Preparation method of L-alanine

Abstract

The invention discloses a preparation method of L-alanine. In particular to a preparation method of L-alanine, which comprises the following steps: inoculating recombinant bacteria seeds for producing L-alanine into a fermentation culture medium, and controlling the concentration of residual sugar to be 5-10g / L and the concentration of ammonia nitrogen to be 0.05-0.15% in the fermentation culture process; according to the control mode, the concentration of residual sugar and the concentration of ammonia nitrogen are detected through the online near-infrared technology, and the concentration is adjusted by adding a supplemented culture medium. According to the preparation method, an online near-infrared technology is adopted, and the concentration of residual sugar and ammonia nitrogen in the fermentation process can be accurately detected and controlled in real time, so that the conversion rate of sugar to a product and the content of L-alanine discharged from a tank are improved, and the stability of a fermentation result is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a method for preparing L-alanine. Background Art

[0002] L-alanine is one of the smallest chiral molecules and has a wide range of uses. In the field of daily chemicals, L-alanine is the main raw material for the production of phosphorus-free detergent methylglycine diacetic acid (MGDA); in the food field, L-alanine can be used as a flavoring agent and acid corrector to improve the utilization rate of protein in food and beverages; in the pharmaceutical field, L-alanine is a good diuretic and the main raw material for synthesizing substances such as vitamin B6 and aminopropanol; due to the increasingly widespread application of L-alanine, its demand is also increasing.

[0003] L-alanine can be prepared by chemical synthesis and biological methods. The chemical synthesis method is less used due to its disadvantages such as long synthesis route, low yield, high cost, and high environmental pollution. The biological method for synthesizing L-alanine has more production advantages due to its green environmental protection, energy saving and carbon reduction. The biological method includes enzyme catalysis and microbial fermentation. Among them, the raw material fumaric acid for the enzyme catalysis method to synthesize L-alanine comes from non-renewable petroleum resources, which is expensive and unstable, resulting in high production costs. The microbial fermentation method for producing L-alanine uses renewable glucose as raw material, which is relatively low in cost and easy to obtain, and the process is mild and environmentally friendly. It has become the current mainstream production method of L-alanine.

[0004] At present, the fermentation method for producing L-alanine usually adopts the strategy of continuous or intermittent sugar supplementation to control the residual sugar in the fermentation broth within a certain lower range, such as 10g / L for production. However, the fermentation process inevitably requires frequent sampling and frequent offline detection, which is labor-intensive. The measured results often have a certain hysteresis, making it difficult to timely and accurately control the residual sugar in the fermentation process within the optimal range. The results of parameter control also fluctuate greatly, and the fermentation results are not very stable. In fact, in addition to carbon sources such as glucose, the fermentation process also has nitrogen sources. For example, the concentration of ammonia nitrogen in the fermentation broth often has a great impact on fermentation, especially for products such as enzymes and amino acids whose molecular structures contain a large proportion of nitrogen components. However, due to the lack of an effective method for simply and quickly determining ammonia nitrogen in the fermentation process, the current fermentation regulation is still mainly limited to the regulation of glucose concentration. Therefore, it is urgent and of great practical significance to seek an effective method that can simultaneously and accurately detect and feedback control the residual sugar concentration and ammonia nitrogen concentration in the L-alanine fermentation process in real time. Summary of the invention

[0005] In view of the defects of difficulty in timely and accurate control of parameters in the fermentation process and unstable fermentation results in the production of L-alanine by fermentation, the present invention provides a method for preparing L-alanine. The preparation method adopts online near-infrared technology, which can detect and control the residual sugar and ammonia nitrogen concentrations in the fermentation process in real time and accurately, thereby improving the conversion rate of sugar to product, the content of L-alanine in the tank, and improving the stability of the fermentation results.

[0006] A method for preparing L-alanine, comprising the following steps: inoculating L-alanine-producing recombinant bacterial seeds into a fermentation medium, and during the fermentation culture process, controlling the residual sugar concentration to be 5-10 g / L and the ammonia nitrogen concentration to be 0.05-0.15%;

[0007] The control method is to use online near infrared technology to detect the residual sugar concentration and the ammonia nitrogen concentration, and adjust the concentration by adding feed culture medium.

[0008] In one embodiment, the online near infrared technology detection specifically includes the following steps:

[0009] S1: Establishing the linear equation

[0010] The near infrared spectra at different time points in the fermentation system are continuously collected by online near infrared analysis equipment (the collection frequency is one point per minute), and then the collected near infrared spectrum data are preprocessed by computer software and chemometric methods after removing abnormal spectra to obtain preprocessed near infrared spectrum data; at the same time, samples are taken at the collection time point corresponding to each spectrum, and process parameters are measured offline (after centrifugal sampling, they are tested separately) to obtain physical and chemical test data; finally, the preprocessed near infrared spectrum data and physical and chemical test data are linearly analyzed to establish a fermentation model; wherein the process parameters include glucose concentration and ammonia nitrogen concentration;

[0011] S2: Real-time feedback control of the fermentation process

[0012] The near infrared spectrum in the fermentation system is continuously fitted by online near infrared analysis equipment, and then the collected near infrared spectrum data is preprocessed by computer software and chemometric methods after removing abnormal spectra to obtain preprocessed near infrared spectrum data, and then the preprocessed near infrared spectrum data is coupled with the fermentation model established in step S1 to convert the process parameters in the fermentation system, and the peristaltic pump is controlled to perform intermittent feeding according to the process parameters; wherein the process parameters include glucose concentration and ammonia nitrogen concentration.

[0013] In a certain embodiment, the online near-infrared analysis equipment is preferably a Fourier transform near-infrared spectrometer.

[0014] In a certain embodiment, the ammonia nitrogen concentration is controlled to be 0.05-0.1% or 0.1-0.15%; for example, 0.1-0.15%.

[0015] In the present invention, the residual sugar concentration is the concentration of sugar in the fermentation medium that is not consumed by the recombinant bacterial seeds during the fermentation culture process. In a certain scheme, the residual sugar concentration is adjusted by the feed medium 1. During the fermentation culture process, when the detected residual sugar concentration is lower than the preset value, the feed medium 1 is added until the detected residual sugar concentration reaches or exceeds the preset value, and the feeding is stopped at this time; the feed medium 1 includes 50-80% sugar; the sugar is glucose and / or sucrose; % represents the mass fraction percentage of the mass of the sugar to the total mass of the feed medium 1; for example, the feed medium 1 includes 60% glucose.

[0016] In the present invention, the ammonia nitrogen concentration is the concentration of ammonia (NH3) or ammonium ions (NH4 + ) in the form of. In a certain embodiment, the ammonia nitrogen concentration is adjusted by feed medium 2. During the fermentation culture process, when the detected ammonia nitrogen concentration is lower than a preset value, feed medium 2 is added until the detected ammonia nitrogen concentration reaches or exceeds the preset value, and then feeding is stopped; the feed medium 2 includes 10-30% of a nitrogen source; % represents the mass fraction percentage of the mass of the nitrogen source to the total mass of the feed medium 2; the nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source, for example, the feed medium 2 includes 20% of an inorganic nitrogen source; preferably, the inorganic nitrogen source is ammonium sulfate, ammonium chloride and / or diammonium phosphate; for example, ammonium sulfate.

[0017] In a certain embodiment, the recombinant bacterial seed is selected from one or more of recombinant Escherichia coli, recombinant Bacillus subtilis, recombinant Corynebacterium glutamicum and recombinant yeast. For example, the recombinant bacterial seed is recombinant Escherichia coli. Preferably, the recombinant Escherichia coli genotype is E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2 △frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI; for another example, the recombinant Escherichia coli is a strain with a deposit number of CCTCC M 20241584.

[0018] The fermentation medium, operation and conditions used in the fermentation culture can all be conventional in the art, and the preferred embodiments of the present invention are as follows.

[0019] In a certain embodiment, the components of the fermentation medium include: 5-15 g / L sugar, 1-10 g / L yeast extract, 1-10 g / L peptone, 1-10 g / L ammonium sulfate, 1-5 g / L potassium dihydrogen phosphate, 1-10 g / L sodium hydrogen phosphate, 1-5 g / L magnesium sulfate and 0.1-1 g / L defoamer; the sugar is glucose and / or sucrose; g / L represents the ratio of the mass of each component to the total volume of the culture medium; preferably, the components of the fermentation medium include: 10 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 8 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L sodium hydrogen phosphate, 3 g / L magnesium sulfate and 0.5 g / L defoamer.

[0020] In a certain embodiment, the magnesium sulfate in the fermentation medium is preferably magnesium sulfate heptahydrate.

[0021] In a certain embodiment, the defoaming agent in the fermentation medium is preferably a polyether defoaming agent.

[0022] In a certain embodiment, the fermentation culture comprises the following steps: inoculating the recombinant bacterial seed solution into a fermentation tank containing the fermentation medium, and performing anaerobic fermentation.

[0023] In a certain embodiment, the inoculation ratio of the recombinant bacterial seed solution to the fermentation medium is 5%-15%, for example, 10%.

[0024] In one embodiment, the temperature of the anaerobic fermentation is 30-40°C; for example 37°C.

[0025] In a certain embodiment, in the fermentation culture, the concentration of total sugar is 142-155 g / L, for example, 150 g / L; the total sugar is total glucose and / or total sucrose.

[0026] In a certain embodiment, the anaerobic fermentation time is such that the glucose and / or sucrose in the fermentation medium is completely consumed, that is, the residual sugar is ≦0.5 g / L; for example, 34 hours.

[0027] In a certain embodiment, during the anaerobic fermentation, aqueous ammonia is used to adjust the pH to 6-7, for example, 6.8.

[0028] In a certain embodiment, the tank pressure of the fermentation tank is 0.02-0.1 MPa, for example 0.05 MPa.

[0029] In a certain embodiment, the rotation speed of the fermenter is 100-300 rpm, such as 150 rpm.

[0030] In one embodiment, the recombinant bacterial seed liquid also includes seed preparation before fermentation culture, and the seed preparation includes the following steps: activating the frozen recombinant bacterial seeds and then shaking culture to obtain shake flask seeds, and then expanding the culture in a seed tank containing seed culture medium.

[0031] The seed culture medium, operations and conditions used in the seed preparation may be conventional in the art, and the preferred embodiments of the present invention are as follows.

[0032] In a certain embodiment, the components of the seed culture medium include: 10-40 g / L sugar, 1-10 g / L yeast extract, 1-10 g / L peptone, 10-30 g / L ammonium sulfate, 1-5 g / L potassium dihydrogen phosphate, 1-10 g / L disodium hydrogen phosphate, 1-5 g / L magnesium sulfate and 0.1-1 g / L defoaming agent; the sugar is glucose and / or sucrose; wherein g / L represents the ratio of the mass of each component to the total volume of the culture medium; preferably, the components of the seed culture medium include: 30 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 20 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, 3 g / L magnesium sulfate and 0.5 g / L defoaming agent.

[0033] In a certain embodiment, the magnesium sulfate in the seed culture medium is preferably magnesium sulfate heptahydrate.

[0034] In a certain embodiment, the defoaming agent in the seed culture medium is preferably a polyether defoaming agent.

[0035] In a certain embodiment, the culture medium used for the activation is LB liquid culture medium; the components of the LB liquid culture medium preferably include 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0036] In a certain embodiment, the pH of the LB liquid culture medium is preferably 7.2.

[0037] In one embodiment, the activation temperature is 30-40°C; for example 37°C.

[0038] In a certain embodiment, the activation time is 10-15 hours, for example 12 hours.

[0039] In a certain embodiment, the shaking culture is cultured in a shaker at a rotation speed of 150-250 rpm, and the rotation speed is preferably 200 rpm.

[0040] In one embodiment, the shaking culture temperature is 30-40°C; for example, 37°C.

[0041] In one embodiment, the shaking culture time is 1-6 hours, such as 2-3 hours, preferably controlling OD 600(Absorbance at 600nm wavelength) is 2-3.

[0042] In a certain embodiment, the inoculation ratio of the shake flask seeds inoculated into the seed tank is 0.3-2%, for example 1%.

[0043] In a certain embodiment, the temperature of the expanded culture is 30-40°C; for example, 37°C.

[0044] In a certain embodiment, the time of the expansion culture is 3-5 hours, for example 4 hours, preferably controlling the OD 600 (optical density at 600nm wavelength) is 5-10, for example, to control OD 600 is 8.

[0045] In one embodiment, during the expanded culture, ammonia water is used to adjust the pH to 6.5-7.2, for example, 6.8.

[0046] In a certain embodiment, the tank pressure of the seed tank is 0.02-0.1 MPa, for example 0.05 MPa.

[0047] In a certain embodiment, the rotation speed of the seed tank is 300-700 rpm, and the rotation speed is preferably controlled to control DO (dissolved oxygen concentration) ≥ 30%.

[0048] In a certain embodiment, the ventilation ratio of the expanded culture is 0.5-2 VVM, such as 1 VVM.

[0049] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0050] The reagents and raw materials used in the present invention are commercially available.

[0051] The positive progressive effects of the present invention are: the present invention realizes real-time accurate detection and feedback control of residual sugar and ammonia nitrogen in the L-alanine fermentation process through online near-infrared technology, which can reduce manual labor, save time and cost; by accurately controlling key nutritional parameters within their optimal ranges, that is, the residual sugar concentration is 5-10g / L, and the ammonia nitrogen concentration is 0.05-0.15%, the growth and metabolism of Escherichia coli are in an optimal state, thereby improving the conversion rate of sugar to product, the content of L-alanine in the tank, and the stability of the fermentation results.

[0052] Biomaterial Deposit Information

[0053] The Escherichia coli A129 strain of the present invention was deposited in the China Type Culture Collection on July 17, 2024, with a deposit address of Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, postal code 430072, and its deposit number is CCTCCNO: M 20241584, and its classification name is Escherichia coli A129. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a liquid phase detection diagram of the fermentation results of Example 6.

[0055] Figure 2 This is the liquid phase detection diagram of the fermentation result of Comparative Example 1.

[0056] Figure 3 This is the liquid phase detection diagram of the fermentation result of Comparative Example 2.

[0057] Figure 4 This is the liquid phase detection diagram of the fermentation results of Comparative Example 3.

[0058] Figure 5 This is the liquid phase detection diagram of the fermentation results of Comparative Example 8.

[0059] Figure 6 This is a diagram of the glucose model.

[0060] Figure 7 This is the ammonia nitrogen model diagram. DETAILED DESCRIPTION

[0061] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0062] The coding genes involved in the present invention, the expression cassettes and plasmids containing these genes, and the transformants containing the plasmids can all be obtained by genetic engineering construction methods well known to those skilled in the art.

[0063] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned are all mass percentages unless otherwise specified.

[0064] The molecular biology experiments involved in the examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are performed with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0065] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium plus 20 g / L agar powder).

[0066] UV mutagenesis plate culture medium: 30 g / L glucose, 5 g / L yeast extract powder, 5 g / L peptone, 20 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, 3 g / L magnesium sulfate heptahydrate, containing 2% agar powder.

[0067] The components of the shake flask fermentation medium are: 30 g / L glucose, 5 g / L yeast extract powder, 5 g / L peptone, 8 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, and 3 g / L magnesium sulfate heptahydrate.

[0068] The composition of the seed tank culture medium is: glucose 30g / L, yeast extract powder 5g / L, peptone 5g / L, ammonium sulfate 20g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate 5g / L, magnesium sulfate heptahydrate 3g / L, and defoamer 0.5g / L.

[0069] The fermentation tank culture medium composition is: glucose 10g / L, yeast extract powder 5g / L, peptone 5g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate 5g / L, magnesium sulfate heptahydrate 3g / L, defoamer 0.5g / L.

[0070] The composition of feed medium 1 is: glucose 600 g / kg.

[0071] The composition of feed medium 2 is: ammonium sulfate 200 g / kg.

[0072] Alanine dehydrogenase activity detection method: The enzyme activity of alanine dehydrogenase is determined based on the absorbance at 595 nm. Specifically, take a suitable dilution of alanine dehydrogenase enzyme solution, add it to the reaction mixture (including 50 mM L-alanine, 50 mM Tris-HCl pH9.0, 0.625 mM NAD+, 0.064 mM 5-methylphenazine methylsulfate (PMS), 0.24 mM nitro blue tetrazolium chloride (NBT)), keep it at 37°C for 10 min, and then measure the absorbance at 595 nm in a spectrophotometer.

[0073] Conversion rate (%) calculation: the ratio of the total mass of L-alanine obtained by fermentation to the total mass of glucose consumed, that is, conversion rate (%) = concentration of L-alanine in the tank sample (g / kg) × tank weight (Kg) / total mass of glucose consumed (g) × 100%.

[0074] HPLC determination conditions of L-alanine:

[0075] Instrument: Shimadzu LC-20AD high performance liquid chromatography

[0076] Column: ODS-3, 5μm, 4.6×250mm (UP)

[0077] Mobile phase: Mobile phase A: 0.006 M potassium dihydrogen phosphate (adjust the pH to 3 with phosphoric acid, then filter with a 0.45 μm water filter and ultrasonically degas for 20 minutes before use); Mobile phase B: methanol (chromatographic grade, ultrasonically degas for 20 minutes before use)

[0078] HPLC detection conditions: detection time: 30 min; detection wavelength: 210 nm; column oven temperature: 35°C; pump mode: low pressure gradient, total flow rate of 0.5 ml / min, mobile phase A: mobile phase B 9:1; injection volume: 10 μl.

[0079] Glucose physicochemical detection: Offline glucose was measured using Shenzhen Hillman M-100 biosensor as a reference.

[0080] Physical and chemical detection of ammonia nitrogen: Phenol-hypochlorite spectrophotometry was used for offline detection of ammonia nitrogen as a reference.

[0081] Near-infrared detection and analysis: The residual sugar and ammonia nitrogen in the fermentation process were detected in real time using the Fourier transform near-infrared spectrometer of Shanghai Yingsa Optical Instrument Co., Ltd. to obtain the near-infrared spectrum of the fermentation parameters. The fermentation model was established by fitting equations with the offline physical and chemical detection data, and the changes in residual sugar and ammonia nitrogen in the fermentation process were measured. The fermentation model was Figure 6 and Figure 7 shown.

[0082] Example 1: Construction of a wild-type alanine dehydrogenase recombinant Escherichia coli expression strain

[0083] For the alanine dehydrogenase PmAlaD from Priestia megaterium, the GenBank accession number is WP_328233963.1, and the amino acid sequence is SEQ ID NO: 1. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform full gene synthesis and optimized according to the codon preference of Escherichia coli. The optimized gene sequence is SEQ ID NO: 2. After the gene synthesis, it was cloned between the NcoI and EcoRI restriction sites of the vector pTrc99A (purchased from Biowind) to obtain the recombinant plasmid pTrc99A-PmAlaD.

[0084] The recombinant plasmid pTrc99A-PmAlaD was transformed into the expression host Escherichia coli W3110 (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) by chemical transformation to obtain the recombinant Escherichia coli PmAlaD-WT capable of expressing wild-type alanine dehydrogenase.

[0085] Example 2: Construction of mutation point library by semi-rational design

[0086] Discovery Studio software was used to predict the substrate binding site in the PmAlaD protein pocket. Using pTrc99A-PmAlaD as the starting plasmid, single-point saturation mutation and iterative saturation mutation were performed on the amino acid sites of E48, T180, H295, A304, and N344, respectively. The screening method of mutant proteins was as follows: the recombinant plasmid containing the point mutation PmAlaD protein was transformed into Escherichia coli W3110 competent cells to obtain mutant transformants. The recombinant Escherichia coli PmAlaD-WT expressing wild-type alanine dehydrogenase was used as a control and inoculated into 5 mL LB medium (containing ampicillin at a final concentration of 50 μg / ml), and cultured at 200 rpm in a 37°C shaker for 16 hours to obtain seed liquid. Each sub-liquid was inoculated into fresh LB medium (containing ampicillin at a final concentration of 50 μg / ml) at a ratio of 1%, and cultured at 200 rpm in a 37°C shaker until OD 600 0.6-0.8, add IPTG with a final concentration of 0.1 mM, cool to 25℃ and induce culture for 24 hours, centrifuge at 4℃, 10000 rpm for 5 min, and collect the bacteria.

[0087] Example 3: Screening of alanine dehydrogenase mutants

[0088] The enzyme activity of each alanine dehydrogenase mutant obtained in Example 2 was determined. 0.5 g of the same mass of cells was taken, 2 mL of 50 mM Tris-HCl pH 9.0 buffer was added for full suspension, and the cells were crushed using an ultrasonic crusher (Shunma Instrument SM-150D) with a crushing power of 5% and a crushing time of 5 min (working for 5 s and stopping for 10 s). After crushing, the cells were centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was used for enzyme activity determination. The enzyme activity of the wild-type PmAlaD was defined as 100%, and Table 1 lists the single-point mutants and five-point mutants with the highest enzyme activity among the screened mutants.

[0089] Table 1 Screening of alanine dehydrogenase mutants

[0090]

[0091] As can be seen from Table 1, we obtained alanine dehydrogenase mutant protein PmAlaD-m6-92, the amino acid sequence of which is shown in SEQ ID NO: 3, which also contains five point mutations, namely E48T, T180I, H295A, A304E and N344I, and its coding sequence is shown in SEQ ID NO: 4. Compared with the wild-type enzyme SEQ ID NO: 1, the enzyme activity of the mutant protein PmAlaD-m6-92 is increased by 8.2 times.

[0092] Example 4: Construction of genetically engineered recombinant strain A0

[0093] In this example, the Escherichia coli CRISPR gene editing tool (Yu Jiang et al. 2015 Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied Environmental Microbiology. 81: 2506-2514) was used to first integrate the mutant alanine dehydrogenase mutant PmAlaD-m6-92 encoding gene from Priesteria gigantea into the pyruvate oxidase poxB gene site and the fumarate reductase frdBC gene site of the Escherichia coli W3110 chromosome, and then knock out the competing pathways of the host Escherichia coli's own pyruvate precursors, including the lactate dehydrogenase encoding gene ldhA, the pyruvate formate lyase gene pflB, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase mgsA, the alanine racemase gene dadX and the transcriptional regulatory factor LacI encoding gene lacI, to obtain the genetically engineered recombinant strain A0.

[0094] 4.1 Integration of the mutant alanine dehydrogenase PmAlaD-m6-92 gene into the pyruvate oxidase poxB locus

[0095] Escherichia coli W3110 (purchased from Biowind) was used as the starting strain to prepare electroporation competent cells, and the pCas9 plasmid (Addgene: #62225) was transformed into W3110 competent cells to obtain the W3110 strain carrying the pCas9 plasmid and further cultured to prepare electroporation competent cells using L-arabinose induction.

[0096] Using the genomic DNA of Escherichia coli W3110 as a template, primers poxB-UF / poxB-UR were used for amplification; using pTrc99A-PmAlaD-m6-92 as a template, primers Ptrc-F / T1T2-R were used for amplification; using the genomic DNA of Escherichia coli W3110 as a template, the upstream homologous arm of the poxB gene, the mutant alanine dehydrogenase Ptrc-PmAlaD-m6-92-T1T2 expression frame and the downstream homologous arm of the poxB gene were amplified using the poxB-DF / poxB-DR combination, and then the primers poxB-UF / poxB-DR combination were used for fusion PCR amplification to obtain the homologous fragment poxB-U-PmAlaD-mD that integrated the PmAlaD-m6-92 coding gene into the poxB site.

[0097] The pTargetF-poxB plasmid targeting the poxB gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-poxB-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-poxB plasmid was obtained. The homologous fragments poxB-U-PmAlaD-mD and pTargetF-poxB plasmid were co-transformed into W3110 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were simultaneously verified by PCR using primers poxB-UUF / PmAlaD-yz-R and PmAlaD-yz-F / poxB-DDR. If a 1746 bp and a 2021 bp band were amplified respectively, they were positive transformants. The positive transformants were induced to eliminate the pTargetF-poxB plasmid, and the strain WA-001 carrying the pCas9 plasmid was obtained. The mutant PmAlaD-m6-92 encoding gene was integrated into the poxB gene site on its genome.

[0098] 4.2 Integration of the mutant alanine dehydrogenase PmAlaD-m6-92 encoding gene into the fumarate reductase frdBC locus

[0099] The strain WA-001 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as a template and primers frdBC-UF / frdBC-UR were used for amplification; pTrc99A-PmAlaD-m6-92 was used as a template and primers Ptrc-F / T1T2-R were used for amplification; the genomic DNA of Escherichia coli W3110 was used as a template and frdBC-DF / frdBC-DR was used to amplify the upstream homologous arm of the frdBC gene, the mutant alanine dehydrogenase PmAlaD-m6-92 and the downstream homologous arm of the frdBC gene, and then the primers frdBC-UF / frdBC-DR were used for fusion PCR amplification to obtain the homologous fragment frdBC-U-PmAlaD-mD that integrated the PmAlaD-m6-92 coding gene into the frdBC site.

[0100] The pTargetF-frdB plasmid targeting the frdB gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-frdB-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-frdB plasmid was obtained. The homologous fragments frdBC-U-PmAlaD-mD and pTargetF-frdB plasmid were co-transformed into WA-001 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were simultaneously verified by PCR using frdBC-UUF / PmAlaD-yz-R and PmAlaD-yz-F / frdBC-DDR. If a 1608 bp and a 1855 bp band were amplified respectively, they were positive transformants. The positive transformants were induced to eliminate the pTargetF-frdB plasmid, and the strain WA-002 carrying the pCas9 plasmid was obtained, in which the frdBC gene site on its genome was integrated with a second copy of the mutant PmAlaD-m6-92 gene.

[0101] 4.3 Knockout of the lactate dehydrogenase encoding gene ldhA

[0102] The strain WA-002 carrying the pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as a template, and the primers ldhA-UF / ldhA-UR; ldhA-DF / ldhA-DR were used to amplify the upstream homology arm and downstream homology arm of the ldhA gene, and then the primers ldhA-UF / ldhA-DR were used to perform fusion PCR amplification of the ldhA gene knockout homology fragment ldhA-UD.

[0103] The pTargetF-ldhA plasmid targeting the ldhA gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-ldhA-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-ldhA plasmid was obtained. The homologous fragments ldhA-UD and pTargetF-ldhA plasmid were co-transformed into WA-002 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the ldhA-UUF / ldhA-DDR combination. If a band of 2145 bp was amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-ldhA plasmid, and the strain WA-003 carrying the pCas9 plasmid was obtained, and the ldhA gene on its genome was knocked out.

[0104] 4.4 Knockout of the pyruvate formate lyase pflB gene

[0105] The strain WA-003 carrying the pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the pflB gene were amplified using the primers pflB-UF / pflB-UR; pflB-DF / pflB-DR combination, and then the primers pflB-UF / pflB-DR combination was used for fusion PCR amplification to obtain the pflB gene knockout homology fragment pflB-UD.

[0106] The pTargetF-pflB plasmid targeting the pflB gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-pflB-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-pflB plasmid was obtained. The homologous fragments pflB-UD and pTargetF-pflB plasmids were co-transformed into WA-003 competent cells carrying the pCas9 plasmid, spread on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening, and the grown transformants were verified by PCR using pflB-UUF / pflB-DDR. If a band of 1793 bp was amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-pflB plasmid, and the strain WA-004 carrying the pCas9 plasmid was obtained, and the pflB gene on its genome was knocked out.

[0107] 4.5 Knockout of the alcohol dehydrogenase adhE gene

[0108] The strain WA-004 carrying the pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as a template, and the upstream homology arm and downstream homology arm of the adhE gene were amplified using the primers adhE-UF / adhE-UR; adhE-DF / adhE-DR combination, and then the primers adhE-UF / adhE-DR combination was used for fusion PCR amplification to obtain the ahdE gene knockout homology fragment adhE-UD.

[0109] The pTargetF-adhE plasmid targeting the adhE gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-adhE-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-adhE plasmid was obtained. The homologous fragments adhE-UD and pTargetF-adhE plasmid were co-transformed into WA-004 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the adhE-UUF / adhE-DDR combination. If a band of 2000 bp could be amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-adhE plasmid, and the strain WA-005 carrying the pCas9 plasmid was obtained, and the adhE gene on its genome was knocked out.

[0110] 4.6 Knockout of the methylglyoxal synthase gene mgsA

[0111] The strain WA-005 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the mgsA gene were amplified using the primers mgsA-UF / mgsA-UR; mgsA-DF / mgsA-DR combination, and then the mgsA gene knockout homology fragment mgsA-UD was obtained by fusion PCR amplification using the primers mgsA-UF / mgsA-DR combination.

[0112] The pTargetF-mgsA plasmid targeting the mgsA gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-mgsA-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-mgsA plasmid was obtained. The homologous fragments mgsA-UD and pTargetF-mgsA plasmid were co-transformed into WA-005 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the mgsA-UUF / mgsA-DDR primer combination. If a band of 2249 bp could be amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-mgsA plasmid, and the strain WA-006 carrying the pCas9 plasmid was obtained, and the mgsA gene on its genome was knocked out.

[0113] 4.7 Knockout of alanine racemase gene dadX:

[0114] The strain WA-006 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the dadX gene were amplified using the primers dadX-UF / dadX-UR; dadX-DF / dadX-DR combination, and then the dadX gene knockout homology fragment dadX-UD was obtained by fusion PCR amplification using the primers dadX-UF / dadX-DR combination.

[0115] The pTargetF-dadX plasmid targeting the dadX gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-dadX-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-dadX plasmid was obtained. The homologous fragments dadX-UD and pTargetF-dadX plasmid were co-transformed into WA-006 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the dadX-UUF / dadX-DDR primer combination. If a band of 2196 bp could be amplified, it was a positive transformant. The obtained positive transformants were induced to eliminate the pTargetF-dadX plasmid, and the strain WA-007 carrying the pCas9 plasmid was obtained, and the dadX gene on its genome was knocked out.

[0116] 4.8 lacI gene knockout

[0117] The strain WA-007 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the lacI gene were amplified using the primers lacI-UF / lacI-UR; lacI-DF / lacI-DR combination, and then the dadX gene knockout homology fragment dadX-UD was obtained by fusion PCR amplification using the primers lacI-UF / lacI-DR combination.

[0118] The pTargetF-lacI plasmid targeting the lacI gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-lacI-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-lacI plasmid was obtained. The homologous fragments lacI-UD and pTargetF-lacI plasmid were co-transformed into WA-007 competent cells carrying the pCas9 plasmid, and the cells were plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR. The grown transformants were verified by PCR using primers lacI-UUF / lacI-DDR. If a band of 2041 bp could be amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF- plasmid, and the strain WA-008 carrying the pCas9 plasmid was obtained, and the lacI gene on its genome was knocked out.

[0119] The WA-008 strain was further inoculated into LB liquid culture medium without any antibiotics and cultured overnight at 42°C and 200 rpm in a shaking incubator to eliminate the pCas9 plasmid and obtain a genetically engineered recombinant strain A0 that does not carry a plasmid and has no resistance marker. The genotype of the A0 strain is E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2 △frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI.

[0120] Table 2. Primers and sequences

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] Example 5: A0 strain was subjected to ultraviolet mutagenesis shake flask screening to obtain A129 strain

[0127] The genetically engineered recombinant strain A0 was cultured in LB medium to OD600=0.4-0.6, and after gradient dilution with physiological saline, it was spread on UV mutagenesis plate medium for UV mutagenesis. The UV wavelength was 254nm, the power was 15W, the mutagenesis distance was 15-20 cm, and different UV irradiation times (10s, 20s, 30s, 40s, 50s, 60s) were set to control the lethality to be above 90%. The plate was wrapped with tin foil and placed in a 37°C incubator for culture. 24 monoclones were selected from the grown monoclones for shake flask culture screening. First, 20 monoclones were inoculated into a 10 mL LB liquid test tube, cultured at 37°C, 200 rpm to OD = 2-3 to obtain seed liquid, and further inoculated into shake flask fermentation medium at a ratio of 1%. The 250 mL shake flask was filled with 220 mL of liquid, and the bottle mouth was plugged with a rubber stopper. It was cultured at 37°C, 150 rpm for 24 hours. The fermentation liquid cell OD, residual glucose concentration and L-alanine concentration were measured. The screening results are shown in Table 3. Among them, the monoclone with the highest OD and the highest L-alanine concentration was A129. The A129 strain was deposited in the China Center for Type Culture Collection on July 17, 2024, and its deposit number is CCTCC M 20241584.

[0128] Table 3 OD, residual glucose concentration and L-alanine concentration of each mutant strain cultured in UV-induced shake flasks

[0129]

[0130] Example 6: Residual sugar controlled at 5-10 g / L, ammonia nitrogen controlled at 0.1%-0.15%

[0131] (1) Seed preparation: Inoculate the frozen A129 Escherichia coli glycerol tube onto the LB plate, activate and culture at 37°C for 12 h, scrape a loopful and inoculate into a 500 ml Erlenmeyer flask containing 100 ml of liquid LB medium, and culture at 37°C and 200 rpm for 2.5 h until the OD reaches 0. 600 The shake flask seeds were inoculated into a 15L seed tank containing 10L seed culture medium at 1% inoculation rate, at 37°C, tank pressure 0.05Mpa, ventilation ratio 1VVM, ammonia water to control pH 6.8, 300-700rpm, DO ≥ 30% by speed control, and cultured for about 4h until OD 600 is 8, and seed liquid is obtained.

[0132] (2) Fermentation culture: The seed liquid was inoculated at 10% into a 15L fermenter containing a fermentation medium with a feed volume of 10L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, a rotation speed of 150 rpm, and no ventilation. The residual sugar and ammonia nitrogen content in the fermentation liquid were detected in real time by online near-infrared. The residual sugar was controlled at 5-10 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.1%-0.15% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≦ 0.5 g / L), the tank was released. The fermentation time was 34 hours. The liquid phase was used to detect the L-alanine content in the released tank, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Tables 4 and Figure 1 .

[0133] Example 7: Residual sugar is controlled at 5-10 g / L, and ammonia nitrogen is controlled at 0.05%-0.1%

[0134] (1) Seed preparation: same as Example 6

[0135] (2) Fermentation culture: The seed liquid was inoculated at 10% in a 15-L fermenter containing a fermentation medium with a feed volume of 10 L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, and a rotation speed of 150 rpm. The residual sugar and ammonia nitrogen contents in the fermentation broth were detected in real time by online near-infrared. The residual sugar was controlled at 5-10 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.05%-0.1% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was released. The fermentation time was 38 hours. The L-alanine content of the released tank was detected by liquid phase detection, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Table 4.

[0136] Comparative Example 1: Residual sugar is controlled at 5-10g / L, and ammonia nitrogen is not controlled

[0137] (1) Seed preparation: same as Example 6

[0138] (2) Fermentation culture: The seed liquid was inoculated at 10% into a 15L fermenter containing a fermentation medium with a feed volume of 10L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, a rotation speed of 150 rpm, and no ventilation. The residual sugar and ammonia nitrogen content in the fermentation liquid were detected in real time by online near-infrared. The residual sugar was controlled at 5-10 g / L by feeding medium 1. Ammonia nitrogen was not controlled during the process. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≦ 0.5 g / L), the tank was released. The total fermentation time was 42 h. The L-alanine content of the released tank was detected by liquid phase detection, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Tables 4 and Figure 2 .

[0139] Comparative Example 2: No residual sugar control, ammonia nitrogen control 0.1%-0.15%

[0140] (1) Seed preparation: same as Example 6

[0141] (2) Fermentation culture: The seed liquid was inoculated at 10% into a 15L fermenter containing a fermentation medium with a feed volume of 10L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, a rotation speed of 150 rpm, and no ventilation. The residual sugar and ammonia nitrogen contents in the fermentation liquid were detected in real time by online near-infrared. The feed medium 1 was replenished at a uniform speed. The residual sugar concentration was not controlled during the process. The ammonia nitrogen was controlled at 0.1%-0.15% by feed medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≦ 0.5 g / L), the tank was released. The total fermentation time was 47 hours. The liquid phase was used to detect the L-alanine content in the released tank, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Tables 4 and Figure 3 .

[0142] Comparative Example 3: Residual sugar controlled at 5-10g / L, ammonia nitrogen controlled at 0.01%-0.05%

[0143] (1) Seed preparation: same as Example 6

[0144] (2) Fermentation culture: The seed liquid was inoculated at 10% into a 15L fermenter containing a fermentation medium with a feed volume of 10L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, a rotation speed of 150 rpm, and no ventilation. The residual sugar and ammonia nitrogen content in the fermentation liquid were detected in real time by online near-infrared. The residual sugar was controlled at 5-10 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.01%-0.05% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≦ 0.5 g / L), the tank was released. The total fermentation time was 39 hours. The liquid phase was used to detect the L-alanine content in the released tank, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Tables 4 and Figure 4 .

[0145] Comparative Example 4: Residual sugar controlled at 15-20g / L, ammonia nitrogen controlled at 0.1%-0.15%

[0146] (1) Seed preparation: same as Example 6

[0147] (2) Fermentation culture: The seed liquid was inoculated at 10% in a 15-L fermenter containing a fermentation medium with a feed volume of 10 L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, and a rotation speed of 150 rpm without ventilation. The residual sugar and ammonia nitrogen contents in the fermentation broth were detected in real time by online near-infrared. The residual sugar was controlled at 15-20 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.1%-0.15% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was drained. The total fermentation time was 40 hours. The L-alanine content of the drained tank was detected by liquid phase detection, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Table 4.

[0148] Comparative Example 5: Residual sugar controlled at 20-30g / L, ammonia nitrogen controlled at 0.1%-0.15%

[0149] (1) Seed preparation: same as Example 6

[0150] (2) Fermentation culture: The seed liquid was inoculated at 10% in a 15-L fermenter containing a fermentation medium with a feed volume of 10 L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, and a rotation speed of 150 rpm without ventilation. The residual sugar and ammonia nitrogen contents in the fermentation broth were detected in real time by online near-infrared. The residual sugar was controlled at 20-30 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.1%-0.15% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was released. The total fermentation time was 39 hours. The L-alanine content of the released tank was detected by liquid phase detection, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Table 4.

[0151] Comparative Example 6: Residual sugar controlled at 1-5g / L, ammonia nitrogen controlled at 0.1%-0.15%

[0152] (1) Seed preparation: same as Example 6

[0153] (2) Fermentation culture: The seed liquid was inoculated at 10% in a 15-L fermenter containing a fermentation medium with a feed volume of 10 L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, and a rotation speed of 150 rpm without ventilation. The residual sugar and ammonia nitrogen contents in the fermentation broth were detected in real time by online near-infrared. The residual sugar was controlled at 1-5 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.1%-0.15% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was discharged. The total fermentation time was 40 hours. The L-alanine content of the discharged tank was detected by liquid phase detection, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Table 4.

[0154] Comparative Example 7: Residual sugar controlled at 5-10g / L, ammonia nitrogen controlled at 0.15%-0.2%

[0155] (1) Seed preparation: same as Example 6

[0156] (2) Fermentation culture: The seed liquid was inoculated at 10% in a 15-L fermenter containing a fermentation medium with a feed volume of 10 L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control the pH at 6.8, and a rotation speed of 150 rpm without ventilation. The residual sugar and ammonia nitrogen contents in the fermentation broth were detected in real time by online near-infrared. The residual sugar was controlled at 5-10 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.15%-0.2% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was discharged. The total fermentation time was 41 hours. The L-alanine content of the discharged tank was detected by liquid phase detection, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Table 4.

[0157] Comparative Example 8: Base sugar 150g / L, no ammonia nitrogen control

[0158] (1) Seed preparation: same as Example 6

[0159] (2) Fermentation culture: The seed liquid was inoculated at 10% into a 15L fermentation tank containing a fermentation medium with a calculated volume of 10L. The fermentation was carried out at 37°C, a tank pressure of 0.05 MPa, ammonia water to control pH 6.8, a rotation speed of 150 rpm, and no ventilation. The initial base sugar was directly 150 g / L. Sugar and ammonia nitrogen were not controlled during the process. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was released. The total fermentation time was 56 hours. The liquid phase was used to detect the L-alanine content of the released tank, and the conversion rate of glucose to L-alanine was calculated. The results are shown in Tables 4 and Figure 5 .

[0160] Comparative Example 9: Fermentation and culture of strain A0 in a 15L fermenter to accumulate L-alanine

[0161] The A0 strain was subjected to seed culture and fermentation culture. The seed preparation and fermentation culture conditions were the same as those in Example 6. When all the sugar was consumed (residual sugar ≦ 0.5 g / L), the tank was released. The fermentation time was 45 hours. The liquid phase detection showed that the L-alanine content in the tank was 129.5 g / kg, and the sugar-acid conversion rate was 95.0%.

[0162] Table 4 Fermentation results of Examples 6-7 and Comparative Examples 1-8

[0163]

[0164] It can be seen from the experimental results of the embodiments and comparative examples that the present invention realizes real-time and accurate detection and feedback control of residual sugar and ammonia nitrogen in the L-alanine fermentation process through online near-infrared technology, thereby improving the L-alanine discharge content and the conversion rate of sugar to product.

[0165] Sequence information

[0166] SEQ ID NO: 1 Wild-type alanine dehydrogenase PmGlaD from Priestia megaterium, GenBank accession number WP_328233963.1

[0167] MKIGVPKEVKNNENRVAITPAGVTALVNGGHDVYIETQAGEGSGFHDEDYTNAGASIVNTAKEAWDAEMVMKVKEPISEEYGYFREGLILFTYLHLAAEEALTKALIDKKVVGIAYETVQLSNKSLPLLTPMSEVAGRMSSQIGAQFLEKTKGGKGILLSGVPGVQRGKVTIIGGGVAGTNAAKIA VGLGADVTIIDLNADRLRQLDDLFGKEVTTLMSNHYNIAQSVKESDLVIGAVLIPGAKAPKLVTEEMVQTMSKGSVVVDIAIDQGGIFETTDRITTHDNPTYSKHEVLHYAVANMPGAVPRTSTLALTNVTVPYALQIANQGYKKACLKNPSLLQGINTLNGFVTYAAVADAHGLTYADATTLLEQA

[0168] SEQ ID NO: 2 The encoding gene of wild-type alanine dehydrogenase PmGlaD from Priestia megaterium, which is optimized according to the codon preference of Escherichia coli

[0169]

[0170] SEQ ID NO: 3 Amino acid sequence of alanine dehydrogenase mutant PmAlaD-m6-92 obtained by screening

[0171] MKIGVPKEVKNNENRVAITPAGVTALVNGGHDVYIETQAGEGSGFHDTDYTNAGASIVNTAKEAWDAEMVMKVKEPISEEYGYFREGLILFTYLHLAAEEALTKALIDKKVVGIAYETVQLSNKSLPLLTPMSEVAGRMSSQIGAQFLEKTKGGKGILLSGVPGVQRGKVTIIGGGVAGINAAKIA VGLGADVTIIDLNADRLRQLDDLFGKEVTTLMSNHYNIAQSVKESDLVIGAVLIPGAKAPKLVTEEMVQTMSKGSVVVDIAIDQGGIFETTDRITTHDNPTYSKHEVLAYAVANMPGEVPRTSTLALTNVTVPYALQIANQGYKKACLKNPSLLQGIITLNGFVTYAAVADAHGLTYADATTLLEQA

[0172] SEQ ID NO: 4 The coding gene sequence of the screened alanine dehydrogenase mutant PmAlaD-m6-92

[0173]

Claims

1. A method for preparing L-alanine, characterized in that: It includes the following steps: The L-alanine-producing recombinant bacterial seeds are inoculated into the fermentation medium. During the fermentation culture process, the residual sugar concentration is controlled to be 5-10 g / L and the ammonia nitrogen concentration is controlled to be 0.05-0.15%; The control method is to use online near infrared technology to detect the residual sugar concentration and the ammonia nitrogen concentration, and adjust the concentration by adding feed culture medium.

2. The preparation method according to claim 1, characterized in that It meets one or more of the following conditions: (1) The online near infrared technology detection specifically includes the following steps: S1: Establishing the linear equation The near infrared spectra at different time points in the fermentation system are continuously collected by online near infrared analysis equipment, and then the collected near infrared spectrum data are preprocessed by computer software and chemometric methods after removing abnormal spectra to obtain preprocessed near infrared spectrum data; at the same time, samples are taken at the collection time point corresponding to each spectrum, and process parameters are measured offline to obtain physical and chemical test data; finally, linear fitting is performed based on the preprocessed near infrared spectrum data and physical and chemical test data to establish a fermentation model; wherein the process parameters include glucose concentration and ammonia nitrogen concentration; S2: Real-time feedback control of the fermentation process The near infrared spectrum in the fermentation system is continuously collected online by using an online near infrared analysis device, and then the collected near infrared spectrum data is preprocessed by removing abnormal spectra using computer software and chemometric methods to obtain preprocessed near infrared spectrum data, and then the preprocessed near infrared spectrum data is coupled with the fermentation model established in step S1 to convert the process parameters in the fermentation system, and the peristaltic pump is controlled to perform intermittent feeding according to the process parameters; wherein the process parameters include glucose concentration and ammonia nitrogen concentration; (2) Control the ammonia nitrogen concentration to 0.05-0.1% or 0.1-0.15%; (3) The residual sugar concentration is adjusted by feed medium 1. During the fermentation culture process, when the detected residual sugar concentration is lower than a preset value, feed medium 1 is added until the detected residual sugar concentration reaches or exceeds the preset value, at which time the feeding is stopped; the feed medium 1 comprises 50-80% of sugar, and the sugar is glucose and / or sucrose; % represents the mass fraction percentage of the mass of the sugar to the total mass of the feed medium 1; (4) The ammonia nitrogen concentration is adjusted by feed medium 2. During the fermentation culture process, when the detected ammonia nitrogen concentration is lower than a preset value, feed medium 2 is added until the detected ammonia nitrogen concentration reaches or exceeds the preset value, at which time the feeding is stopped; the feed medium 2 comprises 10-30% of a nitrogen source, where % represents the mass fraction of the nitrogen source to the total mass of the feed medium 2; the nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source; (5) The recombinant bacterial seed is selected from one or more of recombinant Escherichia coli, recombinant Bacillus subtilis, recombinant Corynebacterium glutamicum and recombinant yeast; (6) The components of the fermentation medium include: 5-15 g / L sugar, 1-10 g / L yeast extract, 1-10 g / L peptone, 1-10 g / L ammonium sulfate, 1-5 g / L potassium dihydrogen phosphate, 1-10 g / L disodium hydrogen phosphate, 1-5 g / L magnesium sulfate and 0.1-1 g / L defoaming agent; wherein the sugar is glucose and / or sucrose; g / L represents the ratio of the mass of each component to the total volume of the culture medium.

3. The preparation method according to claim 2, characterized in that: It meets one or more of the following conditions: (1) The online near-infrared analysis equipment is a Fourier transform near-infrared spectrometer; (2) Control the ammonia nitrogen concentration to 0.1-0.15%; (3) the feed medium 1 comprises 60% glucose; (4) the feed medium 2 comprises 20% of an inorganic nitrogen source; (5) The recombinant bacterial seed is a recombinant Escherichia coli; (6) The components of the fermentation medium include: 10 g / L glucose, 5 g / L yeast extract powder, 5 g / L peptone, 8 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, 3 g / L magnesium sulfate and 0.5 g / L defoaming agent.

4. The preparation method according to claim 3, characterized in that: It meets one or more of the following conditions: (1) The inorganic nitrogen source is ammonium sulfate, ammonium chloride and / or diammonium hydrogen phosphate; (2) The genotype of the recombinant Escherichia coli is E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2△frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI; (3) The magnesium sulfate is magnesium sulfate heptahydrate; (4) The defoaming agent is a polyether defoaming agent.

5. The preparation method according to claim 4, characterized in that: It meets one or both of the following conditions: (1) The inorganic nitrogen source is ammonium sulfate; (2) The recombinant Escherichia coli is a strain with a deposit number of CCTCC M 20241584.

6. The preparation method according to claim 1, characterized in that: The fermentation culture comprises the following steps: inoculating the recombinant bacterial seed solution into a fermentation tank containing the fermentation medium, and performing anaerobic fermentation.

7. The preparation method according to claim 6, characterized in that: It meets one or more of the following conditions: (1) The inoculation ratio of the recombinant bacterial seed solution to the fermentation medium is 5%-15%, for example, 10%; (2) The temperature of the anaerobic fermentation is 30-40°C; for example, 37°C; (3) In the fermentation culture, the concentration of total sugar is 142-155 g / L, for example, 150 g / L; the total sugar is total glucose and / or total sucrose; (4) The anaerobic fermentation time is such that the glucose and / or sucrose in the fermentation medium is completely consumed, i.e., the residual sugar is ≤ 0.5 g / L; for example, 34 h; (5) During the anaerobic fermentation, the pH is adjusted to 6-7, for example, 6.8, using aqueous ammonia; (6) The tank pressure of the fermentation tank is 0.02-0.1 MPa, for example 0.05 MPa; (7) The rotation speed of the fermentation tank is 100-300 rpm, for example, 150 rpm.

8. The preparation method according to claim 6 or 7, characterized in that: The recombinant bacterial seed liquid also includes seed preparation before fermentation culture, and the seed preparation includes the following steps: activating the frozen recombinant bacterial seeds and then shaking culture to obtain shake flask seeds, and then expanding the culture in a seed tank containing seed culture medium.

9. The preparation method according to claim 8, characterized in that: It meets one or more of the following conditions: (1) The components of the seed culture medium include: 10-40 g / L sugar, 1-10 g / L yeast extract powder, 1-10 g / L peptone, 10-30 g / L ammonium sulfate, 1-5 g / L potassium dihydrogen phosphate, 1-10 g / L disodium hydrogen phosphate, 1-5 g / L magnesium sulfate and 0.1-1 g / L defoaming agent; the sugar is glucose and / or sucrose; wherein g / L represents the ratio of the mass of each component to the total volume of the culture medium; (2) The culture medium used for the activation is LB liquid culture medium; (3) The activation temperature is 30-40°C; (4) The activation time is 10-15 hours; (5) The shaking culture is carried out in a shaker at a rotation speed of 150-250 rpm; (6) The shaking culture temperature is 30-40°C; (7) The shaking culture time is 1-6 hours; (8) The inoculation ratio of the shake flask seeds to the seed tank is 0.3-2%; (9) The temperature of the expanded culture is 30-40°C; (10) The time of the expanded culture is 3-5 hours; (11) During the expanded culture, ammonia water is used to adjust the pH to 6.5-7.2; (12) The tank pressure of the seed tank is 0.02-0.1Mpa; (13) The rotation speed of the seed tank is 300-700 rpm; (14) The ventilation ratio of the expanded culture is 0.5-2 VVM.

10. The preparation method according to claim 9, characterized in that: It meets one or more of the following conditions: (1) The components of the seed culture medium include: 30 g / L glucose, 5 g / L yeast extract powder, 5 g / L peptone, 20 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, 3 g / L magnesium sulfate and 0.5 g / L defoamer; for example, the magnesium sulfate is magnesium sulfate heptahydrate; for another example, the defoamer is a polyether defoamer; (2) The components of the LB liquid medium include 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride; (3) The pH of the LB liquid medium is 7.2; (4) The activation temperature is 37°C; (5) The activation time is 12 hours; (6) The rotation speed during the shaking culture is 200 rpm; (7) The shaking culture temperature is 37°C; (8) The shaking culture time is 2-3 hours, preferably controlling OD 600 is 2-3; (9) The inoculation ratio of the shake flask seeds to the seed tank is 1%; (10) The temperature of the expanded culture is 37°C; (11) The time of the expanded culture is 4 hours, preferably controlling the OD 600 For example, the control OD is 5-10 600 is 8; (12) During the expanded culture, ammonia water is used to adjust the pH to 6.8; (13) The tank pressure of the seed tank is 0.05Mpa; (14) The rotation speed of the seed tank is controlled to keep DO ≥ 30%; (15) The ventilation ratio of the expanded culture is 1 VVM.

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