Engineering strain of zymomonas mobilis, compound protein and method

Abstract

The invention relates to the technical field of zymomonas mobilis, in particular to an engineering strain of zymomonas mobilis, compound protein and a method. The engineering strain is zymomonas mobilis containing genes for coding exo-glucanase, coding endoglucanase, coding beta-glucosidase, coding colicin E protein and coding protein with affinity interaction with the colicin E protein. The engineering strain can efficiently express exoglucanase, endoglucanase, beta-glucosidase, Escherichia coli E protein and protein with affinity interaction with the Escherichia coli E protein, is beneficial to forming a compound enzyme assembly, improves the enzyme activity of cellulose hydrolysis and the yield of glucose, reduces the energy consumption of cellulose hydrolysis, and improves the yield of glucose. The method has very important industrial application value.

Description

Technical Field

[0001] The present application relates to the technical field of Zymomonas mobilis, and in particular to an engineered strain, a composite protein and a method of Zymomonas mobilis. Background Art

[0002] Zymomonas mobilis is a natural ethanol-producing facultative anaerobic Gram-negative bacterium with a unique ED metabolic pathway and high sugar fermentation efficiency. It also has the characteristics of an ideal industrial cell factory, such as high ethanol production, low product volume, strong ethanol tolerance, high osmotic pressure resistance, and no need for additional oxygen during the fermentation process. At present, the production and fermentation of products such as PHB, 2,3-butanediol, isobutanol and lactic acid have been achieved in Zymomonas mobilis. In addition, Zymomonas mobilis has a high tolerance to lignocellulosic hydrolysate, and the research on the mechanism of tolerance to inhibitors in the hydrolysate is relatively mature. Therefore, Zymomonas mobilis has become a microbial cell factory for the production of a variety of biochemical substances from lignocellulose, such as lignocellulosic ethanol, PHB, lactic acid, acetyl, isobutanol, etc. Summary of the invention

[0003] Zymomonas mobilis contains genes encoding endogenous endoglucanase. However, the activity of this endogenous enzyme is very low and it cannot directly and completely hydrolyze cellulose. In order to improve the ability of Zymomonas mobilis to hydrolyze cellulose, it is necessary to perform corresponding cellulase metabolic engineering on it.

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

[0005] In the first aspect, the embodiment discloses an engineered strain. The engineered strain is a Z.mobilis ZM4ATCC31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon. The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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] In the second aspect, an embodiment discloses an engineered strain. The engineered strain is a mobilizing Zymomonas containing at least one of a first gene encoding an exoglucanase, a second gene encoding an endoglucanase, a third gene encoding a β-glucosidase, a fourth gene encoding a colicin E protein, and a fifth gene encoding a protein having affinity with the colicin E protein. Wherein, the first gene is at least one of any one of SEQ ID NOs: 1 to 4, the second gene is at least one of SEQ ID NOs: 5 to 8, the third gene is as shown in SEQ ID NO: 10, the fourth gene is at least one of SEQ ID NOs: 11 to 14, and the fifth gene is at least one of SEQ ID NOs: 15 to 18. Wherein, the mobilizing Zymomonas is as shown in the first aspect.

[0007] In a third aspect, an embodiment discloses an engineered strain. The engineered strain is Zymomonas 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 connected in sequence. Wherein, the Zymomonas mobilis is as shown in the first aspect.

[0008] In a fourth aspect, an embodiment discloses an engineered strain, wherein the engineered strain is Zymomonas mobilis 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 connected in sequence. The Zymomonas mobilis is as shown in the first aspect.

[0009] In a fifth aspect, an embodiment discloses an engineered strain, wherein the engineered strain is Zymomonas mobilis 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 connected in sequence. The Zymomonas mobilis is as shown in the first aspect.

[0010] In a sixth aspect, an embodiment discloses an engineered strain, wherein the engineered strain is Zymomonas mobilis 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 connected in sequence. The Zymomonas mobilis is as shown in the first aspect.

[0011] In a seventh aspect, an embodiment discloses an engineered strain, wherein the engineered strain is Zymomonas mobilis containing the fifth gene as shown in SEQ ID NO: 18. The Zymomonas mobilis is as shown in the first aspect.

[0012] In the eighth aspect, the embodiment discloses a combination of the engineered strains from the second to seventh aspects to one.

[0013] In a ninth aspect, the embodiments disclose a complex protein, which is a protein as shown in SEQ ID NOs: 50 to 54 or an assembly thereof.

[0014] In the tenth aspect, the embodiment discloses a method for hydrolyzing cellulose. The method comprises: obtaining the engineered strain of any one of the second to seventh aspects; and incubating the composite protein, the engineered strain, the fermentation broth of the engineered strain, or the fermentation broth extract of the engineered strain, respectively, with the cellulose in a liquid system. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The SDS-PAGE diagram of the target proteins GFP-EX1, GFP-EX2, GFP-EX3 and EX4 provided in the examples. In the figure, lane M is a marker, lanes A1-A8 are all eluates containing GFP-EX2, lanes B1-B8 are all eluates containing GFP-EX3; lanes C1-C8 are all eluates containing GFP-EX1; lanes D1-D9 are all eluates containing EX4.

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

[0017] Figure 3 These are diagrams showing the results of measuring the enzyme activity (A) and relative 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 diagram of the target proteins EN1, EN2, EN4, and EN5 provided in the examples. In the diagram, lane M is a marker, lanes A1-A9 are eluates containing EN1; lanes B1-B15 are eluates containing EN2; lanes C1-C8 are eluates containing EN5, and lanes D1-D16 are eluates containing EN4.

[0019] Figure 5 The results of the enzyme activity (A) and the relative 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 the enzyme activity (A) and the relative 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 SDS-PAGE diagram of the target proteins I1, I2, I3, I4, C1-GFP, C2-GFP, C3-GFP and C4-GFP provided in the examples. In the figure, lane M is a marker, and the other lanes are eluates containing 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] Fig. 9The SDS-PAGE diagram of the fusion proteins C1-EX1, EX1-C1, C2-EN2, EN2-C2, C3-EN1, EN1-C3, C4-GB, and GB-C4 provided in the examples, in which lane M is a marker, and the other lanes are the eluates of C1-EX1, EX1-C1, C2-EN2, EN2-C2, C3-EN1, EN1-C3, C4-GB, and GB-C4, respectively.

[0024] Fig.10 The enzymatic 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, where "RT" stands for room temperature.

[0025] Fig.11 This is a diagram showing the results of cellulose hydrolysis by the complex enzyme assembly and free enzyme provided in the examples. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0027] The inventors of the present application creatively cloned a first gene encoding exoglucanase, a second gene encoding endoglucanase, a third gene encoding β-glucosidase, a fourth gene encoding colicin E protein, and a fifth gene encoding a protein having affinity with the colicin E protein into Zymomonas mobilis to obtain a class of engineered strains. These engineered strains use Zymomonas mobilis as chassis cells and have a stronger ability to hydrolyze lignocellulose.

[0028] Engineered strains

[0029] The term "engineered strain" refers to a strain obtained by genetic engineering of a wild-type strain, including modification of its genome, or the introduction of an overexpression plasmid into its body. For example, a strain obtained by knocking out, overexpressing, inserting or replacing a gene or region in a genome of a wild-type Zymomonas mobilis with an exogenous gene, or by the introduction of an overexpression plasmid into its body is called an "engineered strain".

[0030] The embodiment discloses an engineered strain. The engineered strain is a zymomonas mobilis containing a first gene encoding an exoglucanase, a second gene encoding an endoglucanase, a third gene encoding a beta-glucosidase, a fourth gene encoding a colicin E protein, and a fifth gene encoding a protein that interacts with the colicin E protein. Among them, endoglucanase (Endoglucanase, EG, EC3.2.1.4) can randomly hydrolyze the glycosidic bonds in the cellulose chain to generate glucan chains of different lengths. Exoglucanase (Exoglucanase, CBH, EC3.2.1.91) acts on the reducing end of the cellulose chain and releases cellobiose. Beta-glucosidase (Glucosidase, BGL, EC3.2.1.21) acts specifically on cellobiose to generate glucose. Under the synergistic action of endoglucanase, exoglucanase and beta-glucosidase, cellulose can be hydrolyzed into glucose.

[0031] In addition, the engineered strain provided in the present application can also assemble the three cellulases, the colicin E protein, and the scaffold formed by the colicin E protein affinity protein into a composite enzyme assembly through the high affinity interaction between proteins, thereby promoting the enzyme-enzyme proximity synergistic effect and the enzyme-substrate-cell composite synergistic effect, thereby giving it a more powerful and efficient cellulose hydrolysis ability than the three free cellulases.

[0032] In addition, to provide a host bacterium capable of expressing the first to fifth genes alone or in fusion, the present application embodiment also provides a background strain. The background strain is a strain obtained by genetically engineering the Z.mobilis ZM4ATCC 31821 strain (hereinafter referred to as the ZM4 strain, which is a commercial strain). Specifically, the four endogenous plasmids pZM32 plasmid, pZM33 plasmid, pZM36 plasmid and pZM39 plasmid of the ZM4 strain are knocked out, and the Cas12a gene is inserted into the ZMO0038 interval of its genome, and the ZMO0028 interval is replaced with the T7 operon and the ZMO1650 interval is replaced with the AraC operon to obtain an engineered strain.

[0033] To this end, the embodiment discloses an engineered strain. The engineered strain is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, and the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon. The engineered strain is applied to express the first to fifth genes alone or in fusion to construct a protein or protein complex capable of expressing cellulose hydrolysis, which is the background strain.

[0034] The genome of the Z.mobilis ZM4 ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in GenBank: 31083..32006nt of CP023715.1, the ZMO0038 interval is shown in GenBank: 39623..40225nt of CP023715.1, and the ZMO1650 interval is shown 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, 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 operon is shown as SEQ ID NO:56.

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

[0037] On the one hand, the embodiment discloses an engineered strain. The engineered strain is a mobilizing Zymomonas containing a first gene encoding an exoglucanase, a second gene encoding an endoglucanase, a third gene encoding a β-glucosidase, a fourth gene encoding a colicin E protein, and a fifth gene encoding a protein that interacts with the colicin E protein. The first gene is at least one of any one of SEQ ID NOs: 1 to 4, the second gene is at least one of SEQ ID NOs: 5 to 8, the third gene is at least one of SEQ ID NOs: 10, the fourth gene is at least one of SEQ ID NOs: 11 to 14, and the fifth gene is at least one of SEQ ID NOs: 15 to 18.

[0038] In some embodiments, the first gene is located on the genome of the Zymomonas mobilis chromosome or is extrachromosomal.

[0039] In some embodiments, the second gene is located on the genome of the Zymomonas mobilis chromosome or is extrachromosomal.

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

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

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

[0043] In one aspect, the embodiment discloses an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NOs: 1 to 4, a second gene as shown in any one of SEQ ID NOs: 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] In one aspect, the embodiment discloses an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NOs: 1 to 4, a second gene as shown in any one of SEQ ID NOs: 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] In one aspect, the embodiment discloses an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NOs: 1 to 4, a second gene as shown in any one of SEQ ID NOs: 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] In one aspect, the embodiment discloses an engineered strain. The engineered strain is a background strain containing a first gene as shown in any one of SEQ ID NOs: 1 to 4, a second gene as shown in any one of SEQ ID NOs: 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] In one aspect, the embodiment discloses an engineered strain, which 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 connected in sequence.

[0048] In one aspect, the embodiment discloses an engineered strain, which is a background strain containing an expression sequence in which a fourth gene as shown in SEQ ID NO: 12 and a second gene as shown in SEQ ID NO: 6 are sequentially connected.

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

[0050] On the one hand, the embodiment discloses an engineered strain, which is a background strain containing an expression sequence in which a third gene as shown in SEQ ID NO: 10 and a fourth gene as shown in SEQ ID NO: 14 are sequentially connected.

[0051] In one aspect, the embodiment discloses an engineered strain, which is a background strain containing a fifth gene as shown in SEQ ID NO:18.

[0052] Preparation of background strains

[0053] In order to obtain the engineered strain provided in this application, the embodiment provides a method for preparing a background strain.

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

[0055] The ZMO0038 region of the ZM4 strain was replaced with the Cas12a gene to obtain the ZM4-Cas12a strain; the pZM32 plasmid and the pZM36 plasmid in the ZM4-Cas12a strain were knocked out to obtain the ZM4-Cas12aΔ32Δ36 strain; the pZM33 plasmid of the ZM4-Cas12aΔ32Δ36 strain was knocked out to obtain the ZM4-Cas12aΔ32Δ33Δ36 strain; the TA gene of the pZM39 plasmid of the ZM4-Cas12aΔ32Δ33Δ36 strain was replaced with the chloramphenicol gene to obtain the ZM4-Cas12aΔ32Δ33Δ36 strain. s12aΔ32Δ33Δ36ΔTA::Cm strain; the pZM39 plasmid containing the chloramphenicol gene of the ZM4-Cas12aΔ32Δ33Δ36ΔTA::Cm strain was knocked out to obtain the ZMNP-Cas12a strain; the ZMO0028 region of the ZMNP-Cas12a strain was replaced with the T7 operon to obtain the ZM-Cas12a-T7 strain; the ZMO01650 region of the ZM-Cas12a-T7 strain was replaced with the AraC operon to obtain the ZM-Cas12a-T7-AraC strain as the background strain.

[0056] Among them, the preparation of ZMNP-Cas12a strain can be specifically referred to CN115806922A.

[0057] Among them, the preparation steps of ZM-Cas12a-T7 strain include:

[0058] 1) Ori-F (shown in SEQ ID NO.36) and Ori-R (shown in SEQ ID NO.37) were amplified from pUC57 plasmid (ZK113, Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.) to obtain the Ori sequence.

[0059] 2) Synthesizing a ZMO0028 replacement fragment (shown in SEQ ID NO. 38) formed by connecting the ZMO0028 upstream sequence, T7 operator, spectinomycin gene and ZMO0028 downstream sequence.

[0060] 3) The ZMO0028 replacement fragment and the Ori sequence were ligated using T5 exonuclease, and the ligated fragments were transformed into competent E. coli DH5α cells, and the positive clones on the plate were verified by PCR, and the ZMO0028 replacement plasmid was extracted from the culture of the positive clones. Among them, the T5 reaction system contained 0.04pM Ori sequence, 0.12pM ZMO0028 replacement fragment, 0.5UT5 ligase, 0.5μL 10×Buffer 4 (Thermo) and the balance of double distilled water in 5μL.

[0061] 4) The ZMO0028 replacement plasmid was transferred into the ZMNP-Cas12a strain, and positive colonies were screened from the transformants, which were verified to be the ZM-Cas12a-T7 strain.

[0062] Wherein, the step of preparing the ZM-Cas12a-T7-AraC strain includes:

[0063] 1) Synthesizing a targeting element targeting the ZMO1650 region, wherein the targeting element is composed of two repeating elements (shown in SEQ ID NO.39) and a guide sequence targeting the ZMO1650 region (shown in SEQ ID NO.40) connected between the two recombinants.

[0064] 2) The plasmid pL2R (MC_0101151, www.molecularcloud.org / ) was digested with restriction endonuclease BsaⅠ to obtain linearized pL2R; the annealing product of the primer pair 1650F (shown in SEQ ID NO.41) and 1650R (shown in SEQID NO.42) was connected to the linearized pL2R using T4 ligase; the ligation product was transformed into Escherichia coli DH5α; positive colonies were screened from the transformants, verified by colony PCR, and the ZMO1650 targeting plasmid was extracted from the culture of the positive colonies. The annealing system contained 1 μL 1650F (10 μM), 1 μL 1650R (10 μM) and 8 μL double distilled water in 10 μL, denatured at 95°C for 5 min, and cooled to room temperature for use. Among them, the T4 reaction system contains 20-40ng linearized pL2R, 2μL guide sequence targeting ZMO1650 interval, 0.5μL T4 ligase, 1μL Buffer and the balance of double distilled water in 10μL. The reaction program of colony PCR is pre-denaturation at 98℃, 3min, 1 cycle; denaturation at 98℃, 10s, annealing at 55℃, 10s, extension at 72℃, set at 10s / kb according to the fragment length, a total of 30 cycles; extension at 72℃, 2min, 1 cycle; storage at 12℃, 2min, 1 cycle.

[0065] 3) Reverse PCR amplification of the ZMO1650 targeting plasmid was performed using primers 15Afk-F (shown in SEQ ID NO.43) and 15Afk-R (shown in SEQ ID NO.44) to obtain the reverse-amplified ZMO1650 targeting plasmid.

[0066] 4) Synthesizing a ZMO1650 replacement fragment formed by connecting the ZMO1650 upstream sequence, the AraC operon and the ZMO1650 downstream sequence (shown in SEQ ID NO.45).

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

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

[0069] Preparation of expression plasmid

[0070] In order to prepare the engineering strain of the present application, the embodiment also prepared a series of expression plasmids. The expression plasmid is a pTZ28a plasmid containing at least one of the first gene, the second gene, the third gene, the fourth gene and the fifth gene. Among them, the pTZ28a plasmid is a basic plasmid. The construction process of the pTZ28a plasmid is referenced to CN114774453A. Among them, the first gene is selected from EX1 to EX4, the second gene is selected from EN1 to EN5, the third gene is GB, the fourth gene is selected from C1 to C4, and the fifth gene is selected from I1 to I4.

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

[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 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) DNA molecules were used as target sequences.

[0074] 2) Preparation of expression plasmid

[0075] The linearized pTZ28a fragments were subjected to T5 ligation reaction with different target sequences, and the ligation products were transformed into Escherichia coli. Positive colonies were screened from the transformants, the positive colonies were cultured, and the expression plasmids were extracted from the culture.

[0076] The expression plasmids obtained in the embodiment 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, pTZ28a- 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 contains 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 of double distilled water in 5 μL. The conditions of the T5 ligation reaction include: reacting on ice for 5 minutes.

[0078] Preparation of engineered strains

[0079] The embodiment also provides a method for preparing an engineered strain. The method includes obtaining a background strain; obtaining at least one expression plasmid containing at least one of the first gene, the second gene, the third gene, the fourth gene, and the fifth gene; transferring the at least one expression plasmid into the background strain; and selecting the engineered strain from the transformants.

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

[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 was 0.4-0.6, the resulting bacterial solution was centrifuged at 4000 rpm and 25°C for 8 min to collect the bacteria, and the bacteria were gently resuspended in sterile water, and the bacteria were centrifuged again at 4000 rpm and 25°C for 8 min to collect the bacteria, and the bacteria were gently resuspended in 10% glycerol to obtain the competent cells of the background strain.

[0083] 2) Electroporation of expression plasmid

[0084] Mix about 500 ng of expression plasmid with 50 μL competent cells in a pre-cooled electroporation cup, and electroporate at 1.8 kV, 25 μF, and 200 Ω. Resuspend the solution in the electroporation cup with 1 mL RMG5, and culture at 30°C for 3 to 6 hours. Spread the culture solution on a resistant plate, and culture at 30°C for 2-3 days before performing colony PCR verification. The positive colonies screened are the engineered strains.

[0085] Screening for the first gene

[0086] In some test cases, expression plasmids pTZ28a-GFP-EX1, pTZ28a-GFP-EX2, pTZ28a-GFP-EX3, and pTZ28a-EX4 were respectively transferred into the background strain, 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°C and 100rpm in RMG5 liquid medium containing 3mg / mL arabinose (Arab) and 0.8μg / mL tetracycline (Tc), and the target proteins were extracted from the fermentation broth, and their exoglucanase activities were detected.

[0087] The exoglucanase activity test steps include: taking 100 μL of phosphate-swollen cellulose solution as a substrate, 50 μL of sodium citrate buffer at pH 4.5, and 50 μL of the target protein solution (EX1 is 1.43 g / L) into a 2 mL centrifuge tube and mixing well, reacting at 30°C or 50°C for 30 min, adding 150 μL of DNS (Solarbio D7800) to terminate the reaction, cooling to room temperature, adding sterile water to 2 mL, mixing well, and precipitating 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 per milligram of protein.

[0088] The target proteins expressed by the four recombinant plasmids were analyzed by SDS-PAGE. Figure 1 As shown in the figure, the expression level of GFP-EX1 reached 1.24g / L, the enzyme activity of GFP-EX1 was 27.16U / mL, and the specific enzyme activity of GFP-EX1 was 21.89U / mg. However, the target proteins GFP-EX2, GFP-EX3 and EX4 were almost not expressed, which may be because the growth of GFP-EX2, GFP-EX3 and EX4 recombinant plasmids slowed down after being transferred into the background strain, affecting the correct folding and expression of their proteins.

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

[0090] Screening for the second gene

[0091] In some test examples, expression plasmids pTZ28a-GFP-EN1, pTZ28a-GFP-EN2, pTZ28a-GFP-EN3, pTZ28a-GFP-EN4, and pTZ28a-GFP-EN5 were respectively transferred 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 fermentation was induced in RMG5 liquid culture medium containing 3 mg / mL Arab and 0.8 μg / mL Tc at 30°C and 100 rpm, respectively, and the target proteins were extracted from the fermentation broth.

[0092] like Figure 2 The SDS-PAGE results shown showed that the sizes of the fusion proteins were consistent with expectations, with the expression levels of GFP-EN3, GFP-EN2, GFP-EN5, GFP-EN1, GFP-EN4, GFP-EN3, GFP-EN2, GFP-EN5, GFP-EN1, GFP-EN4, GFP-EN4 and GFP-EN4 being 3.84 g / L, 3.68 g / L, 2.8 g / L, 1.27 g / L and 1.12 g / L, respectively.

[0093] The enzymatic activities and relative enzymatic activities of proteins GFP-EN1, GFP-EN2, GFP-EN3, GFP-EN4 and GFP-EN5 in hydrolyzing cellulose were tested at 50°C. The cells were directly collected from the fermentation broth, and the target protein was extracted according to the above steps. GFP-EN4 was used as a positive control (Control group). Figure 3 As shown in A, the enzyme activities are from high to low in the order of GFP-EN2, GFP-EN1, GFP-EN5, GFP-EN3, and GFP-EN4, and the highest enzyme activity of GFP-EN2 is 62.55 U / mL. Figure 3 As shown in B, the specific enzyme activity from high to low is 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, which is 39.43 U / mg, about 2.3 times that of GFP-EN2.

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

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

[0096] The cellulolytic enzyme activities and specific enzyme activities of EN1, EN2, EN5 and EN4 were tested at 50°C, and the cells were directly collected from the fermentation broth. The target protein was extracted according to the above steps, and EN4 was used as a positive control (Control group). Figure 5 As shown, at the enzyme activity test temperature of 50°C, the enzyme activity of EN1 was 56.03U / mL, the enzyme activity of EN2 was 53.88U / mL, the enzyme activity of EN5 was 53.71U / mL, and the lowest enzyme activity of EN4 was 17.32U / mL; the specific enzyme activity of EN1 was 40.36U / mg, the specific enzyme activity of EN2 was 14U / mg, the specific enzyme activity of EN5 was 19.09U / mg, and the specific enzyme activity of EN4 was 22.20U / mg.

[0097] The cellulolytic enzyme activities and specific enzyme activities of EN1, EN2, EN5 and EN4 were tested at 30°C, and the cells were directly collected from the fermentation broth and the target protein was extracted according to the above steps. EN4 was used as a positive control (Control group). Figure 6 As shown, at the enzyme activity test temperature of 30°C, the enzyme activity of EN1 was 34.60U / mL, the enzyme activity of EN2 was 31.05U / mL, the enzyme activity of EN5 was 32.77U / mL, and the enzyme activity of EN4 was 7.76U / mL; the specific enzyme activity of EN1 was 24.93U / mg, the specific enzyme activity of EN2 was 8.06U / mg, the specific enzyme activity of EN5 was 11.70U / mg, and the specific enzyme activity of EN4 was 9.96U / mg.

[0098] This shows that no matter under the cellulose hydrolysis conditions of 50°C or 30°C, EN1 has the highest enzyme activity and EN2 has the highest expression level. Therefore, endoglucanases EN1 and EN2 were selected to construct the composite enzyme assembly.

[0099] Screening for the fourth and fifth genes

[0100] Some test cases also tested the in vitro specific binding effects of C1-GFP, C2-GFP, C3-GFP and C4-GFP with I1, I2, I3 and I4 respectively. The test process includes:

[0101] The expression plasmids pTZ28a-I1, pTZ28a-I2, pTZ28a-I3, pTZ28a-I4, pTZ28a-C1-GFP, pTZ28a-C2-GFP, pTZ28a-C3-GFP, and pTZ28a-C4-GFP were respectively transferred into the background strain to obtain recombinant strains expressing I1, I2, I3, I4, C1-GFP, C2-GFP, C3-GFP, and C4-GFP, respectively. Positive clones were screened and fermented in RMG5 liquid culture medium containing 3 mg / mL Arab and 0.8 μg / mL Tc at 30°C and 100 rpm, and 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 each protein was correctly expressed.

[0103] The concentrated solutions of the purified target proteins I1, I2, I3, and I4 (with a concentration of approximately 2.5 g / L) were mixed with equal volumes of phosphoric acid swollen cellulose (PASC) solution, incubated at room temperature for 30 min, and the precipitates were washed three times with TBS buffer to remove unbound proteins; the resulting precipitates (PASC complexes with the target proteins I1, I2, I3, and I4, respectively) were resuspended with TBS buffer, and 2 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 the precipitates were resuspended with 100 μL TBS buffer, and the fluorescence was observed under a blue light instrument.

[0104] Figure 8 The results show that protein I expressed and purified in Zymomonas mobilis can specifically bind to protein C.

[0105] Application of engineered strains

[0106] Based on the engineered strain provided in the present application, the embodiments further provide the use of the engineered strain in at least one of preparing a complex enzyme assembly, degrading cellulose or synthesizing glucose.

[0107] On the one hand, the embodiment provides a method for biosynthesizing a complex enzyme assembly. The complex enzyme assembly is assembled by endoglucanase, exoglucanase, β-glucosidase, colicin E protein and a protein that interacts with the colicin E protein with affinity. The method comprises: inoculating the engineered strain obtained in the embodiment into a liquid containing a culture medium for fermentation; harvesting at least one of the endoglucanase, the exoglucanase, the β-glucosidase, the colicin E protein, a protein that interacts with the colicin E protein with affinity or the complex enzyme assembly from the fermentation liquid.

[0108] In some embodiments, the steps after fermentation of the method specifically include: harvesting cellulosomes composed of the endoglucanase, the exoglucanase and the β-glucosidase, and a protein scaffold composed of the colicin E protein and a protein that interacts with the colicin E protein affinity from the fermentation broth; assembling the cellulosomes and the protein scaffold into the complex enzyme assembly.

[0109] In one aspect, the embodiment provides a method for hydrolyzing cellulose, which comprises obtaining the composite enzyme assembly prepared by the method; and contacting the cellulose with the composite enzyme assembly.

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

[0111] The steps after fermentation specifically include:

[0112] 1) Precool the collected fermentation broth on ice and centrifuge it at 4°C, 4000 rpm for 30 min to collect the bacteria, wash the bacteria 1-2 times with buffer, and resuspend them in a buffer containing 1% PMSF (protease inhibitor); disrupt the bacteria by ultrasonication; centrifuge the resulting disrupted broth at 4°C, 4000 rpm for 30 min, collect the supernatant, and add PMSF to a final concentration of 1% to obtain a solution containing intracellular proteins.

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

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

[0115] Fusion protein testing

[0116] In some test examples, 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 respectively transferred into background strains, positive clones were screened, and fermentation was induced in RMG5 liquid culture medium containing 3 mg / mL Arab and 0.8 μg / mL Tc at 30°C and 100 rpm, and 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 Fig. 9 As shown in the SDS-PAGE image, the size of C1-EX1 and EX1-C1 proteins is about 84 kDa, the molecular weight of C2-EN2 and EN2-C2 proteins is about 60 kDa, the molecular weight of C3-EN1 and EN1-C3 proteins is about 84 kDa, and the molecular weight of C4-GB and GB-C4 proteins is about 73 kDa, all of which are in line with expectations.

[0118] The enzyme activities of the separated 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 were tested at 30°C and 50°C, respectively. The test results are as follows: Fig.10 . At 30℃, the activity of EX1 fused with C1 increased, and the activity of C1-EX1 was slightly higher than that of EX1-C1, while at 50℃, the activity of EX1 fused with C1 decreased. At 30℃, the activity of EN2 fused with C2 increased, and the activity of C2-EN2 was higher than that of EN2-C2. At 50℃, the activity of EN2 fused with C2 was significantly increased, and the activity of C2-EN2 was higher than that of EN2-C2. At 30℃, the activity of C3-EN1 was higher than that of a single enzyme, but the activity of EN1-C3 was lower than that of a single enzyme. At 50℃, the activity of EN1 fused with C3 was significantly increased. At room temperature, the activity of GB fused with C4 was similar to that of the unfused one, and the activity of GB-C4 was slightly higher than that of C4-GB. It can be seen that the 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, the fusion protein provided in the examples is also subjected to an in vitro assembly test. The test process includes:

[0121] 1) Preparation of engineered strains

[0122] The expression plasmids pTZ28a-I4, pTZ28a-C1-EX1, pTZ28a-C2-EN2, pTZ28a-C3-EN1 and pTZ28a-GB-C4 were respectively transferred into the background strain to obtain 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 culture medium containing 3 mg / mL Arab and 0.8 μg / mL Tc and cultured at 30° C. and 100 rpm for 24 h.

[0125] 3) Protein purification

[0126] The fermentation liquid collected in step 2) was precooled on ice and centrifuged at 4°C 4000rpm for 30min to collect the bacteria, which were washed with buffer 1-2 times and resuspended with buffer containing 1% PMSF (protease inhibitor); the bacteria were broken by ultrasound; the resulting broken solution was centrifuged at 4°C and 4000rpm for 30min, the supernatant was collected, and PMSF with a final concentration of 1% was added to obtain a solution containing intracellular proteins. The solution containing intracellular proteins was loaded onto a nickel column, and after standing to allow the target protein to fully bind to the nickel column, the impurities were first removed with a low concentration (10mM, 30mM, 50mM) of imidazole elution solution, and then the target protein was eluted with a high concentration (200mM, 300mM, 500mM) of imidazole elution solution. The collected target protein eluate was concentrated and purified to obtain a sample solution containing the target protein, and SDS-PAGE was performed to detect that it was consistent with the expected size, namely, 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) were obtained.

[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 with a final concentration of 3.52 g / L C1-EX1, C2-EN2, C3-EN1 and GB-C4 proteins, react at 30°C or 50°C for 30 min, add 150 μL DNS (Solarbio D7800) to terminate the reaction, cool to room temperature, add sterile water to 2 mL. Mix well and add 50 μL of sodium citrate buffer 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 per milligram of protein.

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

[0130] The detection steps of 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 a 1.5 mL EP tube, adding distilled water to make up to 600 μL, then adding 150 μL DNS to mix, boiling water bath for 5 min, cooling to room temperature, diluting to 2 mL with distilled water, inverting to mix, and measuring OD with a spectrophotometer. 540nm The value of .

[0131] like Fig.10 As shown, at an in vitro assembly temperature of 30°C, the complex enzyme assembly can produce 0.102 mg of glucose, which is 41.67% higher than the free enzyme system. At an in vitro assembly temperature of 50°C, the complex enzyme assembly produces 0.127 mg of glucose, which is 30.93% higher than the free enzyme system.

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

[0133] The present application provides an engineered strain, a composite enzyme and an assembly thereof, which can hydrolyze cellulose at 30°C and 50°C, and can also increase the enzyme activity and glucose yield relative to the free enzyme. This shows that the engineered strain, the composite enzyme and the assembly thereof provided by the present application can reduce the high temperature reaction problem of hydrolyzing cellulose, can efficiently catalyze the hydrolysis of cellulose at a relatively low temperature, greatly reduce the energy consumption in the hydrolysis process, and have very important process application value, especially for promoting the industrialization of cellulosic fuel ethanol and providing important current significance for energy conservation and emission reduction.

[0134] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. An engineered strain, wherein the engineered strain is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; in, Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

2. An engineered strain containing a first gene encoding an exoglucanase; a second gene encoding an endoglucanase; a third gene encoding β-glucosidase; a fourth gene encoding the colicin E protein; as well as A fifth gene encoding a protein that interacts with the colicin E protein At least one of the following: Zymomonas mobilis; Wherein, the first gene is at least one of any one of SEQ ID NOs: 1 to 4, the second gene is at least one of SEQ ID NOs: 5 to 8, the third gene is at least one of SEQ ID NOs: 10, the fourth gene is at least one of SEQ ID NOs: 11 to 14, and the fifth gene is at least one of SEQ ID NOs: 15 to 18; Wherein, the mobilis Zymomonas is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; Among them, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

3. An engineered strain, the engineered strain being Zymomonas mobilis 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 connected in sequence; in, The mobilis Zymomonas is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; Among them, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

4. An engineered strain, the engineered strain being Zymomonas mobilis 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 connected in sequence; in, The mobilis Zymomonas is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; Among them, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

5. An engineered strain, the engineered strain being Zymomonas mobilis 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 connected in sequence; in, The mobilis Zymomonas is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; Among them, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

6. An engineered strain, the engineered strain being Zymomonas mobilis 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 connected in sequence; in, The mobilis Zymomonas is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; Among them, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

7. An engineered strain, wherein the engineered strain is Zymomonas mobilis containing the fifth gene as shown in SEQ ID NO: 18; in, The mobilis Zymomonas is a Z.mobilis ZM4ATCC 31821 strain in which the pZM32 plasmid is knocked out, the pZM33 plasmid is knocked out, the pZM36 plasmid is knocked out, the pZM39 plasmid is knocked out, the ZMO0038 interval is replaced with the Cas12a gene, the ZMO0028 interval is replaced with the T7 operon, and the ZMO1650 interval is replaced with the AraC operon; Among them, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0028 interval is shown in 31083..32006nt of GenBank: CP023715.1, the ZMO0038 interval is shown in 39623..40225nt of GenBank: CP023715.1, the ZMO1650 interval is shown in 1697900..1699036nt of 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.

8. A combination of engineered strains, formed by combining at least one of the engineered strains described in claims 2 to 7.

9. A complex protein, which is the protein shown in SEQ ID NO: 50 to 54 or an assembly thereof.

10. A method for hydrolyzing cellulose, comprising: Obtaining at least one engineered strain according to claim 1 to 8; The composite protein according to claim 9, or the engineered strain or the fermentation broth of the engineered strain, or the fermentation broth extract of the engineered strain are incubated with the cellulose in a liquid system respectively.

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