Engineered strains of zymomonas mobilis, complex proteins, and methods

Abstract

This application relates to the technical field of *Fermentomonas motilityis*, specifically to engineered strains of *Fermentomonas motilityis*, complex proteins, and methods. The engineered strain is *Fermentomonas motilityis* containing genes encoding exoglucanase, endoglucanase, β-glucosidase, coliformin E protein, and a protein that interacts with the coliformin E protein. This engineered strain can efficiently express exoglucanase, endoglucanase, β-glucosidase, coliformin E protein, and the protein that interacts with the coliformin E protein, and facilitates the formation of complex enzyme assemblies, thereby increasing the enzyme activity and glucose yield in cellulose hydrolysis, and reducing the energy consumption of cellulose hydrolysis, thus possessing significant industrial application value.

Description

Technical Field

[0001] This application relates to the technical field of *Morphozoa motilityis*, specifically to engineered strains, complex proteins, and methods of *Morphozoa motilityis*. Background Technology

[0002] Zymomonas mobilis, a naturally occurring facultative anaerobic Gram-negative bacterium that produces ethanol, possesses a unique ED metabolic pathway and high sugar fermentation efficiency. It also exhibits ideal characteristics for industrial cell factories, including high ethanol yield, low biomass, strong ethanol tolerance, high osmotic pressure tolerance, and fermentation without the need for additional oxygen. Currently, the fermentation production of products such as PHB, 2,3-butanediol, isobutanol, and lactic acid has been achieved using Zymomonas mobilis. Furthermore, Zymomonas mobilis demonstrates high tolerance to lignocellulose hydrolysates, and the mechanisms underlying its tolerance to inhibitors in the hydrolysate are well-established. Therefore, Zymomonas mobilis has become a viable microbial cell factory for producing various biochemical substances from lignocellulose, such as lignocellulose ethanol, PHB, lactic acid, acetyl, and isobutanol. Summary of the Invention

[0003] *Fermentomonas motilityis* contains a gene encoding an endogenous endoglucanase. However, this endogenous enzyme has very low activity and cannot directly and completely hydrolyze cellulose. To improve the ability of *Fermentomonas motilityis* to hydrolyze cellulose, it is necessary to perform corresponding cellulase metabolic engineering modifications.

[0004] Based on this, the embodiments of this application disclose at least the following technical solutions:

[0005] In one aspect, an engineered bacterial strain is disclosed in the embodiments. The engineered strain is *Z. mobilis* ZM4ATCC31821, in which pZM32, pZM33, pZM36, and pZM39 plasmids are knocked out, the ZMO0038 region is replaced with the Cas12a gene, the ZMO0028 region is replaced with the T7 operon, and the ZMO1650 region is replaced with the AraC operon. The *Z. mobilis* ZM4ATCC... The genome of strain 31821 is shown in GenBank:CP023715.1. The ZMO0028 region is shown as 31083..32006nt in GenBank:CP023715.1, the ZMO0038 region is shown as 39623..40225nt in GenBank:CP023715.1, the ZMO1650 region is shown as 1697900..1699036nt in GenBank:CP023715.1, the pZM32 plasmid is shown in GenBank:CP023716.1, the pZM33 plasmid is shown in GenBank:CP023717.1, the pZM36 plasmid is shown in GenBank:CP023718.1, and the pZM39 plasmid is shown in GenBank:CP023719.1.

[0006] Secondly, an engineered bacterial strain is disclosed in the embodiments. The engineered strain is a motile fermentation monoclonal bacterium containing at least one of the following: a first gene encoding an exoglucanase, a second gene encoding an endoglucanase, a third gene encoding a β-glucosidase, a fourth gene encoding an escherichia coli E protein, and a fifth gene encoding a protein that interacts with the escherichia coli E protein. Specifically, the first gene is at least one of any of SEQ ID NO: 1 to 4, the second gene is at least one of any of SEQ ID NO: 5 to 8, the third gene is as shown in SEQ ID NO: 10, the fourth gene is at least one of any of SEQ ID NO: 11 to 14, and the fifth gene is at least one of any of SEQ ID NO: 15 to 18. The motile fermentation monoclonal bacterium is as described in the first aspect.

[0007] Thirdly, the embodiments disclose an engineered bacterial strain. The engineered strain is a motile fermentation monoclonal bacteria containing an expression sequence of a fourth gene as shown in SEQ ID NO:11 and a first gene as shown in SEQ ID NO:1, sequentially linked. The motile fermentation monoclonal bacteria are as described in the first aspect.

[0008] Fourthly, the embodiments disclose an engineered bacterial strain, which is a motile fermentation monoclonal bacteria containing an expression sequence of a fourth gene as shown in SEQ ID NO:12 and a second gene as shown in SEQ ID NO:6, sequentially linked. The motile fermentation monoclonal bacteria is as described in the first aspect.

[0009] Fifthly, the embodiments disclose an engineered bacterial strain, which is a motile fermentation monoclonal bacteria containing an expression sequence of a fourth gene as shown in SEQ ID NO:13 and a second gene as shown in SEQ ID NO:5, sequentially linked. The motile fermentation monoclonal bacteria is as shown in the first aspect.

[0010] Sixthly, the embodiments disclose an engineered bacterial strain, which is a motile fermentation monoclonal bacteria containing an expression sequence of a third gene as shown in SEQ ID NO:10 and a fourth gene as shown in SEQ ID NO:14, sequentially linked together. The motile fermentation monoclonal bacteria is as shown in the first aspect.

[0011] In a seventh aspect, an engineered bacterial strain is disclosed in the embodiments. This engineered strain is a *Morphozoa motifosum* containing the fifth gene as shown in SEQ ID NO:18. The *Morphozoa motifosum* is as described in the first aspect.

[0012] Eighthly, embodiments disclose a combination of the engineered strains from the second to the seventh aspects to one of the embodiments.

[0013] In a ninth aspect, the embodiments disclose a composite protein, which is a protein or an assembly thereof as shown in SEQ ID NO:50-54.

[0014] In a tenth aspect, an embodiment discloses a method for hydrolyzing cellulose. The method includes: obtaining the engineered strain described in the second to seventh aspects; and incubating the complex protein described in the eighth aspect, or the engineered strain, or the fermentation broth of the engineered strain, or an extract of the fermentation broth of the engineered strain, with the cellulose in a liquid system. Attached Figure Description

[0015] Figure 1 The image shows SDS-PAGE images of the target proteins GFP-EX1, GFP-EX2, GFP-EX3, and EX4 provided for the examples. In the image, lane M is the marker, lanes A1-A8 are all elution buffer containing GFP-EX2, lanes B1-B8 are all elution buffer containing GFP-EX3, lanes C1-C8 are all elution buffer containing GFP-EX1, and lanes D1-D9 are all elution buffer containing EX4.

[0016] Figure 2The SDS-PAGE images of the target proteins GFP-EN1, GFP-EN2, GFP-EN3, GFP-EN4, and GFP-EN5 provided for the examples are shown below. In the images, lane M is the marker, lanes A1-A8 are all elution buffer containing GFP-EN1; lanes B1-B8 are all elution buffer containing GFP-EN2; lanes C1-C8 are all elution buffer containing GFP-EN5; lanes D1-D15 are all elution buffer containing GFP-EN3; and lanes E1-E12 are all elution buffer containing GFP-EN4.

[0017] Figure 3 The graph shows the results of enzyme activity (A) and specific enzyme activity (B) of the target proteins GFP-EN1, GFP-EN2, GFP-EN3, GFP-EN4, and GFP-EN5 provided in the examples.

[0018] Figure 4 The SDS-PAGE images of the target proteins EN1, EN2, EN4, and EN5 provided for the examples are shown below. In the images, lane M is the marker, lanes A1-A9 are the elution buffer containing EN1, lanes B1-B15 are the elution buffer for EN2, lanes C1-C8 are the elution buffer for EN5, and lanes D1-D16 are the elution buffer for EN4.

[0019] Figure 5 The results of enzyme activity (A) and specific enzyme activity (B) of the target proteins EN1, EN2, EN4, and EN5 provided in the examples at a test temperature of 30°C are shown.

[0020] Figure 6 The results of enzyme activity (A) and specific enzyme activity (B) of the target proteins EN1, EN2, EN4, and EN5 provided in the examples at a test temperature of 50°C are shown.

[0021] Figure 7 The image shows SDS-PAGE images of the target proteins I1, I2, I3, I4, C1-GFP, C2-GFP, C3-GFP, and C4-GFP provided for the examples. In the image, lane M is the marker, and the other lanes are the elution buffers containing the target proteins C1-GFP, C2-GFP, C3-GFP, C4-GFP, I1, I2, I3, and I4, respectively.

[0022] Figure 8 Fluorescence interaction diagrams of the target proteins I1, I2, I3, I4, C1-GFP, C2-GFP, C3-GFP, and C4-GFP provided in the examples.

[0023] Figure 9The SDS-PAGE images of the fusion proteins C1-EX1, EX1-C1, C2-EN2, EN2-C2, C3-EN1, EN1-C3, C4-GB, and GB-C4 provided for the examples are shown. In the images, lane M is the marker, and the other lanes are the elution buffers for C1-EX1, EX1-C1, C2-EN2, EN2-C2, C3-EN1, EN1-C3, C4-GB, and GB-C4, respectively.

[0024] Figure 10 The enzyme activity test results of the fusion proteins EX1, EX1-C1 and C1-EX1 (A), EN2, EN2-C2 and C2-EN2 (B), EN1, EN1-C3 and C3-EN1 (C), GB, GB-C4 and C4-GB (D) provided in the examples are shown in the figure. "RT" represents room temperature.

[0025] Figure 11 The diagram shows the results of the hydrolysis of cellulose by the complex enzyme assembly and free enzyme provided in the example. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0027] The inventors of this application have creatively cloned a first gene encoding exoglucanase, a second gene encoding endoglucanase, a third gene encoding β-glucosidase, a fourth gene encoding coliformin E protein, and a fifth gene encoding a protein that interacts with said coliformin E protein into *Fermentomonas motilityis* to obtain a class of engineered strains. These engineered strains utilize *Fermentomonas motilityis* as chassis cells and possess a stronger hydrolytic ability towards lignocellulose.

[0028] engineered strains

[0029] The term "engineered strain" refers to a strain obtained by genetically modifying a wild-type strain, including modifying its genome or introducing overexpression plasmids into it. For example, strains obtained by knocking out, overexpressing, inserting or replacing a gene or region in the genome of a wild-type *Mammotrophic motilityis*, or introducing overexpression plasmids into it are all called "engineered strains".

[0030] The embodiments disclose engineered bacterial strains. These engineered strains are *Syntrophus motile* containing a first gene encoding an exoglucanase, a second gene encoding an endoglucanase, a third gene encoding a β-glucosidase, a fourth gene encoding an escherichia coli protein E, and a fifth gene encoding a protein that interacts with the escherichia coli protein E. The endoglucanase (EG, EC3.2.1.4) randomly hydrolyzes glycosidic bonds within cellulose chains to generate glucan chains of varying lengths. The exoglucanase (CBH, EC3.2.1.91) acts on the reducing end of the cellulose chain and releases cellobiose. The β-glucosidase (BGL, EC3.2.1.21) specifically acts on cellobiose to generate glucose. Through the synergistic action of the endoglucanase, exoglucanase, and β-glucosidase, cellulose can be hydrolyzed into glucose.

[0031] Furthermore, the engineered strain provided in this application can assemble the three cellulases and the coliform E protein, along with the coliform E protein affinity protein, into a complex enzyme assembly through high affinity between proteins, thereby promoting enzyme-enzyme proximity synergistic effect and enzyme-substrate-cell complex synergistic effect, thus endowing it with a stronger and more efficient cellulose hydrolysis ability compared to the three free cellulases.

[0032] In addition, to provide a host bacterium capable of expressing the first to fifth genes individually or in fusion, this application also provides a background strain. This background strain is a genetically engineered strain of *Z. mobilis* ZM4ATCC 31821 (hereinafter referred to as the ZM4 strain, a commercial strain). Specifically, it is an engineered strain obtained by knocking out four endogenous plasmids of the ZM4 strain: pZM32, pZM33, pZM36, and pZM39; inserting the Cas12a gene into the ZMO0038 region of its genome; replacing the ZMO0028 region with the T7 operon; and replacing the ZMO1650 region with the AraC operon.

[0033] To this end, an engineered strain is disclosed in this embodiment. This engineered strain is *Z. mobilis* ZM4ATCC 31821, in which pZM32, pZM33, pZM36, and pZM39 plasmids are knocked out, the ZMO0038 region is replaced with the Cas12a gene, the ZMO0028 region is replaced with the T7 operon, and the ZMO1650 region is replaced with the AraC operon. This engineered strain is used to express genes one through five individually or in fusion to construct a protein or protein complex capable of expressing hydrolyzed cellulose, which serves as the background strain.

[0034] The genome of *Z. mobilis* ZM4 ATCC 31821 is shown in GenBank:CP023715.1. The ZMO0028 region is shown as 31083.32006nt in GenBank:CP023715.1, the ZMO0038 region is shown as 39623.40225nt in GenBank:CP023715.1, and the ZMO1650 region is shown in GenBank:CP023715.1. As shown in SEQ ID NO: 55, the pZM32 plasmid is shown in GenBank:CP023716.1, the pZM33 plasmid is shown in GenBank:CP023717.1, the pZM36 plasmid is shown in GenBank:CP023718.1, the pZM39 plasmid is shown in GenBank:CP023719.1, and the Cas12a gene is shown in SEQ ID NO: 55.

[0035] In some embodiments, the T7 operator is as shown in SEQ ID NO:56.

[0036] In some embodiments, the AraC operon is shown as SEQ ID NO:57.

[0037] On one hand, the embodiments disclose an engineered bacterial strain. This engineered strain is a motile fermentation monoclonal organism containing a first gene encoding an exoglucanase, a second gene encoding an endoglucanase, a third gene encoding a β-glucosidase, a fourth gene encoding an escherichia coli E protein, and a fifth gene encoding a protein that interacts with the escherichia coli E protein. Specifically, the first gene is at least one of any of SEQ ID NO:1 to 4, the second gene is at least one of any of SEQ ID NO:5 to 8, the third gene is as shown in SEQ ID NO:10, the fourth gene is at least one of any of SEQ ID NO:11 to 14, and the fifth gene is at least one of any of SEQ ID NO:15 to 18.

[0038] In some embodiments, the first gene is located on the genome of the chromosome of *Mammotrophic fermentum* or is free outside the chromosome.

[0039] In some embodiments, the second gene is located on the genome of the chromosome of *Mammotrophic fermentum* or is free outside the chromosome.

[0040] In some embodiments, the third gene is located on the genome of the chromosome of *Mammotrophic fermentum* or is free outside the chromosome.

[0041] In some embodiments, the fourth gene is located on the genome of the chromosome of *Mammotrophic fermentum* or is free outside the chromosome.

[0042] In some embodiments, the fifth gene is located on the genome of the chromosome of *Mammotrophic fermentum* or is free outside the chromosome.

[0043] On one hand, the embodiments disclose an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NO:1 to 4, a second gene as shown in any one of SEQ ID NO:5 to 8, a third gene as shown in SEQ ID NO:10, a fourth gene as shown in SEQ ID NO:11, and a fifth gene as shown in SEQ ID NO:15.

[0044] On one hand, the embodiments disclose an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NO:1 to 4, a second gene as shown in any one of SEQ ID NO:5 to 8, a third gene as shown in SEQ ID NO:10, a fourth gene as shown in SEQ ID NO:11, and a fifth gene as shown in SEQ ID NO:15.

[0045] On one hand, the embodiments disclose an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NO:1 to 4, a second gene as shown in any one of SEQ ID NO:5 to 8, a third gene as shown in SEQ ID NO:10, a fourth gene as shown in SEQ ID NO:12, and a fifth gene as shown in SEQ ID NO:16.

[0046] On one hand, the embodiments disclose an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NO:1 to 4, a second gene as shown in any one of SEQ ID NO:5 to 8, a third gene as shown in SEQ ID NO:10, a fourth gene as shown in SEQ ID NO:13, and a fifth gene as shown in SEQ ID NO:17.

[0047] On one hand, the embodiments disclose an engineered strain. This engineered strain is a background strain containing an expression sequence of the fourth gene as shown in SEQ ID NO:11 and the first gene as shown in SEQ ID NO:1, sequentially linked together.

[0048] On one hand, the embodiments disclose an engineered strain, which is a background strain containing an expression sequence of a fourth gene as shown in SEQ ID NO:12 and a second gene as shown in SEQ ID NO:6.

[0049] On one hand, the embodiments disclose an engineered strain, which is a background strain containing an expression sequence of a fourth gene as shown in SEQ ID NO:13 and a second gene as shown in SEQ ID NO:5.

[0050] On one hand, the embodiments disclose an engineered strain, which is a background strain containing an expression sequence of a third gene as shown in SEQ ID NO:10 and a fourth gene as shown in SEQ ID NO:14, linked together.

[0051] On one hand, the embodiments disclose an engineered strain, which is a background strain containing the fifth gene as shown in SEQ ID NO:18.

[0052] Preparation of background strains

[0053] To obtain the engineered strain provided in this application, the examples provide a method for preparing a background strain.

[0054] In some embodiments, the preparation method includes:

[0055] The ZMO0038 region of strain ZM4 was replaced with the Cas12a gene to obtain strain ZM4-Cas12a; the pZM32 and pZM36 plasmids in strain ZM4-Cas12a were knocked out to obtain strain ZM4-Cas12aΔ32Δ36; the pZM33 plasmid in strain ZM4-Cas12aΔ32Δ36 was knocked out to obtain strain ZM4-Cas12aΔ32Δ33Δ36; the TA gene in the pZM39 plasmid of strain ZM4-Cas12aΔ32Δ33Δ36 was replaced with the chloramphenicol gene to obtain strain ZM4-Ca The ZM4-Cas12aΔ32Δ33Δ36ΔTA::Cm strain was obtained by knocking out the pZM39 plasmid containing the chloramphenicol gene in the ZM4-Cas12aΔ32Δ33Δ36ΔTA::Cm strain; the ZMNP-Cas12a strain was obtained by replacing the ZMO0028 region with the T7 operon; the ZM-Cas12a-T7 strain was obtained by replacing the ZMO01650 region with the AraC operon; the ZM-Cas12a-T7-AraC strain was obtained as the background strain.

[0056] For details on the preparation of the ZMNP-Cas12a strain, please refer to CN115806922A.

[0057] The preparation steps for the ZM-Cas12a-T7 strain include:

[0058] 1) The Ori sequence was amplified from pUC57 plasmid (ZK113, Beijing Zhuangmeng International Biotechnology Co., Ltd.) using Ori-F (SEQ ID NO.36) and Ori-R (SEQ ID NO.37).

[0059] 2) Synthesize the ZMO0028 replacement fragment (SEQ ID NO.38) formed by linking the upstream sequence of ZMO0028, the T7 operon, the spectinomycin gene and the downstream sequence of ZMO0028.

[0060] 3) The ZMO0028 replacement fragment and the Ori sequence were ligated using T5 exonuclease. The ligated fragment was then transformed into competent E. coli DH5α cells. Positive clones on the plates were verified by PCR, and the ZMO0028 replacement plasmid was extracted from the culture of the positive clones. The T5 reaction system consisted of 5 μL of 0.04 pM Ori sequence, 0.12 pM ZMO0028 replacement fragment, 0.5 μL of T5 ligase, 0.5 μL of 10×Buffer 4 (Thermo), and the remainder being double-distilled water.

[0061] 4) Transform the ZMO0028 replacement plasmid into the ZMNP-Cas12a strain, screen positive colonies from the transformants, and verify that they are ZM-Cas12a-T7 strain.

[0062] The steps for preparing the ZM-Cas12a-T7-AraC strain include:

[0063] 1) Synthesize a targeting element that targets the ZMO1650 region. The targeting element consists of two repeaters (shown in SEQ ID NO.39) and a guide sequence (shown in SEQ ID NO.40) connecting the two repeaters to target the ZMO1650 region.

[0064] 2) Linearized pL2R was obtained by digesting plasmid pL2R (MC_0101151, www.molecularcloud.org / ) with restriction endonuclease BsaⅠ. The annealing products of primer pairs 1650F (SEQ ID NO.41) and 1650R (SEQ ID NO.42) were ligated with linearized pL2R using T4 ligase. The ligation product was transformed into E. coli DH5α. Positive colonies were screened from the transformants and verified by colony PCR. The ZMO1650 targeting plasmid was extracted from the culture of positive colonies. The annealing system contained 1 μL of 1650F (10 μM), 1 μL of 1650R (10 μM), and 8 μL of double-distilled water per 10 μL. The mixture was denatured at 95 °C for 5 min and then cooled to room temperature for later use. The T4 reaction system, in 10 μL increments, contained 20-40 ng of linearized pL2R, 2 μL of a guide sequence targeting the ZMO1650 region, 0.5 μL of T4 ligase, 1 μL of buffer, and the remainder double-distilled water. The colony PCR reaction program was as follows: pre-denaturation at 98°C for 3 min, 1 cycle; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C (set at 10 s / kb according to fragment length), 30 cycles; extension at 72°C for 2 min, 1 cycle; and storage at 12°C for 2 min, 1 cycle.

[0065] 3) The ZMO1650 target plasmid was reverse PCR amplified using primers 15Afk-F (SEQ ID NO.43) and 15Afk-R (SEQ ID NO.44) to obtain the reverse-amplified ZMO1650 target plasmid.

[0066] 4) Synthesize the ZMO1650 replacement fragment (shown as SEQ ID NO.45) formed by linking the upstream sequence of ZMO1650, the AraC operator, and the downstream sequence of ZMO1650.

[0067] 5) The back-amplified ZMO1650 targeting plasmid and the ZMO1650 replacement fragment were assembled using Gibson assay at a ratio of 1:3. The assembly product was then transformed into E. coli DH5α. Positive colonies were screened from the transformants, and the ZMO1650 replacement plasmid was extracted and isolated from the validated positive colonies.

[0068] 6) The ZMO1650 replacement plasmid was transformed into the ZM-Cas12a-T7 strain to obtain the ZM-Cas12a-T7-AraC strain.

[0069] Preparation of expression plasmids

[0070] To prepare the engineered strain of this application, a series of expression plasmids were also prepared in the examples. These expression plasmids are pTZ28a plasmids containing at least one of a first gene, a second gene, a third gene, a fourth gene, and a fifth gene. The pTZ28a plasmid is the basal plasmid. The construction process of the pTZ28a plasmid is described in CN114774453A. The first gene is selected from EX1 to EX4, the second gene from EN1 to EN5, the third gene is GB, the fourth gene from C1 to C4, and the fifth gene from I1 to I4.

[0071] In some embodiments, the method for preparing the expression plasmid includes:

[0072] 1) Obtain the target sequence and basic plasmid

[0073] Synthesis of GFP-EX1 (SEQ ID NO:19), GFP-EX2 (SEQ ID NO:20), GFP-EX3 (SEQ ID NO:21), EX4 (SEQ ID NO:4), GFP-EN1 (SEQ ID NO:23), GFP-EN2 (SEQ ID NO:24), GFP-EN3 (SEQ ID NO:25), GFP-EN4 (SEQ ID NO:25) NO:26), GFP-EN5 (SEQ ID NO:27), EN1 (SEQ ID NO:5), EN2 (SEQ ID NO:6), EN4 (SEQ ID NO:8), EN5 (SEQ ID NO:9), I1 (SEQ ID NO:15), I2 (SEQ ID NO:16), I3 (SEQ ID NO:17), I4 (SEQ ID NO:18), C1-EX1 (SEQ ID NO:28), EX1-C1 (SEQ ID NO:29), C2-EN2 (SEQ ID NO:30), EN2-C2 (SEQ ID NO:31), C3-EN1 (SEQ ID DNA molecules containing the sequences NO:32), EN1-C3 (SEQ ID NO:33), GB-C4 (SEQ ID NO:34), C4-GB (SEQ ID NO:35), C1-GFP (SEQ ID NO:46), C2-GFP (SEQ ID NO:47), C3-GFP (SEQ ID NO:48), and C4-GFP (SEQ ID NO:49) were used as the target sequences.

[0074] 2) Preparation of expression plasmids

[0075] The linearized pTZ28a fragment was ligated with different target sequences using a T5 ligation reaction. The ligation products were transformed into E. coli, and positive colonies were screened from the transformants. The positive colonies were cultured, and expression plasmids were extracted from the cultures.

[0076] The expression plasmids obtained in the examples are: pTZ28a-GFP-EX1, pTZ28a-GFP-EX2, pTZ28a-GFP-EX3, pTZ28a-EX4, pTZ28a-GFP-EN1, pTZ28a-GFP-EN2, pTZ28a-GFP-EN3, pTZ28a-GFP-EN4, pTZ28a-GFP-EN5, pTZ28a-EN1, pTZ28a-EN2, pTZ28a-EN4, pTZ28a-EN5, pTZ28a-I1, pTZ28a-I2, pTZ28a-I3, p TZ28a-I4, pTZ28a-C1-GFP, pTZ28a-C2-GFP, pTZ28a-C3-GFP, pTZ28a-C4-GFP, pTZ28a-EX1, pTZ28a-EX1-C1, pTZ28a-C1-EX1, pTZ28 a-EN2, pTZ28a-EN2-C2, pTZ28a-C2-EN2, pTZ28a-EN1, pTZ28a-EN1-C3, pTZ28a-C3-EN1, pTZ28a-GB, pTZ28a-GB-C4, and pTZ28a-C4-GB.

[0077] The T5 ligation reaction system, in 5 μL volumes, contained 0.12 pM of the ligation sequence, 0.04 pM of the linearized pTZ28a fragment, 0.5 μL of 10×Buffer 4, 0.5 μL of T5 exonuclease, and the remainder double-distilled water. The T5 ligation reaction conditions included: reaction on ice for 5 min.

[0078] Preparation of engineered strains

[0079] The embodiments also provide a method for preparing engineered strains. The method includes obtaining a background strain; obtaining at least one expression plasmid containing at least one of a first gene, a second gene, a third gene, a fourth gene, and a fifth gene; transforming the at least one expression plasmid into the background strain; and screening the engineered strain from transformants.

[0080] In some embodiments, the method for preparing the engineered strain includes:

[0081] 1) Preparation of competent cells of background strain

[0082] The background strain was cultured in RMG5 medium (containing 50 g / L glucose, 10 g / L yeast extract and 2 g / L KH2PO4) at 30 °C and 100 rpm until the OD600 value reached 0.4–0.6. The resulting bacterial suspension was centrifuged at 4000 rpm and 25 °C for 8 min to collect the cells. The cells were gently resuspended in sterile water and centrifuged again at 4000 rpm and 25 °C for 8 min to collect the cells. The cells were then gently resuspended in 10% glycerol to obtain the competent cells of the background strain.

[0083] 2) Electroconversion of expression plasmids

[0084] Approximately 500 ng of expression plasmid was mixed with 50 μL of competent cells in a pre-cooled electroporation cuvette and electroporated at 1.8 kV, 25 μF, and 200 Ω. The solution in the electroporation cuvette was resuspended in 1 mL of RMG5 and incubated at 30 °C for 3–6 h. The culture was then spread onto antibiotic plates and incubated at 30 °C for 2–3 days. Colony PCR was performed to verify the culture, and the positive colonies selected were the engineered strains.

[0085] Screening the first gene

[0086] In some test cases, expression plasmids pTZ28a-GFP-EX1, pTZ28a-GFP-EX2, pTZ28a-GFP-EX3, and pTZ28a-EX4 were transformed into background strains, and positive clones were screened to obtain recombinant strains expressing GFP-EX1, GFP-EX2, GFP-EX3, and EX4, respectively. These recombinant strains were induced to ferment at 30℃ and 100 rpm in RMG5 liquid medium containing 3 mg / mL arabinose (Arab) and 0.8 μg / mL tetracycline (Tc), respectively. The target proteins were extracted from the fermentation broth, and their exoglucanase activity was detected.

[0087] The exoglycosaminoglycanase activity assay includes the following steps: 100 μL of phosphate-swellable cellulose solution as substrate, 50 μL of pH 4.5 sodium citrate buffer, and 50 μL of the target protein solution (EX1 is 1.43 g / L) are placed in a 2 mL centrifuge tube and mixed. The mixture is incubated at 30℃ or 50℃ for 30 min, then 150 μL of DNS (Solarbio D7800) is added to terminate the reaction. After cooling to room temperature, sterile water is added to a final volume of 2 mL, and the mixture is stirred. The reaction is then measured at OD... 540nm Measurement. Enzyme activity (U / mL) = 1000 × glucose production (mg) / target protein solution added (mL) × time (min). Specific activity (U / mg) is the number of enzyme activity units contained per milligram of protein.

[0088] SDS-PAGE analysis was performed on the target proteins expressed by the four recombinant plasmids, such as... Figure 1 As shown, the expression level of GFP-EX1 reached 1.24 g / L, the enzyme activity of GFP-EX1 was 27.16 U / mL, and the specific enzyme activity of GFP-EX1 was 21.89 U / mg. However, the target proteins GFP-EX2, GFP-EX3, and EX4 were almost not expressed. This may be because the recombinant plasmids of GFP-EX2, GFP-EX3, and EX4 grew slowly after being transformed into the background strain, affecting their correct protein folding and expression.

[0089] In addition, the expression plasmid pTZ28a-EX1 obtained in the above examples was transformed into a background strain, positive clones were screened, and fermentation was induced at 30℃ and 100 rpm in RMG5 liquid medium containing 3 mg / mL Arab and 0.8 μg / mL Tc, respectively. The target protein EX1 was extracted from the fermentation broth, and its enzyme activity was detected. The results showed that at enzyme activity test temperatures of 30℃ and 50℃, the enzyme activities of EX1 were 41.16 U / mL and 10.30 U / mL, respectively, and the specific enzyme activities were 28.78 U / mg and 13.05 U / mg, respectively. This indicates that the enzyme activity and specific enzyme activity of EX1 provided by the background strain in this application at 30℃ are higher than those at 50℃. Therefore, expressing EX1 using the background strain provided in this application is more conducive to low-temperature hydrolysis of cellulose.

[0090] Screening for second genes

[0091] In some test cases, expression plasmids pTZ28a-GFP-EN1, pTZ28a-GFP-EN2, pTZ28a-GFP-EN3, pTZ28a-GFP-EN4, and pTZ28a-GFP-EN5 were transformed into background strains to obtain recombinant strains expressing GFP-EN1, GFP-EN2, GFP-EN3, GFP-EN4, and GFP-EN5, respectively. Positive clones were screened and induced to ferment at 30℃ and 100 rpm in RMG5 liquid medium containing 3 mg / mL Arab and 0.8 μg / mL Tc, respectively. The target protein was extracted from the fermentation broth.

[0092] like Figure 2 The SDS-PAGE results shown indicate that the sizes of the fusion proteins are as expected. The expression levels of GFP-EN3 are 3.84 g / L, GFP-EN2 are 3.68 g / L, GFP-EN5 are 2.8 g / L, GFP-EN1 are 1.27 g / L, and GFP-EN4 are 1.12 g / L.

[0093] The enzyme activities and specific enzyme activities of proteins GFP-EN1, GFP-EN2, GFP-EN3, GFP-EN4, and GFP-EN5 in hydrolyzing cellulose were tested at 50℃. Cells were collected directly from the fermentation broth, and the target proteins were extracted following the steps described above. GFP-EN4 was used as a positive control (Control group). Figure 3 As shown in Figure A, the enzyme activities from highest to lowest are GFP-EN2, GFP-EN1, GFP-EN5, GFP-EN3, and GFP-EN4, with GFP-EN2 having the highest activity at 62.55 U / mL. Figure 3 As shown in B, the specific enzyme activities from highest to lowest are GFP-EN1, GFP-EN2, GFP-EN5, GFP-EN4, and GFP-EN3. Although GFP-EN1 has the lowest expression level, its specific enzyme activity is the highest among the five endoglucanases, at 39.43 U / mg, which is approximately 2.3 times that of GFP-EN2.

[0094] In some test cases, expression plasmids pTZ28a-EN1, pTZ28a-EN2, pTZ28a-EN4, and pTZ28a-EN5 were transformed into background strains to obtain recombinant strains expressing EN1, EN2, EN4, and EN5, respectively. Positive clones were screened and induced to ferment at 30°C and 100 rpm in RMG5 liquid medium containing 3 mg / mL Arab and 0.8 μg / mL Tc, respectively. The target protein was extracted from the fermentation broth.

[0095] like Figure 4 The SDS-PAGE results shown indicate that the molecular weight of protein EN1 is approximately 68 kDa, protein EN2 is approximately 43 kDa, protein EN4 is approximately 37 kDa, and protein EN5 is approximately 37 kDa, which is consistent with the expected size.

[0096] Cellulase activity and specific enzyme activity of EN1, EN2, EN5, and EN4 were tested at 50℃. Cells were collected directly from the fermentation broth, and the target protein was extracted following the steps described above. EN4 served as a positive control (Control group). Figure 5 As shown, at an enzyme activity test temperature of 50℃, the enzyme activity of EN1 was 56.03 U / mL, the enzyme activity of EN2 was 53.88 U / mL, the enzyme activity of EN5 was 53.71 U / mL, and the enzyme activity of EN4 was the lowest at 17.32 U / mL; the specific enzyme activity of EN1 was 40.36 U / mg, the specific enzyme activity of EN2 was 14 U / mg, the specific enzyme activity of EN5 was 19.09 U / mg, and the specific enzyme activity of EN4 was 22.20 U / mg.

[0097] Cellulase activity and specific enzyme activity of EN1, EN2, EN5, and EN4 were tested at 30℃. Cells were collected directly from the fermentation broth, and the target protein was extracted following the steps described above. EN4 served as a positive control (Control group). Figure 6 As shown, at an enzyme activity test temperature of 30℃, the enzyme activity of EN1 was 34.60 U / mL, the enzyme activity of EN2 was 31.05 U / mL, the enzyme activity of EN5 was 32.77 U / mL, and the enzyme activity of EN4 was 7.76 U / mL; the specific enzyme activity of EN1 was 24.93 U / mg, the specific enzyme activity of EN2 was 8.06 U / mg, the specific enzyme activity of EN5 was 11.70 U / mg, and the specific enzyme activity of EN4 was 9.96 U / mg.

[0098] This indicates that EN1 has the highest specific enzyme activity and EN2 has the highest expression level under cellulose hydrolysis conditions at both 50℃ and 30℃. Therefore, endoglucanases EN1 and EN2 were selected for constructing the complex enzyme assembly.

[0099] Screening for the fourth and fifth genes

[0100] Some test cases also tested the in vitro specific binding efficacy of C1-GFP, C2-GFP, C3-GFP, and C4-GFP to I1, I2, I3, and I4, respectively. The test procedure included:

[0101] Expression plasmids pTZ28a-I1, pTZ28a-I2, pTZ28a-I3, pTZ28a-I4, pTZ28a-C1-GFP, pTZ28a-C2-GFP, pTZ28a-C3-GFP, and pTZ28a-C4-GFP were transformed into background strains to obtain recombinant strains expressing I1, I2, I3, I4, C1-GFP, C2-GFP, C3-GFP, and C4-GFP, respectively. Positive clones were screened and induced to ferment at 30℃ and 100 rpm in RMG5 liquid medium containing 3 mg / mL Arab and 0.8 μg / mL Tc, respectively. The target proteins were extracted from the fermentation broth.

[0102] like Figure 7 As shown, the extracted target proteins were analyzed by SDS-PAGE electrophoresis, and all proteins were correctly expressed.

[0103] The concentrated solutions of purified target proteins I1, I2, I3, and I4 (approximately 2.5 g / L) were mixed with an equal volume of phosphate-expanded cellulose (PASC) solution and incubated at room temperature for 30 min. The precipitates were washed three times with TBS buffer to remove unbound proteins. The resulting precipitates (PASC conjugates of target proteins I1, I2, I3, and I4, respectively) were resuspended in TBS buffer, and two volumes of 5 g / L C1-GFP, C2-GFP, C3-GFP, and C4-GFP solutions were added respectively. The resulting precipitates were washed with 2 M NaCl solution and resuspended in 100 μL TBS buffer. Fluorescence was observed under a blue light spectrometer.

[0104] Figure 8 The results show that the purified I protein expressed in *Fermentomonas motilityis* can specifically bind to the C protein.

[0105] Application of engineered strains

[0106] Based on the engineered strain provided in this application, the examples also provide the application of the engineered strain in at least one of the preparation of complex enzyme assemblies, degradation of cellulose, or synthesis of glucose.

[0107] On one hand, the embodiments provide a method for biosynthesizing a complex enzyme assembly. This complex enzyme assembly is formed by assembling an endoglucanase, an exoglucanase, a β-glucosidase, an escherichia coli E protein, and a protein that interacts with the escherichia coli E protein. The method includes: inoculating an engineered strain obtained in the embodiments into a liquid containing a culture medium for fermentation; and harvesting at least one of the endoglucanase, the exoglucanase, the β-glucosidase, the escherichia coli E protein, the protein that interacts with the escherichia coli E protein, or the complex enzyme assembly from the fermentation broth.

[0108] In some embodiments, the method specifically includes the following post-fermentation steps: harvesting cellulosomal bodies composed of the endoglucanase, the exoglucanase, and the β-glucosidase from the fermentation broth, and a protein scaffold composed of the coliform E protein and a protein that interacts with the coliform E protein; and assembling the cellulosomal bodies and the protein scaffold into the complex enzyme assembly.

[0109] On one hand, an embodiment provides a method for hydrolyzing cellulose. The method includes obtaining a complex enzyme assembly prepared by the method; and contacting the cellulose with the complex enzyme assembly.

[0110] The step of inoculating the engineered strain into a liquid containing culture medium for fermentation includes: inoculating the bacterial culture of the engineered strain into RMG5 liquid culture medium containing 3 mg / mL Arab and 0.8 μg / mL Tc and incubating it at 30°C and 100 rpm for 24 h on a shaker.

[0111] The specific steps following fermentation include:

[0112] 1) After pre-cooling the collected fermentation broth on ice, centrifuge at 4000 rpm for 30 min at 4℃ to collect the cells. Wash the cells 1-2 times with buffer and resuspend them in buffer containing 1% PMSF (protease inhibitor). Sonicate to break down the bacteria. Centrifuge the resulting lysed solution at 4000 rpm for 30 min at 4℃, collect the supernatant, and add 1% PMSF to obtain a solution containing intracellular proteins.

[0113] 2) Load the solution containing intracellular protein onto a nickel column and allow it to stand to allow the target protein to fully bind. First, elute with low concentration (10mM, 30mM, 50mM) imidazole elution buffer to remove other proteins, and then elute with high concentration (200mM, 300mM, 500mM) imidazole elution buffer to collect the target protein.

[0114] 3) The collected target protein eluent was concentrated and purified to obtain a sample solution containing the target protein, and SDS-PAGE analysis was performed to determine that the size was consistent with the expected size.

[0115] Testing of fusion proteins

[0116] In some test cases, expression plasmids pTZ28a-C1-EX1, pTZ28a-EX1-C1, pTZ28a-C2-EN2, pTZ28a-EN2-C2, pTZ28a-C3-EN1, pTZ28a-EN1-C3, pTZ28a-C4-GB, and pTZ28a-GB-C4 were transformed into background strains. Positive clones were screened and fermented at 30°C and 100 rpm in RMG5 liquid medium containing 3 mg / mL Arab and 0.8 μg / mL Tc, respectively. The target proteins were extracted from the fermentation broth to obtain fusion proteins C1-EX1, EX1-C1, C2-EN2, EN2-C2, C3-EN1, EN1-C3, C4-GB, and GB-C4.

[0117] like Figure 9 The SDS-PAGE images shown indicate that the C1-EX1 and EX1-C1 proteins are approximately 84 kDa, the C2-EN2 and EN2-C2 proteins are approximately 60 kDa, the C3-EN1 and EN1-C3 proteins are approximately 84 kDa, and the C4-GB and GB-C4 proteins are approximately 73 kDa, all in line with expectations.

[0118] Enzyme activity assays were performed on the isolated and purified fusion proteins C1-EX1, EX1-C1, C2-EN2, EN2-C2, C3-EN1, EN1-C3, C4-GB, GB-C4, EX1, EN2, EN1, and GB at 30℃ and 50℃, respectively. The test results are as follows: Figure 10 At 30℃, the EX1 activity of fusion C1 increases, with C1-EX1 activity slightly higher than EX1-C1, while at 50℃, the EX1 activity of fusion C1 decreases. At 30℃, the EN2 activity of fusion C2 increases, with C2-EN2 activity higher than EN2-C2 activity. At 50℃, the EN2 activity of fusion C2 significantly increases, with C2-EN2 activity even higher than EN2-C2 activity. At 30℃, C3-EN1 activity is higher than that of single enzymes, but EN1-C3 activity is lower than that of single enzymes. At 50℃, the EN1 activity of fusion C3 significantly increases. At room temperature, the GB activity of fusion C4 is similar to that of the unfused protein, with GB-C4 activity slightly higher than C4-GB. Therefore, fusion proteins C1-EX1 (N-terminal fusion), C2-EN2 (N-terminal fusion), C3-EN1 (N-terminal fusion), and GB-C4 (C-terminal fusion) are more conducive to the assembly of multi-enzyme complexes.

[0119] Fusion protein assembly test

[0120] In some test cases, in vitro assembly tests were also performed on the fusion proteins provided in the examples. The test procedure included:

[0121] 1) Preparation of engineered strains

[0122] Expression plasmids pTZ28a-I4, pTZ28a-C1-EX1, pTZ28a-C2-EN2, pTZ28a-C3-EN1, and pTZ28a-GB-C4 were transformed into background strains, resulting in engineered strains capable of expressing I4, C1-EX1, C2-EN2, C3-EN1, and GB-C4, respectively.

[0123] 2) Cultivation

[0124] The bacterial suspension (OD600 greater than 0.6) of the engineered strain obtained in step 1) was inoculated into RMG5 liquid medium containing 3 mg / mL Arab and 0.8 μg / mL Tc and incubated at 30°C and 100 rpm for 24 h.

[0125] 3) Protein purification

[0126] The fermentation broth collected in step 2) was pre-cooled on ice and centrifuged at 4000 rpm for 30 min at 4°C to collect the cells. The cells were washed 1-2 times with buffer and resuspended in buffer containing 1% PMSF (protease inhibitor). The cells were then sonicated to break down the bacteria. The resulting lysed solution was centrifuged at 4000 rpm for 30 min at 4°C, and the supernatant was collected. A final concentration of 1% PMSF was added to obtain the solution containing intracellular proteins. The solution containing intracellular proteins was loaded onto a nickel column and allowed to stand to allow the target protein to bind fully. First, low-concentration (10 mM, 30 mM, 50 mM) imidazole elution buffer was used to elute off other proteins, followed by high-concentration (200 mM, 300 mM, 500 mM) imidazole elution buffer to collect the target protein. The collected target protein eluent was concentrated and purified to obtain a sample solution containing the target protein. SDS-PAGE analysis showed that the protein was consistent with the expected size, thus yielding C1-EX1 (SEQ ID NO:50), C2-EN2 (SEQ ID NO:51), C3-EN1 (SEQ ID NO:52), GB-C4 protein (SEQ ID NO:53), and I4 (SEQ ID NO:54).

[0127] 4) In vitro assembly and enzyme activity test

[0128] Incubate 500 μL of a solution containing 3.52 g / L I4 protein and 5.0 g / L PASC on ice for 5 min. Add 50 μL of pH 4.5 sodium citrate buffer containing C1-EX1, C2-EN2, C3-EN1, and GB-C4 proteins to a final concentration of 3.52 g / L. Incubate at 30 °C or 50 °C for 30 min, respectively. Terminate the reaction by adding 150 μL of DNS (Solarbio D7800). After cooling to room temperature, add sterile water to a final volume of 2 mL. Mix well and then incubate at OD... 540nm Measurement. Enzyme activity (U / mL) = 1000 × glucose production (mg) / target protein solution added (mL) × time (min). Specific enzyme activity (U / mg) is the number of enzyme activity units contained per milligram of protein.

[0129] Free control: Incubate 500 μL of 5.0 g / L PASC solution on ice for 5 min, then add 50 μL of pH 4.5 sodium citrate buffer containing C1-EX1, C2-EN2, C3-EN1 and GB-C4 proteins to a final concentration of 3.52 g / L.

[0130] The detection steps for glucose production include: taking 0 μL, 20 μL, 40 μL, 60 μL, 80 μL, 100 μL, 120 μL, and 140 μL of 1 mg / mL glucose standard solution into 1.5 mL EP tubes, adding distilled water to bring the volume to 600 μL, then adding 150 μL of DNS and mixing well. The mixture is then boiled in a water bath for 5 min, cooled to room temperature, and brought to a final volume of 2 mL with distilled water. The mixture is inverted and mixed thoroughly, and the OD is measured using a spectrophotometer. 540nm The value of .

[0131] like Figure 10 As shown, at an in vitro assembly temperature of 30°C, the complex enzyme assembly produced 0.102 mg of glucose, an increase of 41.67% compared to the free enzyme system. At an in vitro assembly temperature of 50°C, the complex enzyme assembly produced 0.127 mg of glucose, an increase of 30.93% compared to the free enzyme system.

[0132] This indicates that the engineered strain provided in this application embodiment can normally express the first to fifth genes, and the expressed protein can be assembled in vitro to obtain a complex enzyme assembly capable of hydrolyzing cellulose, which has a significant hydrolysis-promoting effect compared to the free enzyme.

[0133] This application provides engineered strains, complex enzymes, and their assemblies, capable of hydrolyzing cellulose at 30°C and 50°C, and also improving enzyme activity and glucose yield relative to free enzymes. This demonstrates that the engineered strains, complex enzymes, and their assemblies provided in this application can reduce the high-temperature reaction problem in cellulose hydrolysis, enabling efficient catalytic hydrolysis of cellulose at lower temperatures, significantly reducing energy consumption during the hydrolysis process. This has significant technological application value, especially in promoting the industrialization of cellulosic ethanol and providing important current significance for energy conservation and emission reduction.

[0134] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. An engineered strain, which is Z. mobilis containing an expression sequence of a fourth gene as shown in SEQ ID NO: 11 and a first gene as shown in SEQ ID NO: 1 sequentially connected; wherein the Z. mobilis is Z. mobilis ZM4 ATCC 31821 strain in which a pZM32 plasmid is knocked out, a pZM33 plasmid is knocked out, a pZM36 plasmid is knocked out and a pZM39 plasmid is knocked out, a ZMO0038 interval is replaced by a Cas12a gene, a ZMO0028 interval is replaced by a T7 operon and a ZMO1650 interval is replaced by an AraC operon; wherein the genome of the Z. mobilis ZM4 ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in GenBank: CP023715.1 31083..32006 nt, the ZMO0038 interval is shown in GenBank: CP023715.1 39623..40225 nt, the ZMO1650 interval is shown in GenBank: CP023715.1 1697900..1699036 nt, the pZM32 plasmid is shown in GenBank: CP023716.1, the pZM33 plasmid is shown in GenBank: CP023717.1, the pZM36 plasmid is shown in GenBank: CP023718.1, and the pZM39 plasmid is shown in GenBank: CP023719.

1.

2. A complex enzyme assembly formed by proteins as shown in SEQ ID NO: 50 and SEQ ID NO:

54.

3. A method for hydrolyzing cellulose, comprising: obtaining the engineered strain of claim 1; incubating the complex enzyme assembly of claim 2 or the engineered strain of claim 1 with the cellulose respectively in a liquid system.

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