Engineering strain of zymomonas mobilis as well as preparation method and application of engineering strain

Abstract

The invention relates to the technical field of zymomonas mobilis, in particular to an engineering strain of zymomonas mobilis, a preparation method and application. The engineering strain is zymomonas mobilis containing an hfq gene, a groELS gene and a grpE gene on a genome. The engineering strain can efficiently utilize corncob waste residue hydrolysate to produce ethanol, can tolerate high-concentration ethanol, and provides a biosynthetic solution for industrial production of cellulosic ethanol, cost reduction and efficiency improvement.

Description

Technical Field

[0001] The present application relates to the technical field of Zymomonas mobilis, and in particular to an engineered strain, a preparation method and application 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 cellulosic ethanol production has been commercialized. At the same time, the research on the mechanism of tolerance to inhibitors in hydrolysates is also relatively mature. Therefore, Zymomonas mobilis has become a microbial cell factory for the production of a variety of biochemical substances, such as lignocellulosic ethanol, PHB, lactic acid, acetyl, isobutanol, etc. Summary of the invention

[0003] The inventors of this application modified the genome of Zymomonas mobilis to obtain an engineered strain that can efficiently utilize wood cellulose hydrolyzate (such as corn cob waste). The engineered strain can efficiently utilize corn cob waste to produce ethanol and can tolerate high concentrations of ethanol, providing a biosynthetic solution for the industrial production of cellulosic ethanol and cost reduction and efficiency improvement.

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

[0005] In a first aspect, the embodiment discloses an engineered strain, wherein the engineered strain is Zymomonas mobilis containing at least one of the hfq gene, the groELS gene, and the grpE gene.

[0006] In the second aspect, the embodiment discloses an engineered strain, which is a Z.mobilis ZM4 ATCC 31821 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1650 region of the genome is replaced with the hfq gene. The genome of the Z.mobilis ZM4 ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, and the ZMO1650 is shown in the 1697900..1699036nt interval of GenBank: CP023715.1.

[0007] In a third aspect, the embodiment discloses an engineered strain, which is a Z.mobilis ZM4ATCC 31821 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1933 region of the genome is replaced with the groELS gene and the grpE gene. The genome of the Z.mobilis ZM4ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, and the ZMO1933 is shown in the 1990679..1992058nt interval of GenBank: CP023715.1.

[0008] In a fourth aspect, the embodiment discloses a method for preparing an engineered strain. The method comprises: preparing a first editing plasmid, a second editing plasmid and a third editing plasmid; transferring at least one of the first editing plasmid, the second editing plasmid and the third editing plasmid into the Z.mobilis ZM4ATCC 31821 strain; and selecting positive colonies from the transformants, which are the engineered strains. The first editing plasmid carries a first guide sequence of the ZMO0128 region of the Z.mobilis ZM4ATCC 31821 strain genome that guides the Cas enzyme to target the cleavage, the second editing plasmid carries a second guide sequence of the ZMO1650 region of the Z.mobilis ZM4ATCC 31821 strain genome that guides the Cas enzyme to target the cleavage, and a second donor sequence for replacing the ZMO1650 region, the third editing plasmid carries a third guide sequence of the ZMO1933 region of the Z.mobilis ZM4 ATCC 31821 strain genome that guides the Cas enzyme to target the cleavage, and a third donor sequence for replacing the ZMO1933 region, the Z.mobilis ZM4ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, the ZMO1650 is shown in the 1697900..1699036nt interval of GenBank: CP023715.1, and the ZMO1933 is shown in the 1990679..1992058nt interval of GenBank: CP023715.1.

[0009] In a fifth aspect, an embodiment discloses a method for degrading corn cob waste residue, comprising obtaining an engineered strain of at least one of the first to third items; and fermenting the engineered strain in a corn cob waste residue hydrolyzate or a culture medium containing the corn cob waste residue hydrolyzate.

[0010] In the sixth aspect, the embodiments disclose the application of the engineered strain described in any one of items 1 to 3 or the engineered strain prepared by the preparation method described in the fourth aspect, and the application is selected from at least one of: fermentation of monosaccharides, preparation of ethanol, and degradation of corn cob waste hydrolyzate. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the colony PCR image of the ZE1 strain provided in the examples.

[0012] Figure 2 This is the colony PCR image of the ZE2 strain provided in the examples.

[0013] Figure 3 OD600 curves of the fermentation test of the ZM4 strain, ZE1 strain and ZE2 strain provided in the examples in the RMG5 liquid culture medium containing 75 g / L ethanol.

[0014] Figure 4 The glucose content curve and ethanol production curve of the ZM4 strain, ZE1 strain and ZE2 strain provided in the embodiment in the RMG5 liquid medium containing 75g / L ethanol. In the figure, "G" represents glucose and "E" represents ethanol. In the figure, the left side is the glucose concentration in the fermentation supernatant, and the right side is the difference between the ethanol concentration in the obtained fermentation supernatant and the ethanol concentration (75g / L) put into the fermentation medium. DETAILED DESCRIPTION

[0015] 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.

[0016] The inventors of the present application creatively cloned the hfq gene, groELS gene and grpE gene into the genome of Zymomonas mobilis to obtain a type of engineered strain. The present application uses Zymomonas mobilis as a chassis cell to improve the degradation capacity of cellulose (especially corncob waste), and can also efficiently hydrolyze corncob waste to produce ethanol, and can tolerate high concentrations of ethanol, providing a biosynthetic solution for the industrial production of cellulosic ethanol and cost reduction and efficiency improvement.

[0017] Engineered strains

[0018] The term "engineered strain" refers to a strain obtained by genetically engineering a wild-type strain, such as modifying its genome or introducing an overexpression plasmid into it. For example, a strain obtained by knocking out or overexpressing a gene in a genome of a wild-type Zymomonas mobilis or introducing an overexpression plasmid into it is called an "engineered strain."

[0019] The embodiment discloses an engineered strain. The engineered strain is Zymomonas mobilis containing at least one of the hfq gene, the groELS gene and the grpE gene.

[0020] The engineered strain provided in the present application can significantly improve its ethanol tolerance by integrating three ethanol tolerance genes into its genome, while improving its degradation efficiency of cellulose, especially corn cob waste, thereby providing a biosynthetic solution for the industrial production of cellulosic ethanol and cost reduction and efficiency improvement.

[0021] In some embodiments, the hfq gene is located on the genome of the Molecular Biology Bacteria or is free outside the genome.

[0022] In some embodiments, the groELS gene is located on the genome of the Molecular Biology Bacterium or is free from the genome.

[0023] In some embodiments, the grpE gene is located on the genome of the Molecular Biology Bacteria or is free from the genome.

[0024] In some embodiments, the Zymomonas mobilis is Z. mobilis ZM4ATCC 31821 strain, and the genome of the Z. mobilis ZM4ATCC 31821 strain is shown in GenBank: CP023715.1.

[0025] In some embodiments, the hfq gene is as shown in SEQ ID NO:1, the groELS gene is as shown in SEQ ID NO:2, and the grpE gene is as shown in SEQ ID NO:3.

[0026] On the one hand, the embodiment discloses an engineered strain, which is a Z.mobilis ZM4ATCC 31821 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1650 region of the genome is replaced with the hfq gene. The genome of the Z.mobilis ZM4ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, and the ZMO1650 is shown in the 1697900..1699036nt interval of GenBank: CP023715.1.

[0027] On the one hand, the embodiment discloses an engineered strain, which is a Z.mobilis ZM4ATCC 31821 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1933 region of the genome is replaced with the groELS gene and the grpE gene, the genome of the Z.mobilis ZM4ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, and the ZMO1933 is shown in the 1990679..1992058nt interval of GenBank: CP023715.1.

[0028] On the one hand, the embodiment discloses a method for preparing an engineered strain, which includes: preparing a first editing plasmid, a second editing plasmid, and a third editing plasmid; transferring at least one of the first editing plasmid, the second editing plasmid, and the third editing plasmid into the Z.mobilis ZM4ATCC 31821 strain; and selecting positive colonies from the transformants, which are the engineered strains. The first editing plasmid carries a first guide sequence of the ZMO0128 region of the Z.mobilis ZM4ATCC31821 strain genome that guides the Cas enzyme to target the cleavage, the second editing plasmid carries a second guide sequence of the ZMO1650 region of the Z.mobilis ZM4ATCC 31821 strain genome that guides the Cas enzyme to target the cleavage, and a second donor sequence for replacing the ZMO1650 region, the third editing plasmid carries a third guide sequence of the ZMO1933 region of the Z.mobilis ZM4 ATCC 31821 strain genome that guides the Cas enzyme to target the cleavage, and a third donor sequence for replacing the ZMO1933 region, the Z.mobilis ZM4 ATCC The genome of strain 31821 is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, the ZMO1650 is shown in the 1697900..1699036nt interval of GenBank: CP023715.1, and the ZMO1933 is shown in the 1990679..1992058nt interval of GenBank: CP023715.1.

[0029] In some embodiments, the first editing plasmid further carries a first donor sequence, which is composed of an upstream sequence of the ZMO0128 region and a downstream sequence of the ZMO0128 region. In some embodiments, the first guide sequence is shown in SEQ ID NO: 4. In some embodiments, the first donor sequence is shown in SEQ ID NO: 5.

[0030] In some embodiments, the second donor sequence consists of the upstream sequence of the ZMO1650 region, the hfq gene, and the downstream sequence of the ZMO1650 region. In some embodiments, the second guide sequence is shown in SEQ ID NO: 6. In some embodiments, the second donor sequence is shown in SEQ ID NO: 7.

[0031] In some embodiments, the third donor sequence consists of the upstream sequence of the ZMO1933 region, the groELS gene, the grpE gene, and the downstream sequence of the ZMO1933 region. In some embodiments, the third guide sequence is shown in SEQ ID NO: 8. In some embodiments, the third donor sequence is shown in SEQ ID NO: 9.

[0032] In one aspect, the embodiment discloses a method for degrading corncob waste residues, which comprises obtaining the engineered strain provided in the above embodiment; and fermenting the engineered strain in corncob waste residue hydrolyzate or a culture medium containing corncob waste residue hydrolyzate.

[0033] On the one hand, the embodiments disclose applications of the engineered strains provided in the above embodiments, wherein the applications are selected from at least one of: fermenting monosaccharides, preparing ethanol, and degrading corn cob waste hydrolyzate.

[0034] The present application will be described below in conjunction with more specific embodiments, but this does not constitute a limitation on the implementation methods of the present application.

[0035] Preparation of first to third editing plasmids

[0036] In order to obtain the engineered strain provided in the present application, the embodiments provide methods for preparing the first editing plasmid, the second editing plasmid and the third editing plasmid.

[0037] The method for preparing the first editing plasmid provided in the embodiment comprises:

[0038] 1) Synthesizing a targeting element targeting the ZMO0128 region, wherein the targeting element is composed of two repeating elements (shown in SEQ ID NO: 10) and a first guide sequence (shown in SEQ ID NO: 4) targeting the ZMO0128 region connected between the two recombinants.

[0039] 2) The vector pEZ15Asp was digested with restriction endonuclease BsaⅠ to obtain linearized pEZ15Asp; the annealing product of the primer pair 0128-F (shown in SEQID NO:11) and 0128-R (shown in SEQ ID NO:12) was connected to the linearized pEZ15Asp using T4 ligase; the ligation product was transformed into Escherichia coli DH5α; positive colonies were screened from the transformants and verified by colony PCR using primers pEZ15A-F (shown in SEQID NO:13) and pEZ15A-R (shown in SEQ ID NO:14). The ZMO0128 interval targeting plasmid was extracted from the culture of the positive colony. The annealing system (10 μL) contained: 1 μL 0128-F (10 μM), 1 μL 0128-R (10 μM) and 8 μL double distilled water), denatured at 95°C for 5 minutes, and cooled to room temperature for use. Among them, the T4 reaction system contains 20-40ng linearized pEZ15Asp, 2μL first guide sequence, 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℃ for 3min, 1 cycle; denaturation at 98℃ for 10s, annealing at 55℃ for 10s, extension at 72℃, set at 10s / kb according to the fragment length, for a total of 30 cycles; extension at 72℃ for 2min, 1 cycle; preservation at 12℃ for 2min, 1 cycle. Among them, the spectinomycin knockout plasmid vector (pEZ15Asp) is a spectinomycin encoding gene inserted into pEZ15A as a marker gene. Among them, pEZ15A was obtained by referring to the method of "Yang S, Mohagheghi A, Franden MA, et al. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars [J]. Biotechnol Biofuels, 2016, 9 (1): 189." To obtain pEZ15A (www.molecularcloud.org / ) encoding different genes (such as resistance genes), different marker genes can be inserted into it by referring to "Construction and application of plasmid pUC19-CM-D [J]. Anhui Agricultural Science, 2010, Public Methods, Issue 19".

[0040] 3) Synthesize the first donor fragment (shown in SEQ ID NO:5) for knocking out the ZMO0128 region formed by connecting the upstream sequence of ZMO0128 and the downstream sequence of ZMO0128, and use primers 15A-FK-F (shown in SEQ ID NO:15) and 15A-FK-R (shown in SEQ ID NO:16) to reverse PCR amplify the ZM0128 targeting plasmid to obtain the reverse-amplified ZMO0128 interval targeting plasmid. The reverse-amplified ZMO0128 interval targeting plasmid and the first donor fragment (shown in SEQ ID NO:5) were Gibson assembled at a ratio of 1:3, and the assembled product was transferred into Escherichia coli DH5α. Positive colonies were screened from the transformants, and colony PCR verification was performed using pEZ15A-F and pEZ15A-R primers. The first editing plasmid was extracted and separated from the verified positive colonies.

[0041] The preparation method of the second editing plasmid provided in the embodiment includes:

[0042] 1) Synthesizing a targeting element targeting the ZMO1650 interval, wherein the targeting element is composed of two repeating elements (shown in SEQ ID NO: 10) and a second guide sequence (SEQ ID NO: 6) targeting the ZMO1650 interval connected between the two repeating elements.

[0043] 2) The vector pEZ15Asp was digested with restriction endonuclease BsaⅠ to obtain linearized pEZ15Asp; the annealing product of the primer pair 1650-F (shown in SEQ ID NO: 17) and 1650-R (shown in SEQ ID NO: 18) was connected to the linearized pEZ15Asp using T4 ligase; the ligation product was transformed into Escherichia coli DH5α; positive colonies were selected from the transformants, verified by colony PCR, and the ZMO1650 targeting plasmid was extracted from the culture of the positive colonies. The annealing system, T4 reaction system and colony PCR were the same as those in the above embodiment.

[0044] 3) Synthesize a second donor sequence (shown in SEQ ID NO: 7) formed by connecting the upstream sequence of ZMO1650, the hfq gene and the downstream sequence of ZMO1650, perform Gibson assembly with the reverse extension fragment of the ZMO1650 targeting plasmid, transform the assembly product into Escherichia coli DH5α, screen positive colonies from the transformants, and extract and separate the second editing plasmid from the verified positive colonies. The specific steps are the same as those in the above embodiment.

[0045] The embodiment also provides a method for preparing a third editing plasmid, comprising:

[0046] 1) Synthesizing a targeting element targeting the ZMO1933 region, wherein the targeting element is composed of two repeating elements (shown in SEQ ID NO: 10) and a third guide sequence targeting the ZMO1933 region connected between the two recombinants (shown in SEQ ID NO: 8).

[0047] 2) The vector pEZ15Asp was digested with restriction endonuclease BsaⅠ to obtain linearized pEZ15Asp; the annealing product of the primer pair 1933-F (shown in SEQ ID NO: 19) and 1933-R (shown in SEQ ID NO: 20) was connected to the linearized pEZ15Asp using T4 ligase; the ligation product was transformed into Escherichia coli DH5α; positive colonies were selected from the transformants, verified by colony PCR, and the ZMO1933 targeting plasmid was extracted from the culture of the positive colonies. The annealing system, T4 reaction system and colony PCR were the same as those in the above embodiment.

[0048] 3) synthesizing a third donor sequence (shown in SEQ ID NO: 9) formed by connecting the upstream sequence of ZMO1933, the groELS gene, the grpE gene and the downstream sequence of ZMO1933, performing Gibson assembly with the reverse extension fragment of the ZMO1933 targeting plasmid, transforming the assembly product into Escherichia coli DH5α, screening positive colonies from the transformants, and extracting and isolating the third plasmid from the verified positive colonies. The specific steps are the same as those in the above embodiment.

[0049] Preparation of engineered strains

[0050] Example An engineered strain was prepared using the first, second and third editing plasmids and Z. mobilis ZM4 ATCC 31821 strain (hereinafter referred to as ZM4 strain, which is a commercial strain of ATCC).

[0051] To this end, the embodiment provides a method for preparing an engineered strain, including: preparing a competent ZM4 strain; simultaneously or successively transferring a first editing plasmid and a second editing plasmid into the competent ZM4 strain; and screening positive colonies from the transformants, which are the engineered strains.

[0052] To this end, the embodiment provides a method for preparing an engineered strain, comprising: preparing a competent ZM4 strain; simultaneously or successively transferring a first editing plasmid and a third editing plasmid into the competent ZM4 strain; and screening positive colonies from the transformants, which are the engineered strains.

[0053] Wherein, the method for preparing competent ZM4 strain comprises:

[0054] 1) Pick an appropriate amount of ZM4 stored in a -80℃ refrigerator and streak it on an RMG2 plate (20 g / L glucose, 10 g / L yeast extract, 2 g / L KH2PO4, 15 g / L agar), and incubate the plate in a 33℃ constant temperature incubator for 2 to 3 days.

[0055] 2) Pick up a single activated ZM4 colony into a bacterial bottle containing about 5 mL of RMG5 liquid culture medium (50 g / L glucose, 10 g / L yeast extract, 2 g / L KH2PO4) and culture it at 33°C overnight.

[0056] 3) The next day, transfer an appropriate amount of bacterial culture in the logarithmic phase to a 50 mL conical centrifuge tube containing 40 mL RMG5 liquid medium, so that the initial OD600nm is 0.025-0.05, and culture at 33°C until the OD600nm is between 0.4 and 0.5.

[0057] 4) Collect the bacteria in a centrifuge tube (25°C, 6500 rpm, 5 min). After centrifugation, carefully pour out the supernatant in an ultra-clean workbench.

[0058] 5) Add 20 mL of sterile water and shake well; prepare another centrifuge tube to balance it. After centrifugation, carefully pour out the supernatant in the clean bench.

[0059] 6) Resuspend and wash the cells with 20 mL of 10% glycerol, centrifuge and carefully discard the supernatant in a clean bench. Repeat this step twice.

[0060] 7) Add 400 μL of 10% glycerol, shake evenly, dispense 55 μL into 1.5 mL EP tubes, freeze quickly in liquid nitrogen, and store in a -80°C refrigerator.

[0061] The conversion steps include:

[0062] 1) Add 50ng of any of the first to third plasmids to 50μL of Zymomonas mobilis competent cells, mix gently, and add them together to the electroporation cup. The transformation program is set to 200Ω resistance, 25μF capacitance, 1.6kV voltage, and 1mm distance between the electrodes of the electroporation cup. After electroporation, immediately take 950μL of recovery medium RMG5 to the electroporation cup, blow and evenly transfer the bacterial solution to a 1.5mL E tube, seal the EP tube with a sealing film, and recover in a 30℃ constant temperature incubator for 6-8h.

[0063] 2) Take 100 μL of bacterial solution and spread it evenly on the RM plate with the corresponding resistance, and culture it in a constant temperature incubator at 30°C for 2-3 days.

[0064] Wherein, the step of selecting positive colonies from the transformants includes the step of performing colony PCR on the transformants.

[0065] In some embodiments, the engineered strain provided in the embodiments is ZE1, which is a ZM4 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1650 region of the genome is replaced with the hfq gene.

[0066] The preparation method of the ZE1 strain comprises:

[0067] 1) Prepare a first editing plasmid and a second editing plasmid according to the above embodiment;

[0068] 2) Prepare competent ZM4 strain according to the above example;

[0069] 3) Referring to the above example, the first editing plasmid was transformed into the competent ZM4 strain, and positive colonies were screened from the transformants. The screening process included colony PCR. The primer pair used for colony PCR was 0128-OUT-F (shown in SEQ ID NO: 21) and 0128-OUT-R (shown in SEQ ID NO: 22). The verification result was as follows: Figure 1 Top middle picture.

[0070] 4) Referring to the above example, the second editing plasmid was transformed into the competent strain obtained in step 3, and positive colonies (i.e., ZE1 strain) were screened from the transformants. The screening process included colony PCR. The primer pair used for colony PCR was 1650-OUT-F (shown in SEQ ID NO: 23) and hfq-in-R (shown in SEQ ID NO: 24). The verification result was as follows: Figure 1 Lower middle picture.

[0071] The preparation method of the ZE2 strain comprises:

[0072] 5) Prepare the first editing plasmid and the third editing plasmid according to the above embodiment;

[0073] 6) Prepare competent ZM4 strain according to the above example;

[0074] 7) Referring to the above example, the first editing plasmid was transformed into the competent ZM4 strain, and positive colonies were screened from the transformants. The screening process included colony PCR. The primer pair used for colony PCR was 0128-OUT-F and 0128-OUT-R. The verification result was as follows: Figure 2 Top middle picture.

[0075] 8) Referring to the above example, the second editing plasmid was transformed into the competent strain obtained in step 3, and positive colonies (i.e., ZE2 strains) were screened from the transformants. The screening process included colony PCR. The primer pairs used for colony PCR were 1933-OUT-F (shown in SEQ ID NO: 25) and groELS-grpE-in-R (shown in SEQ ID NO: 26). The verification results were as follows: Figure 2Picture below.

[0076] Fermentation testing of engineered strains

[0077] 1. Fermentation test in RMG5 medium

[0078] Glycerol bacteria of strains ZM4, ZE1, and ZE2 were transferred to a 50mL Erlenmeyer flask containing 40mL RMG5 liquid medium to recover and prepare seed liquid. After overnight culture, it reached the mid-exponential phase and was inoculated into a 50mL Erlenmeyer flask containing 40mL RMG5 liquid medium containing 75g / L ethanol. The initial OD600 nm value was 0.1 and it was placed in a shaker at 33℃ and 100rpm for fermentation. During the fermentation process, the absorbance value of the cells was measured at 600nm by a spectrophotometer every 3h, and 1mL of the supernatant was collected by centrifugation at 12,000rpm for 2min. The glucose concentration and ethanol concentration in the culture supernatant were detected by HPLC through a 0.22μm filter. Among them, the components of RMG5 liquid medium: 50g / L glucose, 10g / L yeast extract, 2g / LKH2PO4, pH 5.8, and sterilized at 108℃ for 30min.

[0079] like Figure 3 As shown, the cell growth of the recombinant strains ZE1 and ZE2 was significantly better than that of the control strain ZM4, and the final OD600nm of the three were 1.85±0.04, 2.25±0.03, and 2.32±0.03, respectively.

[0080] like Figure 4 As shown in the figure, at the 36th hour of fermentation, the control strain ZM4 consumed about 21.67±0.03g / L of glucose and produced 9.43±0.05g / L of ethanol. The recombinant strain ZE1 consumed 9.83±0.02g / L more glucose than the control strain ZM4, but the ethanol production increased by 5.06±0.04g / L. The recombinant strain ZE2 consumed almost all of the glucose and produced 20.09±0.02g / L of ethanol.

[0081] This shows that the ethanol tolerance of the recombinant strains ZE1 and ZE2 is better than that of the control strain ZM4, and the ethanol production at the end point of the fermentation is also significantly improved.

[0082] 2. Fermentation test in corncob waste hydrolyzate

[0083] The glycerol bacteria of strains ZM4, ZE1 and ZE2 were transferred to a 50mL triangular flask containing 40mL RMG5 liquid culture medium for recovery to prepare seed liquid. After overnight culture, the medium reached the mid-exponential phase and was inoculated in a 50mL triangular flask containing 40mL corncob waste residue hydrolyzate. The fermentation test was carried out under the same conditions as above, which will not be described in detail here. HPLC detected the glucose concentration and ethanol concentration in the culture supernatant. The results are shown in Table 2, in which glucose consumption and ethanol production are shown, wherein glucose consumption is the difference between the original glucose content in the corncob waste residue hydrolyzate and the glucose concentration in the obtained fermentation supernatant, and ethanol production is the ethanol concentration in the obtained fermentation supernatant.

[0084] As shown in Table 2, the ethanol production of ZE1 and ZE2 were 85.70±2.97 g / L and 86.51±2.45 g / L, respectively, which were higher than the value of the control strain (76.16±5.62 g / L), an increase of 17%±9%, and the ethanol yield was also increased by 15.5%±10%. This shows that the recombinant strains ZE1 and ZE2 can effectively improve the efficiency of producing cellulosic ethanol from corncob waste in practical applications.

[0085] Table 2

[0086] strain Glucose consumption (g / L) Ethanol production (g / L) ZM4 149.42±8.51 76.16±5.62 ZE1 170.15±9.80 85.70±2.97 ZE2 177.71±0.98 86.51±2.45

[0087] 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, the engineered strain containing hfq gene; groELS gene; and grpE gene of Zymomonas mobilis.

2. According to the engineered strain of claim 1, the hfq gene is located on the genome of the Molecular Mononas or is free outside the genome; Optionally, the groELS gene is located on the genome of the Mobilomonas or is free outside the genome; Optionally, the grpE gene is located on the genome of the Mobilomonas or is free outside the genome; Optionally, the Zymomonas mobilis is a strain of Z. mobilis ZM4ATCC 31821, and the genome of the strain of Z. mobilis ZM4ATCC 31821 is shown in GenBank: CP023715.1; Optionally, the hfq gene is shown as SEQ ID NO:1, the groELS gene is shown as SEQ ID NO:2, and the grpE gene is shown as SEQ ID NO:

3.

3. An engineered strain, which is a Z.mobilis ZM4ATCC 31821 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1650 region of the genome is replaced with the hfq gene, the genome of the Z.mobilis ZM4ATCC 31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, and the ZMO1650 is shown in the 1697900..1699036 nt interval of GenBank: CP023715.

1.

4. An engineered strain, which is a Z.mobilis ZM4ATCC 31821 strain in which the ZMO0128 region of the genome is knocked out and the ZMO1933 region of the genome is replaced with the groELS gene and the grpE gene, the genome of the Z.mobilis ZM4 ATCC31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the 115570..11806 interval of GenBank: CP023715.1, and the ZMO1933 is shown in the 1990679..1992058 nt interval of GenBank: CP023715.

1.

5. A method for preparing an engineered strain, comprising: A first editing plasmid, a second editing plasmid and a third editing plasmid were prepared, wherein the first editing plasmid carried a first guide sequence for guiding the targeted cleavage of the Cas enzyme in the ZMO0128 region of the Z.mobilis ZM4ATCC 31821 strain genome, the second editing plasmid carried a second guide sequence for guiding the targeted cleavage of the Cas enzyme in the ZMO1650 region of the Z.mobilis ZM4ATCC 31821 strain genome and a second donor sequence for replacing the ZMO1650 region, the third editing plasmid carried a third guide sequence for guiding the targeted cleavage of the Cas enzyme in the ZMO1933 region of the Z.mobilis ZM4 ATCC 31821 strain genome and a third donor sequence for replacing the ZMO1933 region, the Z.mobilis ZM4 The genome of ATCC31821 strain is shown in GenBank: CP023715.1, the ZMO0128 is shown in the interval 115570..11806 of GenBank: CP023715.1, the ZMO1650 is shown in the interval 1697900..1699036 nt of GenBank: CP023715.1, and the ZMO1933 is shown in the interval 1990679..1992058 nt of GenBank: CP023715.1; Transforming at least one of the first editing plasmid, the second editing plasmid and the third editing plasmid into the Z.mobilis ZM4ATCC 31821 strain; The positive colonies screened from the transformants are the engineered strains.

6. According to the preparation method of claim 5, the first editing plasmid also carries a first donor sequence, and the first donor sequence consists of an upstream sequence of the ZMO0128 region and a downstream sequence of the ZMO0128 region.

7. The preparation method according to claim 5, wherein the second donor sequence consists of an upstream sequence of the ZMO1650 region, an hfq gene, and a downstream sequence of the ZMO1650 region.

8. The preparation method according to claim 5, wherein the third donor sequence consists of the upstream sequence of the ZMO1933 region, the groELS gene, the grpE gene and the downstream sequence of the ZMO1933 region.

9. A method for producing ethanol using corncob waste residue, comprising: Obtaining at least one engineered strain according to claim 1 to 4; The engineered strain is fermented in corncob waste hydrolyzate or in a culture medium containing corncob waste hydrolyzate.

10. Use of the engineered strain according to any one of claims 1 to 4 or the engineered strain obtained by the preparation method according to any one of claims 5 to 8, wherein the use is selected from: Fermentation of monosaccharides; Preparation of ethanol; Degrade at least one of the hydrolyzate of corncob waste.

Images