Method for improving biosynthesis of propylene glycol by thermoanaerobacter thermosaccharolyticus through metabolic engineering modification

Abstract

The invention discloses a method for improving biosynthesis of propylene glycol by thermoanaerobacter thermosaccharolyticus through metabolic engineering modification, and belongs to the technical field of genetic engineering. According to the invention, the tdk gene, the adhE gene and the Ldh gene are knocked out, so that the propylene glycol synthesis capability of the thermoanaerobacter thermosaccharolyticus is improved, the fermentation yield of propylene glycol is further improved, and compared with an original strain, when domestication fermentation is carried out by taking 10g / L glucose as a substrate, the propylene glycol yield of a wild type strain is 1.07 g / L, the propylene glycol yield of a modified engineering strain is 3.95 g / L, and the propylene glycol yield is improved by 269.2%. In the fermentation test process by increasing the glucose concentration, the yield of propylene glycol consumed by 29.5 g / L glucose can reach 11.62 g / L, the conversion rate from glucose to propylene glycol reaches 39.4%, and the method has industrial application potential.

Description

Technical Field

[0001] The present invention relates to a method for improving the biosynthesis of propylene glycol by Thermoanaerobacterium thermosaccharolyticum through metabolic engineering, belonging to the technical field of genetic engineering. Background Art

[0002] Propylene glycol is an important platform compound and chemical intermediate, and is an important raw material for unsaturated polyester, epoxy resin, polyurethane resin, plasticizer, and surfactant. Propylene glycol has good viscosity and hygroscopicity, and is widely used in the fields of food, medicine, cosmetics, etc., and is a chemical with high industrial demand. In recent years, due to the wide range of biomass resources, safer manufacturing processes, and low impact on the environment, the production of chemicals from biomass through biotechnological approaches has attracted the attention of many researchers. Currently, the research methods for propylene glycol production focus on chemical synthesis and microbial fermentation. Chemical synthesis uses non-renewable petroleum resources as raw materials, or uses heavy metals as catalysts to synthesize 1,2-propylene glycol under high temperature and high pressure conditions, with high environmental and economic costs. Compared with chemical synthesis, microbial fermentation has the advantages of clean process, less pollution, fewer by-products, and lower costs.

[0003] Thermoanaerobacterium thermosaccharolyticum is a thermophilic anaerobic Gram-positive bacterium that can grow at 37-72 degrees Celsius, and it can grow within the pH range of 5.0-7.5, which enables it to greatly reduce the contamination of miscellaneous bacteria. At the same time, Thermoanaerobacterium thermosaccharolyticum has a complete hexose and pentose metabolic system, and can secrete heat-resistant dextranase and glucosidase, effectively promoting the utilization of oligosaccharides. The fermentation of wild Thermoanaerobacterium thermosaccharolyticum mainly secretes metabolic products such as propylene glycol, lactic acid, ethanol, and acetic acid. The variety of metabolic products has seriously affected the application of this bacterium in the production of propylene glycol. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned prior art, the purpose of the present invention is to solve the technical problem that the fermentation metabolic products of wild Thermoanaerobacterium thermosaccharolyticum are diverse (including propylene glycol, lactic acid, ethanol, acetic acid), which has seriously affected the application of Thermoanaerobacterium thermosaccharolyticum in the production of propylene glycol. Through metabolic engineering transformation, the metabolic pathways of ethanol, lactic acid, etc. of wild Thermoanaerobacterium thermosaccharolyticum are blocked to obtain a transformed strain whose main metabolic product is propylene glycol.

[0005] The first technical solution provided by the present invention is a genetically engineered bacterium of Thermoanaerobacterium thermosaccharolyticum with improved propylene glycol synthesis ability, which inhibits the expression of bifunctional ethanol / acetaldehyde dehydrogenase gene adhE, lactate dehydrogenase gene Ldh, and triose phosphate isomerase Tim gene.

[0006] In some embodiments, the inhibition refers to knockout or silencing.

[0007] In some embodiments, the nucleotide sequence of the bifunctional ethanol / acetaldehyde dehydrogenase gene adhE is as shown in SEQ ID NO.2.

[0008] In some embodiments, the nucleotide sequence of the lactate dehydrogenase gene Ldh is as shown in SEQ ID NO.3.

[0009] In some embodiments, the nucleotide sequence of the triosephosphate isomerase gene Tim is as shown in SEQ ID NO.4.

[0010] In some embodiments, the starting strain of the genetically engineered strain of Thermoanaerobacterium thermosaccharolyticum is T. thermosaccharolyticum DSM571.

[0011] The second technical solution provided by the present invention is a method for improving the ability of Thermoanaerobacterium thermosaccharolyticum to biosynthesize propylene glycol, and the method is to knockout or silence the bifunctional ethanol / acetaldehyde dehydrogenase gene adhE, the lactate dehydrogenase gene Ldh, and the triosephosphate isomerase gene Tim.

[0012] In some embodiments, the nucleotide sequence of the bifunctional ethanol / acetaldehyde dehydrogenase gene adhE is as shown in SEQ ID NO.2.

[0013] In some embodiments, the nucleotide sequence of the lactate dehydrogenase gene Ldh is as shown in SEQ ID NO.3.

[0014] In some embodiments, the nucleotide sequence of the triosephosphate isomerase gene Tim is as shown in SEQ ID NO.4.

[0015] The third technical solution provided by the present invention is a method for producing propylene glycol, using the genetically engineered strain of Thermoanaerobacterium thermosaccharolyticum described in the first technique as a fermentation strain to produce propylene glycol.

[0016] In some embodiments, after the genetically engineered strain of Thermoanaerobacterium thermosaccharolyticum described in the first technique is activated and cultured, it is added to a reaction system with glucose as a carbon source for fermentation to prepare a fermentation broth, and propylene glycol is separated and extracted from the fermentation broth.

[0017] In some embodiments, the temperature of the activation culture is 37 - 72 °C, and the time of the activation culture is 12 h.

[0018] In some embodiments, the activation medium comprises the following components: sodium citrate dihydrate 3 g / L, (NH 4 )2 SO 4 1.3 g / L, KH 2 PO 4 1.43 g / L, K 2 HPO 4 ·3H 2 O 1.8 g / L, CaCL 2 0.1 g / L, yeast extract 4.5 g / L, MgCL 2 ·6H 2 O 2.6 g / L, L-cysteine hydrochloride anhydrous 0.50 g / L, FeSO 4 ·7H 2 O 0.0011 g / L, cellobiose 5 g / L, NaHCO 3 5 g / L, 3-(N-morpholino)propanesulfonic acid sodium 10 g / L.

[0019] In some embodiments, in the reaction system, the content of glucose is ≥ 10 g / L.

[0020] In some embodiments, the temperature of the fermentation is 37 - 72 °C, and the time of the fermentation is 1 - 5 days.

[0021] In some embodiments, the culture medium used for the fermentation further comprises the following components: sodium citrate dihydrate 3.00 g / L, (NH 4 ) 2 SO 4 1.30 g / L, KH 2 PO 4 1.43 g / L, K 2 HPO 4 ·3H 2 O 1.80 g / L, CaCl 2 ·2H 2 O 0.13 g / L, MgCl 2 ·6H 2 O 2.60 g / L, yeast extract 4.50 g / L, L-cysteine hydrochloride 0.50 g / L, FeSO 4 ·7H 2 O 0.0011 g / L, 3-(N-morpholino)propanesulfonic acid sodium 10.00 g / L. Among them, a high-concentration glucose mother liquor is prepared in advance, and the glucose concentration added during fermentation depends on the strain domestication situation.

[0022] In some embodiments, the method for separating and extracting propylene glycol from the fermentation broth includes but is not limited to membrane separation, distillation, and extraction.

[0023] The fourth technical solution provided by the present invention is the application of the genetically engineered bacterium of Thermoanaerobacterium thermosaccharolyticum described in the first technical solution, the method described in the second technical solution, or the method described in the third technical solution in the production of propylene glycol.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] By knocking out the adhE gene, Ldh gene, and Tim gene, the present invention improves the ability of Thermoanaerobacterium thermosaccharolyticum to synthesize propylene glycol, thereby increasing the fermentation yield of propylene glycol. Compared with the starting strain, when fermenting with 10 g / L glucose as the substrate, the propylene glycol yield of the wild-type strain (WT) is 1.07 g / L, and the propylene glycol yield of the genetically engineered strain (A3) is 3.95 g / L, with a 269.2% increase in propylene glycol yield. During the fermentation test with increasing glucose concentration, when consuming 29.5 g / L glucose, the propylene glycol yield can reach 11.62 g / L, and the conversion rate of glucose to propylene glycol reaches 39.4%, showing potential for industrial application. Description of the Drawings

[0026] Figure 1 It is the colony PCR result of knocking out the tdk gene in T. thermosaccharolyticum DSM571; Lane M: DNA marker; Lanes 1-8: Monoclonal samples selected from the experimental group colonies; Lane W: Wild-type control.

[0027] Figure 2 It is the electrophoresis diagram of KAN screening verification for knocking out adhE in strain Y0; Lane M: DNA marker; Lanes 1-8: Monoclonal samples selected from the experimental group colonies; Lane W: Wild-type control.

[0028] Figure 3 It is the electrophoresis diagram of FUDR screening verification for knocking out adhE in strain Y0; Lane M: DNA marker; Lanes 1-5: Monoclonal samples selected from the experimental group colonies; Lane W: Wild-type control.

[0029] Figure 4 It is the electrophoresis diagram of KAN screening verification for knocking out Ldh in strain A1; Lane M: DNA marker; Lanes 1-8: Monoclonal samples selected from the experimental group colonies; Lane W: Wild-type control.

[0030] Figure 5 It is the electrophoresis diagram of FUDR screening verification for knocking out Ldh in strain A1; Lane M: DNA marker; Lanes 1-6: Monoclonal samples selected from the experimental group colonies; Lane W: Wild-type control.

[0031] Figure 6It is the electrophoresis diagram for KAN screening verification of Tim knockout in strain A2; Lane M: DNA marker; Lanes 1-8: Monoclonal samples selected from colonies in the experimental group; Lane W: Wild-type control.

[0032] Figure 7 It is the electrophoresis diagram for FUDR screening verification of Tim knockout in strain A2; Lane M: DNA marker; Lanes 1-7: Monoclonal samples selected from colonies in the experimental group; Lane W: Wild-type control.

[0033] Figure 8 It is the fermentation product diagram of strain T. thermosacch DSM571 and the engineered strain.

[0034] Figure 9 It is the propylene glycol production diagram of engineered strain A3 of T. thermosacch DSM571 consuming several glucose concentrations.

[0035] Figure 10 It is the propylene glycol production diagram of strain T. thermosacch DSM571 with single knockout of tdk, adhE, ldh, tim and the engineered strain (A3).

[0036] Figure 11 It is the structure diagram of the knockout plasmid. Detailed implementation manners

[0037] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0038] The information of the strains and plasmids involved in the following embodiments is shown in Table 1:

[0039] Table 1 Strains and plasmids

[0040]

[0041] The main reagents and drugs involved in the following embodiments:

[0042] Potassium citrate, L-cysteine hydrochloride, 3-(N-morpholino)propanesulfonic acid sodium were purchased from Macklin, kanamycin sulfate was purchased from Beijing Solarbio Science & Technology Co., Ltd., FUDR (5-fluorodeoxyuridine) was purchased from (Beijing) Cool Chemical Technology Co., Ltd., palladium particle molecular sieve and desiccant were both purchased from Shanghai Longyue Instrument Equipment Co., Ltd., NaCl, anhydrous CaCl 2 、MgCl 2 ·6H 2 O、FeCl2·4H 2 O、Na 2 SO 4 、KH2 PO 4 、K 2 HPO 4 ·3H 2 O, sulfuric acid, and isopropanol were all purchased from Sinopharm Chemical Reagent Co., Ltd. The nucleic acid dye, 5000 DNA Marker, and 1 kb DNA ladder were purchased from Shanghai Yeasen Biotechnology Co., Ltd., and the gel extraction kit was purchased from OMEGA Company, USA.

[0043] Gibson Premix was purchased from NEB Company, USA, Prime STAR Max Premix (2×) was purchased from TaKaRa Bio Inc., Japan, and 2×Hieff Gold PCR Master Mix was purchased from Shanghai Yeasen Biotechnology Co., Ltd.

[0044] The culture media involved in the following examples are shown in Table 2:

[0045] Table 2 Composition of Culture Media

[0046]

[0047] The components in Table 2 are for the activation culture medium, and the fermentation culture medium uses glucose instead of cellobiose as the carbon source. The instruments and equipment involved in the following examples are shown in Table 3:

[0048] Table 3 Main Instruments and Equipment

[0049]

[0050] The primers involved in the following examples are shown in Table 4 and Table 5:

[0051] Table 4 List of Homologous Arm Primers

[0052]

[0053]

[0054] Table 5 List of Gene Knockout Verification Primers

[0055]

[0056]

[0057] The detection methods involved in the following examples are as follows:

[0058] 1. Fermentation test sample treatment:

[0059] Take 2 mL of each sample. First, centrifuge the sample at 12,000 rpm for 1 min, take 900 μL of the supernatant and add it to an EP tube, then add 100 μL of 10% dilute sulfuric acid and mix well. Prepare 2 portions of the sample and store them in a refrigerator at 4°C. Filter through a 0.22 μm filter membrane, and load about 500 μL of the sample into each injection vial.

[0060] 2. Detection of fermentation test samples:

[0061] Analyze the fermentation samples using high-performance liquid chromatography (HPLC) combined with an RID-10A refractive index detector (Shimadzu, Kyoto, Japan) and an HPX-87H (300 x 7.8 mm, Bio-rad, Hercules, CA, USA) chromatographic column.

[0062] Prepare a mobile phase containing a standard sample of the main product and 5 mM dilute sulfuric acid. Filter the sample through a 0.22 μm filter membrane, load about 500 μL of the sample into each injection vial, and then set the main parameters according to the acid column instruction manual: flow rate 0.5 mL / min, column oven temperature 60°C, detector cell temperature 40°C, injection volume 10 μL, and the collection time for one sample is 30 minutes.

[0063] 3. Gel extraction and purification method:

[0064] Select the omega gel extraction kit for the recovery and purification experiment.

[0065] (1) Run the PCR product on a recovery gel, cut the gel and put it into a 2 mL EP tube, weigh the cut gel to determine its volume (if the mass of the gel block is 0.3 g, then the volume of the gel block is 0.3 mL).

[0066] (2) Add one volume of XP2 binding buffer to the EP tube.

[0067] (3) Heat in a 55°C metal bath for 5 - 7 minutes until the gel block dissolves. Shake the EP tube every 2 minutes during heating to accelerate dissolution.

[0068] (4) Install the centrifugal column in the standard 2 mL collection tube and make a mark.

[0069] (5) Add the solubilized mixture to the centrifugal column. If it exceeds 700 μL, add it in two portions. Centrifuge at 10,000 g for 1 min, pour the centrifugate into the centrifugal column and repeat centrifugation at 10,000 g for 1 min, discard the liquid, and reinstall the centrifugal column in the collection tube.

[0070] (6) Add 300 μL of XP2 Binding Buffer, centrifuge at 13,000 g for 1 min, discard the liquid, and reinstall the centrifugal column in the collection tube.

[0071] (7) Add 700 μL of SPW Buffer, centrifuge at 13,000 g for 1 min, discard the liquid, and reinstall the centrifugal column into the collection tube.

[0072] (8) Repeat step (7) once, discard the liquid, and reinstall the centrifugal column into the collection tube.

[0073] (9) Centrifuge the empty column at 13,000 g for 2 min to remove excess alcohol.

[0074] (10) Transfer the centrifugal column to a new 1.5 mL EP tube and add 30 μL of Elution Buffer.

[0075] (11) After standing at room temperature for 2 min, centrifuge at 13,000 g for 1 min and label.

[0076] (12) Take 1 μL and measure the nucleic acid concentration using a micro-spectrophotometer.

[0077] (13) Store the DNA in a -20 °C refrigerator.

[0078] Example 1: Knockout of the thymidine kinase gene tdk

[0079] (1) Construction of the knockout plasmid

[0080] Amplify fragment 1 from pZJ24htktdk (published in the paper "A markerless gene deletion and integration system for Thermoanaerobacter ethanolicus") using primers P1 and P2 in Table 4, amplify fragment 2 from pTT0 using primers P3 and P4, amplify the upstream homologous arm US of the target gene tdk from the genome of Thermoanaerobacter thermosaccharolyticus DSM571 using primers P5 and P6, and amplify the downstream homologous arm DS of the target gene tdk (the nucleotide sequence of the tdk gene is SEQ ID No. 1) using primers P7 and P8; ligate fragment 1, fragment 2, US, and DS using Gibson assembly 2X mix, transfer the Gibson reactant into Escherichia coli DH5a, after plating, pick monoclonal colonies for verification after 1 - 2 days and extract the plasmid, named PTT_tdk.

[0081] (2) Gene knockout and monoclonal screening

[0082] Add 0.1 - 1 μg of the plasmid PTT_tdk constructed in step (1) to the bacterial cells T. thermosacch DSM571 to be transformed. In an anaerobic culture box, quickly place the anaerobic gas - generating bag into the anaerobic culture box after opening it, quickly cover the lid of the anaerobic culture box, and culture at 37 - 70 °C for 18 - 22 hours. During the culture, detect the OD value with a spectrophotometer. When the OD value reaches between 0.2 - 1.0, collect the bacterial liquid, spread the bacterial liquid on plates for gradient screening, add FUDR to a working concentration of 10 - 100 mg / L, and culture for 3 - 7 days. Use primers P27 and P28 in Table 5 for colony PCR verification, and at the same time use wild - type Thermoanaerobacterium thermosaccharolyticum DSM571 as a control. The results are as Figure 1 shown. Pick positive clones, and select the 3rd monoclonal clone numbered as strain Y0. The construction of strain Y0 mainly lies in that after the tdk gene is knocked out, the resistance marker can be recycled, which is convenient for knocking out other genes.

[0083] Example 2: Knockout of the adhE gene of strain Y0

[0084] (1) Construction of the knockout plasmid

[0085] Amplify fragment 1 from plasmid pZJ24htktdk using primers P1 and P2 in Table 4; amplify fragment 2 from pTT0 using primers P3 and P4; amplify the upstream homologous arm US of the target gene adhE from the genome of Thermoanaerobacterium thermosaccharolyticum DSM571 using primers P9 and P10 in Table 3, amplify the downstream homologous arm DS of the target gene adhE from the genome using primers P11 and P12, and amplify the middle homologous arm INT of the target gene adhE from the genome using primers P13 and P14; ligate fragment 1, fragment 2, US, DS, and INT using Gibson assembly 2X mix. Transfer the Gibson reaction product into Escherichia coli DH5α, spread on plates, and pick monoclonal clones for verification after 1 - 2 days, then extract the plasmid and name it PTT_adhE.

[0086] (2) Gene knockout and monoclonal screening

[0087] Add 0.1 - 1 μg of the plasmid PTT_adhE constructed in step (1) to the bacterial cells Y0 to be transformed constructed in Example 1. In an anaerobic culture box, quickly place the anaerobic gas - generating bag into the anaerobic culture box after opening it, quickly cover the lid of the anaerobic culture box, and culture at 37 - 72 °C for 18 - 22 hours. During the culture, detect the OD value with a spectrophotometer, and when the OD value reaches between 0.2 - 1.0, collect the bacterial liquid for subsequent screening.

[0088] The first round of screening: Add kanamycin to the working concentration of 200 - 400 mg / L, perform gradient screening by spreading on plates, incubate for 3 - 4 days, and observe whether colonies appear during this period. Select colonies, use primer P14 in Table 4 and primer P29 in Table 5 for colony PCR verification, and use wild-type Thermoanaerobacterium thermosaccharolyticum DSM571 as a control. The results are as Figure 2 shown. Monoclonal clones 2 and 3 selected are positive clones. Select monoclonal clone 2 for the next FUDR screening experiment. The second round of screening: Spread the water-soluble bacteria verified correctly in the first round on plates for gradient screening, add FUDR to the working concentration of 10 - 100 mg / L, and incubate for 3 - 7 days. Select colonies, use primer P29 and P30 in Table 5 for colony PCR verification, and use wild-type Thermoanaerobacterium thermosaccharolyticum DSM571 as a control. The results are as Figure 3 shown. Select monoclonal clone 5 and number it as strain A1.

[0089] Example 3 Knockout of the Ldh gene of strain A1

[0090] The specific implementation method is the same as that of Example 2, with the differences being:

[0091] In step (1), upstream homologous arm US of the target gene Ldh was amplified from the genome of Thermoanaerobacterium thermosaccharolyticum DSM571 using primers P15 and P16, downstream homologous arm DS of the target gene Ldh was amplified from the genome using primers P17 and P18, and middle homologous arm INT of the target gene Ldh was amplified from the genome using primers P19 and P20. A knockout plasmid was constructed and named PTT_LDH;

[0092] In step (2), the plasmid PTT_LDH constructed in step (1) was added to the transformable bacterial cells A1 constructed in Example 2. Primer P20 and primer P31 were used for the first round of kanamycin screening colony PCR verification, and primers P31 and P32 were used for the second round of FUDR screening colony PCR verification.

[0093] The results of the first round of kanamycin screening verification are as Figure 4 shown. Monoclonal clones 1, 2, 6, 7, and 8 selected are positive clones. Select monoclonal clone 1 for the next FUDR screening experiment. The results of the second round of FUDR screening verification are as Figure 5 shown. Among the selected monoclonal clones, all except clone 2 are positive clones. Select monoclonal clone 3 and number it as strain A2.

[0094] Example 4 Knockout of the Tim gene of strain A2

[0095] The specific implementation method is the same as that of Example 2, with the differences being:

[0096] Step (1): The upstream homologous arm US of the target gene Tim was amplified from the genome of Thermoanaerobacterium thermosaccharolyticum DSM571 using primers P21 and P22 in Table 4. The downstream homologous arm DS of the target gene Tim was amplified from the genome using primers P23 and P24. The middle homologous arm INT of the target gene Tim was amplified from the genome using primers P25 and P26, and a knockout plasmid was constructed and named PTT_TIM.

[0097] Step (2): The plasmid PTT_TIM constructed in step (1) was added to the to-be-transformed bacterial cells A2 constructed in Example 3. The first round of kanamycin screening colony PCR verification was performed using primers P26 and P33, and the second round of FUDR screening colony PCR verification was performed using primers P33 and P34.

[0098] The results of the first round of kanamycin screening verification were as Figure 6 shown. Among the selected monoclonal colonies, all except No. 1 were positive clones. Monoclonal colony No. 6 was selected for the next FUDR screening experiment. The results of the second round of FUDR screening verification were as Figure 7 shown. Among the selected monoclonal colonies, all except Nos. 3 and 7 were positive clones. Monoclonal colony No. 6 was selected and numbered as strain A3.

[0099] Fermentation production of propylene glycol in Example 5

[0100] The wild-type WT strain and strains Y0, A1, A2, and A3 constructed in Examples 1-4 were activated and cultured at 37 - 72 °C and 200 rpm for 12 h to obtain seed solutions. Then, the seed solutions were transferred to a medium containing 10 g / L glucose at an inoculation amount of 10% (V / V) and continuously transferred 4 - 6 times at 37 - 72 °C and 200 rpm, with each culture lasting 1 - 3 days. The results were as Figure 8 shown. After fermentation using 10 g / L glucose as the substrate, the propylene glycol yield of the wild-type WT strain was 1.07 g / L, and the glucose-to-propylene glycol conversion rate was 10.7%. The propylene glycol yield of strain Y0 was 1.12 g / L, and the glucose-to-propylene glycol conversion rate was 11.2%. There was little change in the propylene glycol yield and conversion rate compared to the wild-type WT strain. The propylene glycol yield of strain A1 was 2.83 g / L, and the glucose-to-propylene glycol conversion rate was 28.3%. The propylene glycol yield and conversion rate increased by 164.5% compared to the wild-type WT strain. The propylene glycol yield of strain A2 was 1.95 g / L, and the glucose-to-propylene glycol conversion rate was 19.5%. The propylene glycol yield and conversion rate increased by 82.2% compared to the wild-type WT strain. The propylene glycol yield of strain A3 was 3.95 g / L, and the glucose-to-propylene glycol conversion rate was 39.5%. The propylene glycol yield and conversion rate increased by 269.2% compared to the wild-type WT strain. During the fermentation test with increased glucose concentration, as Figure 9As shown, when fermentation tests were carried out with glucose contents of 15.1 g / L, 19.0 g / L, and 29.5 g / L, the yields of propylene glycol were 5.70 g / L, 7.04 g / L, and 11.62 g / L respectively, and the conversion rates of glucose to propylene glycol were 37.7%, 37.1%, and 39.4% respectively.

[0101] Comparative Example 1:

[0102] The specific implementation manner was the same as that of Example 2, except that the adhE, Ldh, and Tim genes were knocked out respectively using strain Y0 as the starting strain, and three knockout strains, namely Y0-ΔadhE, Y0-ΔLdh, and Y0-ΔTim, were obtained. The above strains were fermented according to the method of Example 5, and the results were as Figure 10 shown. Taking the fermentation test with 10 g / L glucose as an example, after separately knocking out the adhE, Ldh, and Tim genes, after separately knocking out adhE, the yield of propylene glycol was 2.83 g / L, and the conversion rate of glucose to propylene glycol was 28.3%; after separately knocking out Ldh, the yield of propylene glycol was 1.42 g / L, and the conversion rate of glucose to propylene glycol was 14.2%; after separately knocking out Tim, the yield of propylene glycol was 2.91 g / L, and the conversion rate of glucose to propylene glycol was 29.1%; after sequentially knocking out the three key genes, strain Y0-ΔadhEΔLdhΔTim (strain A3) was obtained, and the yield of propylene glycol was 3.95 g / L; the conversion rate of glucose to propylene glycol was 39.5%.

[0103] The above are only the preferred and feasible embodiments of the present invention, and are not limitations on the present invention. The present invention is not limited to the above examples either. Changes, modifications, additions, or substitutions made by those skilled in the art within the essence of the present invention should also fall within the protection scope of the present invention.

Claims

1. A genetically engineered strain of Thermoanaerobacterium thermosaccharolyticum with improved propylene glycol synthesis ability, characterized in that, it inhibits the expression of the bifunctional ethanol / acetaldehyde dehydrogenase gene adhE, lactate dehydrogenase gene Ldh and triose phosphate isomerase Tim gene of Thermoanaerobacterium thermosaccharolyticum.

2. The genetically engineered strain according to claim 1, characterized in that, the inhibition refers to knockout or silencing.

3. The genetically engineered strain according to claim 1 or 2, characterized in that, the nucleotide sequence of the bifunctional ethanol / acetaldehyde dehydrogenase gene adhE is shown in SEQ ID NO.2, the nucleotide sequence of the lactate dehydrogenase gene Ldh is shown in SEQ ID NO.3, and the nucleotide sequence of the triose phosphate isomerase gene Tim is shown in SEQ ID NO.

4.

4. The genetically engineered strain according to any one of claims 1 to 3, characterized in that, the starting strain of the genetically engineered Thermoanaerobacterium thermosaccharolyticum is T. thermosaccharolyticum DSM571.

5. A method for improving the biosynthesis ability of Thermoanaerobacterium thermosaccharolyticum to produce propylene glycol, characterized in that, the method is to knockout or silence the bifunctional ethanol / acetaldehyde dehydrogenase gene adhE, lactate dehydrogenase gene Ldh and triose phosphate isomerase gene Tim of Thermoanaerobacterium thermosaccharolyticum.

6. A method for producing propylene glycol, characterized in that, using the genetically engineered Thermoanaerobacterium thermosaccharolyticum strain according to claims 1 to 4 as the fermentation strain to produce propylene glycol.

7. The method according to claim 6, characterized in that, after the genetically engineered Thermoanaerobacterium thermosaccharolyticum strain according to claims 1 to 4 is activated and cultured, it is added to a reaction system with glucose as the carbon source for fermentation to prepare a fermentation broth, and propylene glycol is separated and extracted from the fermentation broth.

8. The method according to claim 7, characterized in that, in the reaction system, the content of glucose is ≥10 g / L.

9. The method according to claim 7, characterized in that, the temperature of the fermentation is 37 - 72 °C, and the time of the fermentation is 1 - 5 days.

10. Use of the genetically engineered Thermoanaerobacterium thermosaccharolyticum strain according to claims 1 to 4, the method according to claim 5, or the method according to any one of claims 6 to 9 in the production of propylene glycol.

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