A method for producing lactic acid monomer by mixed fermentation using bagasse as raw material

Abstract

The present invention relates to the technical field of fermentation engineering and genetic engineering, and specifically to a production strain and its application for producing lactic acid monomers using bagasse as raw material. The present invention expresses endoglucanase, exoglucanase and β-glucosidase in strains with lactic acid monomer production capacity to obtain a lactic acid monomer production recombinant strain. After compounding, the strain can metabolize bagasse to synthesize high value-added lactic acid monomers. The fermentation tank fermentation level of lactic acid monomers can reach 82.76g / L, and the conversion rate of bagasse to lactic acid can reach 41.38%. It is helpful to supply raw materials for large-scale production of lactic acid, reduce dependence on food or sugars, and optimize the technology of producing lactic acid from bagasse raw materials.

Description

Technical fields:

[0001] This invention relates to the fields of fermentation engineering and genetic engineering, specifically to a production strain for producing lactic acid monomers from sugarcane bagasse and its application, specifically the cloning and expression of a cellulose hydrolase encoding gene, the hydrolysis of lignocellulose, and the fermentation production of lactic acid monomers using the reducing sugar obtained from the hydrolysis as raw material. Background technology:

[0002] Microorganisms can ferment monosaccharides such as glucose, xylose, and galactose to produce lactic acid. With advancements in strain modification and fermentation technology, starch and other raw materials can also be used for lactic acid production. Starch and other raw materials are indispensable food sources in people's lives, but excessive use may lead to unavoidable ethical issues such as competition with humans for food. Utilizing renewable biomass resources can effectively address this problem and alleviate environmental problems caused by the improper use of biomass. Therefore, microbial fermentation methods have advantages over chemical synthesis and enzymatic conversion methods, including a wide range of raw material sources, high safety, high optical purity of products, and less environmental pollution, promoting the rapid development of microbial fermentation in the field of lactic acid synthesis. The main tool for producing L-lactic acid through fermentation is microorganisms, making the selection and breeding of microorganisms particularly important. Research progress in lactic acid production through fermentation both domestically and internationally involves not only the development and optimization of fermentation processes but, more importantly, the natural selection of fermentation strains and the construction of engineered strains capable of producing lactic acid using genetic engineering methods.

[0003] Lactobacillus and Rhizopus can be used in the industrial production of lactic acid. Because lactic acid bacteria can produce high levels of lactic acid and have high acid resistance, they are often used as a class of strains for lactic acid production. Lactobacillus includes Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus helveticus. In recent years, strain selection and production research in the lactic acid industry have received widespread attention. In 2004, Taniguchi et al. proposed two lactic acid bacteria, Lactobacillus casei and Bacillus coagulans IFO, to hydrolyze lignocellulose to obtain 50 g / L xylose and 100 g / L glucose. The lactic acid yield was 95 g / L through fermentation (Horiuchi, et al. Production of L-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic bacteria[J]. Applied Microbiology and Biotechnology, 2004, 66(2): 160-165.). In 2007, Mass RW used cellulase and strain Bacillus coagulans DSM2314 to hydrolyze 2706 g of lime-pretreated wheat bran. After fermentation at 50℃ for 55 h, the total lactic acid content was 711 g, with a conversion rate of 43% (Jansen, et al. Lactic acid production from lime-treated wheat straw by Bacillus coagulans). Coagulans: Neutralization of acid by fed-batch addition of alkaline substrate[J]. Applied Microbiology & Biotechnology, 2008, 78(5): 751-758.). In 2008, Ge XY et al. directly used Jerusalem artichoke fermentation to produce lactic acid by mixing Lactobacillus and Aspergillus niger. The fermentation method was fed-batch fermentation and simultaneous saccharification fermentation. The lactic acid yield at the end of fermentation was 120.5 g / L. Summary of the Invention:

[0004] The purpose of this invention is to achieve the secretion and expression of cellulose hydrolase molecules in lactic acid monomer producing strains through gene cloning and gene recombination technology, thereby obtaining lactic acid monomer producing strains with the ability to metabolize lignocellulose. The obtained strains can directly and efficiently synthesize lactic acid monomers from sugarcane bagasse, thereby expanding the raw material supply for large-scale production of lactic acid monomers, optimizing the production technology of lactic acid monomers from sugarcane bagasse, and applying it to the large-scale production of lactic acid monomers from sugarcane bagasse.

[0005] To achieve the above objectives, one of the technical solutions provided by the present invention is a recombinant strain for lactic acid monomer production that expresses a gene encoding a cellulose hydrolase. The recombinant strain is a strain with lactic acid monomer production capability as the starting strain, and the cellulose hydrolases are endoglucanase, exoglucanase, and β-glucosidase.

[0006] Furthermore, the encoding genes for the endoglucanase, exoglucanase, and β-glucosidase have the nucleotide sequences shown in SEQ ID NO. 1, 2, or 3;

[0007] Furthermore, expression in the starting bacteria is achieved by constructing a recombinant plasmid or recombinant expression cassette containing the above-mentioned cellulose hydrolase encoding gene. Preferably, the expression vector used for the recombinant plasmid includes, but is not limited to, pUC18, pUC19, pBR322, pBlueScript SK(-), pMD18, pMD19, pMD20, pHY300PLK, pET20a, pET20b, pET22a, pET22b, pET28a, pET28b, pND113, pHY-WZX, or pBL-WZX.

[0008] More preferably, the expression vector used for the recombinant plasmid contains the PamyL amylase promoter derived from Bacillus licheniformis and the signal peptide amyL amylase derived from Bacillus licheniformis.

[0009] Preferably, the recombinant strain used as the starting strain includes, but is not limited to, Escherichia coli, Saccharomyces cerevisiae, Candida tropicalis, Bacillus coagulans, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, and Bacillus licheniformis.

[0010] More preferably, the recombinant strain is obtained by molecular cloning and gene recombination technology using Escherichia coli CGMCC NO.11060, a lactic acid monomer producing strain;

[0011] More preferably, the recombinant bacteria is Escherichia coli 090B-egla, which is obtained by cloning the endoglucanase encoding gene egla shown in SEQ ID NO.1 into the pBL-WZX plasmid and expressing it in Escherichia coli CGMCC NO.11060;

[0012] More preferably, the recombinant bacteria is Escherichia coli 090B-cbhc, which is obtained by cloning the exonuclease encoding gene cbhc shown in SEQ ID NO.2 into the pBL-WZX plasmid and expressing it in Escherichia coli CGMCC NO.11060;

[0013] More preferably, the recombinant bacteria is Escherichia coli 090B-bga, which is obtained by cloning the β-glucosidase encoding gene bga shown in SEQ ID NO.3 into the pBL-WZX plasmid and expressing it in Escherichia coli CGMCC NO.11060;

[0014] Recombinant Escherichia coli 090B-cbhc (hereinafter referred to as strain C), 090B-egla (hereinafter referred to as strain E) and 090B-bga (hereinafter referred to as strain B) have the ability to synthesize and secrete cellulosic enzymes and metabolize lignocellulose, and can metabolize sugarcane bagasse to produce lactic acid.

[0015] The second technical solution provided by the present invention is a fermentation agent for producing lactic acid monomers using sugarcane bagasse as raw material. The fermentation agent includes at least one of recombinant Escherichia coli 090B-bga and recombinant Escherichia coli 090B-cbhc and 090B-egla.

[0016] Furthermore, the fermentation agent is composed of strains 090B-cbhc and 090B-bga added in equal proportions;

[0017] Furthermore, the fermentation agent is composed of strains 090B-egla and 090B-bga added in equal proportions;

[0018] Furthermore, the fermentation agent is composed of bacterial strains 090B-cbhc, 090B-egla, and 090B-bga added in equal proportions;

[0019] Furthermore, the fermentation agent is composed of strains 090B-cbhc, 090B-bga, and 090B-egla added in a ratio of 1:2:1, 1:1:2, or 2:1:1.

[0020] Preferably, the fermentation agent is composed of strains 090B-cbhc, 090B-bga, and 090B-egla added in a ratio of 1:2:1.

[0021] The third technical solution provided by the present invention is the application of the recombinant bacteria described in technical solution one or the fermentation agent described in technical solution two, especially in the production of lactic acid from sugarcane bagasse.

[0022] Furthermore, the bagasse used in this study contains 41.8% cellulose and 21.8% hemicellulose. The specific method for producing lactic acid from bagasse is as follows:

[0023] Shake-flask fermentation method: Using pretreated sugarcane bagasse as the fermentation medium, add 8000-15000 U / g of commercial xylanase (octane-dry weight sugarcane bagasse), inoculate the seed liquid of the production strain into the fermentation medium at an inoculation rate of 5%-10%, add 2.0-10.0% (w / v) calcium carbonate, seal, rotate at 50-150 r / min, ferment at 30-37℃, and ferment for 36-48 h. Furthermore, during the fermentation process, maintain the pH between 6.5 and 7.1 with 5-35 wt% calcium hydroxide suspension until the fermentation is completed.

[0024] The pretreatment method for sugarcane bagasse for shake flasks is as follows: Sugarcane bagasse by absolute dry weight is mixed with a 1.0% to 2.0% sodium hydroxide solution at a material-to-liquid ratio of 1:10-15, and then sterilized at 110℃ to 120℃ under high temperature and pressure for 0.5 to 2 hours. The pH is adjusted to 6.0 to 7.0, and 1 to 1.5 times the volume of sodium hydroxide solution of deionized water is added, mixed well, and sterilized again to obtain the fermentation medium for shake flasks.

[0025] Fermentation method in fermenter: Using pretreated sugarcane bagasse as the fermentation medium, the seed liquid of the production strain is inoculated into the fermentation medium at an inoculation rate of 1-10% (v / v) to start lactic acid fermentation. The temperature is controlled at 37℃~50℃, the aeration is turned off, the stirring speed is adjusted to 100~200r / min, and 5%~35% calcium hydroxide suspension is added to maintain the pH at 6.0~7.5. The fermentation cycle is 70-90h.

[0026] The pretreatment method for sugarcane bagasse for fermentation tanks is as follows: Sugarcane bagasse by absolute dry weight is sterilized at 110℃ to 120℃ under high temperature and high pressure for 0.5 to 2 hours in a 1.0% to 2.0% sodium hydroxide solution at a material-to-liquid ratio of 1:10-15. The pH is adjusted to 6.0 to 7.0, concentrated to 40-50% of the original volume, and sterilized again to obtain the fermentation medium for fermentation tanks.

[0027] Furthermore, the production strain includes at least one of recombinant Escherichia coli 090B-bga and recombinant Escherichia coli 090B-cbhc and 090B-egla;

[0028] Preferably, the production strain is composed of strains 090B-cbhc and 090B-bga added in equal proportions;

[0029] Preferably, the production strain is composed of strains 090B-egla and 090B-bga added in equal proportions;

[0030] Preferably, the production strain is composed of strains 090B-cbhc, 090B-egla, and 090B-bga added in equal proportions;

[0031] Preferably, the production strains are strains 090B-cbhc, 090B-bga, and 090B-egla added in a ratio of 1:2:1, 1:1:2, or 2:1:1.

[0032] More preferably, the production strains are strains 090B-cbhc, 090B-bga, and 090B-egla added in a ratio of 1:2:1.

[0033] Beneficial effects:

[0034] This invention provides three strains for producing lactic acid monomers from sugarcane bagasse. These strains can metabolize sugarcane bagasse to synthesize high-value-added lactic acid monomers. The fermentation level of lactic acid monomers in the fermenter can reach 82.76 g / L, and the conversion rate from sugarcane bagasse to lactic acid can reach 41.38%. This invention contributes to the supply of raw materials for large-scale lactic acid production, reduces dependence on grains or sugars, and optimizes the technology for producing lactic acid from sugarcane bagasse. my country's annual sugarcane bagasse production is 2.7 × 10⁻⁶. 7 Currently, the market price of lactic acid offers a profit margin of 70 times compared to the price of its substrate, sugarcane bagasse. Large-scale production of lactic acid from sugarcane bagasse can further improve the economic benefits and industrial structure of sugarcane bagasse. This invention can also be used to produce lactic acid from other lignocellulosic raw materials. By rationally adjusting the proportion of bacterial strains, the usability of bulk biomass raw materials can be realized. Attached image description:

[0035] Figure 1 Physical maps of recombinant plasmids expressing cellulase secretion: (a) recombinant expression plasmid pND-cbhc; (b) recombinant expression plasmid pND-egla; (c) recombinant expression plasmid pND-bga.

[0036] Figure 2 Growth of recombinant strain 090B-bga on a culture medium with cellobiose as the sole carbon source.

[0037] Figure 3 The appearance of clear zones on Congo red medium with CMC-Na as the substrate for recombinant bacterial suspension.

[0038] Figure 4 The use of recombinant bacteria and original strains to produce lactic acid from sugarcane bagasse. Detailed implementation method:

[0039] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0040] The starting strain used in this invention is Escherichia coli CGMCC NO.11060 (disclosed in ZL201580000781.7), an L-lactic acid monomer producing bacterium, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 7, 2015.

[0041] The cellulase used in this invention is a cellulase derived from Aspergillus niger. Its encoding gene was obtained by chemical synthesis after codon optimization. The endoglucanase encoding gene egla, the exoglucanase encoding gene cbhc, and the β-glucosidase encoding gene bga have nucleotide sequences with the sequence characteristics of SEQ ID NO.1, 2, or 3, respectively.

[0042] The plasmid pBL-WZX used in this invention is prior art, and its sequence information and construction method have been disclosed in Chinese Invention Patent (Wang Zhengxiang, Niu Dandan. Chinese Invention Patent, CN200510081648.7). The method for constructing a strain of bacteria for metabolizing sugarcane bagasse to synthesize lactic acid in this invention involves cloning and recombinant gene technology, cloning and obtaining an expression plasmid of cellulase in an expression vector, and transforming it into a lactic acid producing strain to obtain recombinant bacteria, thereby achieving secretory expression of cellulase; or integrating the cellulase expression cassette into the genome of a lactic acid producing strain through site-directed gene integration to obtain recombinant bacteria, thereby achieving secretory expression of cellulase; furthermore, by establishing and optimizing fermentation conditions and processes, it can be used for the industrial production of lactic acid from sugarcane bagasse.

[0043] The main experimental methods used in this invention are as follows:

[0044] 1. Gene Cloning, Gene Recombination, and Expression Plasmid Construction: Routine molecular cloning procedures were performed according to the methods described in the reference (Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989). The cellulase-encoding gene was used as the target gene in this invention; the basic expression vector was pBL-WZX (Wang Zhengxiang, Niu Dandan. Chinese Invention Patent, CN200510081648.7). After PCR amplification, the cellulase-encoding gene was cloned into the pBL-WZX expression vector to obtain a recombinant plasmid expressing sucrase-cellulase.

[0045] 2. Extraction of chromosomal DNA

[0046] The method for extracting Escherichia coli chromosomal DNA was performed according to the literature (Zhuge Jian and Wang Zhengxiang. Handbook of Industrial Microbiology Experimental Techniques, China Light Industry Press, 1994).

[0047] 3. Plasmid DNA extraction was performed using a plasmid miniprep kit from Sigma-Aldrich after lysing the cell wall with a certain concentration of lysozyme.

[0048] 4. Gene Amplification: DNA amplification was performed in 0.2 mL PCR thin-walled tubes. PCR amplification conditions were: 1× (95℃ 5 min); 30× (94℃ 10 s, 58℃ 30 s, 72℃ 30–300 s); 1× (72℃ 10 min). The extension temperature and time of the PCR reaction varied depending on the amplification length. Unless otherwise specified, all PCR reactions were performed using 2× Hieff DNA polymerase.

[0049] 5. Growth of recombinant bacteria and detection of cellulose hydrolase activity.

[0050] (1) On M9 medium with cellobiose as carbon source (0.5%–1.0% cellobiose, 0.5%–2.5% disodium hydrogen phosphate dodecahydrate, 0.02%–0.1% sodium chloride, 0.1%–1.0% potassium dihydrogen phosphate, 0.01%–0.5% ammonium chloride; pH 7.0), recombinant bacteria were inoculated by streaking or spot inoculation and cultured at 37°C for 24 h to observe growth.

[0051] (2) Recombinant bacteria were inoculated onto LB plates by streaking or spot inoculation and cultured at 37°C for 12-18 h. Then, single colonies were picked and inoculated onto M9 medium (glycerol 0.5%-1.0%, disodium hydrogen phosphate dodecahydrate 0.5%-2.5%, sodium chloride 0.02%-0.1%, potassium dihydrogen phosphate 0.1%-1.0%, ammonium chloride 0.01%-0.5%; pH 7.0) with glycerol as the carbon source and cultured at 150-200 rpm at 37°C for 12 h. The bacterial suspension was then spotted onto Congo red plates with CMC-Na as the substrate, and the clear zone was observed.

[0052] (3) Recombinant bacteria were inoculated onto LB plates by streaking or spot inoculation and cultured at 37°C for 12-18 h. Then, single colonies were picked and inoculated into LB liquid medium at 150-200 rpm and cultured at 37°C for 12 h. Then, the inoculation was transferred to M9 medium with glycerol as the carbon source at an initial OD600 of 0.05 and cultured at 150-200 rpm and 37°C for 12 h. The supernatant was collected by centrifugation and enzyme activity was measured.

[0053] The basic method for detecting exoglucanase activity is as follows: 50 μL of diluted enzyme solution is preheated at 50 °C for 5 min. For the experimental group, 350 μL of 1% CMC-Na solution is added first, and the reaction is allowed to proceed accurately for 30 min, followed by the addition of 600 μL of DNS. For the control group, 600 μL of DNS is added, followed by the addition of 350 μL of 1% CMC-Na solution. After the reaction is complete, the solution is boiled for 7 min, cooled to room temperature, and 2 mL of water is added. The absorbance value at 540 nm is then measured.

[0054] The basic method for detecting endoglucanase activity involves preheating 30 mL of 1% CMC-Na solution at 50°C for 5 min. The experimental group received 500 μL of diluted enzyme solution; the control group received 500 μL of the supernatant from the fermentation broth of *E. coli* (B0013-090B, i.e., the starting strain CGMCC 11060). The reaction was carried out at 50°C for 30 min, then stopped by boiling for 10 min. After cooling to room temperature, the viscosity of the reaction solution was measured multiple times using an NDJ-5S rotational viscometer.

[0055] The basic method for detecting β-glucosidase activity involves preheating 50 μL of diluted enzyme solution at 50°C for 5 min. Experimental group: First, add 350 μL of 10 μmol / L cellobiose solution and allow the reaction to proceed accurately for 30 min. Then, add 400 μL of 0.1 mol / L NaOH solution to terminate the enzymatic reaction. Next, add 400 μL of 0.1 mol / L HCl to adjust the pH to 5–6. The glucose concentration (A1) is measured using an enzyme electrode method. Control group: First, add 400 μL of 0.1 mol / L NaOH and maintain the reaction for the same time. Then, add 350 μL of 10 μmol / L cellobiose solution, mix thoroughly, and then add 400 μL of 0.1 mol / L HCl to adjust the pH to 5–6. The glucose concentration (A2) is measured using an enzyme electrode method. The difference ΔA represents the increase in glucose concentration.

[0056] Enzyme activity definition:

[0057] Definition of exoglucanase activity: Under specific conditions of 50℃ and pH 5.0, the amount of enzyme activity (U) is defined as the amount of 1 μmol of cellobiose released by 1 mL of enzyme solution hydrolyzing CMC-Na per unit time.

[0058] Endoglucanase activity is defined as follows: under specific conditions of 50℃ and pH 5.0, after a reaction of 30 min, the amount of enzyme activity (U) is the amount of 1 mg of CMC-Na hydrolyzed per unit time in 1 mL of enzyme solution.

[0059] β-glucosidase activity is defined as follows: at 50°C and pH 5.0, after a reaction of 30 min, the amount of glucose released by 1 mL of enzyme solution through the hydrolysis of cellobiose is defined as one unit of enzyme activity (U).

[0060] 7. Shake-flask fermentation experiment

[0061] A single colony of the recombinant strain was inoculated into 50 mL of LB liquid medium and cultured on a shaker at 30℃–37℃ and 100–250 rpm for 5–15 h to obtain the primary seed culture. The primary seed culture was then inoculated at 5% onto 50 mL of M9 medium (0.5%–2.5% disodium hydrogen phosphate dodecahydrate, 0.02%–0.1% sodium chloride, 0.1%–1.0% potassium dihydrogen phosphate, 0.01%–0.5% ammonium chloride, 0.5%–1.0% glycerol; pH 7.0) in a 250 mL Erlenmeyer flask. The production strain was then inoculated and cultured at 37℃ and 200 rpm for 12 h to obtain the secondary seed culture.

[0062] Pretreated bagasse was used as the fermentation medium. 8000-15000 U / g of commercial xylanase (octane-dry weight bagasse) was added. The seed culture of the production strain was inoculated into the fermentation medium at an inoculation rate of 5%-10%. 2.0-10.0% (w / v) calcium carbonate was added, the medium was sealed, and the fermentation was carried out at a speed of 50-150 r / min and a temperature of 30-37℃ for 36-48 h. During the fermentation process, the pH was maintained between 6.5 and 7.1 with a 5-35 wt% calcium hydroxide suspension until the fermentation was completed.

[0063] Pre-treated sugarcane bagasse as fermentation medium: Sugarcane bagasse by absolute dry weight is treated with 1-2% sodium hydroxide solution at a material-to-liquid ratio of 1:10-15, and sterilized at 110-120℃ under high temperature and pressure for 0.5-2 hours. The pH is adjusted to 6.0-7.0, and 1-1.5 times the volume of sodium hydroxide solution of deionized water is added. The mixture is stirred and sterilized at 121℃ for 15 minutes to obtain the fermentation medium.

[0064] 8. Fermentation experiment in a fermenter

[0065] Take a single colony of the fermentation strain and inoculate it into 50 mL of LB liquid medium. Incubate at 30℃–37℃ and 100–250 rpm for 5–15 h on a shaker to obtain the primary seed culture. Distribute the primary seed culture according to the initial OD... 600 Inoculate 0.1–1.0 g of M9 liquid medium with glycerol as the carbon source into a 5L fermenter. The initial volume of the fermenter after inoculation should be 25%–60% of the working volume, with a glycerol concentration of 1.0%–5.0%. During the cell growth phase of fermentation, maintain the temperature at 30℃–37℃, pH at 5.5–7.5, aeration rate of 0.1–2.0 vvm, and a rotation speed of 100–1000 r / min. When the cell concentration reaches OD500... 600 The concentration is 10-50, which is used as a secondary seed solution.

[0066] The secondary seed culture was inoculated at an inoculation rate of 1-10% (v / v) into a fermentation medium prepared from pretreated sugarcane bagasse (sugarcane bagasse, by dry weight, was mixed with a 1.0%-2.0% sodium hydroxide solution at a material-to-liquid ratio of 1:10-15, sterilized at 110℃-120℃ under high pressure for 0.5-2 hours, the pH was adjusted to 6.0-7.0, concentrated to 40-50% of the original volume, and steam-sterilized at 121℃ for 45 minutes). Lactic acid fermentation was initiated, with the temperature controlled at 37℃-50℃, aeration shut off, and the stirring speed adjusted to 100-200 r / min. A 5%-35% calcium hydroxide suspension was added to maintain the pH at 6.0-7.5, and the fermentation cycle was 70-90 hours. Samples were taken to determine the residual sugar and lactic acid content in the fermentation broth.

[0067] 9. Fermentation process analysis

[0068] Sample preparation: Take 1 mL of the fermentation broth to be tested, centrifuge at 8000 r / min for 5 min, and take an appropriate amount of supernatant to dilute to a suitable ratio.

[0069] Determination of glucose and lactic acid content: After appropriate dilution of the sample, the glucose and lactic acid concentrations were measured using an SBA-40C biosensor, and the average value of three parallel data points was taken.

[0070] In this invention, the starting strain used is CGMCC NO.11060, also known as B0013-090B or 090B. Based on the starting strain CGMCC NO.11060, recombinant bacteria obtained by expressing SEQ ID NO.1, 2, or 3 were named: Recombinant Escherichia coli 090B-egla (strain E), 090B-cbhc (strain C), and 090B-bga (strain B), respectively.

[0071] The present invention will be further explained and illustrated below through specific embodiments.

[0072] Example 1: Free expression of cellulose hydrolase

[0073] To achieve secretory expression of the target protein, pBL-WZX was used as the expression plasmid. The pBL-WZX plasmid contains the PamyL amylase promoter derived from Bacillus licheniformis and the amyL amylase signal peptide derived from Bacillus licheniformis.

[0074] The encoding genes for endoglucanase, exoglucanase, and β-glucosidase from *Aspergillus niger* (as shown in SEQ ID NO. 1, 2, and 3, respectively) were chemically synthesized. The synthesized gene sequences were amplified by PCR (primers are shown in Table 1). The PCR amplification product of the exoglucanase encoding gene was digested with BamHI and cloned into the plasmid pBL-WZX, which was double-digested with SmaHI and BamHI, to obtain the recombinant plasmid pND-cbhc (see attached table). Figure 1 a) The PCR amplification products of the endoglucanase and β-glucosidase gene sequences were digested with XbaI and cloned into the plasmid pBL-WZX, which was double-digested with SmaI and XbaI, to obtain the recombinant plasmid pND-egla (see attached image). Figure 1 b) and pND-bga (attached) Figure 1 c).

[0075] Expression plasmids pND-egla, pND-cbhc, and pND-bga were electroporated into lactic acid monomer producing strain CGMCC NO.11060, respectively. The transformations were verified using solid LB medium containing KmR resistance, and the corresponding recombinant strains 090B-egla (strain E), 090B-cbhc (strain C), and 090B-bga (strain B) were obtained by screening.

[0076] Table 1 Primer sequences used in this invention

[0077]

[0078] (1) The recombinant bacteria 090B-bga were inoculated onto M9 medium with cellobiose as the carbon source by spot inoculation and cultured at 37℃ for 24 h. The growth was then observed. The results are as follows: Figure 2 As shown, 090B-bga can grow well with cellobiose as the sole carbon source.

[0079] (2) Using recombinant bacteria 090B-cbhc, 090B-egla and 090B-bga as production strains, the recombinant bacteria were inoculated onto LB plates by streaking or spotting and cultured at 37°C for 12 h. Then, single colonies were picked and inoculated onto M9 medium (1.51% disodium hydrogen phosphate dodecahydrate, 0.05% sodium chloride, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.5% glycerol; pH 7.0) with glycerol as the carbon source and cultured at 37°C for 12 h at 200 rpm. The bacterial suspension was then spotted onto Congo red plates with CMC-Na as the substrate, and the clear zone was observed.

[0080] The results are as follows Figure 3As shown, all three bacterial strains exhibited distinct clear zones around their sample wells, indicating the activity of endoglucanase, exoglucanase, and β-glucosidase. This demonstrates that cellulase was secreted and expressed in the lactate monomer-producing strains. This further proves that the lactate monomer-producing strains expressing cellulase possess metabolic capabilities.

[0081] (3) Enzyme activity assay

[0082] Recombinant strains 090B-cbhc, 090B-egla, and 090B-bga were used as production strains. The recombinant bacteria were inoculated onto LB agar plates via streaking or spot inoculation and cultured at 37°C for 12 h. Single colonies were then picked and inoculated into LB liquid medium at 200 rpm and cultured at 37°C for 12 h. Finally, the initial OD was determined. 600 The inoculum was transferred at a rate of 0.05 to M9 medium (1.51% disodium hydrogen phosphate dodecahydrate, 0.05% sodium chloride, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.5% glycerol; pH 7.0) with glycerol as the carbon source, and cultured at 200 rpm and 37°C for 12 h. The bacterial culture was prepared by shake-flask fermentation, and the supernatant was collected by centrifugation to determine the enzyme activity.

[0083] The results are shown in the table below:

[0084] 090B-cbhc (U / mL) 090B-egla (U / mL) 090B-bga (U / mL) Exoglucanase 125±4.325 — — Endoglucanase — 203±5.017 — β-glucosidase — — 241±10.252

[0085] Example 2: Production of lactic acid from sugarcane bagasse using recombinant bacteria

[0086] Single colonies of recombinant strains 090B-cbhc, 090B-egla, and 090B-bga, as well as the originating strain CGMCC NO.11060, were inoculated into 50 mL of LB liquid medium and cultured at 37 °C and 250 r / min for 12 h to obtain the primary seed culture.

[0087] The primary seed liquid was prepared according to its initial OD. 600 0.05% of the medium was inoculated into 50 mL of M9 medium (1.51% disodium hydrogen phosphate dodecahydrate, 0.05% sodium chloride, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.5% glycerol; pH 7.0) in a 250 mL Erlenmeyer flask and cultured at 37 °C and 200 rpm for 12 h as a secondary seed culture.

[0088] Different combinations of bacterial strains were transferred to fermentation medium (2.0 g of oven-dry sugarcane bagasse mixed with 1.5% sodium hydroxide solution at a material-to-liquid ratio of 1:10, treated at 115℃ for 1 h under high temperature and high pressure, pH adjusted to 6.5, 27.5 mL of deionized water added, mixed well, and sterilized at 121℃ for 15 min) at a total inoculation rate of 5% (v / v). 10000 U / g of commercial xylanase (oven-dry sugarcane bagasse) was added, followed by 3.0 g of calcium carbonate. The medium was then sealed with sealing film and entered the anaerobic fermentation stage. The fermentation was carried out at 100 r / min and 37℃ for 36 h. During the fermentation process, the pH was maintained between 6.5 and 7.1 with 25 wt% calcium hydroxide suspension. Samples were taken to determine the content of residual sugar and lactic acid in the fermentation broth.

[0089] The different combinations of strains included: ①C+B+CGMCC NO.11060; ②E+B+CGMCC NO.11060 and ③C+E+B. In combinations ①, ②, and ③, the three strains were added in equal proportions. In combinations ① and ②, an equal amount of the original strain CGMCC NO.11060 was used instead of the third strain to further determine whether the addition of the third strain affected lactic acid production when the amounts of the first two strains were the same and the lactic acid production capacity was consistent. The results are shown in Table 2.

[0090] Table 2 Different bacterial cell combinations

[0091]

[0092] It can be seen that adding E+B+CGMCC NO.11060 resulted in a lactic acid yield of 13.5 g / L, a corresponding residual sugar content of 0.10 g / L, a lactic acid conversion rate of 39.2%, and a lactic acid conversion rate of 0.375 g / (L·h). Adding C+B+CGMCC NO.11060 resulted in a total lactic acid yield of 12.5 g / L, a corresponding residual sugar content of 0.41 g / L, a lactic acid conversion rate of 36.3%, and a lactic acid conversion rate of 0.347 g / (L·h). The experimental group adding C+B+E produced a lactic acid yield of 14.5 g / L, a corresponding residual sugar content of 0.11 g / L, a lactic acid conversion rate of 42.1% (based on bagasse weight), and a lactic acid conversion rate of 0.415 g / (L·h). To promote lactic acid yield and conversion efficiency, the optimal combination of strains is C+B+E.

[0093]

[0094] Lactic acid conversion rate A: Conversion rate (%) from sugarcane bagasse content to lactic acid.

[0095] Note: C lac Concentration of lactic acid in fermentation broth (g / L)

[0096] V cultureTotal volume of fermentation broth (mL)

[0097] M 甘蔗渣 Mass of the substrate, sugarcane bagasse (g)

[0098]

[0099] Note:

[0100] Lactic acid conversion rate B: Conversion rate from glucose to lactic acid (%)

[0101] C lac Concentration of lactic acid in fermentation broth (g / L)

[0102] V culture Total volume of fermentation broth (mL)

[0103] M 甘蔗渣 Mass of the substrate, sugarcane bagasse (g)

[0104] 0.418: The cellulose content in sugarcane bagasse is 41.8%.

[0105] 1.05: Conversion coefficient from cellulose to glucose

[0106] Based on this, the addition ratio of strains C, B, and E was further optimized. Using the same fermentation method and conditions, the fermentation strains were replaced with compound strains in different addition ratios (C:B:E = 1:1:1, 1:2:1, 1:1:2, and 2:1:1) for fermentation. After fermentation, samples were taken to determine the content of residual sugar and lactic acid in the fermentation broth. The results are shown in Table 3.

[0107] Table 3 Different bacterial cell mixture ratios

[0108]

[0109] Based on the data in Tables 2 and 3, the optimal combination is C+B+E, with an optimal blending ratio of C:B:E = 1:2:1. Under these fermentation conditions, lactic acid production reaches 15 g / L, the conversion rate from glucose to lactic acid is as high as 99.14%, the conversion rate from sugarcane bagasse to lactic acid is 43.5%, and the average lactic acid production rate is 0.417 g / (L·h). Among these, the recombinant strain 090B-bga hydrolyzes cellobiose in the fermentation broth into glucose, which serves as the carbon source for fermentation. 090B-bga is essential in the fermentation system; therefore, at least two combinations are required in this example, as a single strain cannot complete the fermentation.

[0110] Example 3: Recombinant bacteria mixed fermentation for lactic acid production from sugarcane bagasse

[0111] Single colonies of recombinant strains 090B-cbhc, 090B-egla, and 090B-bga, as well as the originating strain CGMCCNO.11060, were inoculated into 50 mL of LB liquid medium and cultured at 37 °C and 250 r / min for 12 h to obtain the primary seed culture.

[0112] The primary seed liquid was prepared according to its initial OD. 600 0.05% of the medium was inoculated into 50 mL of M9 medium (1.51% disodium hydrogen phosphate dodecahydrate, 0.05% sodium chloride, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.5% glycerol; pH 7.0) in a 250 mL Erlenmeyer flask and cultured at 37 °C and 200 rpm for 12 h as a secondary seed culture.

[0113] At a total inoculum of 5% (v / v), the production strain was transferred into the fermentation medium (2.0 g of dry sugarcane bagasse treated with 1.5% sodium hydroxide solution at a ratio of 1:10, subjected to high temperature and high pressure treatment at 115℃ for 1 h, pH adjusted to 6.5, 27.5 mL of deionized water added, mixed well, and sterilized at 121℃ for 15 min). 10000 U / g of commercial xylanase (dry weight sugarcane bagasse) and 3.0 g of calcium carbonate were added. The mixture was then sealed with sealing film and entered the anaerobic fermentation stage. The fermentation was carried out at 100 r / min and 37℃ for 36 h. During the fermentation process, the pH was maintained between 6.5 and 7.1 using a 25 wt% calcium hydroxide suspension. Samples were taken to determine the content of residual sugar and lactic acid in the fermentation broth.

[0114] The production strains in experimental group 1 were: 090B-cbhc, 090B-bga, and 090B-egla, added in a compound ratio of 1:2:1.

[0115] The strain produced in experimental group 2 was CGMCC NO.11060.

[0116] from Figure 4 The results showed that the lactic acid yield from mixed fermentation of recombinant strains 090B-cbhc, 090B-egla, and 090B-bga was 15.0 g / L, the conversion rate from sugarcane bagasse to lactic acid was 43.5%, and the conversion rate from glucose to lactic acid was 99.14%. The lactic acid yield from fermentation using the starting strain CGMCC NO.11060 was 11.5 g / L, the conversion rate from sugarcane bagasse to lactic acid was 33.4%, and the conversion rate from glucose to lactic acid was 75.79%, which was 30.43% higher than the control group.

[0117] Example 4: Fermentation of lactic acid from sugarcane bagasse in a fermenter

[0118] Single colonies of fermentation strains 090B-cbhc, 090B-egla, and 090B-bga were inoculated into 50 mL of LB liquid medium and cultured at 37°C and 250 rpm for 12 h on a shaker to obtain the primary seed culture. The primary seed culture was then divided according to the initial OD... 600 An inoculum of 0.5 g / L was inoculated into a 5L fermenter in M9 liquid medium (1.51% disodium hydrogen phosphate dodecahydrate, 0.05% sodium chloride, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 3.0% glycerol; pH 7.0) using glycerol as the carbon source. The initial volume of the fermenter after inoculation was 60% of the working volume, and the glycerol concentration was 3.0%. During the cell growth phase of fermentation, the temperature was controlled at 37℃, the pH was maintained at 5.5–7.5, the aeration rate was 0.1–2.0 vvm, and the rotation speed was 1–1000 r / min. When the cell concentration reached OD500... 600 The concentration is 10-50, which is used as a secondary seed solution.

[0119] The secondary seed culture was inoculated into the fermentation medium (10 kg of dry sugarcane bagasse was mixed with 1.5% sodium hydroxide solution at a material-to-liquid ratio of 1:10, treated at 115℃ and high pressure for 1 h, adjusted to pH 6.0-7.0, concentrated to 47.5 L, and steam sterilized at 121℃ for 45 min) at an inoculation rate of 5% (v / v) to a 5% (v / v) inoculation rate of 0.90B-cbhc, 090B-bga and 090B-egla in a compound ratio of 1:2:1) to start lactic acid fermentation. The fermentation temperature was 37℃, the aeration was turned off, the stirring speed was adjusted to 100 r / min, and 25% calcium hydroxide suspension was added to maintain the pH at 7.0. Anaerobic fermentation was maintained under these conditions for 80 h.

[0120] After fermentation, the concentration of lactic acid in the fermentation broth was measured to be 82.76 g / L, the glucose content was 0.83 g / L, the conversion rate of glucose to lactic acid was 94.3% (i.e., lactic acid yield of 0.94 g / g glucose), and the lactic acid yield was 0.413 g / g sugarcane bagasse.

[0121] The endoglucanase encoding gene egla, the exoglucanase encoding gene cbhc, and the β-glucosidase encoding gene bga involved in this invention have nucleotide sequences that have the sequence characteristics of SEQ ID NO. 1, 2, or 3, respectively, as follows:

[0122] SEQ ID NO.1 egla nucleotide sequence 765bp

[0123]

[0124] SEQ ID NO.2 cbhc nucleotide sequence 1341bp

[0125]

[0126]

[0127] SEQ ID NO.3 bga nucleotide sequence 2694bp

[0128]

[0129]

Claims

1. A fermentation agent, characterized in that: The fermentation agent is strain 090B- cbhc , strain 090B- bga , strain 090B- Egla Add in a ratio of 1:2:1; The strain 090B- Egla , is to encode the endoglucanase gene shown in SEQ ID NO. 1 Egla The gene was cloned into pBL-WZX plasmid and expressed in E. coli CGMCC No.11060; The strain 090B- cbhc , is to encode the exoglucanase gene shown in SEQ ID NO. 2 cbhc The gene was cloned into pBL-WZX plasmid and expressed in E. coli CGMCC No.11060; The strain 090B- bga , is to encode the β-glucosidase gene shown in SEQ ID NO. 3 bga The gene was cloned into pBL-WZX plasmid and expressed in E. coli CGMCC No.11060.

2. Use of the fermentation agent according to claim 1 in the production of lactic acid using bagasse as raw material.

3. The use according to claim 2, characterized in that The shake flask fermentation method is as follows: using pretreated bagasse as fermentation medium, adding commercial xylanase 8000-15000 U / g absolute dry weight bagasse, inoculating the production strain seed liquid into the fermentation medium at an inoculation rate of 5%-10%, adding 2.0-10.0% calcium carbonate, sealing, rotating speed 50-150 r / min, fermentation temperature 30-37°C, and fermentation cycle 36-48h; The pretreatment method of sugarcane bagasse for shake flask is as follows: the absolute dry weight of sugarcane bagasse is mixed with 1.0%~2.0% sodium hydroxide solution at a solid-liquid ratio of 1:10-15, sterilized at 110℃~120℃ for 0.5~2 h, the pH is adjusted to 6.0-7.0, deionized water with a volume of 1-1.5 times that of the sodium hydroxide solution is added, mixed, and sterilized again to obtain the fermentation medium for shake flask.

4. The use according to claim 2, characterized in that The fermentation method of the fermentation tank is as follows: using pretreated bagasse as a fermentation medium, inoculating the production strain seed liquid into the fermentation medium at an inoculation rate of 1-10%, starting lactic acid fermentation, controlling the temperature at 37°C-50°C, closing the ventilation, adjusting the stirring speed to 100-200 r / min, adding 5%-35% calcium hydroxide suspension to maintain the pH at 6.0-7.5, and the fermentation period is 70-90h; The pretreatment method of sugarcane bagasse for fermentation tank is as follows: the absolute dry weight of sugarcane bagasse is sterilized at 110℃~120℃ for 0.5~2 h with 1.0%~2.0% sodium hydroxide solution at a solid-liquid ratio of 1:10-15, the pH is adjusted to 6.0-7.0, concentrated to 40-50% of the original volume, and sterilized again to obtain the fermentation medium for the fermentation tank.

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