Method for improving L-lactic acid synthesized by thermoanaerobacter thermolyticus

Abstract

The invention discloses a method for improving L-lactic acid synthesized by thermoanaerobacter thermosaccharolyticus, and belongs to the technical field of genetic engineering. According to the invention, a thymidine kinase gene tdk, a phosphate acetyltransferase-acetokinase gene ptaack, an ethanol dehydrogenase gene adhE, a methylglyoxal synthase gene mgs, a lactic acid racemase race1 and a lactic acid racemase race2 are knocked out, so that the thermoanaerobacter thermolyticus engineering bacterium with high yield of L-lactic acid and reduced by-product accumulation is constructed. The thermoanaerobacter thermolyticus engineering bacterium can be used for producing L-lactic acid with high optical purity through fermentation by taking straw sugar as a carbon source, and has industrial application potential.

Description

Technical Field

[0001] The invention relates to a method for improving the synthesis of L-lactic acid by thermoanaerobic bacterium pyroglycolyticum, belonging to the technical field of genetic engineering. Background Art

[0002] Lactic acid is widely used in food, beverage, medicine, cosmetics and other fields. In recent years, due to the increasingly stringent global environmental protection, bio-based polymers, especially fully biodegradable, healthy and safe polylactic acid materials, have attracted much attention. Therefore, the market demand for lactic acid monomer, the raw material of polylactic acid, has grown rapidly. At present, lactic acid production mainly uses corn and other grains as raw materials, while the use of non-grain raw materials (such as straw) can save food, and the raw materials are widely available and cheap. Raw materials such as straw contain glucan and a large amount of pentose such as xylan. After these polysaccharides are hydrolyzed into monosaccharides, they need to be fully fermented and converted to produce lactic acid and other products economically and efficiently.

[0003] Lactic acid exists in the form of two stereoisomers: L-lactic acid and D-lactic acid. Most of the lactic acid in nature is L-lactic acid. When L-lactic acid and D-lactic acid are mixed in the same proportion, the rotation of polarized light cancels each other out, which is called racemic lactic acid or DL-lactic acid. There is no difference in the application of L-lactic acid, D-lactic acid and DL-lactic acid in food additives or solvents, but the application in the fields of polylactic acid, medicine and pesticides requires high optical purity L-lactic acid or D-lactic acid. D-lactic acid may cause metabolic problems, and D-lactic acid is banned in infant food in Europe and the United States. Lactic acid can be produced by chemical synthesis or microbial fermentation. Chemical methods can usually only synthesize racemic DL-lactic acid. Due to the specificity of biological enzyme catalysis, biological fermentation methods can synthesize high optical purity L-lactic acid or D-lactic acid. At present, the global market demand is mainly for L-lactic acid, which is mainly used in the production of pesticides, insecticides, herbicides, etc. in addition to polylactic acid applications. The production of polylactic acid requires lactic acid monomers with extremely high chemical purity and high optical purity, and the L-lactic acid content is usually required to be greater than 96-99%. Lactic acid fermentation is divided into homolactic fermentation, heterolactic fermentation and bifidobacterium fermentation. The theoretical conversion rate of sugar and acid in homolactic fermentation is 100%, while the latter two are only 50%. In homolactic fermentation, glucose and other substances are converted into pyruvate through the glycolysis pathway, and then pyruvate is converted into lactic acid under the catalysis of lactate dehydrogenase.

[0004] Thermoanaerobacterium thermosaccharolyticum DSM571 can grow at 37℃-68℃, and it can grow in the pH range of 4.0-8.5, which makes it possible to reduce the contamination of foreign bacteria to a great extent. At the same time, Thermoanaerobacterium thermosaccharolyticum has a complete hexose and pentose metabolic system, which can effectively utilize the hexose and pentose in the straw hydrolyzed sugar. Therefore, Thermoanaerobacterium thermosaccharolyticum is an ideal host for producing lactic acid from straw sugar. However, the main product of wild Thermoanaerobacterium thermosaccharolyticum fermentation is ethanol, and it also secretes metabolites such as acetic acid and lactic acid. How to construct a system that can efficiently synthesize L-lactic acid and reduce the accumulation of by-products is a technical problem that needs to be solved for industrial applications. Summary of the invention

[0005] The present invention blocks the metabolic pathways of ethanol, acetic acid, propylene glycol, etc. of wild thermoanaerobic bacillus pyrolysate through metabolic engineering, and obtains a modified strain whose metabolites are mainly lactic acid. On this basis, two lactate racemases are knocked out, and the optical purity of L-lactic acid produced by the strain fermentation exceeds 99%.

[0006] The invention provides an engineered thermophilic anaerobic bacillus strain for producing L-lactic acid with reduced byproducts, wherein the thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase gene mgs, the lactate racemase race1 and the lactate racemase race2 of a starting strain of the thermophilic anaerobic bacillus strain are knocked out.

[0007] In one embodiment, the nucleotide sequence of the thymidine kinase gene tdk is shown as SEQ ID NO.1; the nucleotide sequence of the phosphate acetyltransferase-acetate kinase gene ptaack is shown as SEQ ID NO.2; the nucleotide sequence of the alcohol dehydrogenase gene adhE is shown as SEQ ID NO.3; the nucleotide sequence of the methylglyoxal synthase gene mgs is shown as SEQ ID NO.4; the nucleotide sequence of the lactate racemase race1 is shown as SEQ ID NO.5; and the nucleotide sequence of the lactate racemase race2 is shown as SEQ ID NO.6.

[0008] In one embodiment, the starting strain of the engineered thermoanaerobic bacterium is Thermoanaerobacterium thermosaccharolyticum DSM571.

[0009] The present invention also provides a method for producing L-lactic acid, wherein the thermophilic anaerobic bacillus engineered bacteria seed solution is inoculated into a culture medium for fermentation, the fermentation temperature is 37-68° C., and the fermentation is performed for at least 12 hours.

[0010] In one embodiment, the carbon source that can be utilized by the engineered thermoanaerobic bacterium includes glucose, xylose or arabinose.

[0011] In one embodiment, the seed liquid is prepared as follows: the engineered thermophilic anaerobic bacteria are inoculated into a culture medium containing different carbon sources for fermentation, and continuous transfer is performed after the carbon source in the culture medium is exhausted; the inoculation amount and culture conditions of each transfer are kept consistent, and the transfer is performed at least 15 times; the carbon source includes glucose or xylose.

[0012] The present invention also provides a method for improving the optical purity of L-lactic acid produced by thermoanaerobic bacillus pyrolyticus, wherein lactate racemase race1 and lactate racemase race2 are knocked out in thermoanaerobic bacillus pyrolyticus engineering bacteria; the thermoanaerobic bacillus pyrolyticus engineering bacteria is based on thermoanaerobic bacillus pyrolyticus DSM571 as a starting strain, and the thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE and the methylglyoxal synthase gene mgs are knocked out; the nucleotide sequence of the thymidine kinase gene tdk is shown in SEQ ID NO.1; the nucleotide sequence of the phosphate acetyltransferase-acetate kinase gene ptaack is shown in SEQ ID NO.2; the nucleotide sequence of the lactate dehydrogenase gene ldh is shown in SEQ ID NO.3; the nucleotide sequence of the methylglyoxal synthase gene mgs is shown in SEQ ID NO.4; the nucleotide sequence of the lactate racemase race1 is shown in SEQ ID NO.5; the nucleotide sequence of the lactate racemase race2 is shown in SEQ ID NO. As shown in IDNO.6.

[0013] The present invention also provides a method for reducing the accumulation of by-products in the process of producing L-lactic acid by Thermoanaerobic Bacillus pyrolyticus. The thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase gene mgs, the lactate racemase race1 and the lactate racemase race2 are knocked out in the Thermoanaerobic Bacillus pyrolyticus. The nucleotide sequence of the thymidine kinase gene tdk is shown in SEQ ID NO.1; the nucleotide sequence of the phosphate acetyltransferase-acetate kinase gene ptaack is shown in SEQ ID NO.2; the nucleotide sequence of the lactate dehydrogenase gene ldh is shown in SEQ ID NO.3; the nucleotide sequence of the methylglyoxal synthase gene mgs is shown in SEQ ID NO.4; the nucleotide sequence of the lactate racemase race1 is shown in SEQ ID NO.5; and the nucleotide sequence of the lactate racemase race2 is shown in SEQ ID NO.6.

[0014] The present invention also provides the use of the thermophilic anaerobic bacillus engineered bacteria in the preparation of L-lactic acid or products containing L-lactic acid.

[0015] In one embodiment, the product includes polylactic acid, food, food additives, medicines, pharmaceutical adjuvants, health products, cosmetics, skin care products, feed additives, forage maturation agents, preservatives, slow-release pesticides, descaling agents, pH regulators, water-based coating synthesizers, photosensitive material cleaning agents, acid-base buffers, leather treatment agents, fiber treatment agents or tobacco treatment agents.

[0016] Beneficial effects:

[0017] The present invention constructs a thermoanaerobic bacterium of thermosaccharolyticum with high L-lactic acid production and reduced by-product accumulation by knocking out the thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase gene mgs, the lactate racemase race1 and the lactate racemase race2. The thermoanaerobic bacterium of thermosaccharolyticum can ferment and produce L-lactic acid with glucose, xylose or arabinose as carbon sources. When glucose is used as the carbon source, 60 g / L of glucose can be consumed in 48 hours of fermentation, the glucose conversion rate is 99.9%, the L-lactic acid yield is 91.1%, and the optical purity of L-lactic acid is 99.6%; when xylose is used as the carbon source, 39.5 g / L of xylose can be consumed in 48 hours of fermentation, the xylose conversion rate is 98.8%, the L-lactic acid yield is 90.6%, and the optical purity of L-lactic acid is 99.4%; When an equal proportion mixture of glucose and xylose was used as the carbon source, the conversion rates of glucose and xylose after fermentation for 48 hours were 99.9%, the yield of L-lactic acid was 90.8%, and the optical purity of L-lactic acid was 99.5%; when glucose, xylose and arabinose were used as the carbon sources, the conversion rate of glucose after fermentation for 48 hours was 99.9%, the conversion rate of xylose was 99.5%, the conversion rate of arabinose was 98.3%, the yield of L-lactic acid was 90.5%, and the optical purity of L-lactic acid was 99.5%; in the above fermentation, the by-products ethanol, acetic acid and propylene glycol were not accumulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony PCR results of tdk knockout in T. thermosacch DSM571, lanes 1-8 are parallel experiments.

[0019] Figure 2 This is the colony PCR result of the first knockout of ptaack in the L0 strain, and lanes 1-7 are parallel experiments.

[0020] Figure 3 The colony PCR results of knocking out the resistance screening marker in the L0 strain, and lanes 1-12 are parallel experiments.

[0021] Figure 4 This is the colony PCR result of the first knockout of adhE in L1, and lanes 1-8 are parallel experiments.

[0022] Figure 5 The colony PCR results of knocking out the resistance screening marker in the L1 strain, and lanes 1-10 are parallel experiments.

[0023] Figure 6 This is the colony PCR result of the first knockout of mgs in the L2 strain, and lanes 1-7 are parallel experiments.

[0024] Figure 7The colony PCR results of knocking out the resistance screening marker in the L2 strain, and lanes 1-4 are parallel experiments.

[0025] Figure 8 This is the colony PCR result of the first knockout of adhE in L0, and lanes 1-3 are parallel experiments.

[0026] Fig. 9 The colony PCR results of knocking out the resistance screening marker in the L0 strain, and lanes 1-8 are parallel experiments.

[0027] Fig.10 This is the colony PCR result of the first knockout of race1 in the L3 strain, and lanes 1-6 are parallel experiments.

[0028] Fig.11 The colony PCR results of knocking out the resistance screening marker in the L3 strain, and lanes 1-8 are parallel experiments.

[0029] Fig.12 This is the colony PCR result of the first knockout of race2 in the LA0 strain, and lanes 1-8 are parallel experiments.

[0030] Fig.13 The colony PCR results of knocking out the resistance screening marker in the LA0 strain, and lanes 1-8 are parallel experiments.

[0031] Fig.14 Fermentation results of different genetically engineered bacteria: A: fermentation products and concentrations; B: lactic acid yield.

[0032] Fig.15 This is a diagram of the knockout plasmid structure. DETAILED DESCRIPTION

[0033] 1 Materials and methods

[0034] 1.1 Materials

[0035] 1.1.1 Strains and plasmids

[0036] Table 1 Strains and plasmids

[0037]

[0038]

[0039] 1.1.2 Main reagents and drugs

[0040] Potassium citrate, L-cysteine ​​salt, and sodium 3-(N-morpholinoline) propanesulfonate were purchased from MacLean, kanamycin was purchased from NEB, USA, kanamycin sulfate was purchased from Beijing Solebao Technology Co., Ltd., FUDR (5-fluorouracil deoxynucleoside) was purchased from (Beijing) Kuer Chemical Technology Co., Ltd., palladium particle molecular sieves and desiccant were purchased from Shanghai Longyue Instrument Equipment Co., Ltd., NaCl, anhydrous CaCl2, MgCl2·6H2O, FeCl2·4H2O, Na2SO4, KH2PO4, K2HPO4·3H2O, sulfuric acid, and isopropanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Nucleic acid dyes, 5000DNA Marker, and 1kb DNA ladder were purchased from Shanghai Yisheng Biotechnology Co., Ltd., and gel recovery kits were purchased from OMEGA, USA.

[0041] 1.1.3 Instrumental enzymes

[0042] Gibson The premix was purchased from NEB Biotech, USA; Prime STAR Max Premix (2×) was purchased from TaKaRa Biotech Co., Ltd., Japan; and 2× Hieff Gold PCR Master Mix was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0043] 1.1.4 Instruments and Equipment

[0044] Table 2 Main instruments and equipment

[0045]

[0046] 1.2 Basic Methods

[0047] 1.2.1 Gum recovery and purification

[0048] For recovery and purification experiments, use the omega gel recovery kit.

[0049] 1. Run the PCR product recovery gel and cut the gel into a 2mL EP tube, weigh the cut gel and determine the gel volume. (The mass of the gel block is 0.3g, so the volume of the gel block is 0.3mL.)

[0050] 2. Add one volume of XP2 binding buffer into the EP tube.

[0051] 3. Heat in a 55℃ metal bath for 5-7 minutes until the rubber block dissolves. Shake the EP tube every 2 minutes during heating to accelerate dissolution.

[0052] 4. Place the spin column in a standard 2 mL collection tube and label it.

[0053] 5. Add the sol mixture to the centrifuge column. If it exceeds 700 μL, add it twice. Centrifuge at 10,000 g for 1 min, pour the centrifuge liquid into the centrifuge column, repeat the centrifugation at 10,000 g for 1 min, discard the liquid, and put the centrifuge column back into the collection tube.

[0054] 6. Add 300 μL XP2 Binding Buffer, centrifuge at 13000 g for 1 min, discard the liquid, and put the centrifuge column back into the collection tube.

[0055] 7. Add 700 μL SPW Buffer, centrifuge at 13000 g for 1 min, discard the liquid, and put the centrifuge column back into the collection tube.

[0056] 8. Repeat step 8 once, discard the liquid, and put the centrifuge column back into the collection tube.

[0057] 9. Centrifuge the empty column at 13000g for 2 min to remove excess alcohol.

[0058] 10. Transfer the spin column to a new 1.5 mL EP tube and add 30 μL Elution Buffer.

[0059] 11. After standing at room temperature for 2 minutes, centrifuge at 13000g for 1 minute and mark.

[0060] 12. Take 1 μL and measure the nucleic acid concentration using a micro-spectrophotometer.

[0061] 13. DNA should be stored in a -20℃ refrigerator.

[0062] 1.2.2 Gibson assembly method

[0063] Gibson The premix was purchased from NEB Biotech, USA. The Gibson assembly (20 μL) reaction system was prepared as follows: Gibson assembly 2×mix 10 μL, and sterile water was added to make up to 20 μL after adding DNA. This process was carried out on ice, and the reactants were transferred to the PCR instrument and kept at 50°C for 20 minutes. After the reaction program was completed, the reaction was taken out and frozen for storage.

[0064] 1.2.3 PCR reaction system

[0065] Prepare PCR detection system (50 μL): (primer concentration 10 μm) 2 μL of forward primer; 2 μL of rear primer; 1 μL of Gibson reaction solution; 25 μL of Primer Star 2×mix; make up to 50 μL with sterile water.

[0066] The PCR amplification program was as follows: 98°C pre-denaturation, 2 min; 98°C denaturation, 10 s; 58°C annealing, 10 s; 72°C extension, 1 min; 72°C re-extension, 2 min; 12°C insulation, 20 min. The number of cycles was 30.

[0067] The program is completed and the gel run is performed. If everything is correct, the gel is recovered and purified to obtain relatively clean DNA fragments. Samples are taken for nucleic acid concentration determination and stored in a -20℃ refrigerator for subsequent experiments.

[0068] 1.2.4 Plasmid construction

[0069] The plasmids and primers used are shown in Tables 1 and 4. All plasmids were assembled using the Gibson assembly method. The following are the designed primers.

[0070] Fragment 1 was amplified from pTT0 using primers P1 and P2, fragment 2 was amplified from pTT0 using primers P3 and P4, US, DS and INT of the target gene were amplified from the genome using the corresponding primers in Table 4, fragment 1, fragment 2, US, DS and INT were connected using Gibson assembly 2×mix, the Gibson reaction was transferred into Escherichia coli DH5α, single clones were picked out for verification after 1-2 days after plating, and then the plasmid was extracted.

[0071] Table 3 Primer list

[0072]

[0073]

[0074] 1.2.5 Gene knockout

[0075] The gene knockout steps are as follows. The knockout verification primers are shown in Table 5. The PCR system procedure is shown in 1.2.3.

[0076] 1. Prepare the experimental group and the control group. Add 0.1-1μg of the plasmid to be transformed and the bacterial solution to the experimental group, and only add the bacterial solution to the control group. Put all the experimental samples into the anaerobic culture box, open the anaerobic gas production bag and quickly put it into the anaerobic culture box, quickly cover the lid of the anaerobic culture box, and culture at 55-68℃ for 18-22 hours. The OD value should be controlled between 0.2-1.0.

[0077] 2. Gene knockout and monoclonal screening verification. First round of screening: Add kanamycin to a working concentration of 200-400 mg / L, apply gradient screening on the plate, and culture for 3-4 days. During this period, observe whether there are colonies. Select colonies for PCR verification. If the band is correct, save it for the next round of screening. Second round of screening: Apply the water-soluble bacteria that were correctly verified in the first round of screening on the plate for gradient screening, add FUDR to a working concentration of 10-100 mg / L, and culture for 3-7 days. Use T.thermo wild type as a control for colony PCR verification.

[0078] Table 4 List of primers for gene knockout verification

[0079]

[0080]

[0081] 1.2.6 Strain culture and fermentation methods

[0082] The wild-type WT strain and L0, L1, L2, L3, L4, L5, and L6 strains in Table 1 were subjected to fermentation tests. First, the modified strains were activated and cultured for 12 hours at 55-68°C and 200rpm. Then, 1 mL of the bacterial solution was transferred to a medium containing glucose, and the culture was continued at 55-68°C and 200rpm for 1-3 days before sampling.

[0083] 1.2.7 Fermentation product detection methods

[0084] Fermentation test sample processing

[0085] Take 2 mL of sample each time, centrifuge the sample at 12000 rpm for 1 min, take 900 μL of supernatant and add it to EP tube, add 100 μL of 10% dilute sulfuric acid and mix well. Prepare 2 samples and store them in a refrigerator at 4°C. Filter through a 0.22 μm filter membrane and put about 500 μL of sample into each injection bottle.

[0086] Fermentation test sample detection

[0087] In this study, high performance liquid chromatography (HPLC) was used to analyze the fermentation samples with a RID-10A differential refractometer (Shimadzu, Kyoto, Japan) and a HPX-87H (300 × 7.8 mm, Bio-rad, Hercules, CA, USA) column.

[0088] Prepare standard samples containing the main product and 5 mM dilute sulfuric acid as mobile phase. Filter the samples through a 0.22 μm filter membrane. Put about 500 μL of sample into each injection bottle. Then set the main parameters according to the acid column manual: flow rate 0.5 mL / min, column oven 60°C, detection cell 40°C, injection volume 10 μL, and collection time for one sample is 30 minutes.

[0089] In this study, high performance liquid chromatography (HPLC) was used to analyze the fermentation samples with detector A: LC-2030UV Detector and a chiral liquid chromatography column (Chirex 3126 (D)-Penicillamine 150×4.6 mm).

[0090] Prepare a standard sample containing the main product and a mobile phase of 5mM copper sulfate. Filter the sample through a 0.22μm filter membrane. Place about 500μL of sample in each injection bottle. Then set the main parameters according to the chiral column manual: flow rate 1.0mL / min, column oven 30℃, detection cell 40℃, injection volume 10μL, and collection time for one sample 20 minutes. Finally, use the peak height to refer to the height of the standard sample to calculate the sample quantification.

[0091] Calculation method of lactic acid yield:

[0092] Lactic acid yield = (mass of lactic acid after fermentation - mass of lactic acid before fermentation) / (mass of sugar before fermentation - mass of sugar after fermentation) × 100%.

[0093] Calculation method of sugar conversion rate:

[0094] Sugar conversion rate = (mass of sugar before fermentation - mass of sugar after fermentation) / mass of sugar before fermentation × 100%

[0095] Calculation method of L-lactic acid optical purity:

[0096] Purity = mass of L-lactic acid produced / (mass of L-lactic acid produced + mass of D-lactic acid produced) × 100%.

[0097] Example 1 Construction of Thermoanaerobic Bacillus Engineering Bacteria

[0098] 1. Knockout of thymidine kinase gene

[0099] (1) Construction of knockout plasmid

[0100] Fragment 1 was amplified from pZJ24htktdk (published in the paper "A markerless gene deletion and integration system for Thermoanaerobacter ethanolicus") using primers P1 and P2 in Table 4; fragment 2 was amplified from pZJ24htktdk using primers P3 and P4; the upstream homology arm US of the target gene tdk was amplified using primers P5 and P6, the downstream homology arm DS of the target gene tdk was amplified using primers P7 and P8, and the INT of the target gene tdk was amplified using primers P9 and P10. Fragment 1, fragment 2, US, DS and INT were connected using Gibson assembly 2×mix, and the Gibson reaction was transferred into Escherichia coli DH5α. After plating, a single clone was picked and verified 1-2 days later, and the plasmid was extracted and named pTT_TDK.

[0101] (2) Gene knockout and monoclonal screening

[0102] The plasmid constructed in step (1) is added to the bacterial cells Thermoanaerobacteriumthermosaccharolyticum DSM571 to be transformed at a concentration of 0.1-1ug. In the anaerobic culture box, open the anaerobic gas production bag and quickly put it into the anaerobic culture box. Quickly cover the lid of the anaerobic culture box and culture at 55-68°C for 18-22 hours. During the culture period, the OD value is detected with a spectrophotometer to make the OD value reach between 0.2-1.0. The bacterial solution is collected and plated for gradient screening. FUDR is added to the working concentration of 10-100 mg / L and cultured for 3-7 days. Colony PCR verification is performed using primers P41 and P42 in Table 5, and wild-type Thermoanaerobic Bacillus thermosaccharolyticus is used as a control. The results are as follows: Figure 1 As shown, the positive clones were picked and the correct strains were named L0.

[0103] 2. Knockout of phosphate acetyltransferase gene and acetate kinase gene

[0104] (1) Construction of knockout plasmid

[0105] Fragment 1 was amplified from plasmid pZJ24htktdk using primers P1 and P2 in Table 4; fragment 2 was amplified from pZJ24htktdk using primers P3 and P4; the upstream homology arm US of the target gene ptaack was amplified using primers P11 and P12 in Table 4, the downstream homology arm DS of the target gene ptaack was amplified from the genome using primers P13 and P14 in Table 4, and the midstream homology arm INT of the target gene ptaack was amplified from the genome using primers P15 and P16 in Table 4. Fragment 1, fragment 2, US, DS and INT were connected using Gibson assembly 2×mix, and the Gibson reaction was transferred into Escherichia coli DH5α. After plating, a single clone was picked and verified 1-2 days later, and the plasmid was extracted and named pTT_ptaack.

[0106] (2) Gene knockout and monoclonal screening

[0107] The plasmid constructed in step (1) is added to the bacterial cell L0 to be transformed in the above-constructed manner at a concentration of 0.1-1 ug. In an anaerobic culture box, the anaerobic gas production bag is opened and quickly placed in the anaerobic culture box. The lid of the anaerobic culture box is quickly closed and cultured at 55-68° C. for 18-22 hours. During the culture period, the OD value is detected by a spectrophotometer and the OD value is maintained between 0.2 and 1.0. The bacterial solution is collected for subsequent screening.

[0108] First round of screening: Add kanamycin to a working concentration of 200-400 mg / L, apply gradient screening, and culture for 3-4 days. During this period, observe whether there are colonies. Select colonies and use primers P43 and P44 in Table 5 to perform PCR verification on the colonies. At the same time, use wild-type Thermoanaerobic Bacillus thermosaccharolyticus as a control. The results are as follows Figure 2 As shown, positive clones were selected for the next round of screening. Second round of screening: The water-soluble bacteria that were verified in the first round of screening were plated for gradient screening, and FUDR was added to a working concentration of 10-100 mg / L and cultured for 3-7 days. Colonies were selected and verified by colony PCR using primers P43 and P44 in Table 5, and wild-type thermosaccharolytic thermoanaerobic bacillus was used as a control. The results are shown in Figure 3 As shown, the positive clones were picked and the correct strains were named L1.

[0109] 3. Knockout of alcohol dehydrogenase gene

[0110] The specific implementation is the same as above, except that:

[0111] Step (1) using primers P17 and P18 in Table 4 to amplify the upstream homology arm US of the target gene adhE from the genome of Thermoanaerobic Bacillus saccharolyticus, using primers P19 and P20 in Table 4 to amplify the downstream homology arm DS of the target gene adhE from the genome of Thermoanaerobic Bacillus saccharolyticus, using primers P21 and P22 in Table 4 to amplify the midstream homology arm INT of the target gene adhE from the genome of Thermoanaerobic Bacillus saccharolyticus, and construct a knockout plasmid named pTT_adhE;

[0112] Step (2) The plasmid pTT_adhE was transformed into strain L1, and the first round of kanamycin screening colony PCR verification was performed using primers P45 and primer P46 in Table 5, and the second round of FUDR screening colony PCR verification was performed using primers P45 and P46 in Table 5.

[0113] The first round of kanamycin screening verification results are as follows Figure 4 As shown, positive clones were selected for the next round of screening. The results of the second round of FUDR screening verification are shown in Figure 5 As shown, the positive clones were picked and named as L2.

[0114] 4. Knockout of methylglyoxal synthase gene

[0115] The specific implementation is the same as above, except that:

[0116] Step (1) using primers P23 and P24 in Table 4 to amplify the upstream homology arm US of the target gene mgs from the T. thermo genome, using primers P25 and P26 in Table 4 to amplify the downstream homology arm DS of the target gene mgs from the T. thermo genome, using primers P27 and P28 in Table 4 to amplify the midstream homology arm INT of the target gene mgs from the T. thermo genome, and construct a knockout plasmid named pTT_mgs;

[0117] Step (2) The plasmid pTT_mgs was transformed into strain L2, and the first round of kanamycin screening colony PCR verification was performed using primers P47 and primer P48 in Table 5, and the second round of FUDR screening colony PCR verification was performed using primers P47 and P48 in Table 5.

[0118] The first round of kanamycin screening verification results are as follows Figure 6 As shown, positive clones were selected for the next round of screening. The results of the second round of FUDR screening verification are shown in Figure 7 As shown, the positive clones were picked and named as L3.

[0119] 5. Knockout of race1

[0120] The specific implementation is the same as above, except that:

[0121] Step (1) using primers P29 and P30 in Table 4 to amplify the upstream homology arm US of the target gene race1 from the T. thermo genome, using primers P31 and P32 in Table 4 to amplify the downstream homology arm DS of the target gene race1 from the T. thermo genome, using primers P33 and P34 in Table 4 to amplify the midstream homology arm INT of the target gene race1 from the T. thermo genome, and construct a knockout plasmid named pTT_race1;

[0122] Step (2) The plasmid pTT_race1 was transformed into strain L3, and the first round of kanamycin screening colony PCR verification was performed using primers P49 and primer P50 in Table 5, and the second round of FUDR screening colony PCR verification was performed using primers P49 and P50 in Table 5.

[0123] The first round of kanamycin screening verification results are as follows Fig.10 As shown, positive clones were selected for the next round of screening. The results of the second round of FUDR screening verification are shown in Fig.11 As shown, the positive clone was picked and named as L4.

[0124] 6. Knockout of race2

[0125] The specific implementation is the same as above, except that:

[0126] Step (1) using primers P35 and P36 in Table 4 to amplify the upstream homology arm US of the target gene race2 from the T. thermo genome, using primers P37 and P38 in Table 4 to amplify the downstream homology arm DS of the target gene race2 from the genome, using primers P39 and P40 in Table 4 to amplify the midstream homology arm INT of the target gene race2 from the genome, and constructing a knockout plasmid named pTT_race2;

[0127] Step (2) The plasmid pTT_race2 was transformed into strain L4, and the first round of kanamycin screening colony PCR verification was performed using primers P51 and P52 in Table 5, and the second round of FUDR screening colony PCR verification was performed using primers P51 and P52 in Table 5.

[0128] The first round of kanamycin screening verification results are as follows Fig.12 As shown, positive clones were selected for the next round of screening. The results of the second round of FUDR screening verification are shown in Fig.13 As shown, the positive clone was picked and named as L5.

[0129] Example 2 Application of recombinant strains to produce lactic acid

[0130] The wild-type WT strain and the L0, L1, L2, L3, L4, and L5 strains constructed in Example 1 were activated and cultured in a medium containing 5 g / L glucose at 55-68° C. and 200 rpm for 12 h to obtain seed solution, and then the seed solution was transferred to a medium containing 10 g / L glucose (20 ml) at an inoculum amount of 10% (V / V); other components of the medium were: 3.00 g / L sodium citrate dihydrate, 1.30 g / L (NH4)2SO4, 1.43 g / L KH2PO4, 1.80 g / L K2HPO4·3H2O, 0.13 g / L CaCl2·2H2O, and 0.6 g / L MgCl2·6H2O. 2.60g / L, yeast extract 4.50g / L, L-cysteine ​​hydrochloride 0.50g / L, FeSO4·7H2O 0.0011g / L, sodium 3-(N-morpholine)propanesulfonate 10.00g / L.

[0131] After continuous cultivation at 60±2℃ and 200rpm for 48h, the sample was taken for analysis. Fig.14 As shown. The wild-type strain can use 10g / L glucose to produce 0.54g / L lactic acid, 1.85g / L acetic acid, 0.92g / L propylene glycol and 1.93g / L ethanol, and the lactic acid yield is 5.4%. The strain L0 with the tdk gene knocked out can ferment 10g / L glucose to produce 0.45g / L lactic acid, 1.95g / L acetic acid, 0.85g / L propylene glycol and 1.85g / L ethanol, and the lactic acid yield is 4.5%, which is not much different from the wild type. The strain L1 obtained by knocking out the ptaack gene on L0 can use 10g / L glucose to produce 3.82g / L lactic acid, 0.72g / L propylene glycol and 1.75g / L ethanol, and the lactic acid yield is 38.2%, which is 607% higher than the wild type. The strain L2 obtained by knocking out the adhE gene on L1 can produce 8.22g / L lactic acid and 0.69g / L propylene glycol from 10g / L glucose, and the lactic acid yield is 82.2%, which is 1422% higher than the wild type. The strain L3 obtained by knocking out the mgs gene on L2 can produce 9.11g / L lactic acid from 10g / L glucose, and the lactic acid yield is 91.1%, which is 1587% higher than the wild type. The L4 and L5 strains were obtained by further knocking out race1 and race2 on L3, and fermented 10g / L glucose to produce 9.16g / L and 9.17g / L lactic acid, respectively, with lactic acid yields of 91.6% and 91.7%, respectively. No glucose was detected at the end of the above fermentations, and the conversion rate of glucose was 100%.

[0132] Comparative Example 1 Fermentation of recombinant bacteria with single knockout of alcohol dehydrogenase gene

[0133] The specific implementation is the same as that of Example 1, except that:

[0134] Step (1) using primers P17 and P18 in Table 4 to amplify the upstream homology arm US of the target gene adhE from the genome of Thermoanaerobacterium saccharolyticum, using primers P19 and P20 in Table 4 to amplify the downstream homology arm DS of the target gene adhE from the genome, using primers P21 and P22 in Table 4 to amplify the midstream homology arm INT of the target gene adhE from the genome, and constructing a knockout plasmid named pTT_adhE;

[0135] Step (2) Plasmid pTT_adhE was transformed into strain L0. Primers P45 and P46 in Table 5 were used for the first round of kanamycin screening colony PCR verification, and primers P45 and P46 in Table 5 were used for the second round of FUDR screening colony PCR verification.

[0136] The first round of kanamycin screening results are as follows Figure 8 As shown, positive clones were selected for the next round of screening. The results of the second round of FUDR screening verification are shown in Fig. 9 As shown, the positive clone was picked and named as L6.

[0137] The fermentation test of L6 strain was carried out according to the method of Example 2. The results are as follows Fig.14 As shown, strain L6 obtained by knocking out the adhE gene alone on L0 can use 10 g / L glucose (fermentation volume 20 ml) to produce 1.85 g / L lactic acid, 1.93 g / L acetic acid and 0.62 g / L propylene glycol, with a lactic acid yield of 18.5%. Compared with the wild type, the lactic acid yield increased by 243%. The result was 107% lower than the lactic acid yield of single knockout of ptaack gene on L0, 344% lower than the double knockout of ptaack and adhE, and 392% lower than the triple knockout of ptaack, adhE and mgs.

[0138] Example 3 Fermentation of glucose by recombinant strain to produce L-lactic acid

[0139] The L5 strain was activated and cultured in a medium containing 5 g / L glucose at 55-68° C. and 200 rpm for 12 hours to obtain a seed solution, and then the seed solution was transferred to a fermentation medium (20 ml) containing 10 g / L glucose at an inoculum amount of 10% (V / V); other components of the fermentation medium were: 3.00 g / L sodium citrate dihydrate, 1.30 g / L (NH4)2SO4, 1.43 g / L KH2PO4, 1.80 g / L K2HPO4·3H2O, 0.13 g / L CaCl2·2H2O, 2.60 g / L MgCl2·6H2O, 4.50 g / L yeast extract, 0.50 g / L L-cysteine ​​hydrochloride, 0.0011 g / L FeSO4·7H2O, and 10.00 g / L sodium 3-(N-morpholine)propanesulfonate. The culture was carried out at 60±2°C and 200 rpm. After the glucose in the culture medium was exhausted, the inoculation amount was continuously transferred at 10% (V / V). The inoculation amount and culture conditions of each transfer were kept consistent, and the glucose concentration was gradually increased to 70 g / L. The transfer was carried out 25 times in total to obtain the seed solution. The seed solution was inoculated into a fermentation medium (20 ml) containing 60 g / L glucose at an inoculation amount of 10% (V / V), and cultured at 60±2°C and 200 rpm for 48 hours. The fermentation results showed that L5 could consume 60 g / L glucose to generate 54.7 g / L lactic acid, with a lactic acid yield of 91.1%, a glucose conversion rate of 99.9%, and an L-lactic acid optical purity of 99.6%.

[0140] Example 4: Fermentation of xylose by recombinant strain to produce L-lactic acid

[0141] The glucose in the culture medium used in Example 3 was replaced with xylose as the fermentation medium of this example. The activated L5 strain was inoculated into a fermentation medium (20 ml) containing 10 g / L xylose at an inoculum of 10% (V / V), and cultured at 60 ± 2 ° C, 200 rpm. After the xylose in the culture medium was exhausted, the inoculum was continuously transferred at an inoculum of 10% (V / V). The inoculum and culture conditions of each transfer were kept consistent, and the xylose concentration was gradually increased to 40 g / L. The transfer was performed 15 times in total to obtain the seed liquid. The seed liquid was inoculated into a fermentation medium (20 ml) containing 40 g / L xylose at an inoculum of 10% (V / V), and cultured at 60 ± 2 ° C, 200 rpm for 48 h. The fermentation results showed that L5 could consume 39.5 g / L xylose to generate 35.8 g / L lactic acid, with a lactic acid yield of 90.6%, a xylose conversion rate of 98.8%, and an L-lactic acid optical purity of 99.4%.

[0142] Example 5 Fermentation of glucose and xylose by recombinant strain to produce L-lactic acid

[0143] The xylose in the culture medium used in Example 4 was replaced with a 1:1 mixture of glucose and xylose as the fermentation medium of this example. The seed solution after transfer in Example 5 was inoculated into the fermentation medium (20 ml) at an inoculum amount of 10% (V / V), and cultured at 60±2°C, 200rpm for 48h. The fermentation results showed that L5 could consume 25g / L glucose and 25g / L xylose to generate 45.4g / L lactic acid, with a lactic acid yield of 90.8%, a glucose and xylose conversion rate of 99.9%, and an L-lactic acid optical purity of 99.5%.

[0144] Example 6 Fermentation of straw sugar by recombinant strain to produce L-lactic acid

[0145] The xylose in the culture medium used in Example 5 was replaced with straw hydrolyzed sugar, which contained 9.3 g / L glucose, 31.1 g / L xylose and 4.9 g / L arabinose, as the fermentation medium of this example. The seed liquid after transfer in Example 5 was inoculated into the above fermentation medium (20 ml) at an inoculum amount of 10% (V / V). After fermentation at 60±2°C and 200 rpm for 48 hours, the glucose conversion rate was 99.9%, the xylose conversion rate was 99.5%, the arabinose conversion rate was 98.3%, 40.8 g / L lactic acid was generated, the lactic acid yield was 90.5%, and the optical purity of L-lactic acid was 99.5%.

[0146] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An engineered thermophilic anaerobic bacillus for producing L-lactic acid with reduced by-products, characterized in that: Using Thermoanaerobacterium thermophilum as the starting strain, the thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase gene mgs, the lactate racemase race1 and the lactate racemase race2 were silenced or deleted.

2. The thermophilic anaerobic bacillus engineered bacteria according to claim 1, characterized in that The thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase gene mgs, the lactate racemase race1 and the lactate racemase race2 were knocked out.

3. The thermophilic anaerobic bacillus engineered bacteria according to claim 2, characterized in that: The starting strain of the thermoanaerobic bacillus engineering bacteria is Thermoanaerobacterium thermosaccharolyticum DSM 571.

4. A method for producing L-lactic acid, characterized in that: The seed solution of the thermophilic anaerobic bacillus engineered bacteria according to any one of claims 1 to 3 is inoculated into a culture medium for fermentation at a temperature of 37-68° C. for at least 12 hours.

5. The method according to claim 4, characterized in that The thermophilic anaerobic bacillus engineered bacteria uses glucose, xylose or arabinose as a carbon source.

6. The method according to claim 5, characterized in that: The seed solution is prepared by the following method: the thermophilic anaerobic bacillus engineered bacteria are inoculated into a culture medium containing different carbon sources for fermentation, and continuous transfer is performed after the carbon source in the culture medium is exhausted; the inoculation amount and culture conditions of each transfer are kept consistent, and the transfer is performed at least 15 times; the carbon source includes glucose or xylose.

7. A method for improving the optical purity of L-lactic acid produced by Thermoanaerobic Bacillus pyroglycolyticus, characterized in that: Knock out lactate racemase race1 and lactate racemase race2 of an engineered bacterium of thermoanaerobic bacterium saccharolyticum; the engineered bacterium of thermoanaerobic bacterium saccharolyticum is based on thermoanaerobic bacterium saccharolyticum DSM571 as a starting strain, and knocks out thymidine kinase gene tdk, phosphate acetyltransferase-acetate kinase gene ptaack, alcohol dehydrogenase gene adhE and methylglyoxal synthase gene mgs; the nucleotide sequence of the thymidine kinase gene tdk is shown in SEQ ID NO.1; the nucleotide sequence of the phosphate acetyltransferase-acetate kinase gene ptaack is shown in SEQ ID NO.2; the nucleotide sequence of the lactate dehydrogenase gene ldh is shown in SEQ ID NO.3; the nucleotide sequence of the methylglyoxal synthase gene mgs is shown in SEQ ID NO.4; the nucleotide sequence of the lactate racemase race1 is shown in SEQ ID NO.5; and the nucleotide sequence of the lactate racemase race2 is shown in SEQ ID NO.

6.

8. A method for reducing the accumulation of byproducts in the process of producing L-lactic acid by Thermoanaerobic Bacillus pyrolyticus, characterized in that: In the thermoanaerobic bacillus saccharolyticus, the thymidine kinase gene tdk, the phosphate acetyltransferase-acetate kinase gene ptaack, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase gene mgs, the lactate racemase race1 and the lactate racemase race2 are knocked out; the nucleotide sequence of the thymidine kinase gene tdk is shown in SEQ ID NO.1; the nucleotide sequence of the phosphate acetyltransferase-acetate kinase gene ptaack is shown in SEQ ID NO.2; the nucleotide sequence of the lactate dehydrogenase gene ldh is shown in SEQ ID NO.3; the nucleotide sequence of the methylglyoxal synthase gene mgs is shown in SEQ ID NO.4; the nucleotide sequence of the lactate racemase race1 is shown in SEQ ID NO.5; and the nucleotide sequence of the lactate racemase race2 is shown in SEQ ID NO.

6.

9. Use of the engineered thermophilic anaerobic bacillus according to any one of claims 1 to 3 in the preparation of L-lactic acid or products containing L-lactic acid.

10. The use according to claim 9, characterized in that: The products include polylactic acid, food, food additives, medicines, pharmaceutical adjuvants, health products, cosmetics, skin care products, feed additives, forage maturation agents, preservatives, slow-release pesticides, descaling agents, pH adjusters, water-based coating synthesizers, photosensitive material cleaning agents, acid-base buffers, leather treatment agents, fiber treatment agents or tobacco treatment agents.

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