A strain for producing lactic acid monomer using glycerol and its application

Abstract

The present invention relates to the technical fields of fermentation engineering and genetic engineering, and relates to a production strain capable of synthesizing lactic acid monomers with high conversion rate of glycerol and a method for obtaining the same, specifically, cloning, expressing, deleting key regulatory genes related to cell growth, and fermentation production of lactic acid monomers with glycerol as raw material. The engineering bacteria provided by the present invention use a strain with lactic acid production ability as a host, express the coding gene of the key enzyme in the PDH complex in the host via a temperature-sensitive plasmid, and then delete the coding gene of the key enzyme on the host chromosome, so as to obtain a new lactic acid monomer production strain whose cell growth and proliferation can be regulated by the culture temperature, and the conversion rate of glycerol to lactic acid of the strain is increased to more than 96% under the matching variable temperature microaerobic fermentation process conditions.

Description

Technical field:

[0001] The present invention relates to the technical field of fermentation engineering and genetic engineering, and relates to a production strain capable of synthesizing lactic acid monomers with high conversion rate of glycerol and a method for obtaining the same, specifically to the cloning, expression and deletion of key regulatory genes related to cell growth and the fermentation production of lactic acid monomers using glycerol as raw material. Background technology:

[0002] Glycerol, a byproduct of the oil hydrolysis industry and biodiesel, is one of the ideal bulk raw materials for the fermentation industry. Glycerol used as a raw material in the fermentation production of lactic acid not only helps to solve the problem of comprehensive utilization of byproducts of the oil hydrolysis industry and biodiesel industry, but also helps to enrich the source of industrial raw materials for lactic acid monomer fermentation production. Compared with sugar substances such as glucose, xylose, and lactose, glycerol has higher reducibility (Yazdani SS, Gonzalez R, Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry [J]. Curr Opin Biotechnol, 2007, 18 (3): 213-219). Therefore, when glycerol is used as a raw material for product synthesis, how to provide sufficient reducing power receptors and achieve a balance of redox power is the key to ensuring high conversion efficiency of glycerol to target products. One of the methods to solve the imbalance of glycerol metabolic reducing power is to select strains suitable for producing lactic acid by glycerol fermentation under anaerobic conditions by reconstructing the glycerol metabolic pathway (Mazumdar et al. Engineered Escherichia coli strains for the homofermentative production of D-lactic acid from glycerol [J]. Appl Environ Microbiol, 2010, 76 (13): 4327-4336; Mazumdar et al. Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli. Microbial Cell Factories 2013, 12: 7). There are two problems in the implementation of this strategy. First, the conversion rate of glycerol to D-lactic acid is low, and the fermentation cycle is long and the lactic acid synthesis rate is low. In addition to the strategy of modifying the glycerol metabolic pathway, the goal of rapidly synthesizing lactic acid monomers can be achieved by limiting the oxygen supply during the fermentation acid production process and combining it with the enhancement of the lactic acid synthesis pathway and the variable temperature fermentation process (Tian Kangming, et al. Using temperature regulation to achieve efficient conversion of glycerol to D-lactic acid by new recombinant bacteria [J]. Chinese Journal of Biotechnology, 2013, 29(1): 111-114; Tian Kangming, et al. Metabolic engineering of Escherichia coli to efficiently synthesize L-lactic acid from glycerol [J]. Chinese Journal of Biotechnology, 2013, 29(9): 1-10).

[0003] The present invention selects to regulate the expression of key enzymes of the PDH complex. The key enzyme encoding gene of the PDH complex is expressed by a temperature-sensitive free plasmid, and the original key enzyme encoding gene of the PDH complex on the chromosome is deleted. When the free plasmid exists at a low culture temperature, the expression of the key enzyme of the PDH complex can proceed normally, and after the culture temperature is increased, with the loss of the free plasmid, the key enzyme of the PDH complex is no longer expressed, thereby achieving the restriction of cell growth and proliferation. Even under the ventilation conditions of fermenting and synthesizing lactic acid monomers with glycerol as a raw material, the growth and proliferation of the cells are still restricted due to the lack of key enzymes of the PDH complex. Glycerol flows to the synthesis of lactic acid to the maximum extent, achieving the production of lactic acid monomers by fermentation with glycerol as a raw material at a high conversion rate and a high synthesis rate, and the conversion rate of glycerol to lactic acid monomers in the fermentation acid production stage is above 96%. Summary of the invention:

[0004] The purpose of the present invention is to regulate the key enzyme expression process of the growth of lactic acid monomer production strains by means of free temperature-sensitive plasmids and strong promoters in lactic acid monomer production strains through gene cloning and gene recombination technology, so as to achieve the maximum restriction of cell growth under microaerobic fermentation conditions by increasing the fermentation temperature when glycerol is used as the fermentation raw material, and to synthesize lactic acid monomers with glycerol at a high conversion rate. In addition, a fermentation production technology suitable for efficiently synthesizing lactic acid monomers using glycerol as a raw material is established in conjunction with a variable temperature microaerobic fermentation process.

[0005] One of the technical solutions provided by the present invention is a genetically engineered bacterium that uses glycerol to produce lactic acid. The engineered bacterium uses a strain with lactic acid production ability as a host, expresses the coding gene of the key enzyme in the PDH complex in the host through a temperature-sensitive plasmid, and then deletes the coding gene of the key enzyme on the host chromosome, thereby obtaining a new lactic acid monomer production strain whose cell growth and proliferation can be regulated by the culture temperature. The conversion rate of glycerol to lactic acid of the strain is increased to more than 96% under the matching variable temperature microaerobic fermentation process conditions;

[0006] Furthermore, the key enzymes in the PDH complex are enzymes that catalyze the conversion of pyruvate to acetyl-CoA, including pyruvate dehydrogenase (E1p), dihydrolipoamide transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3). The aceE, aceF, and lpdA genes encode these three proteins, respectively.

[0007] Preferably, the key enzyme in the PDH complex is dihydrolipoamide acetyltransferase E2p, whose amino acid sequence is shown in SEQ ID NO.2; E2p is encoded by aceF gene, whose nucleotide sequence is shown in SEQ ID NO.1;

[0008] Furthermore, the E2p coding gene aceF is cloned into a temperature-sensitive plasmid for free expression in a host, and the temperature-sensitive plasmid has the function of encoding three proteins: Exo, Beta, and Gam;

[0009] Furthermore, the temperature-sensitive plasmid is selected from pKD3, pKD4, pKD46, pKD20 or pKD13;

[0010] Preferably, the temperature-sensitive plasmid is pKD46;

[0011] Furthermore, the thermosensitive plasmid also contains promoter and terminator sequences for controlling the expression of key enzymes in the PDH complex; the sources of the promoter and terminator include but are not limited to the promoter PldhA of Escherichia coli lactate dehydrogenase (LdhA), the promoter PamyL of Bacillus licheniformis amylase (Niu DD&Wang ZX, 2007, 34:357-362), T7 promoter (GenBank accession number: M38302.1), Tet promoter (GenBank accession number: K01791.1), Bacillus subtilis xylose promoter Pxyl promoter, Bacillus megaterium xylose Pxyl promoter or Bacillus licheniformis promoter Pxyl (Wang Shanying. Jiangnan University, 2016) or lactose-inducible promoter Plac (Dickson RC. 1975, 187:27-35), and the terminators corresponding to the above promoters;

[0012] Preferably, the temperature-sensitive plasmid contains a promoter PamyL and a terminator TamyL derived from Bacillus licheniformis amylase, the nucleotide sequence of PamyL is shown in SEQ ID NO.17, and the nucleotide sequence of TamyL is shown in SEQ ID NO.18;

[0013] Furthermore, the expression of the coding gene of the key enzyme in the PDH complex is deleted, including but not limited to gene knockout, gene inactivation, etc., wherein the gene inactivation includes the inactivation of its function by deletion, knockout or insertion of a fragment;

[0014] Preferably, the expression of the coding gene of the key enzyme in the PDH complex is deleted by gene knockout;

[0015] More preferably, the gene encoding the key enzyme in the PDH complex on the host chromosome is knocked out by utilizing the function of thermosensitive plasmid-assisted homologous recombination;

[0016] Furthermore, the host strain capable of producing lactic acid monomers includes but is not limited to Escherichia coli, Saccharomyces cerevisiae, Candida tropicalis, Bacillus coagulans, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Bacillus licheniformis, etc.;

[0017] Preferably, the host bacteria is Escherichia coli CGMCC NO.11059 or CGMCC NO.11060;

[0018] More preferably, the genetically engineered bacteria for producing lactic acid monomers using glycerol are based on Escherichia coli CGMCC 11059 or CGMCC11060 as the host, the coding gene aceF of dihydrolipoamide acetyltransferase, the promoter PldhA and the terminator TldhA are cloned into the thermosensitive plasmid pKD46 to obtain the recombinant plasmid pKD-PldhA-aceF, and the recombinant plasmid pKD-PldhA-aceF is transformed into the above host, and the aceF gene on the chromosome of the host bacteria is knocked out to obtain recombinant Escherichia coli GSFD and GSFL with high production of D-lactic acid and L-lactic acid; the recombinant Escherichia coli GSFD and GSFL are cultured at 42°C to lose the recombinant plasmid pKD-PldhA-aceF, and the recombinant bacteria GDFD are obtained. and GDFL; and then using the recombinant Escherichia coli GDFD or GDFL as a host, cloning the dihydrolipoamide acetyltransferase encoding gene aceF, the promoter PamyL and the terminator TamyL into the simplified plasmid pKDM obtained by removing part of the sequence from the pKD46 plasmid (compared with the plasmid pKD46, the pKDM plasmid deleted the nucleic acid sequence fragment encoding the three proteins Exo, Beta and Gam at sequence positions 3391-6329), obtaining the recombinant plasmid pKDM-PamyL-aceF, and transforming the recombinant plasmid pKDM-PamyL-aceF into the above-mentioned host GDFD or GDFL to obtain the recombinant Escherichia coli;

[0019] Furthermore, using E. coli CGMCC NO.11060 as the initial host, E. coli GYSFL was obtained which could metabolize glycerol and synthesize L-lactic acid monomers with high conversion rate;

[0020] Furthermore, using E. coli CGMCC NO.11059 as the initial host, E. coli GYSFD was obtained which can metabolize glycerol and synthesize D-lactic acid monomers with high conversion rate;

[0021] Both E. coli GYSFL and GYSFD have the characteristic that the cell growth process can be precisely regulated by temperature changes.

[0022] The second technical solution provided by the present invention is the use of the genetically engineered bacteria described in the first technical solution in the production of lactic acid;

[0023] Furthermore, the method for producing lactic acid by fermentation in a fermenter using the genetically engineered bacteria is as follows:

[0024] Add 2-5 wt.% glycerol to the inorganic salt medium, inoculate the production strain at an inoculum of 0.1%-10%, and culture for 4-16 hours at 25-35°C, ventilation of 0.1-3 vvm and 200-1000 r / min to control the dissolved oxygen concentration to be no less than 20%. During the process, add 5-25 wt.% ammonia water to maintain the pH of the fermentation liquid at 6.0-7.5. When the bacterial OD 600 After reaching 10-50, the ventilation volume is adjusted to 0.1-1.5vvm and the rotation speed is reduced to 100-600r / min to maintain the dissolved oxygen concentration at 1-10%, and the fermentation temperature is increased to 35-55°C to enter the lactic acid synthesis stage, during which glycerol is added, and the total amount of glycerol added is 200-270g / L fermentation liquid (calculated based on the initial fermentation volume); in the lactic acid synthesis stage, 5-30wt.% calcium hydroxide suspension is added to maintain the pH of the fermentation liquid at 6.0-7.5; when the glycerol concentration is lower than 0.5g / L, or the glycerol consumption rate is lower than 1g / (L·h), or the solid content of the fermentation liquid is too high to be stirred, the fermentation is terminated, and the entire fermentation cycle is 30-40h;

[0025] Further, glycerol is added by batch-wise adding a glycerol solution having a concentration of 3 to 70 wt.% or by adding a 10 to 70 wt.% glycerol solution at a flow rate of 3 to 30 g / (L·h);

[0026] Furthermore, the inorganic salt culture medium is composed of (w / v): 0.5% to 2.5% disodium hydrogen phosphate dodecahydrate, 0.02% to 0.1% sodium chloride, 0.1% to 1.0% potassium dihydrogen phosphate, 0.01% to 0.5% ammonium chloride, and the rest is water;

[0027] Preferably, 3-4 wt.% glycerol is added to an inorganic salt culture medium, the production strain is inoculated with an inoculum of 5%-8%, and the culture is carried out at 30-35° C., ventilation of 0.3-2 vvm and 200-1000 r / min for 6-8 h. During the process, 15-25 wt.% ammonia water is added to maintain the pH value of the fermentation liquid at 6.5-7.0, and then the ventilation volume is adjusted to 0.3-1.0 vvm and the rotation speed is reduced to 100-400 r / min, and the fermentation temperature is increased to 40-50° C., and then a glycerol solution with a concentration of 50-60 wt.% is added in batches or a 50-60 wt.% glycerol solution is added at a flow acceleration of 8-20 g / (L·h) so that the total amount of glycerol added is 230-240 g / L of fermentation liquid (calculated based on the initial fermentation). During this process, 20-25 wt.% calcium hydroxide suspension is added to maintain the pH value of the fermentation liquid at 6.5-7.0.

[0028] Furthermore, the method for producing lactic acid by shake flask fermentation using the above genetically engineered bacteria is as follows:

[0029] The seed liquid is inoculated into the shake flask fermentation medium at an initial OD600 of 0.05-0.2, and culture is carried out in an aerobic stage at 25-37° C. and 100-250 r / min for 8-16 hours to allow the bacteria to grow, and then the microaerobic lactic acid production stage is entered. In the microaerobic lactic acid production stage, 10-80 g of glycerol and 20-60 g of CaCO3 are added per L of fermentation liquid (initial volume of fermentation liquid), and the culture is carried out at 37-50° C. and 10-100 r / min for 12-50 hours.

[0030] Furthermore, the shake flask fermentation medium is composed of: 0.5% to 2.5% disodium hydrogen phosphate dodecahydrate, 0.02% to 0.1% sodium chloride, 0.1% to 1.0% potassium dihydrogen phosphate, 0.01% to 0.5% ammonium chloride, and 0.1% to 1.0% glycerol; pH 7.0.

[0031] Beneficial effects:

[0032] The recombinant bacteria provided by the present invention have an obvious ability to synthesize lactic acid monomers using glycerol at a high conversion rate. Under the large-scale fermentation production system, the fermentation level of lactic acid monomers reaches more than 155g / L, the optical purity is more than 99.9%, the chemical purity is more than 98%, and the conversion rate of glycerol to lactic acid in the fermentation acid production stage is increased from about 90% of the starting strain to more than 96%. The technology provided by the present invention helps to improve the conversion rate of raw materials to lactic acid monomers in the fermentation production process of lactic acid monomers, and effectively reduces the fermentation production cost and product extraction and refining cost. At the same time, the technology of the present invention realizes the precise control of cell growth and proliferation in the production process of lactic acid monomers by using fermentation temperature as a control means, without the need for exogenous restrictive addition of nutrients. The simple control process helps to realize the intelligent control of the lactic acid monomer fermentation production system and the establishment of an unmanned factory. The present invention can also be directly applied to the fermentation production of lactic acid monomers using crude glycerol as a raw material, or after appropriate adjustment, can be applied to the fermentation production of lactic acid monomers using other raw materials such as glucose, sucrose, starch, molasses, Jerusalem artichoke, burdock, sugarcane juice, beet juice, bagasse hydrolyzate, straw hydrolyzate, bamboo leaf hydrolyzate, and corncob hydrolyzate. Description of the drawings:

[0033] Figure 1 Physical map of plasmid pKD-PldhA-aceF.

[0034] Figure 2 Electrophoresis pattern of plasmid pKD-PldhA-aceF after linearization with BamHI.

[0035] Figure 3 Electrophoresis pattern of plasmid pMD-aceF(Ed)-difGm after linearization with HindIII.

[0036] Figure 4Schematic diagram of the aceF gene deletion process on the chromosome genome.

[0037] Figure 5 The gel electrophoresis patterns of the recombinant bacteria GSFL and GSFD were verified by colony PCR using primers aceFE-p1 and aceFd-p2.

[0038] Figure 6 Comparison of the recombinant bacteria GSFL, GDFL (left) and GSFD, GDFD (right) cultured on LB plates at 30°C for 24 hours.

[0039] Figure 7 Physical map of plasmid pKDM-PamyL-aceF.

[0040] Figure 8 Electrophoresis pattern of plasmid pKDM-PamyL-aceF after linearization with BamHI. Specific implementation method:

[0041] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0042] The preferred starting strains of the present invention are Escherichia coli CGMCC No. 11059 and CGMCC No. 11060 (disclosed in ZL201580000781.7), which are D-lactic acid monomer producing bacteria and L-lactic acid monomer producing bacteria, respectively, and have been deposited in the China General Microbiological Culture Collection Center (referred to as CGMCC), and the preservation date is July 7, 2015.

[0043] The preferred key enzyme (PDH complex) for converting pyruvate into acetyl-CoA in the present invention is the aceF gene encoding dihydrolipoamide transacetylase (E2p), the encoding gene of which is obtained by PCR, and the nucleotide sequence of which has the sequence characteristics of SEQ ID NO.1, and the amino acid sequence of which has the sequence characteristics of SEQ ID NO.2.

[0044] The plasmid pKD46 used in the present invention is a prior art and can be purchased through commercial channels. Its construction method has also been disclosed in the literature (Datsenko and Wanner, PNAS. 2000, 97 (12): 6640-6645). The PamyL promoter and TamyL terminator sequences used in the present invention have been functionally verified in previous studies (Niu DD & Wang ZX, 2007, 34: 357-362), and their nucleotide sequences are shown in SEQ ID NO. 17 and SEQ ID NO. 18.

[0045] The invention uses gene cloning and gene recombination technology to first clone a dihydrolipoamide acetyltransferase encoding gene aceF (SEQ ID NO.1) from Escherichia coli, and a promoter PldhA (SEQ ID NO.19) and a terminator TldhA (SEQ ID NO.20) of D-lactate dehydrogenase into a temperature-sensitive expression vector pKD46 to obtain an expression plasmid pKD-PldhA-aceF, and transform it into a lactic acid monomer production strain, and then completely delete the original aceF gene on the chromosome of the strain through homologous recombination to obtain a recombinant bacterium capable of synthesizing lactic acid monomers and capable of regulating cell growth through culture temperature, and obtain an aceF gene-deficient strain by losing the plasmid pKD-PldhA-aceF through subculture. The aceF gene, as well as the promoter PamyL (SEQ ID NO.17) and terminator TamyL (SEQ ID NO.18) of amylase were cloned into a temperature-sensitive expression vector pKDM with redundant structures (genes encoding Exo, Beta, and Gam proteins) removed to obtain an expression plasmid pKDM-PamyL-aceF, which was then transformed into the above-mentioned aceF gene-deficient strain to obtain a recombinant bacterium that synthesizes lactic acid monomers and whose cell growth can be regulated by culture temperature; further, fermentation conditions and processes were established and optimized, and by controlling the fermentation temperature, a highly active cell factory was obtained in the bacterial growth stage, and cell proliferation was restricted in the lactic acid fermentation stage, so as to guide the carbon source to product synthesis to the maximum extent, thereby improving the conversion rate of industrial manufacturing of lactic acid monomers.

[0046] The main experimental methods used in the present invention are as follows:

[0047] 1. Gene cloning, gene recombination and construction of expression plasmid Conventional molecular cloning operations are performed according to the reference method (Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989). The dihydrolipoamide acetyltransferase encoding gene aceF is used as the target gene in the present invention; the basic expression vector is pKD46, and the Escherichia coli genomic DNA is used as a template to amplify the target gene sequence. The obtained product is digested with a suitable enzyme, connected to a vector with a suitable promoter and transformed, and a sequence with a specific promoter is obtained by selecting a suitable restriction site for restriction or by PCR amplification, and cloned into the plasmid pKD46.

[0048] 2. Extraction of Chromosomal DNA

[0049] The method for extracting Escherichia coli chromosomal DNA was carried out according to the literature (Zhuge Jian and Wang Zhengxiang. Manual of Industrial Microbiology Experimental Technology, China Light Industry Press, 1994).

[0050] 3. Extraction of plasmid DNA

[0051] Plasmid DNA was extracted using a plasmid extraction kit from Sigma after cell walls were lysed with a certain concentration of lysozyme.

[0052] 4. Gene Amplification

[0053] DNA amplification was performed in a 0.2 mL PCR thin-wall tube. The PCR amplification conditions were: 11 (95°C for 5 min); 301 (94°C for 30 s, 58°C for 30 s, 68°C for 30-300 s); 11 (68°C for 10 min). The extension temperature and time of the PCR reaction were different depending on the amplification length.

[0054] 5. Site-specific integration in E. coli

[0055] The method described in the literature (Zhou Li et al., Bulletin of Microbiology, 2010, 37: 923-928) was used. The main steps are as follows: (1) Using Escherichia coli genomic DNA as a template, PCR amplify the target gene aceF sequence and clone it into a suitable vector, select a suitable restriction site for restriction digestion or remove part of the gene sequence by reverse PCR amplification, and simultaneously clone into the difGm fragment to obtain a mutation cassette fragment (i.e., homology arm 1-dif-Gm-dif-homology arm 2). (2) The above-mentioned site-specific integration mutation cassette sequence was prepared by PCR amplification technology, and the fragment was purified and then electroporated into the recipient strain containing the auxiliary plasmid pKD46. (3) Under the action of the Red recombinase produced by the auxiliary plasmid pKD46, the above-mentioned DNA fragment and the target gene on the chromosome were double-exchanged to replace the target gene sequence. The recombinant transformant can be screened out using the gentamicin resistance marker on the mutation cassette. The mutant strain then undergoes recombination of the two dif sites under the action of the Xer recombinase produced by itself, and the antibiotic resistance gene is removed by cyclization. The correct transformants were verified by colony PCR verification, fermentation verification function and other methods.

[0056] 6. Shake flask fermentation test

[0057] Inoculate the strain from the glycerol tube stored at -70℃ onto the corresponding resistance LB plate and place it in a 25-37℃ incubator for 18-24h. Pick a single colony with uniform growth and place it in 50mL liquid LB medium (Zhuge Jian and Wang Zhengxiang Industrial Microbiology Experimental Technology Manual, Beijing: China Light Industry Press, 1994), shake it at 25-37℃, 100-250r / min, for 12-24h, and grow it until the bacterial OD 600 3.0 or above. Take about 50-150 μL of bacterial suspension and inoculate it into 50 mL of shake flask fermentation medium (0.5%-2.5% disodium hydrogen phosphate dodecahydrate, 0.02%-0.1% sodium chloride, 0.1%-1.0% potassium dihydrogen phosphate, 0.01%-0.5% ammonium chloride, 0.1%-1.0% glycerol; pH 7.0), and control the inoculation amount so that the initial OD600 is 0.05-0.2. First, incubate at 25-37°C and 100-250 r / min for 8-16 hours in the aerobic stage to allow the bacteria to grow, and then transfer to the microaerobic lactic acid production stage. In the microaerobic lactic acid production stage, add glycerol with a final concentration of 10-80 g / L and 20-60 g / L CaCO3, and incubate at 37-50°C and 10-100 r / min for 12-50 hours.

[0058] 7. Fermentation test in fermenter

[0059] The plate culture, liquid LB culture and shake flask inorganic salt culture process of the fermentation strain are the same as the shake flask operation. Take 100-500μL of bacterial suspension and inoculate it into 150mL of inorganic salt culture medium with glycerol as the carbon source, shake it at 25-37℃, 100-250r / min, 12-24h, and inoculate a 50L fermenter as a secondary seed, with an initial working volume of 25L. Add 2-5wt.% glycerol to the inorganic salt culture medium, inoculate the production strain at an inoculation rate of 0.1%-10%, control the dissolved oxygen to not less than 20% at 25-35℃, ventilation 0.1-3vvm and 200-1000r / min, and culture for 4-16h. During the process, maintain the pH of the fermentation liquid at 6.0-7.5 by adding 5-25wt.% ammonia water. When the bacterial OD 600 After reaching 10-50, ventilation is performed at 0.1-1.5 vvm and the rotation speed is reduced to 100-600 r / min to maintain a dissolved oxygen concentration of 1-10%, the fermentation temperature is increased to 35-55° C., and then 3-70 wt.% glycerol solution is added in 4 batches or 10-70 wt.% glycerol solution is added at a flow acceleration of 3-30 g / (L·h) (calculated based on the initial fermentation volume, the total amount of glycerol added is 200-270 g / L fermentation liquid). In this process, the pH value of the fermentation liquid is maintained at 6.0-7.5 by adding 5-30 wt.% calcium hydroxide suspension.

[0060] 8. Fermentation process analysis

[0061] (1) Glycerol concentration determination: During the fermentation process, samples were taken to determine the glycerol concentration. The cells were centrifuged and the supernatant was discarded. The supernatant was appropriately diluted to within the detection range of the glycerol determination kit (FG0100-1KT; Sigma-Aldrich, Inc., St. Louis, USA). The average of the three parallel data was taken.

[0062] (2) Determination of L-lactic acid, D-lactic acid and other fermentation products

[0063] Fermentation broth sample treatment method: deacidify the fermentation broth at a ratio of 1 mL of fermentation broth plus 50 μL of concentrated sulfuric acid to release the neutralized organic acid, then centrifuge to obtain the supernatant and remove the generated CaSO4 precipitate. Mix the treated sample with an equal volume of 100 g / L trichloroacetic acid, place at 4°C for 2 hours, centrifuge at 12000 r / min for 6 minutes, dilute the supernatant appropriately, filter with a 0.22 μm microporous membrane, and then perform high performance liquid chromatography analysis.

[0064] Determination of L-lactic acid, D-lactic acid, pyruvic acid, formic acid, acetic acid and succinic acid content: The samples were detected by HPLC UV detector, the mobile phase used was 5mM dilute sulfuric acid solution, the mobile phase flow rate was set to 0.8mL / min, the HPX-87H organic acid analysis column was used, the wavelength was 210nm, the column temperature was 65℃, and the injection volume was 10μL. All data are the average of 3 parallel test results.

[0065] Optical purity of lactic acid monomer was determined by HPLC, with the mobile phase being 5 mM CuSO4 solution, the mobile phase flow rate being 1 mL / min, and the optical purity chiral analytical column Astec CLC-L (D-lactic acid eluted first, L-lactic acid eluted later), the column temperature being 25°C, UV detection being at a wavelength of 254 nm, and the injection volume being 10 μL.

[0066] The primer comparison table involved in the present invention and embodiments is as follows:

[0067]

[0068] The present invention is further explained below by means of specific examples.

[0069] Example 1 Construction of aceF gene expression plasmid and recombinant Escherichia coli

[0070] The aceF gene (GenBank accession number: 944794, nucleic acid sequence SEQ ID No.1) encoding dihydrolipoamide transacetylase derived from Escherichia coli was amplified by PCR using the chromosome genome of Escherichia coli CGMCC No.11059 as a template (upper and lower reaches primers are SEQ ID No.3 and SEQ ID No.4, aceFp1 and aceFp2). The PCR product obtained, which is about 1.9 kb in size, was digested with BamHI and EcoRI and cloned into the downstream of the PldhA promoter of the pLDHex plasmid (the plasmid construction process is disclosed in the document Niu et al. Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth, Microbial Cell Factories 2014, 13: 78) and the BglII and EcoRI sites upstream of the TldhA terminator to obtain the recombinant plasmid pT-PldhA-aceF.

[0071] Using plasmid pT-PldhA-aceF as a template, PCR amplification (upstream and downstream primers are SEQ ID No. 5 and SEQ ID No. 6, ldhA2-p1 / ldhARew-p2) was performed to obtain an expression cassette PldhA-aceF fragment of about 2.5 kb, including the promoter and terminator of the D-lactate dehydrogenase encoding gene and the aceF gene. The obtained expression cassette PldhA-aceF fragment was digested with BamHI and cloned into the BamHI site of the pKD46 plasmid to obtain the recombinant expression plasmid pKD-PldhA-aceF (physical map see Figure 1 ). After BamHI linearization, the recombinant plasmid was confirmed by agarose gel electrophoresis. Figure 2 As shown, two bands with fragment sizes of 6.3 kb and 2.5 kb were obtained, indicating that the recombinant expression plasmid pKD-PldhA-aceF was successfully constructed.

[0072] The aceF gene expression plasmid pKD-PldhA-aceF constructed above was transformed into the D-lactic acid monomer high-yield strain CGMCC No.11059 and the L-lactic acid monomer high-yield strain CGMCC No.11060 respectively by electroporation to obtain the corresponding recombinant bacteria GOFD and GOFL.

[0073] Example 2 Construction of aceF gene mutation cassette and deletion of aceF gene on recombinant Escherichia coli chromosome

[0074] The chromosomal genome of Escherichia coli CGMCC No.11059 was used as a template for PCR amplification (upstream and downstream primers were SEQ ID No.7 and SEQ ID No.8, aceFE-p1 and aceFd-p2, respectively) of the aceF gene and its upstream and downstream fragments of about 500 bp each. The obtained PCR product of about 2.9 kb was digested with HindIII and cloned into the HindIII site of the pMD18-T plasmid (the plasmid can be purchased from Takara Biotech) to obtain the recombinant plasmid pMD-aceF (Ed).

[0075] Using the pMD-aceF(Ed) plasmid as a template, the aceF gene fragment was removed by PCR reverse amplification (upstream and downstream primers were SEQ ID No.9 and SEQ ID No.10, RaceFE-p1 and RaceFd-p2, respectively), and the plasmid RpMD-aceF(Ed) containing the upstream and downstream homologous arms of the aceF gene was obtained. The fragment RpMD-aceF(Ed) obtained by reverse PCR was connected with the difGm fragment (the fragment was purchased from the Escherichia coli gene deletion kit of Jiangsu Ruiyang Biology) to obtain the recombinant plasmid pMD-aceF(Ed)-difGm( Figure 3 , electrophoresis pattern of plasmid pMD-aceF(Ed)-difGm after HindIII linearization). Using plasmid pMD-aceF(Ed)-difGm as template, PCR amplification (primers aceFE-p1 and aceFd-p2) was performed to obtain the aceF gene mutation cassette aceF(Ed)::difGm (including the upstream and downstream homology arms of the aceF gene and the difGm fragment: homology arm 1-dif-Gm-dif-homology arm 2).

[0076] The mutation cassette aceF(Ed)::difGm was transformed into the recombinant bacteria GOFL and GOFD constructed in Example 1 by electroporation, and the aceF gene on the chromosome genome was deleted with the assistance of the temperature-sensitive plasmid (under the action of the Red recombinase produced by the auxiliary plasmid pKD46, aceF(Ed)::difGm and the aceF gene on the chromosome were double-exchanged), and the recombinant bacteria GSFL and GSFD after the aceF gene on the chromosome was deleted were obtained respectively (the schematic diagram of the aceF gene deletion process on the chromosome genome is shown in Figure 4 ), the verification diagram of recombinant bacteria GSFL and GSFD is shown in Figure 5 As shown. Further, by subculturing at 42°C, the recombinant bacteria GDFL and GDFD in which the aceF gene expression plasmid pKD-PldhA-aceF was lost were obtained (the comparison of the recombinant bacteria GSFL, GDFL and GSFD, GDFD cultured on LB plates at 30°C for 24h is shown in the figure below). Figure 6 shown).

[0077] Example 3 Reconstruction of aceF gene expression plasmid and recombinant Escherichia coli

[0078] The aceF gene was amplified by PCR using the chromosome genome of Escherichia coli CGMCC No.11059 as a template (upstream and downstream primers are SEQ ID No.3 and SEQ ID No.4, aceFp1 and aceFp2, respectively). The amylase signal peptide SamyL was removed by reverse PCR amplification using plasmid pBL-WZX (Niu DD&Wang ZX, 2007, 34:357-362; disclosed in ZL200510081648.7, pBL-WZX plasmid contains amylase promoter PamyL and terminator TamyL) as a template (upstream and downstream primers are SEQ ID No.11 and SEQ ID No.12, RPamyL-1 and RTamyL-2, respectively) to obtain a linearized plasmid backbone fragment in which the amylase signal peptide SamyL sequence was removed from the plasmid pBL-WZX, which was digested with BamHI and ligated with the aceF gene fragment that was also digested with BamHI to obtain the recombinant plasmid pBL-PamyL-aceF. The plasmid pBL-PamyL-aceF was used as a template for PCR amplification (upstream and downstream primers were SEQ ID No. 13 and SEQ ID No. 14, PamyL-1 and TamyL-2, respectively) to obtain an expression cassette fragment PamyL-aceF containing the amylase promoter PamyL and the terminator TamyL and the aceF gene with a size of 3053 bp. The plasmid pKD46 was PCR amplified (upstream and downstream primers were SEQ ID No. 15 and SEQ ID No. 16, RKD-1 and RKD-2, respectively) to delete the nucleic acid sequence fragment encoding the three proteins Exo, Beta and Gam at positions 3391-6329 of the pKD46 plasmid sequence to obtain a pKDM fragment of 2951 bp. The amplified product was digested with DpnI enzyme template and purified, and then connected to the expression cassette PamyL-aceF fragment to obtain the recombinant expression plasmid pKDM-PamyL-aceF (physical map see Figure 7 ). After BamHI linearization, the recombinant plasmid was confirmed by agarose gel electrophoresis. Figure 8 As shown, a band with a fragment size of about 6.0 kb was obtained, indicating that the recombinant expression plasmid pKDM-PamyL-aceF was successfully constructed.

[0079] The aceF gene expression plasmid pKDM-PamyL-aceF constructed above was transformed into the above constructed strains GDFD and GDFL respectively by electroporation to obtain the corresponding recombinant bacteria GYSFD and GYSFL;

[0080] The aceF gene expression plasmid pKDM-PamyL-aceF constructed above was transformed into the D-lactic acid monomer high-yield strain CGMCC No.11059 and the L-lactic acid monomer high-yield strain CGMCC No.11060 by electroporation, respectively, to obtain the corresponding recombinant bacteria GYOFD and GYOFL.

[0081] Example 4 Regulation of cell growth by temperature in a shake flask culture system

[0082] The starting strains CGMCC 11059 and CGMCC 11060, and the recombinant bacteria GSFD, GDFD, GYOFD, GYSFD and GSFL, GDFL, GYOFL, GYSFL were cultured with glycerol as the carbon source at 30°C and 42°C for 24 h to observe the growth of the bacteria. The results are summarized in Table 1.

[0083] The starting strains CGMCC 11059 and CGMCC 11060 showed significant growth at 30℃ and 42℃. The strains GYOFD and GYOFL after free expression of aceF gene showed significant growth at 30℃ and 42℃. This is because the free plasmid replicated normally at 30℃ and was not lost, and the aceF gene on the chromosome and the free plasmid expressed E2p protein normally. Although the free plasmid was lost at 42℃, the aceF gene on the chromosome expressed E2p protein normally. In addition, the cell accumulation of recombinant bacteria GYOFD and GYOFL was higher than that of the starting strain, indicating that the free expression of aceF driven by the amylase promoter is more conducive to the growth of bacteria. The recombinant bacteria GYSFD and GYSFL that deleted the chromosomal genome aceF gene on the basis of free expression of aceF gene showed significant growth differences at 30℃ and 42℃. Among them, the growth was significant at 30℃, while the cell proliferation was significantly inhibited at 42℃. This is because the free plasmid was replicated normally at 30℃ and was not lost. Although there was no aceF gene on the chromosome, the aceF gene on the free plasmid expressed the E2p protein normally. At 42℃, the free plasmid was lost and the aceF gene on the chromosome was also missing. Finally, the cell growth was restricted because the E2p protein could not be expressed. The cell accumulation of the recombinant bacteria GYOFD and GYOFL was higher than that of the starting strains and strains GYSFD and GYSFL. This further shows that the amylase promoter is more conducive to the expression of the aceF gene. The recombinant bacteria GDFD and GDFL without the aceF gene free expression plasmid and the chromosomal genome aceF gene deleted showed obvious cell proliferation inhibition at 30℃ and 42℃. This is because under the two culture conditions, there was no aceF gene on the chromosome and no free plasmid expressing the aceF gene in the cell, which resulted in the E2p protein not being expressed and the cell growth was restricted.

[0084] Table 1

[0085]

[0086] Example 5 Glyceric acid conversion rate in the acid production stage of the lower fermentation in the shake flask fermentation system

[0087] The starting strains CGMCC 11059 and CGMCC 11060, the recombinant strains GYOFD, GYSFD, GYOFL and GYSFL obtained above, and GSFD and GSFL were inoculated from the glycerol tubes stored at -70°C to the corresponding resistance LB plates, and then placed in a 30°C incubator for 20 hours. Pick a single colony with uniform growth to 50mL liquid LB medium and shake at 30°C, 200r / min, for 12h. Take about 100μL of bacterial suspension and inoculate it in 50mL shake flask fermentation medium (1.5% disodium hydrogen phosphate dodecahydrate, 0.05% sodium chloride, 0.3% potassium dihydrogen phosphate, 0.3% ammonium chloride, 0.5% glycerol; pH 7.0), so that the initial OD600 is 0.05-0.2, and culture at 30°C, 200r / min for 10h, and then enter the microaerobic lactic acid production stage. During the microaerobic lactic acid production stage, 70 g of glycerol and 60 g of CaCO3 were added per liter of fermentation broth and cultured at 42°C and 100 r / min for 36 h.

[0088] After the fermentation, the conversion rates of glycerol to lactic acid are summarized in Table 2 below. The conversion rates of glycerol to lactic acid of the recombinant strains GYSFD, GYSFL, GSFD and GSFL after the aceF gene was lost in a temperature-sensitive manner were significantly improved compared with the starting strains. Among them, the conversion rates of the recombinant strains GYSFD and GYSFL were increased from about 90% of the original strains to more than 96%. The conversion rates of glycerol to lactic acid of the GYOFD and GYOFL strains expressing the aceF gene in a free manner were slightly lower than those of the starting strains. Compared with GSFD and GSFL, the recombinant strains GYSFD and GYSFL had two differences in the promoters of two different combinations of amylase and lactate dehydrogenase and the free expression of aceF genes in the pKD46 plasmid backbone. One is that the amylase promoter itself may have a higher transcription level than the lactate dehydrogenase promoter under ventilation fermentation conditions. The removal of the three protein-coding genes that assist homologous recombination on the pKD46 plasmid backbone reduced the load of the free plasmid during cell growth to a certain extent. This allows for more active cells to be obtained, and for more efficient conversion of glycerol to lactic acid. In addition, the fermentation of glycerol to lactic acid requires minimal ventilation, and the presence of oxygen reduces the conversion rate of glycerol to lactic acid. In this context, compared to the anaerobic fermentation process of acid production when glucose is used as the raw material, the inactivation of E2p protein during the lactic acid production stage of glycerol fermentation is more conducive to limiting the use of glycerol for cell growth, thereby maximizing the high conversion rate of glycerol to lactic acid.

[0089] Table 2

[0090]

[0091] Example 5 Synthesis of lactic acid monomer with high conversion rate under variable temperature fermentation process

[0092] The starting strains CGMCC 11059 and CGMCC 11060, and the recombinant strains GYSFD and GYSFL were inoculated into kanamycin-resistant (50 μg / mL) LB plates and placed in a 30°C incubator for 20 hours. Single colonies with uniform growth were picked and placed in 50 mL of liquid LB medium, shaken at 30°C, 200 r / min, for 12 hours. 5 mL of bacterial suspension was inoculated into 150 mL of inorganic salt medium with glycerol as carbon source (15.113 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 5 g / L glycerol, adjusted to pH 7.0.), shaken at 30°C, 200 r / min, for 10 hours, and used as secondary seeds to inoculate a 50 L fermenter with an initial working volume of 25 L. 3 wt.% glycerol was added to an inorganic salt medium (15.113 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, pH adjusted to 7.0). The dissolved oxygen concentration was controlled to be not less than 20% at 33°C, ventilation 2 vvm and 200-1000 r / min. The culture was carried out for 8 h. During the process, 25 wt.% ammonia water was added to maintain the pH of the fermentation liquid at 7.0. When the OD 600 After reaching 30, ventilate 1vvm and reduce the speed to 100-400r / min to control the dissolved oxygen concentration to 5%, increase the fermentation temperature to 42°C, add the total amount of glycerol (based on the fermentation starting volume, the total amount of glycerol added is about 230g / L fermentation liquid) into the fermentation tank in 4 batches, and add 25wt.% calcium hydroxide suspension to maintain the pH of the fermentation liquid at 7.0. When the residual glycerol concentration is lower than 0.5g / L, end the fermentation;

[0093] Under these fermentation conditions, the fermentation cycle was about 36 h, the production levels of D-lactic acid and L-lactic acid of the recombinant bacteria GYSFD and GYSFL were between 155 and 165 g / L, the average acid production rate reached 4.36 g / (L·h) to 4.55 g / (L·h), the conversion rate of glycerol to lactic acid in the fermentation acid production stage was 96.2% to 96.9%, the optical purity of D-lactic acid and L-lactic acid were both higher than 99.9%, and the chemical purity was higher than 98% (Table 3).

[0094] Table 3

[0095]

[0096] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A genetically engineered bacterium for producing lactic acid using glycerol, characterized in that: The genetically engineered bacteria is a host bacterium, wherein the gene encoding dihydrolipoamide transacetylase is transformed into F , promoter PldhA and terminator TldhA were cloned into the thermosensitive plasmid pKD46 to obtain the recombinant plasmid pKD-PldhA- F , and the recombinant plasmid pKD-PldhA- F Transformed into the above host, and knocked out the chromosome F Gene, to obtain recombinant E. coli GSFD and GSFL producing D-lactic acid and L-lactic acid respectively; recombinant E. coli GSFD and GSFL were cultured at 42℃ and the recombinant plasmid pKD-PldhA- F , and obtain recombinant bacteria GDFD and GDFL; then, using the recombinant Escherichia coli GDFD or GDFL as the host, the gene encoding dihydrolipoamide transacetylase F , promoter PamyL and terminator TamyL were cloned into the simplified plasmid pKDM obtained by removing the Exo, Beta and Gam protein coding sequences from the pKD46 plasmid to obtain the recombinant plasmid pKDM-PamyL- F , and the recombinant plasmid pKDM-PamyL- F Transform into the above host GDFD or GDFL to obtain recombinant Escherichia coli; Among them, E. coli CGMCC NO.11060 was used as the initial host to obtain E. coli GYSFL that can metabolize glycerol to synthesize L-lactic acid monomers; E. coli CGMCC NO.11059 was used as the initial host to obtain E. coli GYSFD that can metabolize glycerol to synthesize D-lactic acid monomers; The coding gene of the dihydrolipoamide acetyltransferase F , the nucleotide sequence is shown in SEQ ID NO. 1; The promoter PamyL, the nucleotide sequence is shown in SEQ ID NO.17; The terminator TamyL, the nucleotide sequence is shown in SEQ ID NO.

18.

2. Use of the genetically engineered bacteria according to claim 1 in producing lactic acid using glycerol.

3. The use according to claim 2, characterized in that The method for producing lactic acid by fermentation in a fermenter is as follows: Add 2-5 wt.% glycerol to the inorganic salt medium, inoculate the production strain at an inoculum rate of 0.1%-10%, and culture for 4-16 h at 25-35°C, ventilation 0.1-3 vvm, and 200-1000 r / min with the dissolved oxygen concentration controlled to be no less than 20%. During the process, maintain the pH of the fermentation liquid at 6.0-7.

5. When the bacterial OD 600 After reaching 10-50, the ventilation volume was adjusted to 0.1-1.5 vvm and the rotation speed was reduced to 100-600 r / min to maintain the dissolved oxygen concentration at 1-10%. The fermentation temperature was increased to 35-55℃ to enter the lactic acid synthesis stage. During this period, glycerol was added, and the total amount of glycerol added was 200-270 g / L of fermentation liquid. During the lactic acid synthesis stage, the pH of the fermentation liquid was maintained at 6.0-7.

5. When the glycerol concentration was lower than 0.5 g / L, or the glycerol consumption rate was lower than 1 g / (L·h), or the solid content of the fermentation liquid was too high to be stirred, the fermentation was terminated. The entire fermentation cycle was 30-40 h.

4. The use according to claim 2, characterized in that The method for producing lactic acid by shake flask fermentation using the above genetically engineered bacteria is as follows: Inoculate the seed liquid into the shake flask fermentation medium at an initial OD600 of 0.05-0.

2. First, culture at 25-37°C and 100-250 r / min for 8-16 h in the aerobic stage to allow the bacteria to grow, and then switch to the microaerobic lactic acid production stage. In the microaerobic lactic acid production stage, add 10-80 g of glycerol and 20-60 g of CaCO3 per liter of fermentation liquid, and culture at 37-50°C and 10-100 r / min for 12-50 h.

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