Escherichia coli genetically engineered bacterium, construction method thereof and application of escherichia coli genetically engineered bacterium in preparation of sialic acid
By constructing an Escherichia coli genetically engineered strain expressing non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase, and using lactic acid and N-acetylglucosamine as substrates, the problem of high production cost of sialic acid was solved, and efficient and low-cost preparation of sialic acid was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-04-07
AI Technical Summary
The production cost of sialic acid in the existing technology is high, mainly because pyruvic acid, as a substrate, is expensive, resulting in excessively high preparation costs.
A genetically engineered Escherichia coli strain was constructed to express non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminidine aldolase. Sialic acid was prepared from lactate and N-acetylglucosamine using a whole-cell catalytic method, avoiding the use of expensive NAD+/NADP+ as electron acceptors.
This technology enables efficient production of sialic acid, reduces production costs, simplifies the operation process, minimizes raw material loss, and has promising prospects for industrial application.
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Figure CN121801786A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a genetically engineered Escherichia coli strain, its construction method, and its application in the preparation of sialic acid. Background Technology
[0002] Sialic acid, also known as N-acetylneuraminic acid (Neu5Ac), is a non-carbon monosaccharide derivative containing an amino group. It mainly exists in the form of oligosaccharides, glycolipids, or glycoproteins. Sialic acid participates in multiple physiological processes such as cell recognition, signal transduction, and tumorigenesis. It can also serve as a precursor for the synthesis of anti-influenza virus drugs. Due to its important biological functions, the demand for sialic acid in the pharmaceutical and biotechnology industries is constantly increasing.
[0003] The production methods of sialic acid include extraction from natural products, chemical synthesis, enzymatic synthesis, and whole-cell biocatalytic synthesis. In 2013, Lin BX et al. constructed a Neu5Ac synthetic pathway in E. coli by co-expressing AGE from the genus Anabaena and NanA from Escherichia coli, and constructed an engineered strain that produces Neu5Ac. .by As a whole-cell catalyst, it catalyzes the production of Neu5Ac from N-acetylglucosamine and pyruvate. Currently, traditional whole-cell catalytic methods for producing Neu5Ac all use pyruvate as a substrate, but the high price of pyruvate undoubtedly increases the production cost of sialic acid. Summary of the Invention
[0004] The purpose of this invention is to provide a genetically engineered Escherichia coli strain, its construction method, and its application in the preparation of sialic acid, so as to achieve efficient production of N-acetylneuraminic acid (sialic acid) and significantly reduce production costs.
[0005] Therefore, the present invention provides the following technical solution.
[0006] The first aspect of the present invention provides a genetically engineered Escherichia coli bacterium comprising expressing genes encoding non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase, wherein the genes encoding the non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase are expressed intracellularly in the genetically engineered bacterium to form active lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase.
[0007] In some embodiments of the present application, the non-NADP-dependent lactate dehydrogenase gene is from Klebsiella pneumoniae strain BH2511, the N-acetylglucosamine-2-epimerase gene is from Anabaena sp. CH1, and the N-acetylneuraminate aldolase gene is from Escherichia coli K12 MG1655.
[0008] In some embodiments of the present application, the nucleotide sequence of the non-NADP-dependent lactate dehydrogenase gene is shown as accession NO. WP_087874898.1 on NCBI, the nucleotide sequence of the N-acetylglucosamine-2-epimerase gene is shown as accession NO. ABG57043.1 on NCBI, and the nucleotide sequence of the N-acetylneuraminate aldolase gene is shown as accession NO. NP_417692.1 on NCBI.
[0009] The second aspect of the present application provides a method for constructing the genetically engineered Escherichia coli strain, which comprises the following steps: inserting the non-NADP-dependent lactate dehydrogenase gene, the N-acetylglucosamine-2-epimerase gene, and the N-acetylneuraminate aldolase gene into expression plasmids respectively by gene recombination technology to obtain recombinant expression plasmids; transforming the obtained recombinant expression plasmids into Escherichia coli in sequence to obtain the genetically engineered Escherichia coli strain.
[0010] In some embodiments of the present application, the expression plasmid comprises pBAD-hisB and pYB1s.
[0011] In some embodiments of the present application, the Escherichia coli is Escherichia coli which is constructed by the laboratory in the prior art according to the literature in the background art.
[0012] In some embodiments of the present application, the Escherichia coli knocks out the echinoid acid transporter (nanT), N-acetylmannosamine kinase (nanK), and N-acetylmannosamine-6-phosphate isomerase (nanE) in the Escherichia coli.
[0013] The third aspect of the present application provides the use of the genetically engineered Escherichia coli strain as described above in the preparation of echinoid acid by whole-cell catalysis.
[0014] The fourth aspect of the present application provides a method for preparing echinoid acid, which comprises the following steps of using the genetically engineered Escherichia coli strain as described above for whole-cell catalytic reaction. S1: culturing the E. coli genetically engineered bacteria to obtain whole cells co-expressing non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase; S2: constructing a whole cell catalytic reaction system by taking lactic acid and N-acetylglucosamine as substrates and the whole cells as catalysts; S3: performing whole cell catalytic reaction to obtain bird's nest acid.
[0015] In some embodiments of the present application, step S1 comprises: S11: sequentially culturing the E. coli genetically engineered bacteria in a first culture and a second culture to obtain a seed liquid; S12: culturing the seed liquid in a third culture from autoinduction medium to obtain whole cells co-expressing non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase.
[0016] In some embodiments of the present application, in step S11, the first culture condition is as follows: culture medium: LB plate, temperature: 35-38℃, time: overnight culture.
[0017] In some embodiments of the present application, in step S11, the second culture condition is as follows: culture medium: LB liquid medium, temperature: 35-38℃, shaking speed: 200-220 rpm, time: 6-8h.
[0018] In some embodiments of the present application, in step S12, the inoculation amount of the seed liquid is 5-10% by volume fraction.
[0019] In some embodiments of the present application, in step S12, the third culture condition is as follows: culture medium: ZYM-5052 autoinduction medium, temperature: 25-30℃, shaking speed: 200-220 rpm, time: 16-18h.
[0020] In some embodiments of the present application, in step S2, the whole cell catalytic reaction system comprises: pH 7-7.5 phosphate buffer, whole cell catalyst concentration 20-40 OD, lactic acid 1.2-1.6 M, N-acetylglucosamine 0.6-0.9 M, and magnesium sulfate 10-20 mM.
[0021] In some embodiments of the present application, in step S3, the whole cell catalytic reaction condition is as follows: temperature 25-35℃, shaking speed 200-220 rpm, and time 1-24h.
[0022] By the above technical solution, the present application has at least the following advantages: The application constructs an E. coli genetically engineered bacterium which simultaneously overexpresses three enzymes of non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase, and the E. coli genetically engineered bacterium can be used as a whole cell catalyst to prepare eduardisinic acid through catalytic reaction. ＋ Compared with the conventional enzyme conversion method, the E. coli genetically engineered bacterium constructed in the application can realize the preparation of eduardisinic acid from lactic acid and N-acetylglucosamine as substrates in a single step of a single bacterium, and NAD ＋ / NADP ＋ is not needed in the reaction process as an electron acceptor, the cost limitation of NAD ＋ / NADP is avoided, the operation is more simple, and the raw material loss is smaller. The selected substrate lactic acid in the application is cheaper and easier to obtain than the traditional substrate pyruvic acid, and has a good industrial application prospect.
[0023] The above description is only a summary of the technical scheme of the application, in order to more clearly understand the technical means of the application, and the content of the description can be implemented, the following will be described in detail with the preferred embodiments of the application. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 The above description is only a summary of the technical scheme of the application, in order to more clearly understand the technical means of the application, and the content of the description can be implemented, the following will be described in detail with the preferred embodiments of the application.
[0025] Figure 2 The above description is only a summary of the technical scheme of the application, in order to more clearly understand the technical means of the application, and the content of the description can be implemented, the following will be described in detail with the preferred embodiments of the application. DETAILED DESCRIPTION
[0026] In order to make the technical means, creative features, purposes and effects of the application easy to understand, the following will combine the embodiments of the application to clearly and completely describe the technical scheme in the embodiments of the application. Obviously, the described embodiments are only a part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the application.
[0027] Considering the expensive cost and market competitiveness of pyruvic acid, the application explores the feasibility of using lactic acid instead of pyruvic acid as raw material to produce eduardisinic acid through co-expression of multiple genes. The E. coli E. coli ΔnanTEK constructed in the early stage of the laboratory is used as a chassis to express non-NADP-dependent lactate dehydrogenase to convert lactic acid into pyruvic acid, and to express N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase to convert pyruvic acid and N-acetylglucosamine into eduardisinic acid, thereby providing a basis for the industrialized biological catalytic production of eduardisinic acid.
[0028] To this end, the present application provides an engineered Escherichia coli strain, which comprises genes encoding non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-epimerase and N-acetylneuraminate aldolase, and the genes are expressed in the intracellular of the engineered Escherichia coli strain to form active lactate dehydrogenase, N-acetylglucosamine-2-epimerase and N-acetylneuraminate aldolase.
[0029] The non-NADP-dependent lactate dehydrogenase gene is from Klebsiella pneumoniae strain BH2511, the N-acetylglucosamine-2-epimerase gene is from Anabaena sp. CH1, and the N-acetylneuraminate aldolase gene is from Escherichia coli K12 MG1655.
[0030] The nucleotide sequence of the non-NADP-dependent lactate dehydrogenase gene is shown in accession NO. WP_087874898.1 on NCBI, the nucleotide sequence of the N-acetylglucosamine-2-epimerase gene is shown in accession NO. ABG57043.1 on NCBI, and the nucleotide sequence of the N-acetylneuraminate aldolase gene is shown in accession NO. NP_417692.1 on NCBI.
[0031] In another aspect, a method for constructing the above-mentioned engineered Escherichia coli strain is also provided, which comprises the following steps: inserting the non-NADP-dependent lactate dehydrogenase gene, the N-acetylglucosamine-2-epimerase gene and the N-acetylneuraminate aldolase gene into expression plasmids respectively by gene recombination technology to obtain recombinant expression plasmids; transforming the obtained recombinant expression plasmids into the engineered Escherichia coli strain in sequence to obtain the engineered Escherichia coli strain.
[0032] The expression plasmids include pBAD-hisB and pYB1s.
[0033] The Escherichia coli is Escherichia coli , the E. coli is constructed by the laboratory in advance according to the literature in the background art (Lin BX, Zhang ZJ, Tao Y, et al. Enhanced production of N-acetyl-D-neuraminic acid by multi-approach whole-cell biocatalyst [J]. Applied Microbiology and Biotechnology, 2013.).
[0034] The E. coli The E. coli is knocked out of the E. coli in the bird's nest acid transporter (nanT), N-acetylmannosamine kinase (nanK), N-acetylmannosamine-6-phosphate isomerase (nanE).
[0035] The application also provides a use of the E. coli genetically engineered bacteria as described above in the preparation of bird's nest acid by whole-cell catalysis.
[0036] The application also provides a preparation method of bird's nest acid, which is whole-cell catalytic reaction by the E. coli genetically engineered bacteria as described above, and the preparation method comprises the following steps: S1: culturing the E. coli genetically engineered bacteria to obtain whole cells co-expressing non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase; S2: constructing a whole-cell catalytic reaction system by taking lactic acid and N-acetylglucosamine as substrates and the whole cells as a catalyst; S3: performing whole-cell catalytic reaction to obtain bird's nest acid.
[0037] Specifically, step S1 comprises: S11: sequentially culturing the E. coli genetically engineered bacteria by first culture, second culture to obtain a seed liquid; S12: transferring the seed liquid from the induction medium for third culture to obtain whole cells co-expressing non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase.
[0038] In step S11, the first culture condition is: culture medium: LB plate, temperature: 35-38℃, time: overnight culture. Suitable temperature may be, for example, 35℃, 36℃, 37℃ or 38℃, preferably 37℃.
[0039] In the above step S11, the second culture condition is: culture medium: LB liquid medium, temperature: 35-38℃, shaker speed: 200-220 rpm, time: 6-8h.
[0040] Suitable temperature can be, for example, 35℃, 36℃, 37℃ or 38℃, preferably 37℃.
[0041] Suitable shaker speed can be, for example, 200 rpm, 210 rpm or 220 rpm, preferably 220 rpm.
[0042] Suitable time can be, for example, 6h, 7h or 8h.
[0043] In the above step S12, the inoculation amount of the seed liquid is 5-10% volume fraction.
[0044] Suitable inoculation amount can be, for example, 5%, 6%, 7%, 8%, 9% or 10%.
[0045] In the above step S12, the third culture condition is: culture medium: ZYM-5052 autoinduction medium, temperature: 25-30℃, shaker speed: 200-220 rpm, time: 16-18h.
[0046] Suitable temperature can be, for example, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃, preferably 30℃.
[0047] Suitable shaker speed can be, for example, 200 rpm, 210 rpm or 220 rpm, preferably 220 rpm.
[0048] Suitable time can be, for example, 16h, 17h or 18h, preferably 16h.
[0049] In the above step S2, the whole-cell catalytic reaction system is composed of: pH 7-7.5 phosphate buffer, whole-cell catalyst concentration 20-40 OD, lactic acid 1.2-1.6 M, N-acetylglucosamine 0.6-0.9 M, magnesium sulfate 10-20 mM.
[0050] Suitable phosphate buffer pH can be, for example, 7, 7.1, 7.2, 7.3, 7.4 or 7.5, preferably 7.
[0051] Suitable whole-cell catalyst concentration can be, for example, 20 OD, 25 OD, 30 OD, 35 OD or 40 OD, preferably 40 OD.
[0052] Suitable lactic acid concentrations can be, for example, 1.2 M, 1.3 M, 1.4 M, 1.5 M or 1.6 M, preferably 1.2 M.
[0053] Suitable N-acetylglucosamine concentrations can be, for example, 0.6 M, 0.7 M, 0.8 M or 0.9 M, preferably 0.68 M.
[0054] Suitable magnesium sulfate concentrations can be, for example, 10 mM, 15 mM or 20 mM, preferably 10 mM.
[0055] In the above step S3, the whole-cell catalytic reaction conditions are: temperature 25-35℃, shaker rotation speed 200-220 rpm, time 1-24 h.
[0056] Suitable temperatures can be, for example, 25℃, 30℃ or 35℃, preferably 30℃.
[0057] Suitable shaker rotation speeds can be, for example, 200 rpm, 210 rpm or 220 rpm, preferably 220 rpm.
[0058] Suitable times can be, for example, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h or 24 h, preferably 24 h.
[0059] Unless otherwise specified, the percentages involved in the present application refer to mass percentages for solid-liquid mixing and solid-solid mixing, and to volume percentages for liquid-liquid mixing.
[0060] Unless otherwise specified, the percentage concentrations involved in the present application refer to final concentrations. The final concentration refers to the proportion of the added component in the system after the component is added.
[0061] Unless otherwise specified, the temperature parameters in the present application allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows fluctuations within the accuracy range controlled by the instrument.
[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0063] The ZYM-5052 self-induction medium used in the following examples is formulated as follows: 100 mL A + 2 mL B + 2 mL C + 200 μL D + 100 μL E (all in mass percentage concentration) unless otherwise specified; wherein, A. ZY: 1% tryptone, 0.5% yeast powder; B. 50xM: 1.25M Na2HPO4, 1.25M KH2PO4, 2.5M NH4Cl and 0.25M Na2SO4; C. 50x5052: 25% glycerol, 2.5% glucose, 10% arabinose; D. 1M MgSO4; E. 1000x trace elements: 50 mM FeCl3, 20 mM CaCl2, 10 mM MnCl2, CoCl2, NiCl2, Na2MoO4, Na2SeO3 and H3BO3 each 2 mM; F. 20% arabinose; other materials, reagents, etc. can be obtained commercially.
[0064] Example 1: Escherichia coli Knockout strain construction nanTEK is a gene cluster composed of three consecutive genes of Streptococcus mutans acid transport protein (nanT), N-acetylmannosamine kinase (nanK) and N-acetylmannosamine-6-phosphate isomerase (nanE). In this example, the CRISPR-Cas9 gene editing method is used to edit the genome of the original strain E. coli BW25113. The knockout operation system comprises: a pTarget plasmid providing an sgRNA fragment, a homologous arm targeting fragment, and a pCas9 temperature-sensitive plasmid providing Cas9 protein and RED recombinase.
[0065] The pTarget plasmid construction method is to select the N20 of the knockout site at the website https: / / crispy.secondarymetabolites.org, design primers to introduce N20 at the 5' end, and perform PCR amplification with the pTargetF plasmid as the template. The primers used are ΔnanTEK-pT-F and ΔnanTEK-pT-R, and the PCR obtains a fragment of about 2100 bp. DpnI enzyme is added and reacted at 37°C for 3h. The PCR product after DpnI enzyme digestion is added to Trans T1 competent cells, transformed by chemical transformation method, and plated on LB plates containing 50 μg / mL streptomycin sulfate and incubated at 37°C overnight. Single colonies are picked and cultured in LB liquid medium containing 50 μg / mL streptomycin sulfate, and the plasmid is extracted and sequenced to verify. After sequencing is correct, it is ready for use.
[0066] The targeting fragment amplification method is to use E. coli BW25113 as the template, and use primer pairs and , and PCR amplification was performed using the primer sequences shown in Table 1; the amplification procedure was as follows: denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and polymerization at 72°C for 30 s, and then polymerization at 72°C for 5 min, to obtain two fragments of about 800 bp in size. and PCR amplification was performed to obtain a targeting fragment of about 1000 bp in size, which contained a 500 bp upstream homology arm and a 500 bp downstream homology arm from upstream to downstream.
[0067] The E. coli BW25113 electrotransformation competent cells containing the pCAS plasmid were prepared by transforming the pCAS plasmid into the previously prepared E. coli BW25113 competent cells, spreading on an LB plate containing 50 μg / mL kanamycin, and culturing overnight at 30°C, inoculating a single colony into an LB liquid medium containing 50 μg / mL kanamycin and 2 g / L arabinose, and culturing to an OD value of 0.6-0.8, and then ice-bathing the bacterial solution for 10 min, followed by centrifugation at 4000 rpm for 5 min in a 4°C centrifuge, discarding the supernatant, washing with pre-cooled 10% glycerol, mixing gently, and then centrifugation at 4000 rpm for 5 min in a 4°C centrifuge, again discarding the supernatant, and repeating the washing step three times, and finally resuspending the bacterial cells with pre-cooled 10% glycerol, and storing for use. 600 The E. coli BW25113 electrotransformation competent cells containing the pCAS plasmid were prepared by transforming the pCAS plasmid into the previously prepared E. coli BW25113 competent cells, spreading on an LB plate containing 50 μg / mL kanamycin, and culturing overnight at 30°C, inoculating a single colony into an LB liquid medium containing 50 μg / mL kanamycin and 2 g / L arabinose, and culturing to an OD value of 0.6-0.8, and then ice-bathing the bacterial solution for 10 min, followed by centrifugation at 4000 rpm for 5 min in a 4°C centrifuge, discarding the supernatant, washing with pre-cooled 10% glycerol, mixing gently, and then centrifugation at 4000 rpm for 5 min in a 4°C centrifuge, again discarding the supernatant, and repeating the washing step three times, and finally resuspending the bacterial cells with pre-cooled 10% glycerol, and storing for use.
[0068] The electrotransformation knockout step was as follows: mixing the pTarget plasmid and the targeting fragment with the E. coli BW25113 electrotransformation competent cells containing the pCas9 plasmid, ice-bathing for 5 min, placing in a 2 mm electrotransformation cup, 2.5 kV electroporation, quickly adding 1 ml of LB liquid medium, repeatedly blowing and sucking several times, and then sucking all the bacterial solution into an LB liquid medium, and culturing at 30°C for 2 h, centrifuging, discarding most of the supernatant, resuspending the remaining portion, and then spreading on an LB plate containing 50 μg / mL kanamycin and 50 μg / mL streptomycin sulfate, and culturing overnight at 30°C, picking a single colony into an LB liquid medium containing a final concentration of 0.1 mM IPTG and Kan, and culturing at 30°C for 6 h, which can eliminate the pTarget plasmid while culturing the bacteria. PCR amplification was performed using the primer pairs and PCR amplification verification was performed. The band of the successfully knocked out strain was 1600bp, and the control which was not successfully knocked out was about 4600bp. The correct knockout strain was inoculated in LB liquid medium without antibiotics, and cultured at 42°C for 6h. A small amount of bacterial liquid was taken and streaked on LB solid plate without antibiotics, and cultured at 42°C overnight to eliminate pCas plasmid, and obtain plasmid-free E. coli .
[0069] Table 1 Primer sequence ; Example 2: Construction of recombinant E. coli 1. Construction of recombinant E. coli NanA-AGE overexpressing NanA and AGE Example 2: Construction of recombinant E. coli The synthesized sialic acid related genes NanA (N-acetylneuraminic acid aldolase, accession NO. NP_417692.1 on NCBI) and AGE (N-acetylglucosamine-2-epimerase, accession NO. ABG57043.1 on NCBI) gene fragments were used as templates for PCR amplification, and the primer sequences used for PCR amplification are shown in Table 2. The amplification program was as follows: denaturation at 95°C for 5min, followed by 30 cycles of denaturation at 95°C for 30s, annealing at 60°C for 30s and polymerization at 72°C for 60s, and then polymerization at 72°C for 5min to obtain the PCR amplification product. The obtained PCR product was electrophoresed in a 1.0% agarose gel, and then the DNA with the obtained NanA gene fragment of 900bp and the AGE gene fragment of about 1200bp was purified. The purified and recovered two DNA fragments were cloned together with the pBAD-hisB vector digested with XhoI and NcoI, and transformed into competent Trans T1. The transformation step was 42°C heat shock for 90s, ice bath for 2min, 37°C incubation on a shaker for 1h, and then plated on LB plates containing 100μg / mL ampicillin for overnight culture. Single bacteria were picked for PCR screening. The primers used were NanA-F and AGE-R. The positive clone was obtained by PCR to obtain a band of about 2100bp. The positive clone was extracted for plasmid sequencing verification, and the recombinant expression plasmid co-expressing NanA and AGE was obtained. The plasmid map is shown in Figure 1 .
[0070] The E. coli glycerol bacteria were streaked on LB plates and cultured at 37°C for 16h. The E. coli Single colonies were incubated in shake flasks containing 5 mL of LB medium at 37°C until the OD600 reached 0.5–0.6. The bacterial culture was transferred to sterile 10 mL centrifuge tubes and incubated on ice for 10 minutes, then centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was discarded, and the bacterial cells were resuspended in 5 mL of pre-chilled solution containing 80 mM CaCl2 and 20 mM MgCl2, then incubated on ice for 30 minutes. After the ice incubation, the cells were centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was discarded, and the bacterial cells were resuspended in 100 μL of solution containing 20 mM CaCl2 and 10% glycerol (v / v) to obtain *Escherichia coli*. Competent cells.
[0071] The successfully constructed recombinant expression plasmid pBAD-NanA-AGE was added to the resulting E. coli. The competent cells were placed on ice for 30 minutes. After the ice bath, they were incubated in a 42°C water bath for 1 minute, then transferred to ice for 2 minutes. 600 μL of LB medium was added, and the cells were incubated at 37°C on a shaker for 1 hour. After incubation, the bacterial culture was centrifuged at 4000 rpm for 3 minutes, most of the supernatant was discarded, and the cells were resuspended in the remaining supernatant and plated onto LB agar plates containing 100 μg / mL ampicillin. The plates were incubated at 37°C for 16 hours. After incubation, single colonies were picked from the plates and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin. The plates were then incubated at 37°C on a shaker until OD (digestive end-expiratory) reached. 600 The concentration was 1.6-1.8, then an equal volume of 50% glycerol (V / V) was added and mixed well. The mixture was then dispensed into preservation tubes at 1 mL / tube to obtain recombinant *E. coli* overexpressing NanA and AGE. The *E. coli* strain was then analyzed using the NanA-AGE / ... express.
[0072] 2. E. coli genetically engineered strain Kp-lld-NanA-AGE co-overexpression of Kp-lld, NanA, and AGE.
[0073] The synthesized pyruvate-related Kp-lld gene fragment (a non-NADP-dependent lactate dehydrogenase, accession NO. WP_087874898.1 on NCBI) was used as a template for PCR amplification. The primer sequences used for PCR amplification are shown in Table 2. The amplification procedure was the same as above, and the PCR amplification product was obtained. The obtained PCR product was electrophoresed on a 1.0% agarose gel, and then the DNA of the approximately 1200 bp band was purified. The purified DNA fragment was seamlessly cloned with the pYB1s vector digested with EcoRI and NcoI, transformed into competent Trans T1 cells, and the transformation procedure was the same as above. The cells were plated on LB plates containing 50 μg / mL streptomycin sulfate and cultured overnight. Single bacteria were selected for PCR screening using primers Kp-lld-F and Kp-lld-R. Clones producing a band of approximately 1200 bp were considered positive. Plasmids were extracted from these positive clones and sequenced for verification, yielding a recombinant expression plasmid overexpressing the Kp-lld gene. The plasmid map is shown below. Figure 2 As shown, it is named pYB1s-Kp-lld.
[0074] The above-obtained Escherichia coli NanA-AGE / Glycerol-treated bacteria were streaked onto LB agar plates and incubated at 37°C for 16 hours. *E. coli* NanA-AGE / Single colonies were cultured in shake flasks containing 5 mL of LB medium at 37°C until OD reached. 600 The concentration was 0.5–0.6. The bacterial culture was transferred to a sterile 10 mL centrifuge tube and incubated on ice for 10 minutes, then centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was discarded, and the bacterial cells were resuspended in 5 mL of pre-chilled solution containing 80 mM CaCl2 and 20 mM MgCl2, then incubated on ice for 30 minutes. After the ice bath, the cells were centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was discarded, and the bacterial cells were resuspended in 100 μL of solution containing 20 mM CaCl2 and 10% glycerol (v / v) to obtain *Escherichia coli* NanA-AGE / Competent cells.
[0075] The successfully constructed recombinant expression plasmid pYB1s-Kp-lld was added to the resulting E. coli NanA-AGE / Competent cells were placed on ice for 30 minutes. After the ice bath, they were incubated in a 42°C water bath for 1 minute, then transferred to ice for 2 minutes. 600 μL of LB medium was added, and the cells were incubated at 37°C on a shaker for 1 hour. After incubation, the bacterial culture was centrifuged at 4000 rpm for 3 minutes, most of the supernatant was discarded, and the cells were resuspended in the remaining supernatant and plated onto LB agar plates containing 100 μg / mL ampicillin and 50 μg / mL streptomycin sulfate. The plates were incubated at 37°C for 16 hours. After incubation, single colonies were picked from the plates and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and 50 μg / mL streptomycin sulfate. The plates were incubated at 37°C on a shaker until OD (dose elapsed). 600 The concentration was 1.6-1.8, then an equal volume of 50% glycerol (V / V) was added and mixed well. The mixture was then dispensed into preservation tubes at 1 mL / tube to obtain *E. coli* genetically engineered bacteria co-overexpressing Kp-lld, NanA, and AGE. The Kp-lld-NanA-AGE / express.
[0076] Table 2 Primer sequences ; Example 3: Screening of reaction systems and conditions for whole-cell catalytic preparation of sialic acid (1) Determination of the optimal substrate concentration The genetically engineered E. coli strain Kp-lld-NanA-AGE / constructed in Example 2 was used. Induction culture shall be performed according to the following method: First, the genetically engineered E. coli strain Kp-lld-NanA-AGE / Streak LB agar plates containing 50 μg / mL streptomycin sulfate and 100 μg / mL ampicillin (the working concentrations of the antibiotics used below are the same), incubate overnight at 37°C, then pick a single colony and inoculate it into LB liquid medium containing 50 μg / mL streptomycin sulfate and 100 μg / mL ampicillin. Incubate at 37°C and 220 rpm for 8 h to obtain the seed culture.
[0077] The resulting seed culture was then transferred to ZYM-5052 self-induction medium at a volume ratio of 5%, and induced at 30°C and 220 rpm for 16 h. After induction, the culture medium was centrifuged at 4°C and 6000 rpm for 10 min, and the cell pellet was collected. This pellet contained whole cells of the co-expressed non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase.
[0078] The obtained whole cells were subjected to whole-cell catalytic reaction to produce sialic acid. The whole-cell catalytic system used consisted of: pH 7.0 phosphate buffer, whole cell concentration of 30 OD, lactate concentrations of 1.0, 1.2, 1.4, and 1.6 M, corresponding to N-acetylglucosamine concentrations of 0.57, 0.68, 0.8, and 0.9 M, and magnesium sulfate of 10 mM. The obtained whole-cell catalytic system was then placed at 30℃ and a shaking speed of 220 rpm for 24 h to obtain the product. Finally, the sialic acid content in the product of each reaction system was detected by high-performance liquid chromatography (HPLC): the chromatographic column was Bio-rad Aminex HPX-87H, the mobile phase was 5 mM sulfuric acid, the column temperature was 35℃, the flow rate was 0.5 mL / min, and the detection wavelength was 205 nm. The detection results are shown in Table 3.
[0079] Table 3 Comparison of sialic acid production with different substrate concentrations ; As shown in Table 3 above, when the lactic acid concentration is between 1.0 and 1.2 M, the mass conversion rate of sialic acid increases with the proportional increase of both lactic acid and N-acetylglucosamine concentrations. When the lactic acid concentration is greater than 1.2 M, the yield of sialic acid from the catalytic reaction does not increase significantly, while the remaining substrate N-acetylglucosamine increases significantly, leading to a decrease in the corresponding mass conversion rate. This indicates that the reaction has reached its upper limit when the lactic acid concentration is greater than 1.2 M. Therefore, the optimal substrate concentration is 1.2 M lactic acid and 0.68 M N-acetylglucosamine.
[0080] (2) Determination of the optimal reaction pH Recombinant Escherichia coli was induced and cultured according to the induction culture method described in (1) above. After the induction culture was completed, the cells were centrifuged at 4°C and 6000 rpm for 10 min, and the cell pellet was collected. This pellet was the whole cell containing the co-expressed non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase.
[0081] The obtained whole cells were subjected to whole-cell catalytic reaction to produce sialic acid. The whole-cell catalytic system used consisted of phosphate buffer solutions at pH 6.5, 7.0, 7.5, and 8.0, with whole cell concentrations of 30 OD, 1.2 M lactate, 0.68 M N-acetylglucosamine, and 10 mM magnesium sulfate, respectively. The obtained whole-cell catalytic system was placed at 30℃ and a shaking speed of 220 rpm for 24 h to obtain the product. Finally, the sialic acid content was determined by high-performance liquid chromatography, and the results are shown in Table 4.
[0082] Table 4 Comparison of Sialic Acid Production at Different pH Levels ; As shown in Table 4 above, the sialic acid content in the product is higher at pH 7.0, while it decreases under slightly acidic (pH 6.5) and slightly alkaline (pH 8.0) conditions. Therefore, the optimal reaction pH is determined to be 7.0.
[0083] (3) Determination of the optimal reaction temperature Recombinant Escherichia coli was induced and cultured according to the induction culture method described in (1). After the induction culture was completed, the cells were centrifuged at 4°C and 6000 rpm for 10 min, and the cell pellet was collected. This pellet was the whole cell that co-expressed non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase.
[0084] The obtained whole cells were subjected to whole-cell catalytic reaction to produce sialic acid. The whole-cell catalytic system consisted of: pH 7.0 phosphate buffer, whole cell concentration of 30 OD, 1.2 M lactate, 0.68 M N-acetylglucosamine, and 10 mM magnesium sulfate. The obtained whole-cell catalytic system was subjected to conversion at 25, 30, and 35℃ and a shaking speed of 220 rpm for 24 h to obtain the product. Finally, the sialic acid content was detected by high-performance liquid chromatography, and the results are shown in Table 5.
[0085] Table 5 Comparison of Sialic Acid Production at Different Temperatures ; As shown in Table 5 above, the sialic acid content in the product is highest at 30℃, while the yield of sialic acid is lower at 25℃ and 35℃. Therefore, the optimal reaction temperature is determined to be 30℃.
[0086] (4) Determination of the optimal whole-cell concentration Recombinant Escherichia coli was induced and cultured according to the induction culture method described in (1). After the induction culture was completed, the cells were centrifuged at 4°C and 6000 rpm for 10 min, and the cell pellet was collected. This pellet was the whole cell that co-expressed non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase.
[0087] The obtained whole cells were subjected to whole-cell catalytic reaction to produce sialic acid. The whole-cell catalytic system used consisted of: pH 7.0 phosphate buffer, whole cell concentrations of 20, 30, and 40 OD, 1.2 M lactate, 0.68 M N-acetylglucosamine, and 10 mM magnesium sulfate. The obtained whole-cell catalytic system was placed at 30℃ and a shaking speed of 220 rpm for 24 h to obtain the product. Finally, the sialic acid content was determined by high-performance liquid chromatography, and the results are shown in Table 6.
[0088] Table 6 Comparison of sialic acid production at different whole-cell concentrations ; As shown in Table 6 above, within the concentration range of 20-40 OD, the sialic acid content and conversion rate in the product increase with the increase of whole cell concentration. However, when the concentration is increased to 50 OD, the upper limit of conversion rate is reached, and further increasing the OD has no significant effect on improving pyruvate production and will also increase the cost of bacterial culture. Therefore, the optimal whole cell concentration is determined to be 40 OD.
[0089] As shown above, the optimal whole-cell catalytic system determined in this embodiment is: pH 7.0 phosphate buffer, whole-cell concentration 40 OD, lactate 1.2 M, N-acetylglucosamine 0.68 M, and magnesium sulfate 10 mM. Reaction conditions: temperature 30℃, time 24 h.
[0090] Example 4: Whole-cell catalytic production of sialic acid under optimal conditions Following the induction culture method described in Example 3, recombinant Escherichia coli Kp-lld-NanA-AGE / Induction culture was performed. After induction culture, the cells were centrifuged at 4°C and 6000 rpm for 10 min, and the cell pellet was collected. This pellet was the whole cell that co-expressed non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase.
[0091] The obtained whole cells were used as catalysts for whole-cell catalytic reaction to produce sialic acid. The whole-cell catalytic system consisted of: pH 7.0 phosphate buffer, whole cell concentration of 40 OD, 1.2 M lactate, 0.68 M N-acetylglucosamine, and 10 mM magnesium sulfate. The obtained whole-cell catalytic system was placed at 30°C and a shaking speed of 220 rpm for 24 h to obtain the product (first batch).
[0092] The catalytic reaction was repeated three times using the same method to obtain the products (second batch and third batch).
[0093] Finally, the sialic acid content in each batch of products was determined by high performance liquid chromatography. The results are shown in Table 7.
[0094] Table 7. Experimental data from three batches of Escherichia coli producing sialic acid. ; As shown in Table 7 above, the conversion rate of sialic acid produced by the Escherichia coli genetically engineered strain constructed using this invention was consistently above 70% in three batches of experiments.
[0095] The above results indicate that the method of producing N-acetylneuraminic acid by using lactic acid and N-acetylglucosamine as raw materials in the catalytic reaction is feasible.
[0096] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, various changes in form and detail can still be made by those skilled in the art within the scope of protection of the present invention.
[0097] The above-described methods for culturing and transforming genetically engineered bacteria are merely preferred embodiments of the present invention and are not intended to limit the invention. Theoretically, other bacteria, filamentous fungi, actinomycetes, and animal cells can also undergo genome modification and be used to generate sialic acid. Any modifications made within the principles and spirit of the present invention are equivalent to substitutions.
[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A genetically engineered Escherichia coli bacterium, characterized in that, The genetically engineered bacteria contain genes that express encoding non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase, and the genes encoding non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase can be expressed intracellularly in the genetically engineered bacteria to form active lactate dehydrogenase, N-acetylglucosamine-2-isomerase, and N-acetylneuraminic acid aldolase.
2. The genetically engineered Escherichia coli according to claim 1, characterized in that, The non-NADP-dependent lactate dehydrogenase gene is from Klebsiella pneumoniae strain BH2511, the N-acetylglucosamine-2-isomerase gene is from Anabaena sp. CH1, and the N-acetylneuraminic acid aldolase gene is from Escherichia coli K12 MG1655.
3. The method for constructing genetically engineered Escherichia coli according to claim 1 or 2, characterized in that, The method includes: The non-NADP-dependent lactate dehydrogenase gene, N-acetylglucosamine-2-isomerase gene, and N-acetylneuraminic acid aldolase gene were inserted into expression plasmids using gene recombination technology to construct recombinant expression plasmids. The obtained recombinant expression plasmid was sequentially transformed into Escherichia coli to obtain Escherichia coli genetically engineered bacteria.
4. The construction method according to claim 3, characterized in that, The expression plasmids include pBAD-hisB and pYB1s; The *E. coli* is *Escherichia coli*. .
5. The application of the genetically engineered Escherichia coli according to claim 1 or 2 in the whole-cell catalytic method for the preparation of sialic acid.
6. A method for preparing sialic acid, characterized in that, The whole-cell catalytic reaction is carried out using the genetically engineered Escherichia coli according to claim 1 or 2, and the preparation method includes the following steps: S1: Culture the genetically engineered Escherichia coli to obtain whole cells that co-express non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminidase; S2: Using lactic acid and N-acetylglucosamine as substrates and the whole cells as catalysts, a whole-cell catalytic reaction system is constructed; S3: A whole-cell catalytic reaction is carried out to obtain sialic acid.
7. The preparation method according to claim 6, characterized in that, Step S1 includes: S11: The genetically engineered Escherichia coli was cultured first and then second to obtain the seed culture; S12: The seed culture was transferred to the induction medium for a third culture to obtain whole cells co-expressing non-NADP-dependent lactate dehydrogenase, N-acetylglucosamine-2-isomerase and N-acetylneuraminic acid aldolase.
8. The preparation method according to claim 7, characterized in that, In step S11, the first culture conditions are: culture medium: LB plates, temperature: 35~38℃, time: overnight culture; The second culture conditions were as follows: culture medium: LB liquid medium, temperature: 35~38℃, shaking speed: 200~220rpm, time: 6~8h; In step S12, the inoculation amount of the seed liquid is 5-10% by volume. The conditions for the third culture were as follows: culture medium: ZYM-5052 self-induction medium, temperature: 25~30℃, shaking speed: 200~220 rpm, time: 16~18 h.
9. The preparation method according to claim 6, characterized in that, In step S2, the whole-cell catalytic reaction system consists of: pH 7-7.5 phosphate buffer, whole-cell catalyst concentration of 20-40 OD, lactic acid of 1.2-1.6 M, N-acetylglucosamine of 0.6-0.9 M, and magnesium sulfate of 10-20 mM.
10. The preparation method according to claim 6, characterized in that, In step S3, the conditions for the whole-cell catalytic reaction are: temperature 25~35℃, shaking speed 200~220 rpm, and time 1~24 h.