Cellulose synthetase high-efficiency expression system based on cell membrane adaptation strategy, and preparation method and application of cellulose microfibrils

Abstract

The invention belongs to the technical field of synthetic biology and biological materials, and particularly relates to a cellulose synthetase high-efficiency expression system based on a cell membrane adaptation strategy, and a preparation method and application of cellulose microfibrils. The method is used for efficient heterologous expression of cellulose synthetase subunits PtCesA1 and PtCesA8 from populus trichocarpa, and in-vivo synthesis of cellulose microfibrils is realized. The key enzyme NMT of a phosphatidylcholine synthesis route is introduced to modify the escherichia coli cell membrane lipid composition, so that the expression efficiency and the cellulose synthesis capability of the eukaryotic-source membrane protein are remarkably improved. Further, the cellulose yield is remarkably increased by systematically optimizing culture conditions such as concentration, temperature and time of the inducer. The system constructed by the invention can be used for directly synthesizing cellulose microfibrils with an ordered structure and high purity in escherichia coli, and a new path is provided for green biological manufacturing of cellulose.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and biomaterials technology, specifically relating to a high-efficiency expression system for cellulose synthase based on a cell membrane adaptation strategy, a method for preparing cellulose microfibrils, and their applications. Background Technology

[0002] Cellulose is the most abundant renewable biopolymer in nature, and its green and controllable biosynthesis is crucial for the development of the sustainable materials industry. Currently, industrial cellulose mainly relies on plant extraction, a process that is energy-intensive, polluting, and produces large amounts of lignin and hemicellulose, increasing the difficulty and cost of subsequent purification.

[0003] Heterologous expression of cellulase in prokaryotic hosts (such as Escherichia coli) using synthetic biology techniques is considered a promising alternative. However, cellulase is a typical eukaryotic membrane protein, and in prokaryotic hosts where phosphatidylethanolamine (PE) is the main membrane lipid, it generally faces severe challenges such as low expression levels, misfolding, and difficulty in correctly integrating into the membrane structure and performing its function. This results in low cellulose synthesis yields and disordered product structures, far from meeting the threshold for industrial application.

[0004] While existing technologies (such as patent ZL 2024 1 0603738.0) have achieved basic expression of the CesA protein, they have failed to solve the fundamental problem of membrane environment incompatibility. Therefore, developing a technical system that can actively regulate the lipid composition of prokaryotic host membranes to adapt to the functional expression of eukaryotic membrane proteins is the key to breaking through the bottleneck in this field. Currently, there are no publicly reported successful applications of such systems in cellulose synthesis. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a high-efficiency expression system for cellulose synthase based on a cell membrane adaptation strategy, a method for preparing cellulose microfibrils, and their applications. This system uses genetic engineering to modify the membrane lipid composition of *E. coli*, creating a novel working environment adapted to the function of eukaryotic cellulose synthase, thereby significantly improving the enzyme's expression level, catalytic efficiency, and the final yield and quality of cellulose microfibrils.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, this invention provides a highly efficient expression system for cellulase synthase based on a cell membrane adaptation strategy, the core of which lies in the construction of a dual-plasmid co-expression system. This system introduces phosphatidylethanolamine N-methyltransferase (NMT) and utilizes its catalyzed three-step methylation biochemical reaction to directionally modify the lipid composition of the host cell membrane.

[0008] The core mechanism lies in the fact that NMT uses intracellular S-adenosylmethionine (SAM) as a methyl donor to perform three consecutive methylation modifications on the ethanolamine group of phosphatidylethanolamine (PE), the main phospholipid in the *E. coli* cell membrane. This process sequentially generates monomethylphosphatidylethanolamine (PMME), dimethylphosphatidylethanolamine (PDME), and finally phosphatidylcholine (PC). Through this PE methylation pathway, this invention systematically transforms the host cell membrane from a prokaryotic composition dominated by PE to a eukaryotic-like membrane composition rich in PC. This adaptively modified membrane environment provides the correct lipid chaperones and functional microenvironment for plant-derived PtCesA1 / PtCesA8 proteins, thereby significantly promoting their proper membrane integration, spatial folding, and ultimately catalytic activity.

[0009] The system includes:

[0010] The first expression vector containing the gene encoding the cellulose synthase subunits PtCesA1 or PtCesA8;

[0011] A second expression vector containing the gene encoding phosphatidylethanolamine N-methyltransferase (NMT); and

[0012] Prokaryotic host cells containing both the first expression vector and the second expression vector;

[0013] In this system, NMT expression can convert PE in the host cell membrane into PC, thereby achieving precise regulation of membrane lipid composition and creating a functional membrane environment that is more adapted to PtCesA1 / PtCesA8 proteins.

[0014] Furthermore, the heterologous expression vector for the cellulose synthase subunit PtCesA1 contains the PtCesA1 gene 174-3428 in SEQ ID NO.1, and the heterologous expression vector for the cellulose synthase subunit PtCesA8 contains the PtCesA8 gene 171-3125 in SEQ ID NO.2.

[0015] Furthermore, the coding gene for NMT is the gene sequence corresponding to GeneBank accession number AB019196.1, and the gene sequence is shown in SEQ ID NO.7.

[0016] Furthermore, the prokaryotic host cell is *Escherichia coli*;

[0017] The first expression vector is an IPTG-inducible vector, and the second expression vector is an arabinose-inducible vector, forming a dual-inducible synergistic expression system.

[0018] Furthermore, the culture conditions of the system are as follows: induction temperature 28-36℃, final IPTG concentration 0.4-0.8 mM, final arabinose concentration 7-13 mM, and induction time 4-8 h.

[0019] In a second aspect, the present invention provides a carrier combination for the system, comprising the first expression carrier and the second expression carrier.

[0020] A third aspect of the present invention provides a method for constructing the above-mentioned system, the key of which is to co-transform the first expression vector and the second expression vector into competent Escherichia coli cells to construct an engineered strain capable of simultaneously achieving membrane lipid regulation and cellulase expression.

[0021] A fourth aspect of the present invention provides a method for synthesizing cellulose microfibrils using the above-described system, comprising the following steps:

[0022] (1) Propagate the prokaryotic host cells under suitable culture conditions;

[0023] (2) The co-expression of the cellulase subunit and NMT was initiated by simultaneous or sequential induction with IPTG and arabinose;

[0024] (3) Cellulose microfibers are synthesized by the cellulose synthase subunit in a NMT-modified, PC-rich membrane environment.

[0025] Furthermore, the culture conditions were optimized through single-factor experiments and orthogonal experiments. The optimal conditions were: induction temperature 32℃, final IPTG concentration 0.8 mM, final arabinose concentration 13 mM, and induction time 4 h. Under these optimized conditions, the cellulose yield reached 0.168 ± 0.006 mg / mL, an increase of approximately 40% compared to the baseline conditions.

[0026] In a fifth aspect, the present invention provides cellulose microfibrils prepared by the above method, the product having a diameter of 5-15 nm, and the high crystallinity and chemical structure similar to plant cellulose confirmed by FTIR, TEM and other techniques.

[0027] In a sixth aspect, the present invention provides the application of the above-described system, carrier combination or method in the preparation of highly crystalline cellulose materials or in increasing the biosynthetic yield of cellulose microfibrils.

[0028] The present invention has the following advantages over the prior art:

[0029] This invention is based on the invention patent "A heterologous expression in vivo synthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 and its application" patent number: ZL 2024 1 0603738.0, authorization announcement number: CN 118406707 B. Target gene fragments were selected from PtCesA1 and PtCesA8, respectively. Primers were designed at both ends of the target gene, and restriction enzyme sites Sma I and Hind III were introduced. The promoter and target gene were ligated to the corresponding restriction enzyme sites Sma I and Hind III on the stable copy plasmid pQE-80L containing a six-histidine (6×His) tag, thus constructing a heterologous expression vector for cellulose synthase subunits. XL1-blue *E. coli* competent cells were used as the engineered bacteria. However, when using prokaryotes as hosts, given the importance of plant cell membranes for cellulose synthesis and the differences in lipid composition between plant and prokaryotic cell membranes (the main component of phospholipids in eukaryotic membranes is phosphatidylcholine (PC), while the main component of phospholipids in prokaryotic cell membranes is phosphatidylethanolamine (PE), constructing a cell membrane similar to that of plants is crucial in the artificial synthesis of cellulose. Two known PC biosynthetic pathways exist in bacteria: the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine uses S-adenosylmethionine (SAM) as a methyl donor and is methylated three times in a reaction catalyzed by one or more phospholipid N-methyltransferases (NMTs) to produce PC. In the PCS pathway, choline directly condenses with CDP-diglycerides in a PCS-catalyzed reaction to form PC. In this study, to improve the expression of the target gene and obtain more cellulose products after heterologous expression, the membrane of *E. coli* was modified to mimic the components of eukaryotic cell membranes. A methylation pathway was used, and NMT DNA was co-transferred into competent *E. coli* cells along with the DNA of the target genes PtCesA1 and PtCesA8 via heat shock. The NMT DNA was obtained from Kyoto University, Japan.

[0030] Compared to in vitro synthesis methods, this invention utilizes in vivo synthesis to obtain structurally ordered and complete cellulose microfibrils, effectively improving the quality of the cellulose microfibrils. The crystal structure of the PtCesA1 product is close to that of cellulose II, while the crystal structure of the PtCesA8 product is close to that of cellulose I. The cellulose synthesized from E. coli in this invention possesses a chemical structure similar to plant cellulose, and both the cellulose-PtCesA1 and PtCesA8 products synthesized from E. coli are nanoscale cellulose.

[0031] In existing technologies, it is difficult to obtain structurally ordered and complete cellulose products through in vivo biosynthesis using individual cellulose synthase subunits. However, this invention uses PtCesA1 or PtCesA8 cellulose synthase subunits to catalyze the synthesis of cellulose, resulting in products with ordered and complete structures and high expression levels, which have excellent prospects for industrial applications.

[0032] This invention enables in vivo biosynthesis of cellulose by heterologously expressing plant cellulose synthase subunits in Escherichia coli to directly synthesize a cellulose product with high purity, similar to that of plants, that is free of lignin and hemicellulose. Attached Figure Description

[0033] Figure 1 Western blot pattern of recombinant Escherichia coli cell membrane protein gene NMT (A) and internal control (B);

[0034] Figure 2 Western blot pattern of the CesA gene in recombinant Escherichia coli cell membrane protein (A) and internal control (B);

[0035] Figure 3 The Fourier transform infrared (FTIR) spectrum of the product purified by the SDS / NaOH method is shown below.

[0036] Figure 4 Transmission electron microscopy (TEM) images of PtCesA1 (AC) and PtCesA8 (DF) products, showing the nanoscale microfibril structure. pBAD (GI) is a control electron microscopy image of the product expressed in E. coli without the target gene.

[0037] Figure 5 A quantitative comparison of glucose content in cellulose synthesized by PtCesA1 and PtCesA8 engineered bacteria before and after cell membrane adaptation.

[0038] Figure 6 The graph shows the changes in lipid composition of the engineered bacterial membrane analyzed by LC-MS, illustrating the conversion of PE to PC.

[0039] Figure 7 This is a graph showing the effect of different final IPTG concentrations on the glucose content of the product in a single-factor experiment.

[0040] Figure 8 The figure shows the effect of different final arabinose concentrations on the glucose content of the product in a single-factor experiment.

[0041] Figure 9 This is a graph showing the effect of different induction temperatures on the glucose content of the product in a single-factor experiment.

[0042] Figure 10 This is a graph showing the effect of different induction times on the glucose content of the product in a single-factor experiment.

[0043] Figure 11 The graph shows the glucose content of each group in a four-factor, three-level orthogonal experiment based on a single-factor experimental design.

[0044] Figure 12 The glucose standard curve was obtained to verify the optimal combination of culture conditions. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or under conditions recommended by the manufacturer.

[0046] Example 1

[0047] Construction of engineered bacteria based on cell membrane adaptation strategy

[0048] Genes and vectors:

[0049] Cellulose synthase genes: PtCesA1 (SEQ ID NO.1) and PtCesA8 (SEQ ID NO.2) genes from Populus tomentosa were introduced into Sma I and Hind III restriction sites through specific primers (SEQ ID NO.3-6), and cloned into pQE-80L vector to construct IPTG-induced expression plasmids.

[0050] SEQ NO:3

[0051] PtCesA1 upstream primer (5'-taccccgggATGGAAGCGAATGCTGG-3'),

[0052] SEQ NO:4

[0053] Downstream primer (5'-ccaagcttCTAGCAATTGACGCCACAT-3');

[0054] SEQ NO:5

[0055] PtCesA8 upstream primer (5'-CCCGGGaATGATGGAATCTGGGGCTC-3');

[0056] SEQ NO:6

[0057] Downstream primer (5'-TGCATTTCTATAGATTGCTGAAAGCTT-3').

[0058] Key gene for membrane adaptation: The phosphatidylethanolamine N-methyltransferase (NMT) gene (GeneBank: AB019196.1) derived from Acetic Acidobacterium was cloned into the arabinose-induced pBAD33 vector to construct an arabinose-induced expression plasmid.

[0059] Construction of engineered bacteria:

[0060] The pQE-80L-PtCesA1 (or PtCesA8) plasmid and pBAD33-NMT plasmid were successfully constructed and co-transformed into Escherichia coli XL1-Blue competent cells by heat shock method. The cells were then plated on LB agar plates containing ampicillin and chloramphenicol for double resistance. Positive clones were screened, and finally, cellulose-synthesizing engineered bacteria with cell membrane adaptation ability were obtained.

[0061] Example 2

[0062] Validation of cell membrane adaptation effect

[0063] The engineered bacteria were cultured under suitable conditions, and samples were taken at 0 h and 6 h after induction. Total lipids were extracted using the Bligh & Dyer method and analyzed by liquid chromatography-mass spectrometry (LC-MS). The collected bacterial cultures before and after induction were centrifuged at 8000 g for 10 min, the supernatant was discarded, and 2 mL of chloroform, 4 mL of methanol, and 1.6 mL of water (chloroform:methanol:water = 1:2:0.8) were added to the precipitate. The mixture was vortexed, sonicated at room temperature for 30 min, and centrifuged at 4500 rpm for 10 min. The lower chloroform layer was collected, and the extraction was repeated once. The chloroform layers were combined, dried under nitrogen, and finally resuspended in 1 mL of solvent (chloroform:methanol = 2:1) for later use. 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC) were selected as standards. Each phospholipid standard was weighed, dissolved in chloroform:methanol = 2:1 (v / v) solution, and diluted to volume to prepare a single standard stock solution with a concentration of 10 μg / mL. The working standard curve solution was prepared by serial dilution with concentration gradients of 0.01, 0.05, 0.1, 0.5, and 1.0 μg / mL. Results (see...) Figure 6As shown in Table 1, the proportion of eukaryotic phospholipid PC (POPC + DOPC) in the total phospholipids (PE + PC) of the engineered bacterial cell membrane increased significantly after induction. For example, the PC content in the PtCesA1 engineered bacteria increased from 8.71% before induction to 21.67% after induction. This data directly confirms that the PE methylation pathway was successfully activated, and the cell membrane adaptation strategy of this invention has successfully regulated the membrane properties of the host cell from prokaryotic to a state more suitable for the functional expression of eukaryotic membrane proteins. This change in membrane composition, especially the significant increase in PC content, is considered to be a key structural basis for achieving efficient functional expression of PtCesA1 and PtCesA8 proteins and increased cellulose yield (see Example 4) by providing a more suitable membrane fluidity and surface charge environment. Protein expression verification results are shown in... Figure 1 and Figure 2 .

[0064] Table 1. Changes in membrane lipid composition before and after induction with cell membrane-adapted engineered bacteria (LC-MS analysis)

[0065] Example 3

[0066] Efficient Synthesis and Process Optimization of Cellulose Microfibrils

[0067] Basic synthesis verification: Cellulose microfilaments were synthesized by culturing engineered bacteria under unoptimized conditions (28℃, 0.4 mM IPTG, 13 mM arabinose, 6 h). Electron microscopy observation ( Figure 4 The FTIR spectrum clearly shows that cellulose is synthesized directly from the cell surface. Figure 3 This confirms that it has the characteristic functional group absorption peaks of cellulose.

[0068] Systematic process optimization:

[0069] Single-factor experiments: Using PtCesA8 engineered bacteria as a model, the effects of four key factors—IPTG concentration, arabinose concentration, induction temperature, and induction time—on yield were systematically investigated. Glucose content was quantified using the sulfuric acid-anthrone method (relevant data are shown in Tables 2 to 5, results are detailed below). Figures 7 to 10 The optimal levels for each factor were determined as follows: IPTG 0.6 mM, arabinose 10 mM, temperature 32℃, and time 4 h.

[0070] Table 2. Absorbance Measurement Results of Each Sample in the IPTG Group

[0071] Table 3. Absorbance Measurement Results of Each Sample in the Arabinose Group

[0072] Table 4. Absorbance Measurement Results of Each Sample in the Temperature Group

[0073] Table 5. Absorbance Measurement Results of Each Sample in the Time Group

[0074] Orthogonal Experiment: Based on single-factor results, a four-factor, three-level orthogonal experiment (L9(3)) was designed. 4 Range analysis showed that the order of influence of each factor was: induction temperature > induction time > IPTG concentration > arabinose concentration. The optimal combination was ultimately determined to be: A2B3C3D2, i.e., induction temperature 32℃, final IPTG concentration 0.8 mM, final arabinose concentration 13 mM, and induction time 4 h (see Tables 6 and 7; results are available in [reference needed]). Figure 11 ).

[0075] Table 6. Orthogonal Experiment Factor Level Design Table

[0076] Table 7 Results of Orthogonal Experiments

[0077] Optimization effect verification: Three repeated verification experiments were conducted under optimal conditions, and the glucose content in the product was measured to be 0.168 ± 0.006 mg / mL (verification experimental data are shown in Table 8, standard curve is shown in...). Figure 12 Compared to the yield before optimization (approximately 0.12 mg / mL), the yield increased by 40%, demonstrating the effectiveness and significant benefits of the optimization scheme of this invention.

[0078] Table 8. Absorbance Measurement Results of Samples in the Optimal Combination

[0079] Example 4

[0080] Product characterization and yield comparison analysis

[0081] The product synthesized under optimal conditions was characterized in detail and quantitatively analyzed (see comparison figures). Figure 5 Compare the production increase effect before and after the transformation.

[0082] Morphological analysis: Transmission electron microscopy (TEM) observation ( Figure 3 , Figure 4 The results showed that the products were nanoscale microfibrils with diameters of 5-15 nm, and that the PtCesA1 and PtCesA8 products differed in the aggregated structure of the microfibrils.

[0083] Yield comparison: Quantitative analysis by sulfuric acid-anthrone method ( Figure 4 It was confirmed that by simply introducing a cell membrane adaptation strategy (without full-condition optimization), the cellulose synthesis yield of PtCesA1 and PtCesA8 engineered bacteria increased by 9.57% and 13.76%, respectively, compared to the unmodified original system. Combined with full-process optimization, the total improvement rate can reach 40%.

[0084] The above embodiments fully illustrate the significant advantages and industrialization prospects of the expression system based on cell membrane adaptation strategy constructed in this invention in the efficient and high-quality synthesis of cellulose microfibrils.

[0085] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cellulose synthase high-efficiency expression system based on cell membrane adaptation strategy, characterized in that, The system comprises: a first expression vector containing a cellulose synthase subunit PtCesA1 or PtCesA8 encoding gene; a second expression vector containing a phosphatidylethanolamine N-methyltransferase NMT encoding gene; and a prokaryotic host cell containing the first expression vector and the second expression vector simultaneously; wherein the expression of the NMT in the host cell converts phosphatidylethanolamine PE in the cell membrane into phosphatidylcholine PC, thereby changing the membrane lipid composition of the host cell, achieving precise regulation of the membrane lipid composition, and creating a more suitable functional membrane environment for the PtCesA1 / PtCesA8 protein.

2. The system of claim 1, wherein, The cellulose synthase subunit PtCesA1 heterologous expression vector contains the PtCesA1 gene of 174-3428 in SEQ ID NO. 1, and the cellulose synthase subunit PtCesA8 heterologous expression vector contains the PtCesA8 gene of 171-3125 in SEQ ID NO.

2.

3. The system of claim 1 or 2, wherein, The NMT encoding gene is the gene sequence corresponding to GeneBank accession number AB019196.

1.

4. The system according to any of claims 1-3, characterized in that, The prokaryotic host cell is Escherichia coli. The first expression vector is an IPTG inducible vector, and the second expression vector is an arabinose inducible vector.

5. The system according to any of claims 1-4, characterized in that, The culture conditions of the system are as follows: induction temperature 28-36℃, IPTG final concentration 0.4-0.8 mM, arabinose final concentration 7-13 mM, and induction time 4-8 h.

6. A carrier combination for use in the system of any one of claims 1-5, wherein, The system comprises the first expression vector and the second expression vector.

7. A method of synthesizing cellulose microfibrils using the system of any one of claims 1-5, wherein, The method comprises the following steps: (1) Propagating the prokaryotic host cell under suitable culture conditions; (2) Simultaneously or sequentially inducing using IPTG and arabinose to start co-expression of the cellulose synthase subunit and NMT; (3) Catalyzing synthesis of cellulose microfibrils by the cellulose synthase subunit in the NMT modified PC rich membrane environment.

8. The method of claim 7, wherein, The specific induction conditions in step (2) are as follows: adding IPTG with a final concentration of 0.8 mM and arabinose with a final concentration of 13 mM at 32℃, and inducing culture for 4 h.

9. Cellulose microfibrils produced by the method of claim 7 or 8, characterized in that, The diameter is 5-15 nm, and it is confirmed by FTIR, TEM and other techniques that it has high crystallinity and chemical structure similar to plant cellulose.

10. Use of the system of any one of claims 1-5, the combination of vectors of claim 6, or the method of any one of claims 7-8 in the preparation of high crystallinity cellulose material or in the improvement of the biosynthesis yield of cellulose microfibrils.

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