Fermentation method for relieving L-valine crystallization

Abstract

The invention relates to a fermentation method for relieving L-valine crystallization, and belongs to the technical field of fermentation. According to the method, firstly, key genes of multiple competitive pathways are subjected to single knockout, then key enzyme genes of a target pathway are subjected to combined knockout, an optimal combined knockout strategy is explored, enrichment of pyruvic acid is achieved, generation of by-products is reduced, and the yield is increased. Through overexpression of proton translocation NAD (P) + transhydrogenase and NAD + kinase, circulation of NADPH and NADH is constructed, and regeneration circulation of cofactors is realized. In addition, before the concentration of the product is close to a crystallization threshold value, a diluent is accurately supplemented into the fermentation tank to enlarge the volume of the system, so that the product is diluted, and a series of side effects on cells caused by excessive precipitation of the product are relieved. No matter the diluent is sterile water or culture fermentation liquor, the adverse effect of crystals on cells can be obviously relieved, the fermentation time is prolonged, and the production capacity is improved.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, and in particular to a fermentation method for alleviating L-valine crystallization. Background Art

[0002] L-valine is one of the essential glucogenic amino acids for the human body and is classified as a branched-chain amino acid (BCAA) together with L-leucine and L-isoleucine. Its molecular formula is C5H 11 NO2, and its chemical structure is 2-amino-3-methylbutyric acid. Due to its unique physiological effects and metabolic functions, L-valine is widely used in the food, animal feed, and pharmaceutical industries. It can participate in the synthesis of important proteins such as collagen and actin, maintaining the normal functions of body tissues; as a branched-chain amino acid, L-valine has extensive applications and indispensable physiological functions in promoting muscle growth and recovery and enhancing muscle function; since L-valine can improve the activity of lymphocytes and enhance the functions of immune cells, it plays an important role in improving the immune system function. In addition, in the chemical industry, L-valine is widely used as a precursor of amino acid derivatives in the production of pharmaceutical intermediates and high-value-added chemicals.

[0003] The preparation methods of L-valine mainly include chemical synthesis method, enzyme-catalyzed synthesis method and microbial fermentation method. Due to the disadvantages of high production cost, many steps and impurities in the chemical synthesis method and enzyme-catalyzed synthesis method, they have been gradually eliminated by the market. Therefore, constructing an efficient L-valine producing strain can greatly improve the production capacity of L-valine. At present, the mainstream preparation method of L-valine is the microbial fermentation method. Its characteristics of low raw material cost, mild reaction conditions, few by-products and easy large-scale production have been gradually recognized and utilized in industrial production. Usually, L-valine is fermented by the fed-batch method. After primary and secondary amplification cultures, the primary carbon source (such as glucose) is used as the substrate for synthesis. However, the crystallization of L-valine produced by microbial fermentation has an irreversible impact on cell morphology: the sharp edges of the crystals may damage the cell structure, the crystals will cause an increase in shear force during stirring, causing certain mechanical damage to the cells, a high concentration of L-valine will increase the osmotic pressure of the fermentation broth, affecting the normal metabolism of the cells, and the crystal particles interfere with the ventilation efficiency, increasing the energy consumption of the ventilation equipment. Therefore, whether it is repeated fed-batch fermentation, fed-batch fermentation or semi-continuous fed-batch fermentation, the strategy to extend fermentation is to remove the crystals after the formation of L-valine crystals, which will to a certain extent lead to a decrease in cell growth rate, a decrease in product synthesis efficiency and even an early termination of the fermentation process. Moreover, each batch of fermentation broth requires processes such as sterilization, decolorization, concentration and separation at different time points, resulting in an increase in production cost and low utilization rate of fermentation equipment, resulting in an extended fermentation cycle and a significant decrease in production efficiency. To ensure production efficiency, fermentation is immediately stopped whenever the L-valine concentration reaches saturation or supersaturation. If crystals precipitate accidentally, the crystals need to be separated in advance or dissolved by adding water and then diluted before the separation operation can be carried out, which will increase the processing difficulty and production cost. Moreover, the seed liquid needs to be amplified and cultured at multiple levels before each batch of fermentation, which will increase the equipment usage time and labor cost; after the batch is completed, the fermentation equipment needs to be cleaned and disinfected in time, and the operation process is complex, resulting in low production efficiency. Therefore, it is necessary to research and develop a fermentation method to relieve L-valine crystallization and extend the fermentation time to simplify the fermentation cycle process and promote the industrialization process of L-valine. Summary of the Invention

[0004] To this end, the technical problem to be solved by the present invention is to overcome the problem of the lack of a fermentation method for relieving L-valine crystallization in the prior art.

[0005] To solve the above technical problems, the present invention provides a fermentation method for alleviating L-valine crystallization. First, by single knockout of key genes of numerous competing pathways, the competing branches affecting L-valine production are explored, and then by combinatorial knockout of key enzyme genes of the target pathway, the optimal combinatorial knockout strategy is explored to achieve the enrichment of pyruvate, reduce the generation of by-products, and increase the yield. By overexpressing proton-translocating NAD(P)+ transhydrogenase and NAD+ kinase, a cycle of NADPH and NADH is constructed. Since both enzymes are reversible enzymes, substances will maintain a dynamic balance, realizing the recycling of cofactors. Existing crystallization control strategies generally have the defects of passive lag and irreversible damage to the process. Therefore, the present invention proposes a pre-intervention dynamic dilution fermentation technology. By accurately adding a diluent to the fermenter to expand the system volume before the product concentration approaches the crystallization threshold, the dilution of the product is achieved, and a series of side effects brought by the excessive precipitation of the product to the cells are alleviated. Whether the diluent is sterile water or culture fermentation broth, it can significantly alleviate the adverse effects of crystals on cells, extend the fermentation time, and improve the production capacity.

[0006] The first object of the present invention is to provide a fermentation method for alleviating L-valine crystallization, including:

[0007] S1. Inoculate recombinant Escherichia coli into a fermentation system for primary fermentation, and monitor the product concentration of L-valine;

[0008] S2. When the product concentration of L-valine is higher than 70 g / L, add a diluent to dilute so that the product concentration of L-valine is less than or equal to 70 g / L, and perform secondary fermentation;

[0009] Wherein, the diluent includes sterile water or fermentation broth, and the fermentation broth includes a carbon source, a nitrogen source, inorganic salts and metal ions.

[0010] Further, the fermentation broth includes 10 - 20 g / L glucose, 5 - 10 g / L yeast powder, 5 - 10 g / L potassium dihydrogen phosphate, 5 - 10 g / L ammonium sulfate, 2 - 5 g / L magnesium sulfate, 2 - 5 g / L citric acid, 5 - 10 mg / L FeSO4·7H2O and 5 - 10 mg / L MnSO4·H2O.

[0011] Further, in the primary fermentation process in step S1, the initial rotation speed is 300 rpm, the step of increasing the rotation speed is set to 1 - 5 rpm / h from 0 to 4 h, and 100 - 150 mL of 200 g / L ammonium sulfate solution is added at a flow rate of 20 - 30 mL / min; from 4 to 12 h, the step of increasing the rotation speed is set to 30 - 50 rpm / h, and IPTG with a concentration of 0.2 - 1 mM is added at 12 h.

[0012] Further, the dissolved oxygen content in the aerobic fermentation described in step S1 is 20%-30%, and the dissolved oxygen content in the anaerobic fermentation described in step S2 is 5-10%.

[0013] Further, when the diluent is sterile water in step S2, the flow rate of the diluent is 40-50 mL / min; when the diluent is fermentation broth, the flow rate of the diluent is 70-80 mL / min. The present invention has found that when the diluent is sterile water, adding sterile water is equivalent to diluting the fermentation system, and the concentrations of substances such as trace elements inside will decrease. Too fast a flow rate has a negative effect on growth. When the diluent is fermentation broth, the fermentation broth will not cause the concentrations of substances such as trace elements in the fermentation system to decrease, and a large flow rate will not affect fermentation.

[0014] Further, the recombinant Escherichia coli is modified as follows: knocking out 3-methyl-2-oxobutyrate hydroxymethyltransferase panB, formate dehydrogenase pflB, 2-isopropylmalate synthase leuA, valine-pyruvate aminotransferase gene avtA, branched-chain amino acid transporter gene brnQ, regulatory RNA gene gcvB, and D-lactate dehydrogenase gene ldhA; overexpressing acetolactate synthase subunit I gene ilvBN, acetolactate synthase subunit II gene ilvGM, acetolactate synthase subunit III gene ilvIH, dicarboxylic acid reductase isomerase gene ilvC, dicarboxylic acid dehydratase gene ilvD, branched-chain amino acid transaminase gene ilvE, proton-translocating NAD(P)+ transhydrogenase gene pntAB, and NAD+ kinase gene nadK.

[0015] Further, the recombinant Escherichia coli also includes intrinsically disordered protein RGG and intrinsically disordered protein FUS N and the first interacting short peptide RIAD and the second interacting short peptide RIDD;

[0016] The dicarboxylic acid dehydratase gene ilvD and the branched-chain amino acid transaminase gene ilvE are linked to the first interacting short peptide RIAD, and the intrinsically disordered protein RGG and the intrinsically disordered protein FUS N are linked to RIDD, and the first interacting short peptide RIAD and the second interacting short peptide RIDD specifically bind to each other.

[0017] Further, the present invention recruits and localizes the dicarboxylic acid dehydratase gene ilvD and the branched-chain amino acid transaminase gene ilvE to the membraneless organelles RGG and FUS N so that they are assembled at specific spatial positions to achieve spatial regulation, significantly improve the reaction efficiency, and achieve the directional transfer of substrates.

[0018] Further, the proton-translocating NAD(P)+ transhydrogenase gene pntAB is strongly expressed through the Ptac promoter.

[0019] Furthermore, the Gene ID of the 3-methyl-2-oxobutanoate hydroxymethyltransferase gene panB is 944839; the Gene ID of the formate dehydrogenase gene pflB is 945514; the Gene ID of the 2-isopropylmalate synthase leuA is 947465; the Gene ID of the valine-pyruvate aminotransferase gene avtA is 948087; the Gene ID of the branched-chain amino acid transporter gene brnQ is 945042; the Gene ID of the regulatory RNA gene gcvB is 2847720; the Gene ID of the D-lactate dehydrogenase gene ldhA is 946315;

[0020] The acetolactate synthase subunit I gene ilvBN includes the ilvB subunit gene with Gene ID 948182 and the ilvN subunit gene with Gene ID 948183; the acetolactate synthase subunit II gene ilvGM includes the ilvG subunit gene with Gene ID 2847699 and the ilvM subunit gene with Gene ID 948279; the acetolactate synthase subunit III gene ilvIH includes the ilvI subunit gene with Gene ID 948793 and the ilvH gene with Gene ID 947267; the Gene ID of the dicarboxylic acid reductase isomerase gene ilvC is 948286; the Gene ID of the dicarboxylic acid dehydratase gene ilvD is 948277; the Gene ID of the branched-chain amino acid transaminase gene ilvE is 948278; the proton-translocating NAD(P)+ transhydrogenase gene pntAB includes the pntA subunit gene with Gene ID 946628 and the pntB subunit gene with Gene ID 946144; the Gene ID of the NAD+ kinase gene nadK is 947092.

[0021] Furthermore, the amino acid sequence of the intrinsically disordered protein RGG is as shown in SEQ ID NO.1, and the amino acid sequence of the intrinsically disordered protein FUS N is as shown in SEQ ID NO.2, the amino acid sequence of the first interacting short peptide RIAD is as shown in SEQ ID NO.3, and the amino acid sequence of the second interacting short peptide RIDD is as shown in SEQ ID NO.4.

[0022] SEQ ID NO.1:

[0023] MESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDG

[0024] SEQ ID NO.2:

[0025] MASASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGGG

[0026] SEQ ID NO.3:

[0027] MLEQYANQLADQIIKEATE

[0028] SEQ ID NO.4:

[0029] LRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK

[0030] Furthermore, during the secondary fermentation process, the glucose content is less than 0.1 g / L.

[0031] Furthermore, in step S1, the pH of the primary fermentation is 7.0 - 7.2, and in step S2, the pH of the secondary fermentation is 7.0 - 7.2.

[0032] Advantages of the present invention:

[0033] The present invention provides a fermentation method for alleviating L-valine crystallization. First, by knocking out genes at key sites, the enrichment of pyruvate is achieved, the generation of by-products is reduced, and the yield is increased. In addition, the present invention utilizes a pre-intervention dynamic dilution fermentation technique. Before the product concentration approaches the crystallization threshold, a diluent is accurately added to the fermenter to expand the system volume, thereby diluting the product and slowing down a series of side effects brought by the excessive precipitation of the product to the cells. Whether the diluent is sterile water or a culture fermentation broth, it can significantly alleviate the adverse effects of crystals on the cells, extend the fermentation time, and improve the production capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0035] Figure 1 shows the differences between ordinary aerobic fermentation and "stress-type" two-stage anaerobic fermentation;

[0036] Figure 2 is a result diagram of fermentation using different diluents. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following further describes the present invention in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0038] Sample treatment methods involved:

[0039] When the L-valine producing strain needs to detect the L-valine content, 1 mL of the fermentation broth is taken, centrifuged at 12000 rpm for 10 min at room temperature, and the supernatant is filtered through a 0.22 μm aqueous filter membrane and appropriately diluted, and the L-valine content in the sample is detected by HPLC.

[0040] Detection methods involved:

[0041] The L-valine synthesized by the recombinant Escherichia coli of the present invention is detected by a high performance liquid chromatography (HPLC) system (Agilent Technologies 1260 series), and the concentration of L-valine in the fermentation broth is determined by an Infinity Lab Poroshell 120 EC-C18 column (Agilent). The HPLC detector is an ultraviolet absorption detector (UV), and the detection temperature of the chromatographic column is set at 40 °C.

[0042] The sample needs to be detected after pre-column derivatization with OPA boric acid. The preparation methods of related reagents are as follows:

[0043] Boric acid solution, adjusting 0.4 M boric acid solution to pH 10.2 with concentrated NaOH solution.

[0044] OPA solution, taking 9 mL of the above boric acid solution, adding 100 mg of o-phthalaldehyde, 1 mL of acetonitrile, and 100 μL of mercaptopropionic acid.

[0045] During pre-column derivatization, 7 μL of boric acid is drawn and mixed with 1 μL of the sample, then 2 μL of OPA solution is added, 30 μL of water is added, and 20 μL is injected.

[0046] Mobile phase A: Prepare 1 L of 3.01 g / L sodium acetate anhydrous solution, add 200 μL of triethylamine, and adjust the pH to 7.20 ± 0.05 with 5% acetic acid; after suction filtration, add 5 mL of tetrahydrofuran, mix and reserve.

[0047] Mobile phase B: Prepare 0.2 L of 15.05 g / L sodium acetate anhydrous solution; adjust the pH to 7.20 ± 0.05 with 5% acetic acid; after suction filtration, add 400 mL of acetonitrile and 400 mL of methanol to this solution, mix and reserve. The detection flow rate for gradient elution is 0.8 mL / min.

[0048] Example 1: Construction of recombinant bacterium HP4

[0049] The following modifications were made to HP4 in Escherichia coli K12MG1655:

[0050] (1) The genes of acetolactate synthase subunit I (ilvBN), acetolactate synthase subunit II (ilvGM), acetolactate synthase subunit III (ilvIH), dicarboxylic acid reductase isomerase (ilvC), dicarboxylic acid dehydratase (ilvD), and branched-chain amino acid transaminase (ilvE) were strongly expressed.

[0051] When constructing recombinant Escherichia coli, the Ptac promoter was used for gene enhancement in all cases. The overexpressed genes include: ilvBN (NCBI - GeneID: 948182, 948183), ilvGM (NCBI - GeneID: 2847699, 948279), ilvIH (NCBI - GeneID: 948793, 947267), ilvC (NCBI - GeneID: 948286), ilvD (NCBI - GeneID: 948277), and ilvE (NCBI - GeneID: 948278).

[0052] (2) The genes of valine - pyruvate aminotransferase (avtA), branched-chain amino acid transporter (brnQ), and regulatory RNA gene (gcvB) were deleted.

[0053] The deleted genes include avtA (NCBI-GeneID: 948087), brnQ (NCBI-GeneID: 945042), and gcvB (NCBI-GeneID: 2847720).

[0054] (3) To achieve the co-localized recruitment of key enzyme genes in the L-valine metabolic synthesis pathway to membraneless organelles and stable production, in this example, the gene of dicarboxylic acid dehydratase ilvD and the gene of branched-chain amino acid transaminase ilvE were ligated with the interacting short peptide RIAD together, and the endogenous intrinsically disordered proteins RGG and FUSN were ligated to RIDD together.

[0055] Example 2: Verification of the effect of optimized knockout of metabolic pathway combinations on L-valine production

[0056] Taking the knockout of the D-lactate dehydrogenase ldhA gene as an example:

[0057] Determine the upstream and downstream gene sequences of the ldhA gene, and construct the ldhA knockout cassette using fusion PCR. Using the pTarget plasmid as a template, amplify a 2096bp pTarget plasmid fragment with primers pTarget-ldhA-F / pTarget-ldhA-R for constructing pTarget-ldhA.

[0058] The fusion system is: 10 μL of PrimeSTAR Max DNAPolmerase, and other fragments are mixed equally to reach 10 μL, with a total volume of 20 μL.

[0059] The fusion conditions are: pre-denaturation at 98°C for 5 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 1 minute (20 cycles); extension at 72°C for 5 minutes (1 cycle).

[0060] The amplification system is: 25 μL of PrimeSTAR Max DNAPolmerase, 200 ng of DNA template, 1 μL of each primer (10 μM), and 23 μL of distilled water, with a total volume of 50 μL.

[0061] The amplification conditions are: pre-denaturation at 98°C for 5 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 1 minute (34 cycles); extension at 72°C for 5 minutes (1 cycle).

[0062] Transform pEcCas9 into recombinant Escherichia coli by chemical method. Then co-electroporate the above pTarget-ldhA and the ldhA knockout cassette into the HP4 electrocompetent cells containing pEcCas9.

[0063] The transformation conditions are as follows: Use the Competent Cell Preparation Kit to prepare HP4 chemically competent cells, and transform the pEcCas9 plasmid into the HP4 chemically competent cells by chemical transformation. Then prepare HP4 electrocompetent cells containing pEcCas9. Transform the pTarget-ldhA plasmid and the ldhA deletion cassette into the HP4 electrocompetent cells of the pEcCas9 plasmid by electroporation.

[0064] The electroporation conditions are as follows: First, prepare HP4 electrocompetent cells with the pEcCas9 plasmid; place 50 μL of the competent cells on ice, add 500 ng of the pTarget-ldhA plasmid and 1400 ng of the ldhA deletion cassette DNA fragment, place on ice for 30 minutes, and transfer to a 0.1 cm Bio-Rad electroporation cuvette. Use a MicroPulse (Bio-Rad) electroporator with an electrode parameter of 1.8 kV voltage. Immediately after electroporation, transfer 1 mL of LB medium to the electroporation cuvette, pipette 5 times, and then transfer to an EP tube. Incubate at 220 rpm and 37 °C for 2 hours.

[0065] Take 200 μL of the bacterial solution and spread it on an LB plate containing spectinomycin (final concentration 1 mM) and kanamycin (final concentration 1 mM), culture overnight at 37 °C, pick single colonies for PCR verification. The primers used are test-ldhA-F / test-ldhA-R. The correct colony amplification product is a 1053 bp fragment. Pick one correct single colony and inoculate it into a liquid LB medium containing 2.5% (V / V) 1 M L-rhamnose, culture at 37 °C and 220 rpm for 10 - 12 h to eliminate the plasmid pTarget-ldhA. For the strains from which the pTarget plasmid is to be eliminated, pick single colonies and spot them onto solid LB medium containing spectinomycin (final concentration 1 mM) and kanamycin (final concentration 1 mM) respectively to verify the successful elimination of pTarget. And use the primers test-ldhA-F / test-ldhA-R to pick single colonies for PCR verification. Inoculate the correct single colonies onto solid LB medium containing 5 g / L glucose and 10 g / L sucrose, culture at 37 °C for 10 - 12 h to eliminate the plasmid pEcCas9. For the strains from which the pEcCas9 plasmid is to be eliminated, pick single colonies and spot them onto solid LB medium containing kanamycin and solid LB medium without additional antibiotics respectively to verify the successful elimination of pEcCas9. Obtain the recombinant Escherichia coli with completed gene editing and name it HP5.

[0066] Using HP4 as the starting strain, the D-lactate dehydrogenase gene ldhA, 3-methyl-2-oxobutanoate hydroxymethyltransferase gene panB, formate dehydrogenase gene pflB, alcohol dehydrogenase gene adhE (Gene ID: 945837), threonine dehydrogenase gene ilvA, and 2-isopropylmalate synthase gene leuA were knocked out respectively. It was found that only the deletion of genes ldhA, panB, pflB, and leuA had a positive effect on the yield. Subsequently, all possible combinations of the four genes were knocked out, and it was found that the highest yield was obtained after knocking out ldhA, panB, and pflB in combination.

[0067] Using HP4 as the starting strain, the D-lactate dehydrogenase gene ldhA was knocked out from the Escherichia coli genome to obtain the L-valine producing strain HP5. Using HP5 as the starting strain, the 3-methyl-2-oxobutanoate hydroxymethyltransferase gene panB was knocked out from the Escherichia coli genome to obtain the L-valine producing strain HP6. Using HP6 as the starting strain, the formate dehydrogenase gene pflB was knocked out from the Escherichia coli genome to obtain the L-valine producing strain HP7.

[0068] The results of shake flask fermentation experiments showed that the yield of HP5 reached 21.8 g / L, the yield of HP6 reached 22.0 g / L, and the yield of HP7 in shake flasks reached 22.3 g / L.

[0069] Example 2: Verification of the effect of the construction of the cofactor recycling system on the yield of L-valine

[0070] Taking the overexpression of the proton-translocating NAD(P)+ transhydrogenase gene pntAB as an example:

[0071] The upstream and downstream gene sequences of the gapC gene were determined, and the Ptac-pntAB expression cassette was constructed by fusion PCR. Using the pTarget plasmid as a template, a 2096 bp pTarget plasmid fragment was amplified using primers pTarget-gapC-F / pTarget-gapC-R for the construction of pTarget-gapC. The constructed Ptac-pntAB expression cassette and pTarget-gapC were co-electroporated into the electrocompetent cells of HP7 by electroporation to construct the strain HP8. The method of eliminating the tool plasmid was the same as that of deleting genes.

[0072] Using HP7 as the starting strain, an expression cassette composed of the Ptac promoter and the proton-translocating NAD(P)+ transhydrogenase gene pntAB was inserted into the Escherichia coli genome to obtain the L-valine producing strain HP8. Using HP8 as the starting strain, an expression cassette composed of the Ptac promoter and the NAD+ kinase gene nadK was inserted into the Escherichia coli genome to obtain the L-valine producing strain HP9.

[0073] The results of the shake-flask fermentation experiment showed that the yield of HP8 reached 22.6 g / L and the yield of HP9 reached 23.0 g / L.

[0074] Example 3: Verification that "stress-type" two-stage anaerobic fermentation can increase the yield of L-valine

[0075] Fermentation process of "stress-type" two-stage anaerobic fermentation: Streak the strain HP9 onto an LB solid agar medium and incubate it at a constant temperature of 37 °C for 12 h. Pick a monoclonal strain and inoculate it into an LB liquid medium containing 5-10 g / L glucose, and prepare a seed solution at 37 °C and 220 rpm. Finally, inoculate the seed solution into the fermentation tank medium at 10-20% (v / v). The fermentation medium composition consists of the following parts (g / L): 10-20 glucose, 5-10 yeast powder, 5-10 potassium dihydrogen phosphate, 5-10 ammonium sulfate, 2-5 magnesium sulfate, 2-5 citric acid; trace elements (mg / L): 5-10 FeSO4·7H2O, 5-10 MnSO4·H2O. Culture conditions: Control the temperature at a stable 37 °C and the pH between 7.0 and 7.2 during the fermentation process. The "stress-type" two-stage anaerobic fermentation process can be divided into two stages: aerobic growth and anaerobic production. In the aerobic growth stage, the initial rotation speed is 300 rpm, and the dissolved oxygen is controlled at 20-30%. The dissolved oxygen is correlated with the rotation speed within 0-4 h, and the step size of the rotation speed increase is set at 1-5 rpm; and add 100-150 mL of a 200 g / L ammonium sulfate solution at a flow rate of 20-30 mL / min. The dissolved oxygen is correlated with the rotation speed within 4-12 h, and the step size of the rotation speed increase is set at 30-50 rpm. Add IPTG with a final concentration of 0.2-1 mM at 12 h to induce the recruitment and anchoring of related genes to the membrane-free organelles, and realize the spatio-temporal regulation of metabolic pathway genes. In the anaerobic growth stage, when the OD 600 is stable or when it first starts to decline, correlate the rotation speed and the dissolved oxygen, and control the dissolved oxygen at the level of 5-10%. Switch from the growth condition to the production condition. After the bottom sugar is exhausted, keep the glucose content less than 0.1 g / L throughout the process.

[0076] Fermentation process of normal aerobic fermentation: Correlate the dissolved oxygen and the rotation speed, and control the dissolved oxygen at the level of 30-50%. Other conditions are the same as those of the "stress-type" two-stage anaerobic fermentation process.

[0077] Finally, in the case of normal aerobic fermentation, when the fermentation reaches 22 h, that is, 10 h after induction, the yield reaches 51.7 g / L. While the "stress-type" two-stage anaerobic fermentation can produce 100.0 g / L of L-valine at 36 h. This is a 93.4% increase compared to the former (the results are as Figure 1As shown). Moreover, when the dissolved oxygen in the anaerobic fermentation stage is controlled at 0-5%, the yield at 36 h is only 90.1 g / L. If the dissolved oxygen is controlled at 10-15%, the yield at 36 h is only 89.2 g / L.

[0078] Example 4: Prove that adding sterile water as a diluent can relieve L-valine crystallization and extend the fermentation time

[0079] Under the premise of the fermentation process of "stress-type" two-stage anaerobic fermentation, continuously detect the yield of L-valine. When the yield reaches 60-70 g / L, add sterile water to dilute the fermentation broth at a rate of 40-50 mL / min, and ensure that under the premise that the product concentration in the fermenter is less than 70 g / L, to a certain extent, increase the flow rate of the diluent and maintain it until the end of fermentation.

[0080] The experimental results show that starting from 18 h, sterile water is added at a rate of 50 mL / min. From this moment until the end of fermentation, the average change rate of OD 600 increases from -0.49 abs / h to 0.12 abs / h, indicating that the irreversible impact of crystallization on cell growth can be solved by adding a diluent. In addition, if sterile water is supplemented at a rate of 25 mL / min, the average change rate of OD 600 is 0.08 abs / h. If sterile water is supplemented at a rate of 75 mL / min, the average change rate of OD 600 is 0.06 abs / h, both of which are not as high as the advantage of the 50 mL / min supplementation rate. Moreover, adding sterile water at a rate of 50 mL / min can better relieve the impact of L-valine crystallization on growth, and the fermentation time is also extended. The behavior of adding sterile water as a diluent dilutes the fermentation broth by 1.34 times, and the product concentration before dilution is calculated to reach 112.0 g / L, which is 12% higher than that without dilution, and the yield reaches 0.44 g / g glucose.

[0081] Example 5: Prove that adding fermentation broth as a diluent can relieve L-valine crystallization and extend the fermentation time

[0082] Under the premise of the fermentation process of "stress-type" two-stage anaerobic fermentation, continuously detect the yield of L-valine. When the yield reaches 60-70 g / L, add the fermentation culture medium to dilute the fermentation broth at a rate of 70-80 mL / min, and ensure that under the premise that the product concentration in the fermenter is less than 70 g / L, to a certain extent, increase the flow rate of the diluent and maintain it until the end of fermentation.

[0083] The experimental results show that starting from 16 h, the fermentation medium is added at a rate of 50 mL / min. From this moment until the end of fermentation, the average change rate of OD 600The average rate of change is 0.10 abs / h, indicating that the addition of the fermentation medium can alleviate the irreversible impact of crystal formation on cell growth. Moreover, while alleviating the impact of L-valine crystallization on growth, the fermentation time is also extended to 50 h. The act of adding the fermentation medium as a diluent dilutes the fermentation broth by 1.56 times. The product concentration before dilution is calculated to reach 125.4 g / L, which is 25.4% higher than that without dilution, and the yield reaches 0.47 g / g of glucose. This measure takes into account operational compatibility. The low-cost diluent can greatly reduce costs and achieve the purpose of improving production efficiency; process continuity can, to a large extent, extend the production time, reduce the proportion of disinfection time in the fermentation cycle, improve production capacity and reduce production energy consumption; operational simplicity. For the concentration of L-valine, feeding is carried out at an appropriate feeding rate, which can effectively alleviate L-valine crystallization and extend the fermentation time.

[0084] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A fermentation method for alleviating L-valine crystallization, characterized in that, It includes the following steps: S1. Modify Escherichia coli to obtain recombinant Escherichia coli, inoculate the recombinant Escherichia coli into a fermentation system for primary fermentation, and monitor the product concentration of L-valine; S2. When the product concentration of L-valine is higher than 70 g / L, add a diluent to dilute it so that the product concentration of L-valine is less than or equal to 70 g / L, and perform secondary fermentation; Among them, the diluent includes sterile water or fermentation broth, and the fermentation broth includes glucose, yeast powder, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, citric acid, FeSO4·7H2O, and MnSO4·H2O.

2. The fermentation method according to claim 1, wherein The dissolved oxygen content in the primary fermentation in step S1 is 20%-30%, and the dissolved oxygen content in the secondary fermentation in step S2 is 5%-10%.

3. The fermentation method according to claim 1, wherein In step S2, when the diluent is sterile water, the flow rate of the diluent is 40-50 mL / min; when the diluent is fermentation broth, the flow rate of the diluent is 70-80 mL / min.

4. The fermentation method according to claim 1, characterized in that, The recombinant Escherichia coli is modified as follows: Knock out 3-methyl-2-oxobutyrate hydroxymethyltransferase panB, formate dehydrogenase pflB, 2-isopropylmalate synthase leuA, valine-pyruvate aminotransferase gene avtA, branched-chain amino acid transporter gene brnQ, regulatory RNA gene gcvB, and D-lactate dehydrogenase gene ldhA; Overexpress acetolactate synthase subunit I gene ilvBN, acetolactate synthase subunit II gene ilvGM, acetolactate synthase subunit III gene ilvIH, dicarboxylic acid reductase isomerase gene ilvC, dicarboxylic acid dehydratase gene ilvD, branched-chain amino acid transaminase gene ilvE, proton-translocating NAD(P)+ transhydrogenase gene pntAB, and NAD+ kinase gene nadK.

5. The fermentation method according to claim 4, wherein The recombinant Escherichia coli includes the intrinsically disordered protein RGG and the intrinsically disordered protein FUS N as well as a first interacting short peptide RIAD and a second interacting short peptide RIDD; The dicarboxylic acid dehydratase gene ilvD and the branched-chain amino acid transaminase gene ilvE are linked to the first interacting short peptide RIAD, and the intrinsically disordered proteins RGG and the intrinsically disordered protein FUS N are linked to the second interacting short peptide RIDD, and the first interacting short peptide RIAD and the second interacting short peptide RIDD specifically bind to each other.

6. The fermentation method according to claim 4, wherein The proton-translocating NAD(P)+ transhydrogenase gene pntAB is strongly expressed through the Ptac promoter.

7. The fermentation method according to claim 4, characterized in that, The Gene ID of the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB is 944839; the Gene ID of the formate dehydrogenase gene pflB is 945514; the Gene ID of the 2-isopropylmalate synthase leuA is 947465; the Gene ID of the valine-pyruvate aminotransferase gene avtA is 948087; the Gene ID of the branched-chain amino acid transporter gene brnQ is 945042; the Gene ID of the regulatory RNA gene gcvB is 2847720; the Gene ID of the D-lactate dehydrogenase gene ldhA is 946315; The acetolactate synthase subunit I gene ilvBN includes the ilvB subunit gene with Gene ID 948182 and the ilvN subunit gene with Gene ID 948183; The acetolactate synthase subunit II gene ilvGM includes the ilvG subunit gene with Gene ID 2847699 and the ilvM subunit gene with Gene ID 948279; The acetolactate synthase subunit III gene ilvIH includes the ilvI subunit gene with Gene ID 948793 and the ilvH gene with Gene ID 947267; The Gene ID of the dicarboxylic acid reductase gene ilvC is 948286; the Gene ID of the dicarboxylic acid dehydratase gene ilvD is 948277; the Gene ID of the branched-chain amino acid transaminase gene ilvE is 948278; the proton-translocating NAD(P)+ transhydrogenase gene pntAB includes the pntA subunit gene with Gene ID 946628 and the pntB subunit gene with Gene ID 946144; the Gene ID of the NAD+ kinase gene nadK is 947092.

8. The fermentation method according to claim 5, characterized in that The amino acid sequence of the intrinsically disordered protein RGG is shown in SEQ ID NO.1, and the intrinsically disordered protein FUS N has an amino acid sequence shown in SEQ ID NO.

2. The amino acid sequence of the first interacting short peptide RIAD is shown in SEQ ID NO.3, and the amino acid sequence of the second interacting short peptide RIDD is shown in SEQ ID NO.

4.

9. The fermentation method according to claim 1, wherein During the secondary fermentation process, the glucose content is less than 0.1 g / L.

10. The fermentation method according to claim 1, wherein, In the primary fermentation process described in step S1, the initial rotation speed is 300 rpm, the step of increasing the rotation speed is 1 - 5 rpm / h within 0 - 4 h, and 100 - 150 mL of 200 g / L ammonium sulfate solution is added at a flow rate of 20 - 30 mL / min within 0 - 4 h; within 4 - 12 h, the step of increasing the rotation speed is 30 - 50 rpm / h, and isopropyl-β-D-thiogalactoside with a concentration of 0.2 - 1 mM is added at 12 h.

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