to express PtCesA1 protein and PtCesA8 protein, and in the presence of isopropyl-β-D-thiogalactoside (IPTG), the synthesis of cellulose microfibrils is catalyzed.

[0015] The fifth aspect of the present invention is to provide cellulose microfibrils produced by the heterologous expression in vivo synthesis system of the aforementioned cellulose synthase subunits PtCesA1 and PtCesA8 or the aforementioned cellulose microfibril biosynthesis method.

[0016] The sixth aspect of the present invention discloses the use of the aforementioned heterologous expression in vivo synthesis system of the cellulose synthase subunits PtCesA1 and PtCesA8 or the aforementioned heterologous expression vector in the biosynthesis of cellulose microfibrils or in increasing the yield of cellulose microfibrils.

[0017] The invention is based on a double enzyme digestion method, and target gene fragments are selected on PtCesA1 and PtCesA8 respectively, primers are designed at both ends of the target gene and enzyme cutting sites Sma I and Hind III are introduced, a promoter and the target gene are connected to the corresponding enzyme cutting sites Sma I and Hind III on a stable copy plasmid pQE-80L containing six histidine (6×His) tags, a cellulose synthase subunit heterologous expression vector is constructed, XL1-blue Escherichia coli competent cells are used as engineering bacteria, cellulose biosynthesis is achieved, and a cellulose product with high purity similar to plants and free of lignin and hemicellulose is directly synthesized from the bacteria by heterologously expressing plant cellulose synthase subunits in Escherichia coli.

[0018] Compared with the in vitro synthesis method, the present invention adopts the in vivo synthesis method to obtain a structurally ordered and complete cellulose microfibril product, and effectively improves the quality of the cellulose microfibril product. The crystal structure of the PtCesA1 product is close to cellulose II, while the crystal structure of the PtCesA8 product is close to cellulose I. The cellulose synthesized from Escherichia coli in the present invention has a chemical structure similar to that of plant cellulose, and the cellulose-PtCesA1 cellulose product or PtCesA8 cellulose product synthesized from Escherichia coli is nano-scale cellulose.

[0019] In the prior art, it is difficult to obtain a structurally ordered and complete cellulose product through in vivo biosynthesis of a single cellulose synthase subunit. However, the present invention uses PtCesA1 or PtCesA8 cellulose synthase subunits to catalyze the synthesis of cellulose. The obtained product has an ordered and complete structure and a high expression level, which has excellent prospects in industrial applications.

[0020] The present invention realizes biosynthesis of cellulose in vivo, and directly synthesizes plant-like cellulose products with high purity and free of lignin and hemicellulose from the bacteria by heterologously expressing plant cellulose synthase subunits in Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the Western blot map of the recombinant protein expressed in E. coli;

[0022] Figure 2 is the FTIR spectrum of the product purified by SDS / NaOH method;

[0023] Figure 3 Electron microscopic observations of PtCesA1 (A) and PtCesA8 (B) products;

[0024] Figure 4 Electron microscopic observations of PtCesA1 (AC) and PtCesA8 (DF) products;

[0025] Figure 5 Glucose standard curves drawn using the results of Table 1 and Table 2, A: linear fit graph drawn from the data of Group 1; B: linear fit graph drawn from the data of Group 2, C: linear fit graph drawn from the data of Group 3;

[0026] Figure 6 This is a map of the heterologous expression vector for the cellulose synthase subunit PtCesA1;

[0027] Figure 7 This is a map of the heterologous expression vector for cellulose synthase subunit PtCesA8. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0029] Example 1: Obtaining the genes of the cellulose synthase subunits PtCesA1 and PtCesA8 of Populus trichocarpa

[0030] The gene sequences of the cellulose synthase subunits PtCesA1 and PtCesA8 of Populus trichocarpa were obtained from the NIST database, and the amino acid sequence of the target protein was obtained using the online tool for translating DNA sequences into amino acid sequences (https: / / www.novopro.cn / tools / translate.html). The molecular weight of the target protein was calculated using the online tool for calculating protein molecular weight (https: / / www.novopro.cn / tools / protein_mw.html), and the molecular weight of PtCesA1 was 120 kDa, and that of PtCesA8 was 110 kDa.

[0031] Example 2: Construction of heterologous expression vector of poplar cellulose synthase subunit

[0032] A mutant Escherichia coli strain with most of the cellulose synthase genes knocked out was used as a host (e.g., XL1-Blue). Choosing Escherichia coli as a host has the advantages of clear genetic background, low cost, high expression level, and relatively simple separation and purification of the expression product. Based on the double enzyme digestion method, target gene fragments were selected on PtCesA1 and PtCesA8, respectively. The target gene fragment of PtCesA1 contained restriction endonucleases PspX I-BstB I-Dra III-Nru I-Avr II-EcoN I-BmgB I-BsrGI-Csi I-BfuA I-BspM I-Bsu36 I, and the target gene fragment of PtCesA8 contained restriction endonucleases Bgl III-Pml I-Nsi I-Afl II-Csi I-BfuA I-BspM I. Primers were designed at both ends of the target gene, and the upstream primer of PtCesA1 was

[0033] (5'-taccccgggATGGAAGCGAATGCTGG-3'), downstream primer (5'-ccaagcttCTAGCAATTGACGCCACAT-3'); PtCesA8 upstream primer (5'-CCCGGGaATGATGGAATCTGGGGCTC-3'); downstream primer (5'-TGCATTTCTATAGATTGCTGAAAGCTT-3') and introduced restriction sites Sma I and Hind III, and connected the promoter and target gene to the corresponding restriction sites Sma I and Hind III on the stable copy plasmid pQE-80L containing six histidine (6×His) tags to construct a heterologous expression vector for cellulose synthase subunits. The sequence numbers of the above four primers are SEQ ID NO: 3-6 respectively. The structures of the two heterologous expression vectors are shown in Figure 6-7 shown.

[0034] The sequence of the pQE-80L vector into which the target gene of the cellulose synthase subunit PtCesA1 is inserted is shown in SEQ ID NO: 1, and the inserted target gene fragment is located at positions 174 to 3428. The sequence of the pQE-80L vector into which the target gene of the cellulose synthase subunit PtCesA8 is inserted is shown in SEQ ID NO: 2, and the inserted target gene fragment is located at positions 171 to 3125.

[0035] Example 3: Construction of a poplar cellulose synthase recombinant system

[0036] The cellulose synthase expression plasmid (i.e. Figure 6 or Figure 7 The heterologous expression vector, the sequence of which is shown in SEQ ID NO: 1-2, referred to as recombinant plasmid PtCesA1 or PtCesA8) was introduced into Escherichia coli to establish a plant cellulose synthase recombinant system. The PtCesA1 plant cellulose synthase recombinant system and the PtCesA8 plant cellulose synthase recombinant system were obtained respectively.

[0037] Example 4: Transgenic Escherichia coli culture conditions

[0038] The engineered bacteria XL1-blue containing the recombinant plasmid PtCesA1 or PtCesA8 were inoculated into 100 mL of 100 μg / mL penicillin. The cells were cultured overnight at 37°C in an LB resistance medium. The next day, the engineered bacteria XL1-blue containing the recombinant plasmid PtCesA1 or the engineered bacteria XL1-blue containing the recombinant plasmid PtCesA8 were inoculated at an inoculum size of 2% in a 2×YT medium containing 100 μg / mL penicillin, and cultured at 37°C and 200 rpm in an inoculum for 3-4 hours until the OD value of the culture medium reached the expected value (0.5-0.8). 50 mg / mL of isopropyl-β-D-thiogalactoside (IPTG, final concentration of 0.4 mM / L) was used to induce the expression of PtCesA1 and PtCesA8 genes, respectively, and the culture was continued at 28°C and 200 rpm for 6 hours (cellulose was synthesized in situ in Escherichia coli during this process) to obtain mixed solutions (respectively, a mixed solution obtained by in situ synthesis of cellulose catalyzed by PtCesA1 and a mixed solution obtained by in situ synthesis of cellulose catalyzed by PtCesA8).

[0039] Example 5: Purification of biosynthetic cellulose products

[0040] The cellulose product was purified by the SDS / NaOH method, and the specific method was as follows: the mixed solution obtained in Example 4 was centrifuged at 6000 rpm for 5 min, the supernatant was removed, and the precipitate was treated with 2% sodium dodecyl sulfate (SDS) in an autoclave at 120° C. for 1 h; the mixed solution was centrifuged at 7000 rpm for 10 min, and the precipitate was further treated with 2% NaOH at 120° C. for 1 h. Cellulose microfibrils were obtained after repeated washing by centrifugation with pure water. The product characterization is shown below.

[0041] Figure 1 is the Western blot map of the recombinant protein expressed in Escherichia coli (from left to right, respectively, the extracted proteins without PtCesA subunit before and after induction (P0, P2, where P0 is before induction and P2 is 2h after induction), the extracted proteins containing PtCesA1 before induction, 2h after induction, and 6h after induction (A1-0, A1-2 and A1-6), and the extracted proteins containing PtCesA8 before induction, 2h after induction, and 6h after induction (A8-0, A8-2 and A8-6)); Figure 1As shown, all samples showed a band at 30 kDa. The distribution of bands in A1-0 and A8-0 was consistent with that in P0, while new bands appeared in induced A1-2, A1-6, A8-2, and A8-6, indicating that PtCesA1 and PtCesA8 proteins were expressed in E. coli after IPTG induction. For A8-2 and A8-6, the bands were mainly prominent at 75 kDa, 45 kDa, and 20 kDa. Analysis of the structural model of PtCesA8 showed that the PtCesA8 protein consisted of a symmetrical trimer and a cytoplasmic stack aligned with the trimer symmetry axis, and the trimer was formed by the N-terminal domain (NTD) of PtCesA8. It can be inferred that the lysate added during protein extraction destroyed the association between the NTD and the trimer, and the NTD was eluted. Therefore, the Western blot results did not show the 110kDa band corresponding to the complete PtCesA8 protein, but showed the monomer, dimer and trimer bands of PtCesA8. This result is basically consistent with the SDS-page results after the PtCesA8 trimer was thermally denatured to remove the NTD, which also confirms the speculation. Similarly, A1-2 and A1-6 do not have the 122kDa band corresponding to the complete PtCesA1 protein, but the band at 70kDa is more obvious. It can be determined that PtCesA1 also contains NTD, and its molecular weight is slightly larger than PtCesA8. Therefore, it can be inferred that the PtCesA1 protein was also destroyed during the protein extraction process, resulting in inconsistent results with expectations. Based on the above analysis, PtCesA1 and PtCesA8 proteins were expressed in our system, but the expressed molecular weight did not match the complete molecular weight, and the trimer in PtCesA8 was partially depolymerized. It can be inferred that the N-terminal connection of the CesA protein in our system is relatively fragile, and conventional protein extraction processes will inevitably lead to the elution of NTD.

[0042] Figure 2 The FTIR spectrum of the product purified by SDS / NaOH method is shown in Figure 3. As can be seen from the figure, the two images are almost identical. -1 is the stretching vibration peak of -OH, 2921cm -1 807cm-1 is the stretching vibration peak of CH, and 807cm-1 is the C1-H vibration absorption peak of heterocarbon. Both have obvious cellulose characteristics, such as -CH2 at 1440cm-1. -1 The bending vibration peak of cellulose -CH at 1396cm -1 The bending vibration peak at 1154 cm -1 This proves that the chemical structure of these two heterologous expression products is close to that of cellulose. In addition, the infrared results show some impurity peaks, such as the representative group of carbonyl amide group at 1636cm-1 There are some proteins in the product, indicating that some impurities remain after purification. It is speculated that the impurities may mainly come from incomplete bacterial lipids, which is consistent with the TEM results. -1 The CH vibration peak at the A8-NaOH product was split, while this phenomenon was not obvious in the image of the A8-NaOH product. The infrared spectrum results showed that PtCesA1 and PtCesA8 could obtain products with chemical structures similar to natural cellulose in this system, and the functional groups of the two products were not much different. According to the results of the -OH vibration peak, the crystal structures of the two heterologous expression products were different. The crystal structure of the PtCesA1 product was closer to cellulose II, while the crystal structure of the PtCesA8 product was closer to cellulose I. This shows that the cellulose synthesized in Escherichia coli by this method has a chemical structure similar to that of plant cellulose.

[0043] Figure 3 These are electron microscopic observations of the products of PtCesA1 (A) and PtCesA8 (B). The arrows in the figure indicate the synthesized cellulose. It can be clearly seen that the cellulose is synthesized directly from Escherichia coli.

[0044] Figure 4 The electron microscope observation pictures of the products of PtCesA1 (AC) and PtCesA8 (DF) show that the products of PtCesA1 are mainly composed of closely arranged microfibrils with a diameter of about 5 nm ( Figure 4 B), while a small number of relatively intact microfibrils with a slightly larger diameter (15 nm) were also found to aggregate and arrange into bundles with a diameter of about 90 nm ( Figure 4 In addition to the microfibrils, some spherical structures with a diameter of 15nm-35nm were also found. It is speculated that this structure is an engineered bacterial liposome that was not completely removed during the purification process. Similar to the PtCesA1 product, the PtCesA8 product ( Figure 4 DF) also contains disordered microfibrils with a diameter of about 5 nm ( Figure 4 E) and relatively intact microfibrils with a diameter of about 15 nm. Previous studies have reported that the in vitro synthesis products of CesA8 protein are about 4-6 nm in diameter. Although the products are mainly fibrils, there are still some crystalline cellulose products ( Figure 4F in the figure). One possible reason is that the crystal structure of a few cellulose products changed under alkaline conditions during the purification process. The PtCesA8 product has more spherical structures and greater size differences, with diameters ranging from 40-220nm, which may be related to the expression of the two CesAs in E. coli. Both CesA proteins can be heterologously expressed in E. coli to directly synthesize cellulose microfibrils, and there are certain structural differences between the two products. Among them, the microfibrils of the PtCesA1 product are arranged relatively tightly, and there is a bundle structure formed by fiber aggregation. In addition to fibers, there are also some crystalline cellulose products in the PtCesA8 product.

[0045] The specific determination process of the "sulfuric acid-anthrone" method is as follows: 500 mL of the mixed solution obtained by catalyzing the in-situ synthesis of cellulose by PtCesA1 or the mixed solution obtained by catalyzing the in-situ synthesis of cellulose by PtCesA8 (PQE and CESEC refer to Example 4 to prepare the corresponding 500 mL mixed solution respectively), and the cellulose product is purified by the SDS / NaOH method obtained by the method of Example 5, and each is resuspended with an appropriate amount of deionized water and transferred to a glass tube. At the same time, an appropriate amount of the prepared glucose solution of five concentrations is added to five corresponding glass tubes, and acetic nitric acid (the volume ratio of nitric acid to acetic acid is 1:3) reagent is added to each tube at a ratio of 1:1 between the sample and acetic nitric acid, and a glass ball is placed on each glass tube to reduce evaporation and produce reflux, and the mixture is mixed evenly on a vortex oscillator. Then, the metal is heated at 120°C for 30 minutes. The mixture is transferred to a centrifuge tube, centrifuged at 8000 rpm for 10 minutes, the insoluble matter is collected, and it is resuspended with a small amount of deionized water and transferred to a glass tube. Add anthrone sulfate solution to all glass tubes at a ratio of 1:3 between sample and anthrone sulfate solution, mix carefully on a vortex shaker, treat in a metal bath (100°C, 15 min), and then let stand on ice for 10-15 min, keeping the ice surface level with the liquid surface in the glass tube.

[0046] Take 1 mL of the solution from each glass tube and transfer it to a cuvette, and read its absorbance at a wavelength of 625 nm on a UV-visible spectrophotometer. The 0 mg / mL glucose solution is used as a blank control group, and the absorbance of the remaining tubes is quickly measured.

[0047] Table 1 shows the absorbance data of PtCesA1 (abbreviated as A1) and PtCesA8 (abbreviated as A8) measured by the "sulfuric acid-anthrone" method. Cellulose is composed of glucose rings connected by β-1,4-glycosidic bonds. 4C1 chair conformation linked linear polymer compounds, therefore, by measuring the number of glucose molecules can be obtained the content of cellulose in the crude separation product. Deionized water was used to prepare 0mg / mL, 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL concentrations of glucose (Glucose, Glu) solution to construct a standard curve. To reduce experimental errors, three groups of experiments were performed.

[0048] Table 1 Absorbance data of PtCesA1 and PtCesA8 measured by the "sulfuric acid-anthrone" method

[0049]

[0050] Two controls were set up: PQE: an empty vector without the target gene, used as a blank control, and the expression process was the same as that of PtCesA1 and PtCesA8; CESEC: the cesA and B subunits of the CESEC gene were constructed together in a vector. CESEC is the bacterial cellulose synthase gene of Acetobacter xylinum. The cesA or cesB subunit alone cannot catalyze cellulose synthesis. Except for the inserted target gene, the vector used and the expression process were the same as those of PtCesA1 and PtCesA8. For the CESEC gene, please refer to the literature: Tomoya Imai et al., Functional Reconstitution of Cellulose Synthase in Escherichia coli, Biomacromolecules 2014 15(11), 4206-4213.

[0051] Table 2 Glucose standard solution concentration data obtained from three groups of experiments combined with linear fitting graphs

[0052]

[0053] The linear fit graph drawn from the data of "Group 1" ( Figure 5 A), it can be seen that when the absorbance of PQE is 0.122, the concentration of the glucose standard solution is 0.0365 mg / ml, when the absorbance of A1 is 0.508, the concentration of the glucose standard solution is 0.1131 mg / ml, when the absorbance of A8 is 0.700, the concentration of the glucose standard solution is 0.1511 mg / ml, and when the absorbance of CESEC is 0.701, the concentration of the glucose standard solution is 0.1513 mg / ml.

[0054] The linear fit graph drawn from the data of "Group 2" ( Figure 5B), it can be seen that when the absorbance of PQE is 0.103, the concentration of the glucose standard solution is 0.0364 mg / ml, when the absorbance of A1 is 0.498, the concentration of the glucose standard solution is 0.1075 mg / ml, when the absorbance of A8 is 0.684, the concentration of the glucose standard solution is 0.1409 mg / ml, and when the absorbance of CESEC is 0.661, the concentration of the glucose standard solution is 0.1368 mg / ml.

[0055] The linear fit graph drawn from the data of "Group 3" ( Figure 5 C), it can be seen that when the absorbance of PQE is 0.137, the concentration of the glucose standard solution is 0.0347 mg / ml, when the absorbance of A1 is 0.485, the concentration of the glucose standard solution is 0.0990 mg / ml, when the absorbance of A8 is 0.739, the concentration of the glucose standard solution is 0.1459 mg / ml, and when the absorbance of CESEC is 0.698, the concentration of the glucose standard solution is 0.1383 mg / ml.

[0056] Table 2: Glucose standard solution concentration data obtained from three sets of experiments combined with the linear fitting graph. The average value of the three sets of experimental results was taken as the final result. From Table 2, it can be seen that the final glucose standard solution concentration of PQE was 0.0359 mg / ml, the final glucose standard solution concentration of A1 was 0.1065 mg / ml, the final glucose standard solution concentration of A8 was 0.1340 mg / ml, and the final glucose standard solution concentration of CESEC was 0.1301 mg / ml.

[0057] Previously, the prior art had not successfully used PtCesA1 alone to catalyze the synthesis of cellulose. The present invention was the first to successfully use PtCesA1 alone to catalyze the synthesis of cellulose and achieve quantification; and the yield of cellulose catalyzed by PtCesA8 was higher than that of CESEC. This shows that both cellulose synthase subunits heterologously expressed cellulose products, and the products can be directly quantified by the "sulfuric acid-anthrone" method, with a yield higher than that of CESEC, showing the advantages of PtCesA1 and PtCesA8 synthase subunits in the preparation of cellulose; and the yield is higher than that of cellulose produced by inverted cell membranes constructed in vitro, while the amount of cellulose produced by inverted cell membranes constructed in vitro is so small that it cannot be quantified.

[0058] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A heterologous expression in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8, characterized in that: It includes a heterologous expression vector of a cellulose synthase subunit PtCesA1 or a heterologous expression vector of a cellulose synthase subunit PtCesA8; and a heterologous cell containing the vector; the heterologous expression vector of the cellulose synthase subunit PtCesA1 includes the PtCesA1 gene at 174-3428 in SEQ ID NO:1, and the heterologous expression vector of the cellulose synthase subunit PtCesA8 includes the PtCesA8 gene at 171-3125 in SEQ ID NO:2; the heterologous cell includes Escherichia coli, PtCesA1 and PtCesA8 are respectively expressed in Escherichia coli and recombined to form cellulose synthase, and in the presence of isopropyl-β-D-thiogalactoside (IPTG), the cellulose synthase catalyzes the in situ synthesis of cellulose microfibrils.

2. A method for constructing a heterologous expression in vivo synthesis system of the cellulose synthase subunits PtCesA1 and PtCesA8 as described in claim 1.

3. A method for biosynthesis of cellulose microfibrils, comprising synthesizing cellulose microfibrils by using the heterologous expression in vivo synthesis system of the cellulose synthase subunits PtCesA1 and PtCesA8 described in claim 1.

4. The method for biosynthesis of cellulose microfibrils according to claim 3, characterized in that: Escherichia coli cells containing heterologous expression vectors of cellulose synthase subunits PtCesA1 and PtCesA8 were cultured, and PtCesA1 gene and PtCesA8 gene were induced to express PtCesA1 protein and PtCesA8 protein using isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.3-0.5 mM / L. In the presence of isopropyl-β-D-thiogalactoside (IPTG), the synthesis of cellulose microfibrils was catalyzed.

5. Cellulose microfibrils produced by the heterologous expression in vivo synthesis system of the cellulose synthase subunits PtCesA1 and PtCesA8 according to claim 1 or the cellulose microfibril biosynthesis method according to claim 3 or 4.

6. Use of the heterologous expression in vivo synthesis system of the cellulose synthase subunits PtCesA1 and PtCesA8 according to claim 1 in the biosynthesis of cellulose microfibrils or in increasing the yield of cellulose microfibrils.

Images