Method for producing high-concentration ethanol through presaccharification and synchronous enzymolysis and fermentation of high-temperature-resistant fermentation strain and application of high-temperature-resistant fermentation strain

Abstract

The invention relates to a method for producing high-concentration ethanol through presaccharification and synchronous enzymolysis fermentation of a high-temperature-resistant fermentation strain in the field of microbial fermentation and application of the method. The method for producing high-concentration ethanol through pre-saccharification and synchronous enzymolysis fermentation of the high-temperature-resistant fermentation strain comprises the following steps: step 1, adding water into a cellulose raw material to obtain an ingredient liquid; step 2, adding enzyme into the ingredient liquid for pre-saccharification, and then adjusting the pH value to obtain pre-saccharified liquid; step 3, adding a fermentation strain into the pre-saccharified liquid, and adjusting the temperature to carry out synchronous enzymolysis fermentation, so as to obtain fermentation liquid containing ethanol; the fermentation strain is a high-temperature-resistant saccharomyces cerevisiae strain and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.27605, and the preservation name is SEB22. When the specific saccharomyces cerevisiae strain and the specific synchronous enzymolysis fermentation method are used for producing ethanol, the concentration of ethanol in fermentation liquor can reach 70 g / L or above.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and more particularly to a method and application of producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation bacteria. Background Art

[0002] As energy crisis and environmental pollution become increasingly serious, renewable clean energy fuel ethanol has received great attention. Compared with food raw materials such as corn and cassava, the production of fuel ethanol using cellulosic raw materials such as plant straw is the main development direction of fuel ethanol in my country. Straw can be converted into ethanol through pretreatment, enzymatic hydrolysis, fermentation and other steps. In order to improve production efficiency, simultaneous saccharification and fermentation (SSF) is usually used for production. However, there are significant differences between the optimal temperature for enzymatic hydrolysis (45℃~50℃) and the optimal temperature for fermentation (30℃~35℃), resulting in high cooling costs and low fermentation efficiency. Increasing the SSF temperature is an effective way to reduce production costs and improve fermentation efficiency. However, yeast cells face high temperature stress during high-temperature SSF, which seriously hinders cell growth and fermentation. Therefore, after selecting and breeding Saccharomyces cerevisiae strains with excellent high temperature tolerance, the development and utilization of high-temperature fermentation strains for pre-saccharification and simultaneous enzymatic hydrolysis and fermentation to produce ethanol will help increase ethanol production and reduce production costs. Summary of the invention

[0003] In order to solve the above problems existing in the prior art, the present invention provides a method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation strains. Specifically, it relates to a method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation strains and its application.

[0004] One of the purposes of the present invention is to provide a method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of a thermostable fermentation strain, which may include the following steps:

[0005] Step 1, adding water to the cellulose raw material to obtain a batching liquid;

[0006] Step 2: adding enzyme to the batch liquid for pre-saccharification, adding alkali or acid, adjusting the pH, and obtaining a pre-saccharified liquid;

[0007] Step 3, adding fermentation bacteria to the pre-saccharification liquid, and adjusting the temperature to perform simultaneous enzymatic hydrolysis and fermentation to obtain a fermentation liquid containing ethanol;

[0008] The fermentation strain is a thermostable Saccharomyces cerevisiae strain, which is deposited in the General Microbiological Center of China Microbiological Culture Management Committee with a deposit number of CGMCC NO.27605 and a deposit name of SEB22.

[0009] in,

[0010] In step one,

[0011] The weight concentration of cellulose in the batching solution may be 8% to 15%.

[0012] In step 2, the enzyme may be cellulase; the amount of enzyme added may be 5 to 15 fpu / g cellulose, and the pre-saccharification may be performed for 12 to 36 hours at 45 to 55°C.

[0013] The pH can be adjusted to 3-6.

[0014] In step three,

[0015] The fermentation inoculation amount of the fermentation bacteria may be 0.5 to 2 g cells (dry weight) / L fermentation liquid;

[0016] and / or,

[0017] The temperature can be adjusted from 33℃ to 44℃.

[0018] In step three,

[0019] The time of simultaneous enzymatic hydrolysis and fermentation can be 48h to 144h.

[0020] During the simultaneous enzymatic hydrolysis and fermentation process, feed and enzyme supplementation can be carried out in batches;

[0021] Preferably, in batch feeding and enzyme supplementation, the time interval between feeding and enzyme supplementation can be 24-48 hours; the feeding amount is such that the final weight concentration of cellulose in the fermentation broth is 15%-35%; and the batch enzyme supplementation amount can be 5-25 fpu / g cellulose.

[0022] In step three,

[0023] The ethanol concentration in the ethanol-containing fermentation liquid can reach above 70 g / L.

[0024] Among them, the cellulose raw material used in this application may be at least one of corn straw cellulose, corn cob cellulose, rice straw cellulose, wheat straw cellulose, reed cellulose, and bean straw cellulose. Specifically, cellulose commonly used in the art can be selected, or cellulose prepared according to the general method in the art can be used. Cellulose can also be prepared according to the method in the Chinese patent with application number 202310857792.3. The entire contents of the patent application (application number 202310857792.3, subject name: Method and application of co-production of cellulose, xylose and lignin from lignocellulosic raw materials) are introduced here.

[0025] The cellulose preparation method may specifically include the following steps:

[0026] Step 1: After the lignocellulose raw material is crushed and impurities are removed, it is acid-impregnated, and after the acid-impregnation liquid is removed, it is treated by steam explosion to obtain a pretreated material, in which the hemicellulose is hydrolyzed to obtain xylose; the acid-impregnation liquid after removal can be recycled and used in the acid-impregnation step;

[0027] Step 2, washing the pretreated material with water, and then performing solid-liquid separation to obtain a liquid phase 1 containing xylose and a solid material 1 containing cellulose and lignin;

[0028] Step 3, adding the solid material 1 into an alkali solution for alkali treatment, and then separating the solid and the liquid to obtain a liquid phase 2 and a solid material 2 containing cellulose;

[0029] Optionally, in step 4, the solid material 2 is washed with water, and then the solid and liquid are separated to obtain a cellulose wet material and a liquid phase 3;

[0030] Optionally, in step five, drying the wet cellulose material to obtain cellulose;

[0031] Optionally in step six, liquid phase two and optional liquid phase three may be combined, and then acid is added for acid treatment to separate the solid and the liquid to obtain a lignin wet material and a waste liquid;

[0032] Optionally, in step seven, the wet lignin material is dried to obtain lignin.

[0033] The size of the stalks after the lignocellulose raw material is crushed is in the range of 1 to 10 cm.

[0034] The present invention adopts a pretreatment method combining acid impregnation and steam explosion, so that the structures among cellulose, hemicellulose and lignin are destroyed without being excessively decomposed into other impurities, which is more conducive to separation.

[0035] The lignocellulose raw material can be selected from at least one of corn straw, corn cob, rice straw, wheat straw, reed and bean straw.

[0036] Specifically, in the method for preparing cellulose,

[0037] In step one,

[0038] The acid impregnation solution is a sulfuric acid solution, preferably the concentration of the sulfuric acid solution is 0.5wt% to 4wt%; and / or,

[0039] The acid immersion time is 2 to 20 minutes; and / or,

[0040] The acid impregnation temperature is 10 to 90°C; and / or,

[0041] After the acid pickling solution is removed, the acid pickling solution is recovered and used for the next acid pickling treatment;

[0042] The temperature of steam explosion treatment is 120℃~230℃;

[0043] The pressure of steam explosion treatment is 0.3MPaG~1.5MPaG;

[0044] The steam explosion treatment time is 20 minutes to 60 minutes.

[0045] Specifically, in the method for preparing cellulose,

[0046] In step 2 and step 4, the solid-liquid ratio during water washing is independently selected from 1:5 to 1:10, and the number of water washings is independently 2 to 10 times.

[0047] Specifically, in the method for preparing cellulose,

[0048] In step 2, step 3, step 4 and step 6, the solid-liquid separation method is independently selected from at least one of plate and frame filtration, centrifugal separation, screw extrusion dehydration and vacuum belt filtration.

[0049] Specifically, in the method for preparing cellulose,

[0050] In step 3, the alkali solution is an aqueous solution of an inorganic strong base, more preferably an aqueous solution of at least one alkali selected from sodium hydroxide, potassium hydroxide, and calcium hydroxide;

[0051] In the alkali solution, the dry matter concentration of the solid material 1 is 5 to 15% (g / g); and / or,

[0052] The amount of alkali added is 5% to 20% of the dry matter in the solid material; and / or,

[0053] The temperature of the alkali treatment is 20°C to 80°C; and / or,

[0054] The alkali treatment time is 1h to 4h.

[0055] Specifically, in the method for preparing cellulose,

[0056] In step 5 and step 7, the drying methods are independently selected from at least one of tube bundle drying, rotary drying, drum drying, and air flow drying.

[0057] Specifically, in the method for preparing cellulose,

[0058] In step 6, the acid used in the acid treatment is selected from inorganic strong acids, preferably at least one selected from sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid;

[0059] The amount of acid added is 1 to 1.5 times the amount of alkali added in the alkali treatment in step 3; and / or,

[0060] The temperature of the acid treatment is 20 to 50°C; and / or,

[0061] The acid treatment time is 0.5 to 2 hours.

[0062] Specifically, in the cellulose preparation method, in step six, after the acid treatment, heating is performed, and then solid-liquid separation is performed; preferably, the heating temperature is 70 to 90° C., and the heating time is 0.5 to 2 hours.

[0063] In the cellulose preparation method, the separation process of each component is as follows:

[0064] Step 1, pretreating the lignocellulose raw material to obtain a pretreated material, in which the hemicellulose is hydrolyzed to obtain xylose; step 2, washing the pretreated material obtained in step 1 and performing solid-liquid separation to obtain a liquid phase containing xylose and a solid material containing cellulose and lignin; step 3, performing alkali treatment on the solid material containing cellulose and lignin obtained in step 2 to dissolve the lignin therein, and then performing solid-liquid separation to obtain a liquid phase containing lignin and a solid material containing cellulose; optionally step 4, washing the solid material containing cellulose obtained in step 3 and performing solid material separation to obtain a wet cellulose material and a washing liquid containing lignin; optionally step 5, drying the wet cellulose obtained in step 4 to obtain cellulose; optionally step 6, combining the liquid phase containing lignin obtained in step 3 and the washing liquid containing lignin obtained in step 4, and then performing acid treatment to precipitate lignin, and then heating and solid-liquid separation to obtain a wet lignin material, wherein the liquid phase is wastewater; optionally step 7, drying the wet lignin material obtained in step 6 to obtain lignin. The separation method uses fewer types of chemical reagents, has a simple process and has a better separation effect.

[0065] The present application can use the cellulose obtained in step four or step five (preferably step five) of the cellulose preparation method as a raw material for the simultaneous enzymatic hydrolysis and fermentation of the cellulose raw material to produce high-concentration ethanol according to the technical scheme of the present application.

[0066] Among them, regarding the thermostable Saccharomyces cerevisiae strain used in this application,

[0067] During the breeding process of high-temperature resistant strains, the inventor team found that transcription factor CRZ1 and L-asparaginase II encoding gene ASP3 play an important role in affecting the high temperature tolerance of Saccharomyces cerevisiae. After a lot of practice, the inventor found that if SEB4 is used as the starting strain, CRZ1 and ASP3 are jointly regulated at the same time, which will have obvious advantages in improving the high temperature tolerance of the strain. Therefore, this application uses SEB4 (CGMCC11324) as the starting strain, and uses CRISPR / Cas9 gene editing technology to collaboratively knock out ASP3 and highly express CRZ1 to obtain the engineered strain SEB22 (Saccharomyces cerevisiaeSEB22). The high-temperature resistant Saccharomyces cerevisiae strain in the present invention is related to the patent with application number 202311311773.7, and the entire content of the patent application (application number: 202311311773.7, the subject name is: a high-temperature resistant Saccharomyces cerevisiae strain and its construction method and application) is introduced in full here.

[0068] The thermostable Saccharomyces cerevisiae strain is deposited in the General Microbiological Center of China Microbiological Culture Management Committee with a deposit number of CGMCC NO.27605 and a deposit name of SEB22.

[0069] The method for constructing the thermostable Saccharomyces cerevisiae strain is to use SEB4 as the starting strain, and to use a gene editing method to knock out the L-asparaginase II encoding gene ASP3 and the highly expressed transcription factor CRZ1 to obtain the engineered strain SEB22; wherein the starting strain SEB4 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCCNO.11324. wherein the gene editing method can be a CRISPR / Cas9 gene editing method.

[0070] Specifically, the method for constructing the thermostable Saccharomyces cerevisiae strain may include the following steps:

[0071] 1) Construction of gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1;

[0072] 2) construct repair fragments for ASP3 knockout and CRZ1 overexpression;

[0073] 3) Preparation of Cas9-NAT plasmid;

[0074] 4) Perform CRISPR / Cas9 transformation on the starting strain.

[0075] Wherein, the CRISPR / Cas9 transformation may include the following steps:

[0076] 1) introducing the Cas9-NAT plasmid into the starting strain to obtain a yeast strain containing the Cas9-NAT plasmid; the introduction method is preferably a lithium acetate transformation method;

[0077] 2) Knockout of the coding gene ASP3;

[0078] 3) High expression of transcription factor CRZ1.

[0079] Wherein, the method for knocking out the coding gene ASP3 may include the following steps:

[0080] The pMEL13-ASP3 plasmid and the repair fragment for ASP3 knockout were transferred into the yeast strain already containing the Cas9-NAT plasmid, and the plasmid was removed from the transformant to obtain the SEB4 ASP3 knockout strain.

[0081] The method for highly expressing the transcription factor CRZ1 may include the following steps:

[0082] The endogenous promoter of CRZ1 was replaced with the TEF1 promoter to highly express the transcription factor CRZ1;

[0083] Preferably, the method may specifically include the following steps: first, introducing the Cas9-NAT plasmid into the ASP3 knockout strain of SEB4; further introducing the pMEL13-CRZ1 plasmid and a repair fragment for high expression of CRZ1 into the ASP3 knockout strain of SEB4 containing the Cas9-NAT plasmid, and performing plasmid removal on the transformant to obtain the ASP3 knockout and CRZ1 high expression strain SEB22.

[0084] The second object of the present invention is to provide an application of the method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation strains, specifically the application of the method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of cellulose as raw material and high-temperature resistant brewer's yeast strains as fermentation strains as described in one of the objects of the present invention.

[0085] The technical solution of the present invention utilizes a selected brewer's yeast strain with excellent high temperature tolerance to carry out synchronous enzymatic fermentation to produce ethanol, and the ethanol concentration in the fermentation liquid can reach more than 70g / L.

[0086] Strain deposit information

[0087] The thermostable Saccharomyces cerevisiae strain is deposited in the General Microbiology Center of China Microbiological Culture Management Committee, with the deposit number: CGMCC NO.27605, the deposit name: SEB22, the classification name: Saccharomyces cerevisiae, and the deposit date: June 12, 2023. DETAILED DESCRIPTION

[0088] The present invention is described in detail below in conjunction with specific embodiments. It is necessary to point out that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by those skilled in the art based on the contents of the present invention still fall within the scope of protection of the present invention.

[0089] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0090] Source of raw materials

[0091] The raw materials used in the examples and comparative examples, unless otherwise specified, are disclosed in the prior art, for example, they can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0092] Corn stalk cellulose is prepared according to the following method:

[0093] The method comprises the following steps:

[0094] Step 1, using corn stalks as raw materials, crushing and removing impurities from the corn stalks, acid impregnating, removing the acid impregnation liquid, and then pretreating the corn stalk raw materials by steam explosion. Specifically, the corn stalk raw materials are impregnated with 2.5wt% dilute sulfuric acid, the acid impregnation time is 10 minutes, and the acid impregnation temperature is 50°C; after acid impregnation, the acid liquid is removed by centrifugation, and then the pretreated material is treated at a temperature of 160°C and a pressure of 0.6MPaG for 30 minutes to obtain a pretreated material, in which the hemicellulose is hydrolyzed to obtain xylose.

[0095] Step 2: Wash the pretreated material with water at a solid-liquid ratio of 1:7 for 3 times, and then use plate and frame filter press to separate the solid and liquid to obtain a liquid phase containing xylose and a solid material containing cellulose and lignin; dry the liquid phase containing xylose to obtain xylose.

[0096] Step three, adding the solid material containing cellulose and lignin to an alkali solution for alkali treatment, the alkali treatment adopts sodium hydroxide solution treatment, the amount of sodium hydroxide added is 13% (g / g) of the dry matter weight of the solid material, the dry matter concentration of the solid material containing cellulose and lignin in the alkali solution is 9% (g / g), the alkali treatment temperature is 65°C, the alkali treatment time is 2h, the lignin therein is dissolved, and then centrifugation is used for solid-liquid separation to obtain a liquid phase containing lignin and a solid material containing cellulose.

[0097] Step 4: Wash the solid material containing cellulose with water at a solid-liquid ratio of 1:8 for 4 times, and then separate the solid and liquid by centrifugation to obtain a wet cellulose material and a washing liquid containing lignin.

[0098] Step 5: bundle-drying the wet cellulose material obtained in step 4 to obtain corn straw cellulose.

[0099] Step 6: Combine the lignin-containing liquid phase obtained in step 3 and the lignin-containing water washing liquid obtained in step 4, and then perform acid treatment. The acid treatment uses a sulfuric acid solution (50wt%), and the amount of sulfuric acid added is 1.3 times the mass of sodium hydroxide added in step 3. The acid treatment temperature is 30°C and the time is 1h, so that the lignin precipitates, and then heats to 85°C and maintains for 1h, so that the precipitated lignin aggregates into large particles, and then vacuum belt filtration is used for solid-liquid separation to obtain a lignin wet material.

[0100] Step seven, performing air flow drying on the wet lignin material obtained in step six to obtain lignin.

[0101] The yield and purity results of cellulose, xylose and lignin co-produced using corn straw raw materials are shown in the following table (yield and purity results of cellulose, xylose and lignin).

[0102] Cellulose, xylose and lignin yield and purity results

[0103]

[0104] Take the corn stalk cellulose obtained in step 5 and set aside.

[0105] Example 1 Bacterial strain construction

[0106] 1. Starting strain

[0107] The starting strain SEB4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.11324.

[0108] 2. Culture medium

[0109] The culture medium used is shown in Table 1. If it is a solid culture medium, add 2% agar powder before sterilization. The sterilization conditions are 0.1MPa, 121℃, 15min. After the culture medium is cooled to 50-60℃, add antibiotics.

[0110] Table 1 Culture medium composition

[0111]

[0112]

[0113] 3. Plasmids, strains and primers

[0114] The plasmids and strains used are shown in Table 2; the primers used to construct the gRNA plasmids are shown in Table 3; the primers and fragments used for strain transformation are shown in Table 4.

[0115] Table 2 Plasmid and strain information

[0116]

[0117] (Existing literature [1] Mans R, van Rossum HM, Wijsman M, et al. CRISPR / Cas9: amolecular swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae [J]. FEMS Yeast Research, 2015, 15(2): fov004.

[0118] Existing literature [2] Zhang GC, Kong II, Kim H, et al. Construction of a quadrupleauxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease [J]. Applied and Environmental Microbiology, 2014, 80(24):7694-7701.)

[0119] Table 3 Primers for constructing gRNA plasmids

[0120]

[0121]

[0122] Note: F: upstream primer; R: downstream primer; Underline: 20bp recognition sequence.

[0123] The sequence number of CRZ1 tgR F is SEQ ID No.1, the sequence number of CRZ1 tgR R is SEQ ID No.2, the sequence number of ASP3tgR F is SEQ ID No.3, the sequence number of ASP3 tgR R is SEQ ID No.4, the sequence number of 6006-F is SEQ ID No.5, and the sequence number of 6005-R is SEQ ID No.6.

[0124] Table 4 Primers and fragments required for strain transformation

[0125]

[0126] Note: RE: repair fragment; dg: verification primer; F: upstream primer; R: downstream primer

[0127] The sequence number of CRZ1 RE F is SEQ ID No.7, the sequence number of CRZ1 RE R is SEQ ID No.8, the sequence number of CRZ1 dgF is SEQ ID No.9, the sequence number of CRZ1 dg R is SEQ ID No.10, the sequence number of ASP3 RE F is SEQ ID No.11, the sequence number of ASP3 RE R is SEQ ID No.12, the sequence number of ASP3 dg F is SEQ ID No.13, the sequence number of ASP3dg R is SEQ ID No.14, the sequence number of Cas9-dg-F is SEQ ID No.15, and the sequence number of Cas9-dg-R is SEQ ID No.16.

[0128] 4. Construction of strain SEB22

[0129] 4.1 Construction of gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1

[0130] 1) Construction of double-stranded gRNA fragment

[0131] Referring to the genome information of the model yeast S288c, the FASTA sequences of the ASP3 coding region and the CRZ1 promoter region were inputted at the website http: / / www.e-crisp.org / E-CRISP / to obtain the 20 bp gRNA recognition sequence. Genewise was commissioned to synthesize the primer pairs ASP3 tgR F / R and CRZ1tgR F / R (sequences in Table 3) with 20 bp recognition sequences and 50 bp homology arms upstream and downstream. The two single-stranded primers were diluted to 10 μM with sterile water, mixed in equal volumes, heated at 95°C for 5 min, and cooled at room temperature to obtain double-stranded gRNA fragments.

[0132] 2) Amplify the linear backbone of the gRNA plasmid

[0133] The linear backbone was amplified using the pMEL13 plasmid as a template, and the PCR reaction system and reaction conditions were shown in Table 5. The PCR product was purified using the PCR product purification kit of OMEGA to obtain the linear backbone of the gRNA plasmid.

[0134] Table 5 PCR amplification of gRNA linear backbone

[0135]

[0136] The template plasmid was digested with Quick cut Dpn I. The digestion system and reaction conditions are shown in Table 6. The amount of DpnI used was determined by the amount of template plasmid (1 μg plasmid can be added with 1 μL DpnI). The gRNA linear backbone was purified.

[0137] Table 6 gRNA linear backbone template digestion system

[0138]

[0139] 3) Gibson ligation of gRNA fragments and linear backbone

[0140] The gRNA fragment and the pMEL13 linear backbone were connected by homologous recombination using the Gibson ligation kit. The Gibson ligation reaction system and conditions are shown in Table 7. Take 5 μL of the ligation solution and transform it into Escherichia coli. The transformation solution was spread on the LB+Kan plate and cultured at 37°C overnight. The transformant was inoculated in a test tube containing 5 mL of LB+Kan liquid culture medium and cultured for 12 to 16 hours (160 rpm, 37°C), the bacteria were collected, and the gRNA plasmid was extracted using the plasmid extraction kit of Omega Company. The correct gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1 were confirmed by sequencing.

[0141] Table 7 Gibson ligation reaction system

[0142]

[0143] 4.2 Building repair fragments

[0144] 1) Repair fragment for ASP3 knockout

[0145] The repair fragment for ASP3 knockout consists of 60 bp upstream and 60 bp downstream of the ASP3 coding sequence, and was commissioned to be synthesized in the form of primers by Genewise (sequences are shown in Table 4). The two single-stranded primers were diluted to 10 μM with sterile water, mixed in equal volumes, heated at 95°C for 5 min, and cooled at room temperature to obtain the repair fragment for ASP3 knockout, which can be directly used for yeast transformation.

[0146] 2) Repair fragment for high expression of CRZ1

[0147] The endogenous promoter of CRZ1 was replaced with the TEF1 promoter to achieve high expression of CRZ1. Using the genomic DNA of strain SEB4 as a template, primers CRZ1 RE F and CRZ1 RE R were used to amplify the repair fragment containing the TEF1 promoter sequence and homology arms (primer sequence is shown in Table 4, TEF1 promoter sequence is shown in Table 8). The PCR reaction system and reaction conditions are shown in Table 9. The PCR product was purified and used for transformation.

[0148] Table 8 TEF1 promoter sequence

[0149]

[0150] P TEF1 The sequence number is SEQ ID No.17.

[0151] Table 9 PCR amplification repair fragment

[0152]

[0153] 4.3 Cas9-NAT plasmid preparation

[0154] E. coli containing the Cas9-NAT plasmid was inoculated into 5 mL of LB+NAT liquid medium and cultured at 37°C and 160 rpm for 12 to 16 hours. The bacteria were collected by centrifugation, and the Cas9-NAT plasmid was extracted using a plasmid extraction kit from OMEGA.

[0155] 4.4 CRISPR / Cas9 transformation

[0156] 1) Introduction of Cas9-NAT plasmid into Saccharomyces cerevisiae strains by lithium acetate transformation

[0157] a. Streak strain SEB4 on a 2% YPD plate for activation, take an appropriate amount of cells and inoculate into 5 mL 2% YPD liquid medium, and culture at 30°C and 160 rpm for 16 h;

[0158] b. Take 2 mL of bacterial solution and inoculate it into 100 mL of 2% YPD culture medium. Cultivate at 30°C and 160 rpm for 2 to 3 hours. 600 When the concentration reached 0.2-0.3, all the bacteria were collected by centrifugation, the bacterial precipitate was washed twice with sterile water, resuspended in 300 μL sterile water, and placed on ice for later use;

[0159] c. Boil salmon sperm DNA (ssDNA) at 100°C for 5 min and place on ice for later use;

[0160] d. Add 60% PEG4000 (115 μL), 4 M lithium acetate solution (5 μL), ssDNA (10 μL), Cas9-NAT plasmid (100 ng) and yeast cells (50 μL) in a 1.5 mL centrifuge tube, mix well and heat shock at 42°C for 40 min;

[0161] e. Centrifuge at 8,000 × g for 1 min, discard the supernatant, wash the cells twice with sterile water, add 1 mL of 2% YPD culture medium, and culture at 30°C and 160 rpm for 2 to 3 h;

[0162] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, and resuspend the cells in 1 mL of sterile water. Spread 100 μL of the bacterial solution on a 2% YPD+NAT plate and culture at 30°C for 2 to 3 days;

[0163] g. Streak the transformants onto 2% YPD+NAT plates and perform colony PCR verification after culturing for 24 h.

[0164] 2) Colony PCR verification of Cas9-NAT plasmid in yeast transformants

[0165] a. Add 95 μL 1% SDS, 5 μL 4M lithium acetate solution and appropriate amount of bacteria to a 1.5 mL centrifuge tube, vortex and incubate at 75°C for 10 min.

[0166] b. Add 300 μL of anhydrous ethanol to the centrifuge tube and vortex; centrifuge at 13000 rpm for 3 min at room temperature, discard the supernatant, and dry at 37°C with the lid open for 10 min;

[0167] c. Add 100 μL sterile water, vortex, centrifuge at 13000 rpm for 1 min at room temperature, and retain the supernatant;

[0168] d. Determine the concentration of the supernatant, adjust the OD to 10-20 ng / μL, take 1 μL as a template for PCR verification, and the PCR reaction system and reaction conditions are shown in Table 10;

[0169] e. 1.5% agarose gel electrophoresis (100 V, 30 min) was used to verify the target band.

[0170] Table 10 Colony PCR Verification System

[0171]

[0172] 3) ASP3 knockout

[0173] The pMEL13-ASP3 plasmid and the knockout repair fragment were transferred into the yeast strain containing the Cas9-NAT plasmid. The transformation process was the same as 1). Nourseothricin (NAT) was added to the 2% YPD medium to ensure the stable presence of the Cas9-NAT plasmid. The transformation system is shown in Table 11. The cells were resuspended with 200 μL of sterile water, and all the bacterial liquid was spread on a 2% YPD+NAT+G418 plate and cultured at 30°C for 2 to 3 days. The transformants were picked for colony PCR verification. The specific method was the same as 2). The target fragment was amplified using primers ASP3 dg F and ASP3 dg R (Table 4). The PCR reaction system and reaction conditions are shown in Table 10. The extension time and annealing temperature were adjusted according to the characteristics of the primers. The PCR product with the correct band was sequenced for confirmation. The plasmid of the correct transformant was further removed.

[0174] Table 11 Target gene transformation system

[0175]

[0176] 4) Removal of plasmid

[0177] Remove the Cas9-NAT plasmid and gRNA plasmid of the correct transformant. The specific steps are as follows:

[0178] a. Streak the correct transformants onto 2% YPD plates, pick a small amount of cells and inoculate into 10 mL 2% YPD medium, and culture at 30°C for 1 day.

[0179] b. Take 200 μL of bacterial solution and dilute 10 5 times, take 100 μL of bacterial solution and spread it on 2% YPD plate, and culture for 1-2 days;

[0180] c. Take a small amount of cells and resuspend them in 200 μL sterile water (pick 5 to 8 single bacteria for each transformant), take 2 μL of the bacterial suspension and spot them on 2% YPD, 2% YPD+G418 and 2% YPD+NAT plates, and culture them at 30°C for 1-2 days; the strain that can only grow on 2% YPD plates has successfully removed the plasmid, and the ASP3 knockout strain of SEB4 (SEB19) is obtained.

[0181] 5) CRZ1 high expression

[0182] CRZ1 was highly expressed in SEB4 and its ASP3 knockout strain, respectively. First, Cas9-NAT plasmid was introduced into SEB4 and its ASP3 knockout strain, the method was the same as 1). The pMEL13-CRZ1 plasmid and the repair fragment for high expression were further introduced into SEB4 and its ASP3 knockout strain containing Cas9-NAT plasmid, the method was the same as 3). Colony PCR verification was performed using primers CRZ1 dg F and CRZ1 dg R (Table 4). The PCR reaction system and reaction conditions are shown in Table 10, and the PCR products with correct bands were sequenced for confirmation. Plasmid removal was performed on the correct transformants, the method was the same as 4). Finally, the CRZ1 highly expressed strain SEB4-CRZ1 and the ASP3 knockout and CRZ1 highly expressed strain SEB22 of the present invention were obtained. The strain SEB22 was cultured for standby use.

[0183] Example 2

[0184] This embodiment provides a method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria, comprising the following steps:

[0185] Step 1: using corn stalk cellulose as raw material, adding water to the cellulose raw material to prepare a batching liquid with a cellulose weight content of 10%.

[0186] Step 2: adding cellulase to the batch liquid at an enzyme dosage of 12 fpu / g cellulose, pre-saccharifying at 50° C. for 24 h, and adjusting the pH of the batch liquid to 5 after the pre-saccharification.

[0187] Step 3: Add a thermostable Saccharomyces cerevisiae strain (CGMCC NO.27605, deposited name SEB22) to the pre-saccharification liquid, with an inoculation amount of 1 g cell (dry weight) / L fermentation liquid, and perform simultaneous enzymatic fermentation at 35° C. for 120 h.

[0188] During the simultaneous enzymatic fermentation process, feed was added twice at 24h and 72h respectively, so that the final cellulose addition amount was 25% of the weight of the fermentation liquid; and 16 fpu / g cellulose was added at the same time during the two feed additions.

[0189] After the simultaneous enzymatic hydrolysis and fermentation, an ethanol-containing fermentation broth was obtained, and the ethanol concentration in the fermentation broth was 85 g / L.

[0190] Example 3

[0191] This embodiment provides a method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria, comprising the following steps:

[0192] Step 1: using corn stalk cellulose as raw material, adding water to the cellulose raw material to prepare a batching liquid with a cellulose weight content of 10%.

[0193] Step 2: adding cellulase to the batch liquid at an enzyme dosage of 12 fpu / g cellulose, pre-saccharifying at 50° C. for 24 h, and adjusting the pH of the batch liquid to 5 after the pre-saccharification.

[0194] Step three: adding a thermostable Saccharomyces cerevisiae strain to the pre-saccharification liquid, with an inoculation amount of 1 g of cells (dry weight) / L of fermentation liquid, and performing simultaneous enzymatic fermentation at 40° C. for 72 h.

[0195] During the simultaneous enzymatic fermentation process, feed was added once every 24 hours, so that the final cellulose addition amount was 19% of the weight of the fermentation liquid; and enzyme was supplemented at 16 fpu / g cellulose at the same time as the feed.

[0196] After the simultaneous enzymatic hydrolysis and fermentation, an ethanol-containing fermentation broth was obtained, and the ethanol concentration in the fermentation broth was 71 g / L.

[0197] Comparative Example 1

[0198] Step 1: using corn stalk cellulose as raw material, adding water to the cellulose raw material to prepare a batching liquid with a cellulose weight content of 10%.

[0199] Step 2: adding 12 fpu / g cellulase to the batch liquid, pre-saccharifying at 50°C for 24 hours, and adjusting the pH of the batch liquid to 5 after the pre-saccharification.

[0200] Step 3: Add the starting strain SEB4 of the thermostable Saccharomyces cerevisiae strain (which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.11324) to the pre-saccharification liquid, with an inoculation amount of 1 g cell (dry weight) / L fermentation liquid, and perform synchronous enzymatic fermentation at 35°C for 120 hours.

[0201] During the simultaneous enzymatic fermentation process, feed was added twice at 24h and 72h respectively, so that the final cellulose addition amount was 25% of the weight of the fermentation liquid; and 16 fpu / g cellulose was added at the same time during the two feed additions.

[0202] After the simultaneous enzymatic hydrolysis and fermentation, an ethanol-containing fermentation broth was obtained, and the ethanol concentration in the fermentation broth was 62 g / L.

[0203] It can be seen from the above Examples 1, 2 and Comparative Example 1 that by producing ethanol using the specific brewer's yeast strain described in the present application and the specific simultaneous enzymatic fermentation method, the ethanol concentration in the fermentation broth can reach above 70 g / L, which has obvious technical effects and helps to increase ethanol production and reduce production costs.

Claims

1. A method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria, characterized in that: The following steps are involved: Step 1, adding water to the cellulose raw material to obtain a batching liquid; Step 2, adding enzymes to the batch liquid for pre-saccharification, and adjusting the pH to obtain a pre-saccharified liquid; Step 3, adding fermentation bacteria to the pre-saccharification liquid, and adjusting the temperature to perform simultaneous enzymatic hydrolysis and fermentation to obtain a fermentation liquid containing ethanol; The fermentation strain is a thermostable Saccharomyces cerevisiae strain, which is deposited in the General Microbiological Center of China Microbiological Culture Management Committee with a deposit number of CGMCC NO.27605 and a deposit name of SEB22.

2. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature fermentation bacteria according to claim 1, characterized in that: In step one, In the batching liquid, the weight concentration of cellulose is 8% to 15%; and / or, In step 2, The enzyme is cellulase; preferably, the enzyme is added in an amount of 5 to 15 fpu / g cellulose, and the pre-saccharification is carried out at 45 to 55° C. for 12 to 36 hours; and / or, Adjust the pH to 3-6.

3. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation bacteria according to claim 1, characterized in that: In step three, The fermentation inoculation amount of the fermentation bacteria is 0.5-2 g cells (dry weight) / L fermentation liquid; and / or, Adjust the temperature to 33°C to 44°C; and / or, The time of simultaneous enzymatic hydrolysis and fermentation is 48h to 144h; and / or, In step three, The ethanol concentration in the ethanol-containing fermentation liquid reaches above 70 g / L.

4. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria according to claim 1, characterized in that: In step three, Batch feeding and enzyme supplementation are carried out during the simultaneous enzymatic hydrolysis and fermentation process; Preferably, in batch feeding and enzyme supplementation, the time interval between feeding and enzyme supplementation is 24-48 hours; the feeding amount is such that the final cellulose concentration is 15%-35% of the weight concentration of the fermentation liquid; and the enzyme supplement amount is 5-25 fpu / g cellulose.

5. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation bacteria according to claim 1, characterized in that: The cellulose is prepared by a method comprising the following steps: a), crushing and removing impurities from the lignocellulose raw material, acid impregnating, removing the acid impregnation liquid and then treating with steam explosion to obtain a pretreated material; b), washing the pretreated material with water, and then performing solid-liquid separation to obtain a liquid phase containing xylose and a solid material containing cellulose and lignin; c), adding the solid material 1 into an alkali solution for alkali treatment, and then separating the solid and the liquid to obtain a liquid phase 2 and a solid material 2 containing cellulose; Optionally, d), washing the solid material 2 with water, and then separating the solid and the liquid to obtain a cellulose wet material and a liquid phase 3; Optionally, e), drying the wet cellulose material to obtain cellulose; Preferably, the lignocellulosic raw material is selected from at least one of corn straw, corn cob, rice straw, wheat straw, reed and bean straw.

6. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria according to claim 5, characterized in that: In step a), The acid impregnation solution is a sulfuric acid solution, preferably the concentration of the sulfuric acid solution is 0.5wt% to 4wt%; and / or, The acid immersion time is 2 to 20 minutes; and / or, The acid impregnation temperature is 10 to 90°C; and / or, After the acid pickling solution is removed, the acid pickling solution is recovered and used for the next acid pickling treatment; The temperature of steam explosion treatment is 120℃~230℃; The pressure of steam explosion treatment is 0.3MPaG~1.5MPaG; The steam explosion treatment time is 20 minutes to 60 minutes.

7. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation bacteria according to claim 5, characterized in that: In step b) and step d), the solid-liquid ratio during water washing is independently selected from 1:5 to 1:10, and the number of water washings is independently 2 to 10 times; and / or, In step b), step c) and step d), the solid-liquid separation methods are each independently selected from at least one of plate and frame filtration, centrifugal separation, screw extrusion dehydration, and vacuum belt filtration.

8. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria according to claim 5, characterized in that: In step c), the alkali solution is an aqueous solution of an inorganic strong base, more preferably an aqueous solution of at least one alkali selected from sodium hydroxide, potassium hydroxide, and calcium hydroxide; In the alkali solution, the dry matter concentration of the solid material 1 is 5 to 15% (g / g); and / or, The amount of alkali added is 5% to 20% of the dry matter in the solid material; and / or, The temperature of the alkali treatment is 20°C to 80°C; and / or, The alkali treatment time is 1h to 4h.

9. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation bacteria according to any one of claims 1 to 8, characterized in that: The method for constructing a thermostable Saccharomyces cerevisiae strain comprises the following steps: knocking out the L-asparaginase II encoding gene ASP3 and the highly expressed transcription factor CRZ1 in the starting strain to obtain the strain SEB22; the starting strain is a Saccharomyces cerevisiae strain; preferably, the starting strain is strain SEB4, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.11324.

10. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria according to claim 9, characterized in that: The method for constructing the thermostable Saccharomyces cerevisiae strain comprises the following steps: 1) Construction of gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1; 2) construct repair fragments for ASP3 knockout and CRZ1 overexpression; 3) Preparation of Cas9-NAT plasmid; 4) Perform CRISPR / Cas9 transformation on the starting strain.

11. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of high-temperature resistant fermentation bacteria according to claim 10, characterized in that: The CRISPR / Cas9 transformation comprises the following steps: 1) introducing Cas9-NAT plasmid into the starting strain to obtain a yeast strain containing Cas9-NAT plasmid; the introduction method is preferably lithium acetate transformation method; 2) knocking out the coding gene ASP3; 3) over-expressing the transcription factor CRZ1; Preferably, the method for knocking out the coding gene ASP3 comprises the following steps: transferring the pMEL13-ASP3 plasmid and the ASP3 knockout repair fragment into a yeast strain already containing the Cas9-NAT plasmid, and performing plasmid removal on the transformant to obtain the SEB4 ASP3 knockout strain.

12. The method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of thermostable fermentation bacteria according to claim 11, characterized in that: The method for highly expressing the transcription factor CRZ1 comprises the following steps: The endogenous promoter of CRZ1 was replaced with the TEF1 promoter to highly express the transcription factor CRZ1; Preferably, the method comprises the following steps: First, the Cas9-NAT plasmid was introduced into the ASP3 knockout strain of SEB4. Then, the pMEL13-CRZ1 plasmid and the repair fragment for CRZ1 overexpression were introduced into the ASP3 knockout strain of SEB4 containing the Cas9-NAT plasmid, and the plasmid was removed from the transformant to obtain the ASP3 knockout and CRZ1 overexpression strain SEB22.

13. Use of the method for producing high-concentration ethanol by pre-saccharification and simultaneous enzymatic fermentation of a high-temperature-resistant fermentation strain according to any one of claims 1 to 12.

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