Multi-enzyme synergistic expression recombinant genetically engineered bacteria and application thereof

Abstract

The present application relates to a kind of multi-enzyme synergistic expression recombinant genetically engineered bacteria, and its application in the biological catalysis preparation L-2-amino butyric acid.The present application constructs leucine dehydrogenase gene and formic acid dehydrogenase gene co-expression system, and is transformed into expression bacteria to obtain recombinant genetically engineered bacteria, with the live cells of the recombinant genetically engineered bacteria fermented and cultivated as catalyst applied in the catalytic reaction system of preparation L-2-amino butyric acid, production efficiency is high, when L-threonine feed amount is 300g / L, reaction 12h, conversion rate reaches more than 99%, effectively improve the ability of catalytic high concentration substrate to generate L-2-amino butyric acid.The present application uses whole cell as catalyst, avoids the purification of enzyme, avoids broken cell, and operation is simple;Two enzymes in one cell, can reduce steric hindrance, speed up the reaction speed;With deionized water as reaction medium to form reaction system, reaction product is easy to purify;Substrate concentration is high in the reaction process, saves production cost and time.

Description

(I) Technical Field

[0001] This invention relates to a recombinant genetically engineered bacterium that co-expresses multiple enzymes, and its application in the biocatalytic preparation of L-2-aminobutyric acid. (II) Background Technology

[0002] L-2-aminobutyric acid (L-ABA) is a non-natural amino acid that can enhance glucose-6-phospholipase activity, promote brain cell metabolism, and is used clinically for painless caries removal and treatment of sequelae of cerebrovascular diseases. It also has antihypertensive effects. Furthermore, L-2-aminobutyric acid is an important chemical raw material and pharmaceutical intermediate. It can be prepared by amidation to produce (S)-2-aminobutyramide salt, a key intermediate in the synthesis of the antiepileptic drug levetiracetam. It can also be used to prepare (S)-2-aminobutanol by reducing the terminal carboxyl group, and is subsequently used to synthesize ethambutol. The industrial production technology of L-2-aminobutyric acid has become a hot topic in pharmaceutical engineering research.

[0003] The preparation methods for L-2-aminobutyric acid mainly include chemical synthesis and biosynthesis. Chemical methods include desulfurization reactions, ammoniation hydrolysis reactions, and butanone reduction. Chemical synthesis strategies have significant drawbacks, such as poor selectivity, demanding reaction conditions, numerous byproducts, and difficulties in separation and purification.

[0004] The biosynthesis of L-2-aminobutyric acid (L-2-aminobutyric acid) includes three methods: microbial fermentation, chiral resolution by enzymes, and enzymatic conversion. Microbial fermentation uses glucose as a nutrient and modified *E. coli* to produce L-2-aminobutyric acid. Chiral resolution involves oxidizing the racemic L-2-aminobutyric acid with D-amino oxidase to produce keto acid, which is then converted back to L-2-aminobutyric acid by transaminase or amino acid dehydrogenase, transforming the original racemic compound into optically pure L-2-aminobutyric acid. Most research on L-2-aminobutyric acid synthesis focuses on enzymatic conversion. Using L-threonine as a substrate, transaminase converts it to 2-ketobutyric acid, followed by dehydrogenase conversion to L-2-aminobutyric acid. Coupling with a coenzyme cycle system to improve coenzyme utilization can significantly increase yield. Enzymatic conversion is increasingly being researched and applied due to its high conversion efficiency, low raw material cost, absence of byproducts, easy product extraction, and environmental friendliness.

[0005] The ribosome binding site (RBS) is a purine-rich untranslated region upstream of the start codon AUG. The RBS contains the SD (Shine-Dalg-arno) sequence, typically 5 nucleotides long and rich in G and A. This sequence is complementary to the 3' end of the 16S ribosomal rRNA, promoting ribosome binding to the mRNA and facilitating translation initiation. The binding strength of the RBS depends on the structure of the SD sequence and its distance from the start codon AUG. A distance of 4–10 nucleotides between SD and AUG is generally preferred, with 9 nucleotides being optimal. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant genetically engineered bacterium that co-expresses leucine dehydrogenase and formate dehydrogenase with high enzyme expression levels and high substrate conversion rates, and to apply it to the industrial production of L-2-aminobutyric acid. By utilizing a multi-enzyme synergistic expression system, the catalytic reaction of high-concentration substrates can be achieved during the reaction process.

[0007] The technical solution adopted in this invention is:

[0008] A recombinant genetically engineered bacterium for multi-enzyme co-expression is constructed by the following method: a single-plasmid dual-gene expression vector is used, and formate dehydrogenase and leucine dehydrogenase genes are inserted into its multiple cloning site. The RBS sequence before the restriction enzyme site is replaced with CTACCCCCAAGGTTGATAAGGAGGTATTTT. The bacterium is then transformed into host cells to obtain the recombinant genetically engineered bacterium for multi-enzyme co-expression. The nucleotide sequence of the leucine dehydrogenase gene is shown in SEQ ID NO.1, and the nucleotide sequence of the formate dehydrogenase gene is shown in SEQ ID NO.3.

[0009] The single-plasmid dual-gene expression vector is one of pACYCDuet, pCDFDuet, pETDuet, or pRSFDuet. The host cell of the recombinant genetically engineered bacteria is Escherichia coli BL21(DE3).

[0010] Preferably, the single plasmid dual gene expression vector is pRSFDuet, into which formate dehydrogenase gene and leucine dehydrogenase gene are inserted to obtain pRSFDuet-fdh-Leudh, and the RBS sequence of its MCS1 is replaced with CTACCCCCAAGGTTGATAAGGAGGTATTTT.

[0011] This invention also relates to the application of the recombinant genetically engineered bacteria in the biocatalytic preparation of L-2-aminobutyric acid.

[0012] Specifically, the application is as follows: using live cells obtained by fermentation culture of the recombinant genetically engineered bacteria as a catalyst, L-threonine as the starting substrate, ammonium formate as the co-substrate for coenzyme recycling, and adding threonine deaminase, and using deionized water as the reaction medium to form a reaction system, an enzyme-catalyzed reaction is carried out. After the reaction is completed, a reaction solution containing L-2-aminobutyric acid is obtained. The reaction solution is then separated and purified to obtain the L-2-aminobutyric acid.

[0013] The fermentation culture method for recombinant genetically engineered bacteria is as follows: the recombinant genetically engineered bacteria are inoculated into a culture medium containing antibiotics to obtain a seed culture. After expansion culture, a bacterial culture with an OD600 of 0.6 to 0.9 is obtained. IPTG is added as an inducer and induced at 22-28℃ for 10-18 hours. After the fermentation culture is completed, the wet bacterial cells are collected.

[0014] The culture medium can be any culture medium in the art that can enable the cells to grow and produce the present invention, preferably TB medium, which consists of: 12 g / L peptone, 24 g / L yeast extract, 5 g / L glycerol, 2.312 g / L potassium dihydrogen phosphate trihydrate, and 12.54 g / L anhydrous dipotassium hydrogen phosphate, dissolved in distilled water, pH 7.0.

[0015] The seed culture conditions were 37℃ and 180 r / min for 12 h. The scale-up culture conditions were 37℃ and 180 r / min.

[0016] When culturing recombinant genetically engineered bacteria in shake flasks, IPTG is used as the inducer at a final concentration of 0.06-0.12 mmol / L, an induction temperature of 22-30℃, and an induction time of 10-16 h. Preferably, the induction temperature is 28℃, the inducer concentration is 0.1 mmol / L IPTG, and the induction time is 14 h.

[0017] When culturing genetically engineered bacteria in a fermenter, lactose is used as an inducer. Lactose is added to the bacterial culture to a final concentration ≥4 g / L, and the culture is induced for 13-19 hours. Preferably, the optimal lactose concentration is 8 g / L, and the induction time is 17 hours.

[0018] In the reaction system, the mass concentration of L-threonine is 100–500 g / L, the mass concentration of ammonium formate is 100–200 g / L, the concentration of threonine deaminase is 20,000–30,000 U / L, the live recombinant genetically engineered bacterial cells are 10–50 g / L based on the wet weight of the bacterial cells, the enzyme catalysis temperature is 25–40 °C, the reaction time is 2–16 h, and 0.1–1.0 g / L of coenzyme NAD+ is added 20–30 min after the start of the reaction.

[0019] More preferably, in the optimal reaction system, the mass concentration of L-threonine is 300 g / L, the mass concentration of ammonium formate is 158 g / L, the concentration of threonine deaminase is 25000 U / L, the live recombinant genetically engineered bacteria cells are 25 g / L based on the wet weight of the cells, and the amount of coenzyme added is 0.20 g / L; the enzyme catalysis temperature is 35℃, the reaction time is 12 h, the conversion rate is above 99%, the ee value is above 99.5%, and the catalytic efficiency is high.

[0020] The beneficial effects of this invention are mainly reflected in:

[0021] (1) This invention constructs a co-expression system of leucine dehydrogenase gene and formate dehydrogenase gene, transforms it into expression bacteria to obtain recombinant genetically engineered bacteria, and uses the live cells of the fermented recombinant genetically engineered bacteria as a catalyst in the catalytic reaction system for the preparation of L-2-aminobutyric acid. The production efficiency is high; when the L-threonine feed amount is 300 g / L, the conversion rate reaches over 99% after 12 h of reaction. This strain can effectively improve the ability to catalyze the production of L-2-aminobutyric acid from high-concentration substrates.

[0022] (2) The present invention uses whole cells as catalysts, which avoids enzyme purification and cell disruption, and is easy to operate; two enzymes in one cell can reduce steric hindrance and accelerate the reaction rate; the reaction system is constructed with deionized water as the reaction medium, and the reaction products are easy to purify; the substrate concentration is high during the reaction, which saves production costs and time. (iv) Description of the attached drawings

[0023] Figure 1 This is a schematic diagram of the co-expression plasmid construction; where (A) is pACYCDuet-leudh-fdh, (B) is pACYCDuet-fdh-leudh, (C) is pCDFDuet-leudh-fdh, (D) is pCDFDuet-fdh-leudh, (E) is pETDuet-leudh-fdh, (F) is pETDuet-fdh-leudh, (G) is pRSFDuet-leudh-fdh, and (H) is pRSFDuet-fdh-leudh.

[0024] Figure 2 To replace the effect of the RBS sequence on the SDS-PAGE (A) and substrate conversion (B) of pRSFDuet-fdh-leudh, in (A), AG represents the supernatant of cell lysis induced at different predicted translation initiation rates, and ag represents the precipitate corresponding to ag.

[0025] Figure 3To illustrate the effect of induction temperature on SDS-PAGE (A) and substrate conversion (B) of pRSFDuet-fdh-leudh, in (A), AE represents the supernatant of cell disruption cultured at induction temperatures of 22, 24, 26, 28, and 30 °C, and ae represents the corresponding precipitate.

[0026] Figure 4 The effect of inducer concentration on SDS-PAGE (A) and substrate conversion (B) of pRSFDuet-fdh-leudh is shown. In (A), AE are the supernatant of cell lysate induced by inducer concentrations of 0.06, 0.08, 0.1, 0.12 and 0.14 mmol / L, respectively, and ae are the precipitates corresponding to the lysate.

[0027] Figure 5 To illustrate the effect of induction time on SDS-PAGE (A) and substrate conversion (B) of pRSFDuet-fdh-leudh, in (A), AD represents the supernatant of cell disruption cultured at induction times of 10, 12, 14, and 16, respectively, and ad represents the corresponding precipitate.

[0028] Figure 6 The effect of catalytic reaction temperature on the preparation of L-2-aminobutyric acid.

[0029] Figure 7 The effect of inducing agent concentration on the catalytic reaction process in fermenters.

[0030] Figure 8 The effect of fermenter induction time on the catalytic reaction process.

[0031] Figure 9 The reaction process for preparing L-2-aminobutyric acid using the pRSFDuet-fdh-leudh strain. (V) Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] Example 1: Cloning and co-expression recombination system of leucine dehydrogenase gene (LeuDH) and formate dehydrogenase gene (FDH)

[0034] The leucine dehydrogenase gene is derived from the complete genome sequence of *Thermoactinomyces intermedius*. To enable the expression of a His-tag protein after ligation into the vector pET-28b, the stop codon was removed, and the sequence was optimized using the codon preference of *B. subtilis* 168 as a reference, along with the commonly used restriction endonuclease recognition sites BamHI, XhoI, PstI, HindIII, and NcoI. This resulted in a new leucine dehydrogenase gene (nucleotide sequence SEQ ID NO.1, amino acid sequence SEQ ID NO.2), disclosed in patent application (201610867380.8). The new leucine dehydrogenase gene has been ligated into the expression vector pET-28b, becoming pET-28b-leudh.

[0035] The formate dehydrogenase gene (nucleotide sequence SEQ ID NO.3, amino acid sequence SEQ ID NO.4) is derived from Fusarium graminearum and has been ligated into the expression vector pET-28b, namely pET-28b-fdh.

[0036] Using pET-28b-leudh as a template, the leucine dehydrogenase gene fragment was amplified by PCR.

[0037] Upstream primer (primer 1): 5'-CCATGGGTAAAATCTTCGACTACATG-3' (SEQ ID NO.5),

[0038] Downstream primer (primer 2): 5'-AAGCTTTTATTTGTTGTTGAAGTTGA-3' (SEQ ID NO. 6);

[0039] Using pET-28b-fdh as a template, the formate dehydrogenase gene fragment was amplified by PCR.

[0040] Upstream primer (primer 3):

[0041] 5'-CATATGGGTAAAATTGTACTGGTTCTGTATGAC-3' (SEQ ID NO.7),

[0042] Downstream primer (primer 4):

[0043] 5'-CTCGAGTTATTTCTTGTCGTGTTTACCGTAC-3' (SEQ ID NO. 8).

[0044] The PCR reaction system (50 μL) consisted of: 25 μL of 2×Taq polymerase buffer, 2 μL of dNTP™ imprint, 2 μL of Taq polymerase DNA polymerase, 1 μL of template plasmid, 1 μL each of forward and reverse primers, and 18 μL of sterile water.

[0045] The Bio-Rad PCR instrument was used, and the reaction program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 1 min (32 cycles); 72℃ extension for 10 min.

[0046] The PCR amplification products were recovered using a recovery kit (Axygen, USA), and the purified fragments were labeled as fragment leudh and fragment fdh, respectively.

[0047] The pACYCDuet plasmid was linearized using a one-step cloning method and labeled as V-pACYCDuet.

[0048] The obtained enzyme fragment was ligated to a linearized vector using a one-step cloning method.

[0049] The ligation system (20 μL) consisted of: 1 μL of linearized vector V-pACYCDuet, 1 μL of fragment leudh, 4 μL of 5×CEⅡ buffer, 2 μL of Exnase Ⅱ, and 12 μL of sterile water.

[0050] Using a Bio-Rad PCR instrument at 37°C for 30 minutes, a single plasmid recombinant co-expression vector pACYCDuet-leudh-MCS2 containing the leucine dehydrogenase gene fragment of this invention was constructed. The constructed pACYCDuet-leudh-MCS2 was linearized, and then the formate dehydrogenase gene fragment was ligated into the vector using the above ligation system to obtain pACYCDuet-leudh-fdh.

[0051] The single-plasmid co-expression systems constructed using the same strategy are: pACYCDuet-leudh-fdh, pACYCDuet-fdh-leudh, pCDFDuet-leudh-fdh, pCDFDuet-fdh-leudh, pETDuet-leudh-fdh, pETDuet-fdh-leudh, pRSFDuet-leudh-fdh, and pRSFDuet-fdh-leudh.

[0052] The co-expression system was co-transformed into E. coli BL21(DE3) competent cells. The transformation method was as follows: 1-2 μL of plasmid was added to 100 μL of E. coli BL21(DE3) competent cells, mixed thoroughly, and incubated on ice for 30 min; Eppendorf tubes containing the mixture were placed in a 42°C water bath for 90 s heat shock, and then immediately transferred to ice for 5 min cooling; 600 μL of ILB liquid medium was added to the tubes, and the tubes were incubated on a 37°C, 200 rpm constant temperature shaker for 45 min, and then spread on LB solid plates containing the corresponding antibiotics and incubated at 37°C for 12-18 h.

[0053] Example 2: Replacement of the ribosome binding site (RBS) sequence

[0054] As mentioned above, E. coli BL21(DE3)pRSFDuet-fdh-leudh was selected as the preferred engineered bacterium for subsequent research. Experiments showed that fdh was the rate-limiting enzyme in this catalytic reaction. To improve catalytic efficiency, this experiment synthesized seven RBS sequences with different predicted translation initiation rates by performing calculations on the RBS library website. The synthesized RBS sequences were then used to replace the RBS sequence before MCS1 of the pRSFDuet-fdh-leudh vector using a one-step cloning method. The replaced RBS sequences are shown in Table 2.

[0055] Table 2: RBS sequences with different predicted translation initiation rates

[0056]

[0057] Taking RBS-1 as an example, but not limited to this invention, the RBS-1 sequence is synthesized within primers using pRSFDuet-fdh-leudh as a template, and the original RBS sequence is replaced with RBS-1 through a one-step cloning method.

[0058] Upstream primer (primer 5):

[0059] 5'-TGTACCCTCTTTTATAAATTTGAGTTGAGGACCCTTTTTatataccATGGCC-3' (SEQ IDNO.9),

[0060] Downstream primer (primer 6):

[0061] 5'-AAAAAGGGTCCTCAACTCAAATTTATATAAAAGAGGGTACAattaaagttaaaca aaa-3' (SEQ ID NO. 10);

[0062] The PCR reaction system (50 μL) consisted of: 25 μL of 2×Taq polymerase buffer, 2 μL of dNTP™ imprint, 2 μL of Taq polymerase DNA polymerase, 1 μL of template plasmid, 1 μL each of forward and reverse primers, and 18 μL of sterile water.

[0063] The Bio-Rad PCR instrument was used. The reaction program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s (30 cycles); 72℃ extension for 10 min. The RCR product was labeled as RBS-1.

[0064] RBS-1 was transformed into E. coli BL21(DE3) competent cells. The transformation method was as follows: 1-2 μL of plasmid was added to 100 μL of E. coli BL21(DE3) competent cells, mixed thoroughly, and incubated on ice for 30 min; Eppendorf tubes containing the mixture were placed in a 42°C water bath for 90 s heat shock, and then immediately transferred to ice for 5 min cooling; 600 μL of LLB liquid medium was added to the tubes, and the tubes were incubated on a constant temperature shaker at 37°C and 200 rpm for 45 min, and then spread on LB solid plates containing the corresponding antibiotics and incubated at 37°C for 12-18 h.

[0065] On RBS-1LB solid plates, pick bacteria and send them for testing to determine if the preservation was successful. The successfully preserved strain is recorded as E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-1), and the subsequent RBS sequence substitutions are as above.

[0066] The replaced strains were labeled as pRSFDuet-fdh-leudh(RBS-1), pRSFDuet-fdh-leudh(RBS-2), pRSFDuet-fdh-leudh(RBS-3), pRSFDuet-fdh-leudh(RBS-4), pRSFDuet-fdh-leudh(RBS-5), pRSFDuet-fdh-leudh(RBS-6), and pRSFDuet-fdh-leudh(RBS-7).

[0067] Example 3: Expression of leucine dehydrogenase and formate dehydrogenase in different co-expression systems

[0068] Different co-expression strains constructed in Examples 1 and 2 were inoculated into test tubes containing 10 mL of LB liquid medium and corresponding antibiotics were added. The culture was carried out in a shaker at 37°C and 150 rpm for 12 h to obtain seed culture. The seed culture was then transferred to 100 mL of LB liquid medium at a volume fraction of 1% (v / v), and antibiotics were added. The culture was carried out at 37°C and 180 rpm until the OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.1 mmol / L, and the induction temperature was 28°C for 14 h. After fermentation, the cells were centrifuged at 4°C, 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with physiological saline. The collected cells were stored at 4°C for later use.

[0069] Example 4: Efficiency of different co-expression strains in producing L-2-aminobutyric acid

[0070] Sample detection method: Thermo Fisher Scientific high performance liquid chromatograph, Eclipse XD8-C18 column (5μm 4.6mm × 250mm), mobile phase: 0.02mol / L disodium hydrogen phosphate (pH 7.2): acetonitrile = 70:30, flow rate: 1.0mL / min, column temperature: 30℃, UV detection wavelength: 360nm.

[0071] Sample derivatization conditions: Take 100 μL of the sample to be tested, mix it with 100 μL of 0.5 mol / L NaHCO3 solution and 100 μL of 1% (v / v) 2,4-dinitrofluorophenylacetonitrile solution, and incubate at 60℃ in the dark for 1 h. After the reaction is complete, cool to room temperature, and then add 700 μL of NaH2PO4 / Na2HPO4 buffer (0.2 mol / L, pH 7.0). Principle: The free terminal NH2 of amino acids can undergo a nucleophilic aromatic ring substitution reaction with 2,4-dinitrofluorobenzene in an alkaline environment, and the generated dinitrophenyl amino acid derivatives can be quantitatively detected by HPLC. Under the above conditions, the retention times of 2,4-dinitrofluorobenzene and L-2-aminobutyric acid were 4.1 min and 5.8 min, respectively. Figure 2 As shown.

[0072] Screening of dominant strains: The reaction system consisted of 240 g / L substrate L-threonine, 126 g / L co-substrate ammonium formate, 20000 U / L crude threonine deaminase solution, and 20 g / L bacterial cells (each bacterial cell obtained in Example 2), all in 100 mL of deionized water. The enzyme-catalyzed reaction was carried out at 35℃ and 600 r / min for 12 h. After 30 min of reaction, 0.20 g / L NAD+ was added. Samples were taken periodically during the reaction, and the reaction was terminated with concentrated hydrochloric acid. The samples were used for liquid chromatography analysis. The specific results are shown in Table 1.

[0073] The quality of co-expression strains was evaluated based on their efficiency in producing L-2-aminobutyric acid (GABA). The co-expression strains with high catalytic efficiency were: pCDFDuet-leudh-fdh, pRSFDuet-leudh-fdh, pRSFDuet-fdh-leudh, and pRSFDuet-fdh-leudh (RBS-3). Among them, pRSFDuet-fdh-leudh (RBS-3) exhibited the highest catalytic efficiency, with a substrate conversion rate of 99.5%. Therefore, E. coli BL21(DE3)pRSFDuet-fdh-leudh (RBS-3) was selected as the preferred engineered strain for subsequent research.

[0074] Table 1: Catalytic performance of strains constructed using different co-expression systems

[0075]

[0076]

[0077] Note: This indicates that the reaction system consisted of 240 g / L substrate L-threonine, 126 g / L co-substrate ammonium formate, 20000 U / L crude threonine deaminase solution, and 20 g / L bacterial cells (each bacterial cell obtained in Examples 1 and 2), all contained in 100 mL of deionized water. The enzyme-catalyzed reaction was carried out at 35°C and 600 r / min for 12 h, and 0.20 g / L NAD+ was added after 30 min of reaction.

[0078] Example 5: Optimization of shake-flask fermentation conditions for E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3) strain

[0079] The reaction system was the same as the screening conditions for the aforementioned superior strains. The fermentation conditions for E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3) shake-flask culture were optimized, with SDS-PAGE and substrate conversion rate selected as reference indicators. First, the induction temperature was optimized by adding 0.1 mmol / L IPTG during induction and inducing at 22, 24, 26, 28, and 30℃ for 14 h, respectively. The results are shown below. Figure 3 As shown in the figure, 28℃ is the optimal induction temperature.

[0080] To further investigate the effect of IPTG concentration on enzyme production, IPTG was added at concentrations of 0.06, 0.08, 0.1, 0.12, and 0.14 mmol / L during induction, and induction was performed at 28°C for 14 h. The results are shown below. Figure 4 The optimal IPTG concentration was ultimately determined to be 0.1 mmol / L.

[0081] The effect of induction time on enzyme production was further investigated. 0.1 mmol / L IPTG was added during induction, and induction was performed at 28℃ for 10, 12, 14, and 16 h, respectively. Figure 5 It can be seen that the optimal induction time is 14 hours.

[0082] Example 6: Effect of reaction temperature on the preparation of L-2-aminobutyric acid

[0083] The effect of reaction temperature on the preparation of L-2-aminobutyric acid was investigated. The reaction system consisted of 240 g / L L-threonine substrate, 126 g / L ammonium formate co-substrate, 20000 U / L crude threonine deaminase solution, and 20 g / L bacterial cells (E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3)) in deionized water to form a 100 mL reaction system. The enzyme-catalyzed reaction was carried out at 35℃ and 600 r / min for 12 h. After 30 min of reaction, 0.20 g / L NAD+ was added, and the enzyme-catalyzed reaction was carried out at 25, 30, 35, 40, and 45℃ and 600 r / min for 12 h. The results are shown in the figure. Figure 6 Before reaching 35℃, the conversion rate increased with increasing reaction temperature. At 35℃, the conversion rate reached its maximum. Further increasing the reaction temperature caused the conversion rate to drop sharply, possibly because the excessively high reaction temperature inactivated the enzyme. Based on the experimental results, 35℃ was determined to be the optimal reaction temperature.

[0084] Example 7: Optimization of fermentation conditions for E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3) strain in fermenter culture

[0085] Based on shake-flask culture and fermentation, large-scale fermenter culture and fermentation were carried out to verify the industrial application capability of E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3) strain. First, the effect of lactose concentration on the catalytic reaction process was investigated. Recombinant genetically engineered bacteria co-expressing leucine dehydrogenase and formate dehydrogenase were inoculated into shake flasks containing 100 mL LB liquid medium, and kanamycin was added to a final concentration of 50 mg / L. The culture was carried out at 37 °C and 150 r / min for 12 h as seed culture. The seed culture was then transferred to 3 L fermentation medium at an inoculation rate of 10% (v / v), and kanamycin was added to a final concentration of 50 mg / L. The aeration rate was 0.6 vvm, the pH was 7.0 (adjusted with 50% glycerol aqueous solution and ammonia aqueous solution), and the culture was carried out at 37 °C and 450 r / min until the OD600 reached 8-10. Lactose concentrations of 6, 8, 10, and 12 g / L were then added, and the induction temperature was 28 °C for 17 h. After fermentation, centrifuge at 4℃, 8000r / min for 10min, discard the supernatant, wash twice with physiological saline, collect the bacterial cells, and store at 4℃ for later use.

[0086] The reaction system consisted of 240 g / L L-threonine substrate, 126 g / L ammonium formate co-substrate, 20000 U / L crude threonine deaminase solution, and 20 g / L bacterial cells, all dissolved in deionized water to form a 100 mL reaction system. The enzyme-catalyzed reaction was carried out at 35℃ and 600 rpm for 12 h. After 30 min of reaction, 0.20 g / L NAD+ was added. The results are shown in [Figure number missing]. Figure 7 ,Depend on Figure 7 It can be seen that too low a lactose addition is not conducive to the reaction. The minimum lactose concentration required for complete substrate conversion is 8 g / L. Further increasing the lactose concentration has no significant effect on the reaction process. Therefore, the optimal lactose concentration is determined to be 8 g / L.

[0087] The effect of induction time on the catalytic reaction process was further investigated. With prolonged induction time, the expression level of the enzyme protein increased to a certain extent. To determine the optimal induction time, lactose at a concentration of 8 g / L was added during induction at 28℃. Samples were taken and analyzed after 13, 15, 17, and 19 hours of induction at cooling temperature. Other culture and reaction conditions remained the same. The results are shown in the figure. Figure 8 ,Depend on Figure 8 It can be seen that the catalytic activity of the target protease gradually increases with the extension of induction time. After 17 hours of induction, the catalytic activity basically stabilizes, and the subsequent increase is not significant. The induction time was finally determined to be 17 hours.

[0088] Example 8: Reaction process for preparing L-2-aminobutyric acid using E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3) strain

[0089] E. coli BL21(DE3)pRSFDuet-fdh-leudh(RBS-3) was inoculated into shake flasks containing 100 mL LB liquid medium, and kanamycin was added to a final concentration of 50 mg / L. The culture was incubated at 37°C and 150 rpm for 12 h as the seed culture. This seed culture was then transferred to 3 L of fermentation medium at a 10% (v / v) inoculation rate, and kanamycin was added to a final concentration of 50 mg / L. Aeration was maintained at 0.6 vvm, pH 7.0 (adjusted with 50% (v / v) glycerol and ammonia aqueous solutions), and the culture was incubated at 37°C and 450 rpm until the OD600 reached 8–10. Lactose (8 g / L) was then added as an inducer, and the induction temperature was 28°C for 17 h. After fermentation, the cells were centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with physiological saline. The collected cells were stored at 4°C for later use.

[0090] The reaction system (1 L) consisted of 300 g / L L-threonine substrate, 158 g / L ammonium formate co-substrate, 25000 U / L crude threonine deaminase solution, and 25 g / L bacterial cells, all dissolved in deionized water to form a 1000 mL reaction system. The enzyme-catalyzed reaction was carried out at 35℃ and 600 rpm for 12 h. After 30 min of reaction, 0.25 g / L NAD+ was added. Samples were taken periodically during the reaction, and the reaction was terminated with concentrated hydrochloric acid. The samples were then used for liquid chromatography analysis. The reaction progress is shown in [link to reaction details]. Figure 9 L-2-aminobutyric acid is generated rapidly within 2 hours of reaction, after which the reaction rate slows down. The reaction is basically completed after 12 hours, at which point the substrate conversion rate is over 99% and the ee value is over 99.5%.

Claims

1. A multi-enzyme co-expressing recombinant genetically engineered bacterium, constructed by the following method: A single-plasmid dual-gene expression vector is used, into which a formate dehydrogenase gene and a leucine dehydrogenase gene are inserted at the multiple cloning site, and the RBS sequence before the restriction enzyme site is replaced. The vector is then transformed into a host cell to obtain the multi-enzyme co-expressing recombinant genetically engineered bacterium. The nucleotide sequence of the leucine dehydrogenase gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the formate dehydrogenase gene is shown in SEQ ID NO.

3. The single-plasmid dual-gene expression vector is pRSFDuet, into which a formate dehydrogenase gene and a leucine dehydrogenase gene are inserted into MCS1 and MCS2 to obtain pRSFDuet-fdh-Leudh, and the RBS sequence of its MCS1 is replaced with CTACCCCCAAGGTTGATAAGGAGGTATTTT. The host cell of the recombinant genetically engineered bacterium is *Escherichia coli* BL21(DE3).

2. The application of the recombinant genetically engineered bacteria according to claim 1 in the biocatalytic preparation of L-2-aminobutyric acid.

3. The application as described in claim 2, characterized in that... The application is as follows: using live cells obtained by fermentation culture of the recombinant genetically engineered bacteria as a catalyst, L-threonine as the starting substrate, ammonium formate as the co-substrate for coenzyme recycling, and adding threonine deaminase, and using deionized water as the reaction medium to form a reaction system, an enzyme-catalyzed reaction is carried out. After the reaction is completed, a reaction solution containing L-2-aminobutyric acid is obtained. The reaction solution is separated and purified to obtain the L-2-aminobutyric acid.

4. The application as described in claim 3, characterized in that: In the reaction system, the mass concentration of L-threonine is 100-500 g / L, the mass concentration of ammonium formate is 100-200 g / L, the concentration of threonine deaminase is 20000-30000 U / L, the concentration of recombinant genetically engineered bacterial live cells is 10-50 g / L (wet weight), the enzyme catalysis temperature is 25-40℃, the reaction time is 2-16 h, and 0.1-1.0 g / L of coenzyme NAD is added 20-30 min after the start of the reaction. + .

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