Construction method for producing yeast protein recombinant bacteria by using lactic acid-containing waste liquid

Abstract

The invention relates to construction and application of a recombinant saccharomyces cerevisiae strain capable of efficiently producing yeast protein by utilizing lactic acid-containing waste liquid (including L type and D type). The method specifically comprises the following steps: cloning and expressing genes ECL, ECD and ECP derived from escherichia coli; a recombinant saccharomyces cerevisiae strain taking the lactic acid-containing waste liquid as a raw material is constructed, saccharomyces cerevisiae somatic cells are proliferated through fermentation, and mycoprotein is accumulated, so that D-lactic acid, L-lactic acid, glucose and inorganic nitrogen in the lactic acid-containing waste liquid are converted into mycocells rich in crude protein. When fermentation culture is finished, the thallus concentration OD600 can reach about 150, the L-lactic acid content is lower than 1 g / L, the D-lactic acid content is lower than 1 g / L, the glucose content is lower than 1 g / L, and the protein content accounts for 60% or above of total solids.

Description

Technical Field

[0001] The present invention relates to the technical fields of fermentation engineering and genetic engineering, and in particular to a method for constructing a recombinant brewer's yeast capable of efficiently producing bacterial protein by utilizing D-lactic acid, L-lactic acid, glucose and inorganic nitrogen in waste liquid containing lactic acid as raw materials. Background Art

[0002] Lactic acid (LA) is an important organic acid with a wide range of industrial applications. Polylactic acid is considered to be an environmentally friendly alternative to petroleum-based plastics and has a wide range of applications, which has aroused great interest, and the demand for optically pure L- or D-lactic acid has also increased. The research progress of lactic acid production by fermentation at home and abroad is not only the development and optimization of fermentation processes, but more importantly, the natural selection of fermentation strains and the use of genetic engineering methods to construct engineered strains that can produce lactic acid. Similarly, the use of genetic engineering to construct engineered strains that can metabolize and utilize lactic acid is also worthy of attention.

[0003] Lactic acid fermentation broth is a complex system. In addition to lactic acid, it also contains a large amount of bacteria, proteins, residual sugars, pigments, inorganic salts, by-product organic acids and unconverted starch. The presence of these impurities makes it very difficult to separate and purify lactic acid. In actual production, the cost of lactic acid extraction accounts for 50-60% of the total production cost. It is the technical bottleneck and difficulty that restricts lactic acid production, and also determines the quality and yield of lactic acid. (JOGLEKAR HG, RAHMAN I, BABU S, et al. Comparative assessment of downstream processing options for lactic acid[J]. Separation and Purification Technology, 2006, 52(1): 1-17.) The calcium lactate crystallization-acid hydrolysis process is currently a widely used lactic acid separation and extraction process in industry. The process is mature and easy to control, but the whole process is long, with high loss and low yield. Jiang Shaotong et al. improved the traditional calcium salt process by using macroporous adsorption resin for decolorization, reducing the loss of lactic acid, and improving the product extraction rate and purity. (Jiang Shaotong, Pan Lijun, Zheng Zhi, et al. New process for extracting lactic acid from fermentation broth: 160329 A[P]. 2005-04-06.) JOGLEKAR et al. studied the method of preparing pure lactic acid by esterification-hydrolysis. After lactic acid and methanol were esterified, methyl lactate was distilled, and then hydrolyzed and distilled to remove methanol to obtain pure lactic acid solution. Although this method can produce high-purity lactic acid and separate it from other organic acids, the lactic acid yield is low due to the limitation of reaction kinetics. (Jiang Shaotong, Pan Lijun, Zheng Zhi, et al. New process for extracting lactic acid from fermentation broth: 160329 A[P]. 2005-04-06. ) PANW ANA et al. used the method of permeation evaporation to assist esterification reaction to purify lactic acid in fermentation broth, converting most of the lactic acid into ethyl lactate, and then distilling and hydrolyzing to obtain high-purity L-lactic acid with a recovery rate of more than 95%, but the lactic acid yield was low. (PANW ANA K, PAILIN B, DIETMAR H, et al. Purification of L-(+)-lactic acid from pre-treated fermentation broth using vapor permeation-assisted esterification[J]. Process Biochemistry, 2012, 47: 1948-1956. The separation and purification of lactic acid have their own advantages, such as high separation efficiency, good product quality, low energy consumption, less environmental pollution, etc. However, it is difficult to effectively complete the separation of lactic acid with a single technology, and there is a problem of low yield. Therefore, the method of microbial fermentation is adopted to use the waste lactic acid that cannot be purified as a fermentation medium for microbial growth, which can not only solve the problem of recycling waste lactic acid, but also reduce pollution to the environment.

[0004] Microorganisms that metabolize and utilize lactic acid, such as Saccharomyces cerevisiae, have recently received increasing attention due to their acid resistance. Saccharomyces cerevisiae has many advantages, such as a short reproduction cycle, simple nutritional requirements, and easy genetic manipulation. The main method of the present invention is to construct a Saccharomyces cerevisiae that can metabolize and utilize lactic acid while being resistant to low pH. The use of this characteristic of recombinant Saccharomyces cerevisiae to convert residual lactic acid into high-concentration bacterial protein is of great significance for the recycling of waste lactic acid. Summary of the invention

[0005] The purpose of the present invention is to obtain a production strain capable of metabolically utilizing lactic acid through gene cloning and gene recombination technology. The obtained strain can directly use waste lactic acid as a raw material to efficiently supply microbial growth, thereby allowing waste lactic acid to be used in large quantities as a raw material supply, thereby optimizing the production technology of producing high-concentration bacterial protein using waste lactic acid as a raw material.

[0006] In order to achieve the above object, the present invention discloses the following technical contents: A method for constructing a recombinant strain of Saccharomyces cerevisiae that can metabolize and utilize D-lactic acid, L-lactic acid, glucose and inorganic nitrogen in lactic acid-containing waste liquid to accumulate crude protein-rich bacterial cells, characterized in that the recombinant strain is obtained by heterologously expressing the genes ECL, ECD and ECP of Escherichia coli in the Saccharomyces cerevisiae strain SAP0001 that tolerates low pH values; the gene ECD-ECP-ECL is optimized and conventionally synthesized according to the codon preference of Saccharomyces cerevisiae to achieve expression; the ECL, ECD and ECP encoding genes have the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0007] The expression vector used for the coding gene is pYES2; the recombinant Saccharomyces cerevisiae utilizes the pGK1p promoter, and the nucleotide sequence of the pGK1p promoter is shown in SEQ ID NO.4.

[0008] The starting strain used in the recombinant strain is Saccharomyces cerevisiae SAP0001 (deposited in this laboratory) that can tolerate pH 3.0-8.5. The recombinant strain (obtained by coding gene synthesis) SAP0001-ECD-ECP-ECL is synthesized by synthesizing the coding gene ECD, coding gene ECP and coding gene ECL shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.1, adding 5' (H restriction site) and 3' (X restriction site), cloning the gene into the vector pYES2 through 5' H and 3' X, and expressing it in Saccharomyces cerevisiae SAP0001.

[0009] The present invention further discloses the application of the recombinant strain for metabolic utilization of lactic acid in converting waste liquid containing lactic acid into high value-added bacterial protein, wherein the application is to use waste liquid containing lactic acid as raw material to metabolically utilize D-lactic acid, L-lactic acid, glucose and inorganic nitrogen. The experimental results show that the waste liquid containing lactic acid (red lactic acid) can reach the level of L-lactic acid content less than 1 g / L, D-lactic acid content less than 1 g / L, glucose content less than 1 g / L, and OD value of 150 after fermentation in a fermenter.

[0010] The protein content of the obtained recombinant strain is increased to more than 60% of the solid content at the end of fermentation. The fermentation method of the fermenter is as follows: the waste liquid containing lactic acid from the lactic acid production enterprise is used as raw material, and the seed liquid of the recombinant strain (obtained by synthesizing the encoding gene) is inoculated into the fermentation medium at an inoculation rate of 1-10%, and the fermentation is started. The temperature is controlled at 30°C, oxygen is introduced, the stirring speed is adjusted to 200-950 r / min, and the speed is adjusted according to the dissolved oxygen. The fermentation cycle is 36 hours; at the end of fermentation, the lactic acid content is less than 1 g / L, and the glucose content is less than 1 g / L, that is, the fermentation is over, the bacterial concentration reaches 150, and the proportion of protein to solids reaches 60%.

[0011] The present invention is described in more detail as follows: The present invention provides a recombinant strain capable of metabolizing and utilizing lactic acid, wherein the recombinant bacteria uses Saccharomyces cerevisiae that does not metabolize and utilize lactic acid but is resistant to low pH as a starting bacterium; further, the ECL, ECD, and ECP encoding genes derived from Escherichia coli have nucleotide sequences shown in SEQ ID NOs. 1, 2, and 3; The expression of the ECL, ECD, ECP encoding genes can be achieved by integrating the encoding genes shown in SEQ ID NOs. 1, 2 and 3 into the host genome, or by transferring the expression vector containing the encoding genes shown in SEQ ID NOs. 1, 2 and 3 into the host cell; Preferably, the expression vector used for the encoding gene includes but is not limited to pYES2; Preferably, the recombinant strain is based on the cerevisiae SAP0001 strain that does not metabolize lactic acid but tolerates low pH; Preferably, the recombinant bacterium is Saccharomyces cerevisiae SAP0001-ECD-ECP-ECL, and the strain synthesizes the coding gene ECD, the coding gene ECP and the coding gene ECL according to the order shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.1, adds 5' (H restriction site) and 3' (X restriction site), clones the gene into the vector pYES2 through 5' H and 3' X, and expresses it in Saccharomyces cerevisiae SAP0001.

[0012] The obtained Saccharomyces cerevisiae SAP0001-ECD-ECP-ECL has the ability to ferment, metabolize and utilize lactic acid.

[0013] The application of the recombinant bacteria provided by the present invention is particularly to produce high-concentration bacterial protein using waste lactic acid as raw material; the waste lactic acid used in this study contains 5-40 g / L of L-lactic acid, 5-40 g / L of D-lactic acid, and 1-10 g / L of glucose. The specific method for producing bacterial protein using waste lactic acid as raw material is as follows: By optimizing the cultivation of recombinant brewer's yeast and providing nutrients and suitable temperature conditions, it can be fermented to synthesize protein, and then protein feed is obtained through extraction, purification and other steps; Fermentation tank fermentation method: waste lactic acid is used as fermentation medium, the production strain seed liquid is inoculated into the fermentation medium at an inoculation rate of 5-10%, and aerobic culture is carried out at 30°C and a rotation speed of 200-950 r / min for 36 hours. Furthermore, the rotation speed is adjusted according to the change of dissolved oxygen during the fermentation process. The biosensor detects that the L-lactic acid content is less than 1 g / L, the D-lactic acid content is less than 1 g / L, and the glucose content is less than 1 g / L; the spectrophotometer measures the OD value to be about 150. The treatment method of fermentation tank fermentation is: high-pressure sterilization of lactic acid waste liquid at 115°C for 20 minutes, and the fermentation medium for the fermentation tank is obtained.

[0014] The beneficial effects of the method for constructing a recombinant yeast protein producing bacteria using waste liquid containing lactic acid disclosed in the present invention are: (1) The present invention constructs a recombinant Saccharomyces cerevisiae capable of metabolizing and utilizing lactic acid by tandemly expressing the ECL gene, ECD gene and ECP gene derived from Escherichia coli.

[0015] (2) The recombinant strain for metabolic utilization of lactic acid constructed by the present invention can convert lactic acid waste liquid into high value-added bacterial protein, and the fermentation level in the fermenter can reach an L-lactic acid content of less than 1 g / L, a D-lactic acid content of less than 1 g / L, a glucose content of less than 1 g / L, and an OD value of 150. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Map and linearization map of the recombinant plasmid pYES2-ECD-ECP-ECL that secretes and expresses the genes encoding ECL, ECD, and ECP from E. coli; Figure 2 Metabolism and utilization of L-lactic acid by recombinant bacteria; Figure 3 The metabolic utilization of D-lactic acid by the recombinant bacteria; Figure 4 Glucose consumption; Figure 5 OD of recombinant bacteria; Figure 6 Growth control on plates with L-lactic acid as carbon source; Figure 7 Growth control using D-lactic acid as carbon source. DETAILED DESCRIPTION

[0017] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.

[0018] The starting strain used in the present invention is Saccharomyces cerevisiae SAP0001 (commercially available); The ECD-ECP-ECL encoding gene derived from Escherichia coli used in the present invention is synthesized after codon optimization, and the nucleotide sequences of the ECL encoding gene, the ECD encoding gene and the ECP encoding gene have the sequence characteristics of SEQ ID NO.1, 2 or 3 respectively.

[0019] The invention connects three genes, ECL, ECD and ECP, from Escherichia coli, optimizes and synthesizes the ECL, ECD and ECP genes shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, adds 5' (H restriction site) and 3' (X restriction site), clones the genes into a vector pYES2 through 5' H and 3' X, obtains a recombinant plasmid pYES2-ECD-ECP-ECL, and expresses them in cerevisiae SAP0001, thereby obtaining cerevisiae SAP0001-ECD-ECP-ECL.

[0020] The main experimental methods used in the present invention are as follows: 1. The ECD-ECP-ECL fragment was transformed into the Saccharomyces cerevisiae SAP0001 strain by the lithium acetate transformation method, which specifically includes the following steps: (1) Pick 3-4 single colonies of Saccharomyces cerevisiae SAP0001 strain and inoculate them into 5 mL of conventional Saccharomyces cerevisiae culture medium and culture them at 30°C for 12 h. (2) Measure the OD of the bacterial solution using a spectrophotometer. 600 Value (3) Initial OD 600 The bacterial solution was transferred to 50 mL of fresh YPD medium (4) and activated for 4-5 hours until the OD value of the bacterial solution reached 0.2. 600The value reaches 0.8-0.9 (5) Pre-boil ssDNA for 10 min (6) Collect the bacteria by centrifugation at 12000 rpm for 5 min, and wash twice with 25 mL ddH2O (7) Resuspend the yeast with 1 mL ddH2O in a sterile 1.5 mL centrifuge tube (8) Collect the bacteria by centrifugation at 12000 rpm for 30 s (9) Resuspend the yeast with 1 mL water and divide it into 100 μl bacterial solution per tube for transformation. Centrifuge the divided bacterial solution in a centrifuge for 20 s and discard the supernatant, then add the transformation system (see Table 1).

[0021] Table 1: Lithium acetate conversion system ratio The mixed transformation system was evenly mixed with the bacteria, kept in a 30°C incubator for 20 min; heat-shocked at 42°C for 40 min, then spread on a recovery plate, inverted and cultured in a 30°C incubator for 3 days. After the transformants grew out, single clones were selected and genomic DNA was extracted for sequencing verification to obtain the SAP0001-ECD-ECP-ECL recombinant strain.

[0022] 2. Shake flask fermentation experiment Take a single colony of the recombinant strain and inoculate it into 50-100 mL of YPD liquid medium, and culture it in a shaking incubator at 30°C and 100-250 r / min for 16 h as the first-level seed liquid. Inoculate 5% of the first-level seed liquid into YPD liquid medium at 30°C and 100-250 r / min for 16 h as the second-level seed liquid.

[0023] The treated lactic acid waste liquid is used as the fermentation medium. The recombinant bacterial seed liquid is inoculated into the fermentation medium at an inoculation rate of 5%-10%. It is aerobically cultured at 30°C and a speed of 100-250 r / min for 36 hours. The fermentation is determined by detecting the glucose content, lactic acid content, pH value and OD value. Pre-treatment of lactic acid waste liquid as fermentation medium: Take a certain volume of lactic acid waste liquid and sterilize it at 115°C for 20 minutes to obtain the fermentation medium; 3. Fermentation experiment in fermenter Take a single colony of the recombinant strain and inoculate it into 50-100 mL of YPD liquid medium. Incubate it at 30°C, 100-250 r / min for 16 h as the first-level seed liquid. Inoculate 5% of the first-level seed liquid into YPD liquid medium at 30°C, 100-250 r / min for 16 h as the second-level seed liquid. 600The inoculum size of 0.1-1.0 is inoculated into the fermentation medium with lactic acid waste liquid as the carbon source in a 5 L fermenter. The initial volume of the fermenter after inoculation is 25-60% of the working volume. During the bacterial growth stage of fermentation, the temperature is controlled at 30°C, the ventilation volume is 0.1-2.0 vvm, and the rotation speed is 200-950 r / min.

[0024] The secondary seed liquid was inoculated into the treated lactic acid waste liquid fermentation medium at an inoculation rate of 1-10% (v / v), and the fermentation tank was sterilized at 121℃ for 20 min. The fermentation tank with fermentation medium was steam sterilized at 115℃ for 20 min, and fermentation began. The temperature was controlled at 30℃, the ventilation volume was 0.1-2.0 vvm, the stirring speed was adjusted to 200-950 r / min, and the fermentation cycle was 36 h. Samples were taken to determine the content of residual sugar and lactic acid, pH value and OD value in the fermentation liquid to determine whether the fermentation was over.

[0025] In the present invention, the starting strain used is Saccharomyces cerevisiae SAP0001. Based on the starting strain Saccharomyces cerevisiae SAP0001, SEQ ID NOs. 2, 3 and 1 are expressed to obtain a recombinant strain named: Recombinant Saccharomyces cerevisiae SAP0001-ECD-ECP-ECL.

[0026] The cloning vector is a commercially available cloning vector, or any cloning vector with equivalent functions. In the present invention, the cloning vector pYES2 is preferred.

[0027] The promoter may be any promoter that can be used in Saccharomyces cerevisiae. Preferably, the promoter used in the present invention is pGK1p, and the terminator may be any terminator that can be used in Saccharomyces cerevisiae.

[0028] The present invention is further explained below by means of specific examples. Example

[0029] Expression of ECL, ECD, ECP The ECL, ECD and ECP encoding genes (as shown in SEQ ID NO.1, 2 and 3, respectively) derived from Escherichia coli were synthesized; the ECD encoding gene, ECP encoding gene and ECL encoding gene shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.1 were optimized and synthesized, 5' (H restriction site) and 3' (X restriction site) were added, and the gene was cloned into the vector pYES2 plasmid through 5' H and 3' X to obtain the recombinant plasmid pYES2-ECD-ECP-ECL.

[0030] The expression plasmid pYES2-ECD-ECP-ECL was transformed into the Saccharomyces cerevisiae strain SAP0001 to obtain the recombinant bacteria SAP0001-ECD-ECP-ECL. Example

[0031] Utilization of lactic acid wastewater by shake flask fermentation with recombinant bacteria Pick out 3-4 single colonies of the starting strain SAP0001 and the recombinant strain SAP0001-ECD-ECP-ECL, inoculate them into 50 mL of YPD liquid medium, and culture them at 30°C and 200 r / min for 16 h as the primary seed liquid. 600 The concentration of 0.05 was inoculated into 50 mL YPD liquid medium in a 250 mL Erlenmeyer flask at 30°C and 200 r / min; the culture was used as the secondary seed solution for 16 h.

[0032] According to the inoculation amount of 10% (v / v), the recombinant strain SAP0001-ECD-ECP-ECL and the starting strain SAP0001 were transferred to the fermentation medium (115℃, high temperature and high pressure sterilization for 20 min), 200 r / min, fermentation temperature 30℃, and continued to culture for 36 h. Samples were taken to determine the content of lactic acid and glucose in the fermentation broth, OD value, and Kjeldahl nitrogen analyzer to determine the protein content. In the shake flask, the recombinant bacteria SAP0001-ECD-ECP-ECL fermented, the initial lactic acid content was 50 g / L, and the lactic acid content was 33.4 g / L at the end of the fermentation; the lactic acid content in the fermentation system of the starting strain SAP0001 did not change. The results of the recombinant bacteria shake flask fermentation are shown in Table 2: Table 2 Changes in raw material content in shake flask fermentation strains Initial lactic acid (g / L) Lactic acid after fermentation (g / L) Initial glucose (g / L) Glucose after fermentation (g / L) Initial protein (%) Protein after fermentation (%) SAP0001 50.5 52.8 5.0 0.24 10.34 14 SAP0001-ECD-ECP-ECL 50.0 33.4 5.0 0.12 10.34 35 Example 3 Fermentation and metabolism of recombinant bacteria in a 5 L fermenter to utilize lactic acid wastewater Pick out 3-4 single colonies of the starting strain SAP0001 and the recombinant strain SAP0001-ECD-ECP-ECL, inoculate them into 50 mL of YPD liquid medium, and culture them at 30°C and 200 r / min for 16 h as the primary seed liquid. 600 The concentration of 0.05 was inoculated into 50 mL YPD liquid medium in a 250 mL Erlenmeyer flask at 30°C and 200 r / min; the culture was used for 16 h as the secondary seed solution.

[0033] The secondary seed solution was added according to the initial OD 600The inoculation amount of 0.1-1.0 was inoculated into the fermentation medium with lactic acid waste liquid as the carbon source in a 5 L fermenter, and the fermenter was sterilized at 121℃ and high temperature and high pressure for 20 min. The fermenter containing the fermentation medium was steam sterilized at 115℃ for 20 min. The initial volume of the fermenter after inoculation was 25-60% of the working volume. During the bacterial growth stage of fermentation, the temperature was controlled at 30℃, the ventilation volume was 0.1-2.0 vvm, the speed was 200-950 r / min, and the fermentation cycle was 36 h. Samples were taken to determine the residual sugar and lactic acid content and OD value in the fermentation broth, and the protein content was determined by Kjeldahl nitrogen analyzer.

[0034] The recombinant bacteria SAP0001-ECD-ECP-ECL were fermented in a fermenter. The initial lactic acid content was 50 g / L and the lactic acid content was 0.01 g / L at the end of the fermentation. The lactic acid content in the fermentation system of the starting strain SAP0001 did not change.

[0035] The results of the fermentation and metabolism of lactic acid by the recombinant bacteria are shown in Table 3. The recombinant bacteria SAP0001-ECD-ECP-ECL has a good ability to metabolize and utilize lactic acid, and the protein content is increased to about 60%. Table 3 Changes in raw material content in fermentation tank The culture medium involved in the following examples is as follows: YPD liquid medium: 1% yeast powder, 2% tryptone, 2% glucose.

[0036] Fermentation medium: lactic acid waste liquid fermentation broth.

[0037] The detection methods involved in the following embodiments are as follows: Recombinant Saccharomyces cerevisiae OD 600 Detection method: Inoculate 3-6 single colonies from a 24-hour-old YPD plate into YPD liquid medium and culture at 30°C and 220 rpm for about 16 hours. When sampling, dilute the plate in an appropriate ratio and measure the OD using a UV spectrophotometer. 600 .

[0038] Determination of glucose and L-lactic acid content: 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 the sample to an appropriate multiple. After the sample is appropriately diluted, use the SBA-40C biosensor to measure the glucose concentration and L-lactic acid concentration, and take the average of three parallel data.

[0039] Detection of total lactic acid content during fermentation: The fermented bacterial liquid was collected and centrifuged. The fermented liquid was mixed with concentrated sulfuric acid at a ratio of 1:0.05 for 10 minutes, centrifuged at 12000 r / min for 5 minutes, and the supernatant was taken. The supernatant was mixed with 10% trichloroacetic acid at a ratio of 1:1, allowed to stand at 4°C for more than 2 hours, centrifuged at 12000 r / min for 5 minutes, and the supernatant was taken. The supernatant was filtered through a 0.22 μl water filter membrane and placed in a liquid phase vial. The lactic acid content was detected by high performance liquid chromatography (HPLC); the high performance liquid chromatography (HPLC) conditions were: FL 2200 HPLCUVDetector was used for high performance liquid chromatography detection, and the chromatographic column was Aminex®HPX-87H (300 mm×7.8 mm (catalog#1250140) (BIORAD)). Mobile phase: 5 mM H2SO4 (take 277 μl concentrated H2SO4, dilute to 1 L ultrapure water, filter with 0.45 μm water filter membrane, and degas by ultrasonication for 20 min), flow rate 0.6 mL / min, column temperature 65℃, wavelength UV@210 nm, injection volume 20 μl.

[0040] The nucleotide sequences of ECL, ECD and ECP derived from Escherichia coli involved in the present invention have the sequence characteristics of SEQ ID NO. 1, 2 or 3 respectively.

[0041] The promoter pGK1p involved in the present invention has a nucleotide sequence with the sequence characteristics of SEQ ID NO.4.

Claims

1. A method for constructing a recombinant strain of Saccharomyces cerevisiae that can metabolize and utilize D-lactic acid, L-lactic acid, glucose and inorganic nitrogen in lactic acid wastewater to accumulate crude protein-rich bacterial cells, characterized in that: The recombinant strain is obtained by heterologously expressing the E. coli genes ECL, ECD and ECP in the low pH-tolerant Saccharomyces cerevisiae strain SAP0001; the gene ECD-ECP-ECL is optimized and conventionally synthesized according to the codon preference of Saccharomyces cerevisiae to complete the expression; the ECL, ECD and ECP encoding genes have the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.

3.

2. The construction method according to claim 1, characterized in that The expression vector used for the coding gene is pYES2.

3. The construction method according to claim 1, characterized in that The recombinant Saccharomyces cerevisiae utilizes the pGK1p promoter, and the nucleotide sequence of the pGK1p promoter is shown in SEQ ID NO.

4.

4. The construction method according to claim 1, characterized in that The recombinant strain is obtained by separating and purifying the starting strain from the waste liquid of lactic acid; it is Saccharomyces cerevisiae SAP0001 which can tolerate pH 3.0-8.5, preferably pH 4.

0.

5. The construction method according to claim 1, characterized in that The recombinant strain SAP0001-ECD-ECP-ECL is obtained by synthesizing the coding genes ECD, ECP and ECL shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.1, adding 5' (H restriction site) and 3' (X restriction site), cloning the genes into the vector pYES2 through 5'H and 3'X, and expressing them in Saccharomyces cerevisiae SAP0001.

6. The use of the recombinant strain for metabolic utilization of lactic acid according to any one of claims 1 to 5; the use is to use waste liquid containing lactic acid as raw materials for metabolic utilization of D-lactic acid, L-lactic acid, glucose and inorganic nitrogen; characterized in that The lactic acid waste liquid contains 5-40 g / L of L-lactic acid, 5-40 g / L of D-lactic acid, and 1-10 g / L of glucose; preferably 25 g / L of L-lactic acid, 25 g / L of D-lactic acid, and 5 g / L of glucose.

7. The use according to claim 6, characterized in that The obtained recombinant strain increases the protein content to more than 60% of the solid content at the end of fermentation.

8. The use according to claim 6, characterized in that The fermentation method in the fermenter is as follows: waste liquid containing lactic acid from lactic acid production enterprises is used as raw material, and the seed liquid of the recombinant strain is inoculated into the fermentation medium at an inoculation rate of 1-10%, and fermentation is started. The temperature is controlled at 30°C, oxygen is introduced, the stirring speed is adjusted to 200-950 r / min, and the speed is adjusted according to the dissolved oxygen. The fermentation cycle is 36 hours; at the end of fermentation, the lactic acid content is lower than 1 g / L, and the glucose content is lower than 1 g / L, that is, the fermentation is ended, the bacterial concentration reaches 150, and the proportion of protein to solids reaches 60%.

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