Engineering escherichia coli for producing L-fucose and application thereof

Abstract

The invention relates to engineering Escherichia coli for producing L-fucose and application of the engineering Escherichia coli, the production of the L-fucose is enhanced through a metabolic engineering strategy by utilizing a mode of combining genome integration and plasmid expression path genes, and the efficient production of the L-fucose is realized by optimizing a double-plasmid system and enhancing precursor supply. In addition, a hok / sok system and a cysC auxotroph system are introduced, and the plasmid-based engineering strain without antibiotic dependence is successfully constructed. The strain produces 7.99 g / L of L-fucose and 61.91 g / L of L-fucose through shake-flask culture and fed-batch culture under the condition that antibiotics are not added, the highest L-fucose yield reported so far is provided, and the use of antibiotics is completely not needed in the fermentation process.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to an L-fucose-producing engineered Escherichia coli and application thereof. Background Art

[0002] L-fucose (6-deoxy-L-galactose) has prebiotic activity, can protect intestinal barrier function, improve lipid metabolism, and also exhibit anti-tumor activity and slow skin aging. Therefore, it has the potential to be used as a bioactive ingredient in the pharmaceutical, health care and cosmetic industries. Compared with algae extraction and chemical synthesis, the synthesis of L-fucose by synthetic biology has the advantages of being green, environmentally friendly and low-cost, and is a very promising way to synthesize L-fucose.

[0003] At present, the synthesis of L-fucose by metabolically engineered microorganisms usually starts from a metabolically engineered Escherichia coli strain for the synthesis of 2′-fucosyllactose, uses exogenously added lactose as a glycosyl receptor to synthesize 2′-fucosyllactose, and further introduces a specific α-L-fucosidase to hydrolyze 2′-fucosyllactose into L-fucose and lactose. However, the yield of this method reported in current research is low. For this reason, our company has further transformed and optimized the strain, as described in the Chinese patent application number CN202310014321.6_, but its L-fucose yield can only reach 51.05 g / L. Although it has been greatly improved, it still needs to be improved. In addition, the constructed strain carries two plasmid vectors, and two antibiotics need to be added for fermentation and culture of the strain, which is not conducive to large-scale industrial production. In addition, the antibiotics need to be removed during the extraction of L-fucose, and there is a problem of antibiotic residues.

[0004] Therefore, constructing an engineered Escherichia coli that produces L-fucose at high yield without the need for antibiotics is crucial to improving the industrial production process of L-fucose and simplifying the extraction process of L-fucose. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide an engineered Escherichia coli producing L-fucose and its application, which does not require the addition of antibiotics during the fermentation process, greatly improves the yield of L-fucose, and promotes the industrialization process of L-fucose biosynthesis.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] An engineered Escherichia coli producing L-fucose is obtained by using Escherichia coli as a starting strain and performing the following transformations AF:

[0008] A, knockout of β-galactosidase gene lacZ and UDP-glucose lipid carrier transferase gene wcaJ;

[0009] B. Using a strong constitutive promoter P J23119 Promote the expression of phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB

[0010] C. Using a strong constitutive promoter P J23119 Promote expression of the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG;

[0011] D. Integrate the α-1,2-fucosyltransferase gene BKHT at the recA site, arsB site, and ldhA site.

[0012] E, knockout of the fucose isomerase-L-fucokinase gene cluster fucI-fucK;

[0013] F. Integration of α-L-fucosidase gene afcA.

[0014] As a further technical solution, the α-L-fucosidase gene afcA is integrated into the fucIK site, wherein fucIK is an abbreviation of fucI-fucK.

[0015] As a further technical solution, the Escherichia coli is also transformed in the following G and F items:

[0016] G. Plasmid pRSFDuet-1 was used to express the phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB and the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG;

[0017] H. Use plasmid pETDuet-1 to express the positive transcriptional regulatory factors rcsA and rcsB and the α-1,2-fucosyltransferase gene BKHT.

[0018] As a further technical solution, the positive transcription regulatory factors rcsA and rcsB are derived from Escherichia coli K-12MG1655.

[0019] As a further technical solution, the Escherichia coli is also transformed in the following item 1:

[0020] I: using the plasmid pETDuet-1 to enhance the expression of one or more of the phosphomannose mutase gene manC, the mannose-1-phosphate guanyl transferase gene manB, the GDP-mannose-6-dehydrogenase gene gmd, and the GDP-fucose synthase gene wcaG, preferably, the mannose-1-phosphate guanyl transferase gene manB is enhanced. The rcsA, rcsB positive transcription regulatory factors and BKHT derived from Escherichia coli K-12MG1655 expressed in the present invention are cloned into the plasmid of pETDuet-1 to obtain the pET-rcsAB-BKHT plasmid, and the precursor supply is enhanced by plasmid optimization to obtain a stable inherited plasmid.

[0021] As a further technical solution, the Escherichia coli is also transformed as follows J and K:

[0022] J: Insert the hok / sok system between the CDF ori and lacI sites of the plasmid pRSFDuet-1; the present invention solves the antibiotic dependence problem in the fermentation process by inserting the hok / sok system. The hok / sok system is a toxin-antitoxin system that causes irreversible membrane structure damage by encoding the highly toxic transmembrane protein hok, and the sok encoded by the antisense RNA cis can block the expression of the hok protein. In cells into which the plasmid containing the hok / sok system is introduced, sokRNA binds to the activated hokmRNA, thereby blocking the binding of hokmRNA to the ribosome, thereby inhibiting the expression of the highly toxic transmembrane protein hok. When there is no plasmid in the cell, sokRNA is rapidly degraded, and the hokmRNA, due to its decay, allows the toxic protein to be fully expressed and aggregated, ultimately leading to cell death.

[0023] K: knock out the thioadenosine kinase gene cysC on the Escherichia coli, and clone the thioadenosine kinase gene cysC into the plasmid pETDuet-1; CysC is a key enzyme in the bacterial sulfur metabolism pathway, responsible for catalyzing the conversion of inorganic sulfate into active sulfate, and then participating in the biosynthesis of cysteine. The present invention constructs a cysC gene-deficient strain by knocking out the cysC gene to maintain plasmid stability. The nutritional deficiency system is an effective plasmid maintenance strategy, which involves knocking out or mutating a key gene in the synthesis pathway of an essential nutritional factor in the host strain, so that the strain cannot synthesize the nutritional factor, and thus cannot grow in a culture medium without external supplementation of the factor. By inserting the corresponding wild-type gene on the plasmid, the presence of the plasmid enables the strain to compensate for its nutritional deficiency, thereby growing in a culture medium without supplementing the nutritional factor. In this way, the maintenance of the plasmid is directly related to the growth and survival of the host strain, thereby significantly improving the stability of the plasmid. Without the use of antibiotics, this auxotrophic system provides an effective selection mechanism for the stable maintenance of plasmids, ensuring the stable performance of engineered strains during long-term cultivation and large-scale fermentation.

[0024] As a further technical solution, the first multiple cloning site on the plasmid pETDuet-1 expresses the transcriptional regulatory factors rcsA and rcsB;

[0025] As a further technical solution, the second multiple cloning site on the plasmid pETDuet-1 expresses the α-1,2-fucosyltransferase gene BKHT;

[0026] As a further technical solution, the gene cysC encoding thioadenosine kinase in the host cell is knocked out, and the plasmid pETDuet-1 also carries the gene cysC.

[0027] As a further technical solution, the α-1,2-fucosyltransferase gene BKHT is derived from Helicobacter pylori sp.13S00401-1.

[0028] As a further technical solution, the phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB is derived from Escherichia coli, wherein the GenBank number of the phosphomannose mutase gene manB is NP_416552.1, and the GenBank number of the mannose 1-guanylyltransferase encoding gene manC is NP_416553.1; the specific nucleotide sequence thereof is known to the public and will not be described in detail here;

[0029] As a further technical solution, the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG is derived from Escherichia coli, wherein the GenBank number of the GDP-D-mannose-4,6-dehydratase gene gmd is NP_416557.1, and the GenBank number of the GDP-fucose synthase gene wcaG is NP_416556.1; the specific nucleotide sequences thereof are known to the public and will not be described in detail here;

[0030] As a further technical solution, the strong constitutive promoter P J23119 The nucleotide sequence is as shown in SEQ ID NO.1 or has at least 98% identity with SEQ ID NO.1;

[0031] As a further technical solution, the nucleotide sequence of the hok / sok system is as shown in SEQ ID NO.2 or has at least 98% identity with SEQ ID NO.2;

[0032] As a further technical solution, the α-1,2-fucosyltransferase gene BKHT is derived from Helicobacter sp.11S02629-2, and the GenBank number of the α-1,2-fucosyltransferase gene BKHT is PAF50342.1. The specific nucleotide sequence is known to the public and will not be described in detail here;

[0033] As a further technical solution, the α-L-fucosidase gene afcA is derived from Bifidobacterium bifidum, and the GenBank number of the α-L-fucosidase gene afcA is NP_417959.4; its specific nucleotide sequence is known to the public and will not be repeated here;

[0034] As a further technical solution, the fucose isomerase-L-fucokinase gene cluster fucI-fucK is derived from Escherichia coli BL21 (DE3), and the nucleotide sequence of the fucose isomerase-L-fucokinase gene cluster fucI-fucK is shown in SEQ ID NO.3 or has at least 98% identity with SEQ ID NO.3;

[0035] As a further technical solution, the thioadenosine kinase gene cysC is derived from Escherichia coli BL21 (DE3), and the NCBI sequence number of the thioadenosine kinase gene cysC is CAD5999255.1.

[0036] As a further technical solution, the nucleotide sequence of the thioadenosine kinase gene cysC is as shown in SEQ ID NO.4 or has at least 98% identity with SEQ ID NO.4;

[0037] As a further technical solution, the NCBI sequence numbers of the β-galactosidase gene lacZ and the UDP-glucose lipid carrier transferase gene wcaJ are NP_416551.1, the NCBI sequence number of the recA site is ACT44368.1, the NCBI sequence number of the arsB site is CAD5999887.1, and the NCBI sequence number of the ldhA site is WP_000762236.1.

[0038] As a further technical solution, the Escherichia coli includes one or more of E. coli BL21 (DE3), E. coli K-12, and E. coli Top10.

[0039] The L-fucose-producing engineered Escherichia coli is used in producing L-fucose.

[0040] A method for producing L-fucose comprises fermenting and culturing the L-fucose-producing engineered Escherichia coli to produce L-fucose.

[0041] As a further technical solution, the fermentation culture uses glycerol as a carbon source and lactose as a substrate.

[0042] As a further technical solution, the seed solution of the plasmid-based antibiotic-free and high-L-fucose-producing engineered Escherichia coli was added to a shake flask fermentation medium containing 20 g / L glycerol, and cultured at 35-38° C., 180-220 rpm until OD 600 =0.8±0.1, add IPTG with a final concentration of 0.05-1.0 mM, and at the same time add lactose with a final concentration of 3-15 g / L, and continue inducing culture at 25°C, 180-220 rpm for no less than 72 h.

[0043] As a further technical solution, the seed liquid of the plasmid-based, antibiotic-free and high-yield L-fucose engineered Escherichia coli is added to a fermentation medium for a fermenter containing 20 to 40 g / L glycerol, the temperature of the fermentation system is controlled at 35 to 38°C, the stirring speed is 100 to 800 r / min, the ventilation volume is 2 to 8 vvm, the pH is controlled at 6.8 ± 0.2, and the fermentation is continued until OD 600 12-16, add lactose with a final concentration of 5-10 g / L and IPTG with a final concentration of 0.05-0.2 mM, and induce culture at 25-28°C for not less than 70 h.

[0044] As a further technical solution, glycerol is added during the reaction of the fermentation tank system to ensure that the glycerol is not completely consumed, and the glycerol content in the fermentation system does not exceed 10 g / L.

[0045] As a further technical solution, glycerol is fed by flow addition. Glycerol is fed at a certain rate. The feeding frequency is increased when glycerol is insufficient, and is reduced when glycerol is excessive, so as to maintain the growth of the bacteria.

[0046] In the present invention, 8 g / L of lactose is initially added as a synthetic substrate of 2′-fucosyllactose, and 2′-fucosyllactose is decomposed to produce lactose. Therefore, the concentration of lactose as a running substrate is kept stable to a certain extent.

[0047] As a further technical solution, the shake flask fermentation medium or the fermentation medium for the fermenter also contains 4.0 g / L diammonium hydrogen phosphate, 13.5 g / L potassium dihydrogen phosphate, 1.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid and 10 ml / L trace metal elements; the trace metal elements include: 0.35 g / L manganese sulfate monohydrate, 10 g / L ferrous sulfate, 1.0 g / L anhydrous cupric sulfate, 2.25 g / L zinc sulfate heptahydrate, 2.0 g / L calcium chloride dihydrate, 0.23 g / L sodium borate decahydrate and 0.11 g / L ammonium molybdate.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The present invention uses the α-1,2-fucosyltransferase gene BKHT derived from Helicobacter sp.13S00401-1 to replace the traditional α-1,2-fucosyltransferase gene wbgL, and adds additional arsB site and ldhA site to integrate the α-1,2-fucosyltransferase gene BKHT, which greatly improves the yield of L-fucose;

[0050] 2. The present invention utilizes plasmid pETDuet-1 to express positive transcriptional regulatory factors rcsA and rcsB and α-1,2-fucosyltransferase gene BKHT in a free manner and to enhance the expression of mannose-1-phosphate guanyltransferase gene manB, and utilizes plasmid pRSFDuet-1 to express phosphomannose mutase-mannose-1-phosphate guanyltransferase gene cluster manC-manB and GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG in a free manner, thereby further increasing the yield of L-fucose;

[0051] 3. In order to solve a series of problems caused by the need to add antibiotics for fermentation culture of plasmid-based engineering strains, the present invention inserts the hok / sok system into the plasmid pRSFDuet-1, knocks out the thioadenosine kinase gene cysC of Escherichia coli, and clones the gene into the plasmid pETDuet-1. Without affecting the L-fucose production, the plasmid is able to stably express L-fucose-engineered Escherichia coli, thereby solving the problem of its dependence on antibiotics in fermentation culture.

[0052] In summary, the present invention has carried out a series of metabolic engineering modifications on the synthesis pathway of L-fucose in Escherichia coli, and the L-fucose engineered Escherichia coli strain finally obtained has achieved stable maintenance of plasmid under antibiotic-free conditions. The engineered strain has achieved an L-fucose production of 7.99 g / L in shake flask culture, and through fed-batch culture in a 5L bioreactor, the L-fucose production is as high as 61.91 g / L within 65 hours, which greatly improves the L-fucose production and demonstrates its excellent potential in industrial-scale production.

[0053] In addition, the system does not rely on antibiotics, reducing production costs, while avoiding the impact of antibiotic residues on the environment and the safety of the final product, meeting the market's demand for green and sustainable production processes. Therefore, the present invention not only improves production efficiency and product quality, but also helps to promote the microbial fermentation industry to develop in a more environmentally friendly and healthy direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The graph is a graph showing the production of 2′-fucosyllactose and L-fucose by the engineered strain constructed in Example 1 under shake flask culture;

[0055] exist Figure 1 Middle, A: 2′-fucosyllactose, B: L-fucose;

[0056] Figure 2 The effect of optimizing the dual plasmid system on the yield of L-fucose in Example 2;

[0057] Figure 3 This is a diagram showing the effect of introducing the hok / sok system plasmid on L-fucose fermentation in Example 3;

[0058] Figure 4 This is a diagram showing the effect of introducing the cysC auxotrophic system on L-fucose fermentation in Example 4;

[0059] Figure 5 This is the monitoring diagram of each component of the fermentation broth of the recombinant Escherichia coli strain BBA3C-5HC under batch fed culture conditions;

[0060] exist Figure 5In the figure, A: 2′-fucosyllactose and L-fucose, B: lactose and glycerol. DETAILED DESCRIPTION

[0061] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0063] In the present invention,

[0064] 1. The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits and DNA gel recovery kits used were commercial products, and the specific operations were carried out in accordance with the instructions of the kits. The preparation of competent cells, nucleic acid agarose gel electrophoresis and heat shock transformation and other experimental operations were carried out in accordance with conventional methods. The primer synthesis and plasmid sequencing were completed by Suzhou Genewise Biotechnology Co., Ltd.

[0065] 2. Culture medium

[0066] (1) LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0067] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0068] (3) Fermentation medium for shake flasks: 20 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

[0069] (4) Fermentation medium for fermenter: 20 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous cupric sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

[0070] (5) Fed-batch fermentation Feed solution: 600 g / L glycerol, 200 g / L lactose, 20 g / L magnesium sulfate heptahydrate and 0.2 g / L thiamine. pH control: 14% ammonia water (v / v).

[0071] 3. L-fucose fermentation

[0072] (1) L-fucose shake flask fermentation process: The constructed strain was inoculated into LB liquid medium, cultured overnight at 37°C, 200 rpm for 12 h to obtain seed solution, 2 mL of seed solution was inoculated into 100 mL of fermentation medium, and cultured at 37°C, 200 rpm until OD 600 The concentration of lactose was 0.8-1.0, 8 g / L lactose was added, and the induction culture was continued at 25°C and 200 rpm for 72 h.

[0073] (2) L-fucose fed-batch fermentation process: 4 mL LB seed solution was cultured overnight, then expanded in a shake flask and transferred to a 5 L fermenter. Fed-batch fermentation was carried out in a 5 L fermenter containing 2 L of culture medium. The initial temperature was maintained at 37 °C. After the 30 g / L glycerol in the initial culture medium was completely consumed, feeding of carbon source was started to meet cell growth. When OD 600 When the temperature reaches about 18, the temperature is slowly lowered to 25°C, and lactose is added as a substrate. The pH is maintained at 6.8±0.2 throughout the process, and the foam is controlled by adding a defoamer. The dissolved oxygen is controlled by adjusting the stirring speed (100-900 rpm) and the ventilation volume (2-8 vvm).

[0074] 4. L-fucose detection:

[0075] Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0076] HPLC detection conditions: by high performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: RezexROA-Organic acid H +(8%); detector: Waters 2414RIDetector differential detector; mobile phase: 5MmH2SO4; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL.

[0077] 5. The strains involved are shown in Table 1, and the plasmids involved are shown in Table 2;

[0078] Table 1: Strains involved in the present invention

[0079]

[0080]

[0081] Table 2: Plasmids involved in the present invention

[0082]

[0083]

[0084] 6. The primers involved in the present invention are shown in Table 3;

[0085] Table 3

[0086]

[0087]

[0088]

[0089]

[0090] Example 1: Construction of engineered Escherichia coli producing L-fucose

[0091] Using the CRISPR-Cpf1 system, an engineered Escherichia coli that produces L-fucose was constructed, including:

[0092] (1) Using Escherichia coli BL21 (DE3) as the initial strain, the β-galactosidase gene lacZ and the UDP-glucose lipid carrier transferase gene wcaJ on E. coli BL21 (DE3) were knocked out, and the strong constitutive promoter P J23119 Replace the original promoter of the phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB and integrate the strong promoter P at the recA site. J23119 The activated α-1,2-fucosyltransferase gene BKHT (from Helicobacter sp., locus 13S00401-1) was obtained; the strain BWLBC was obtained;

[0093] (2) Using Escherichia coli BWLBC as the initial strain, the strong constitutive promoter P J23119 Strain BB1 was obtained by replacing the original promoter of the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG;

[0094] (3) Using strain BB1 as the initial strain, the gene arsB on strain BB1 was knocked out and a strong promoter P was integrated at the arsB site. J23119 The activated α-1,2-fucosyltransferase gene BKHT (from Helicobacter sp., site 13S00401-1) was obtained to obtain strain BB2;

[0095] (4) Using strain BB2 as the initial strain, the gene ldhA on strain BB2 was knocked out, and a strong promoter P was integrated at the ldhA site. J23119 The activated α-1,2-fucosyltransferase gene BKHT (from Helicobacter sp., site 13S00401-1) was obtained to obtain strain BB3;

[0096] (5) Using strain BWLBC, strain BB1, strain BB2, and strain BB3 as the initial strains, the L-fucose decomposition pathway fucose isomerase-L-fucokinase gene cluster fucI-fucK in the initial strains was knocked out, and a strong promoter P was integrated at the fucIK site. J23119 The activated α-L-fucosidase gene afcA resulted in strains BWLBCA, strain BBA1, strain BBA2, and strain BBA3;

[0097] The specific operations of this step are as follows:

[0098] Using the E. coli BL21 (DE3) genome as a template, using fucIK-UH-F / R and fucIK-DH-F / R as primers (Table 3), and then using the synthetic codon-optimized afcA gene as a template, using primers afcA-F / R (Table 3), amplify the afcA gene fragment by PCR and then use gel recovery to purify the fragment. The three fragments amplified by overlapping PCR were connected as donor DNA fragments. PCR amplification technology was used to amplify the N on pcrEG 23 The sequence was replaced with N complementary to the gene cluster fucI-fucK 23Sequence, obtain pcrEG plasmid pcrEG-FucIK with targeting fucI-fucK gene cluster. pcrEG-FucIK plasmid and donor DNA fragment were electroporated into competent cells, coated on LB plate containing kanamycin and spectinomycin, cultured at 37℃ for 24h, and PCR colony verification was performed; the pcrEG-FucIK plasmid and pEcCpf1 plasmid were removed from the successfully verified recombinant strain to obtain a recombinant strain with strong promoter P integrated at the fucIK site. J23119 The host bacteria that activated the α-L-fucosidase gene afcA.

[0099] The strains BWLBC, BB1, BB2, BB3, BWLBCA, BBA1, BBA2, and BBA3 were cultured in shake flasks without plasmids. Figure 1 Before knocking out fucI and fucK and integrating afcA, the L-fucose production of strain BB3 was the highest. After knocking out the gene cluster fucI-fucK and integrating afcA, the L-fucose production of strain BBA3 was the highest, at 3.50 g / L.

[0100] Therefore, subsequent studies were conducted based on strain BBA3.

[0101] Example 2: L-fucose biosynthesis pathway enhanced by plasmid

[0102] The biosynthesis pathway of L-fucose was enhanced by additional plasmid expression, including the following steps:

[0103] (1) The precursor genes for GDP-L-fucose synthesis, phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB and GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG, were cloned into the plasmid pRSFDuet-1 for enhanced expression to obtain the plasmid pRSF-CBGW;

[0104] The rcsA and rcsB positive transcriptional regulatory factors and BKHT from Escherichia coli K-12MG1655 were cloned into the pETDuet-1 plasmid for enhanced expression, and the plasmid pET-rcsAB-BKHT was obtained;

[0105] (2) Plasmid pRSF-CBGW, plasmid pET-rcsAB-BKHT, plasmid pRSF-CBGW and plasmid pET-rcsAB-BKHT were respectively introduced into the host strain BBA3 to obtain strain BBA3-1, strain BBA3-2 and strain BBA3-3;

[0106] (3) Optimizing the dual-plasmid system of strain BBA3-3 and strengthening the precursor supply, specifically: cloning gene manC, gene manB, gene manC and gene manB, gene gmd, gene wcaG, gene gmd and gene wcaG into plasmid pET-rcsAB-BKHT, respectively, to obtain optimized plasmids, namely plasmid PET-rcsAB-BKHT-C, plasmid PET-rcsAB-BKHT-B, plasmid PET-rcsAB-BKHT-CB, plasmid PET-rcsAB-BKHT-G, plasmid PET-rcsAB-BKHT-W, plasmid PET-rcsAB-BKHT-GW, and using the above optimized plasmids to replace the original plasmid pET-rcsAB-BKHT in strain BBA3-3, to obtain strains BBA3-4, strains BBA3-5, strains BBA3-6, strains BBA3-7, strains BBA3-8, strains BBA3-9;

[0107] In a medium containing lactose, the plasmid was stabilized by antibiotics and the strain was induced to express using IPTG. The results are shown in Figure 2 Among them, strains BBA3-3 and BBA3-5 had the best effects. The L-fucose production of strain BBA3-5 was 7.27 g / L. Therefore, strain BBA3-5 was used for subsequent studies.

[0108] Example 3: Introducing the hok / sok system to reduce reliance on antibiotics

[0109] (1) The hok / sok gene cassette was inserted between the CDF ori and lacI sites of the plasmid pRSF-CBGW to obtain the plasmid pRSF-CBGW-OI.

[0110] (2) Plasmid pRSF-CBGW-OI and plasmid PET-rcsAB-BKHT, plasmid pRSF-CBGW-OI and plasmid PET-rcsAB-BKHT-B were introduced into host strain BBA3-3 to obtain strain BBA3-3H and strain BBA3-5H;

[0111] In a lactose-containing medium, the expression was induced by IPTG, and only ampicillin was added without kanamycin or kanamycin and ampicillin were added at the same time. The strains BBA3-3, BBA3-3H, BBA3-5, and BBA3-5H were fermented in shake flasks to observe the effect of reducing antibiotics on L-fucose production. The results are shown in Figure 3After the introduction of the hok / sok system, the dependence on antibiotics can be reduced. The hok / sok system does have the effect of stabilizing the genetic stability of the plasmid. When the corresponding antibiotics are not added, the same yield can be obtained as when the antibiotics are added. Among them, after the introduction of the hok / sok system, the L-fucose yield of strain BBA3-5H is the highest, which is 7.58g / L.

[0112] Therefore, subsequent studies were performed with strain BBA3-5H.

[0113] Example 4: Introducing an auxotrophic system to further reduce reliance on antibiotics

[0114] (1) Using the CRISPR / Cpf1 gene editing system, the cysC gene in strain BBA3-3H and strain BBA3-5H was knocked out to obtain strains BBA3C-3H and BBA3C-5H;

[0115] (2) The cysC gene was inserted into the plasmid pET-rcsAB-BKHT-B to construct the plasmid

[0116] pET-rcsAB-BKHT-B-cysC;

[0117] (3) Plasmid pET-rcsAB-BKHT-B-cysC was used to replace plasmid pET-rcsAB-BKHT-B in strain BBA3C-3H and strain BBA3C-5H to obtain strain BBA3-3HC and strain BBA3C-5HC;

[0118] The strains BBA3-3HC and BBA3C-5HC were cultured in shake flasks using medium containing antibiotics and medium without antibiotics, respectively, to evaluate the effect of the nutrient deficiency system on L-fucose production. The results are shown in Figure 4 After the introduction of the nutrient deficiency system, the yield of the strain did not decrease when it was fermented in a medium without antibiotics. Among them, the yield of strain BBA3C-5HC increased slightly when it was fermented in a medium without antibiotics compared with that in a medium with antibiotics, and the L-fucose yield could reach 7.99 g / L. Therefore, it was shown that the cysC nutrient deficiency system does have the effect of stabilizing the genetic stability of the plasmid, and the same yield can be obtained when the corresponding antibiotics are not added as when the antibiotics are added.

[0119] Example 5: Synthesis of L-fucose by fed-batch culture of strain BBA3C-5HC in a fermenter

[0120] (1) A fed-batch fermentation experiment of L-fucose in a 5-L fermenter was conducted on strain BBA3C-5HC. 200 mL of secondary seed solution was inoculated into a 5-L fermenter containing 2 L fermenter medium without antibiotics for fermentation. The initial temperature was maintained at 37°C. When OD 600 When the temperature reached about 18°C, the temperature was lowered to 25°C, and 0.2 mM IPTG and a final concentration of 8 g / L lactose were added as substrates.

[0121] (2) Feeding conditions: After the 30 g / L glycerol in the initial culture medium is completely consumed, feed carbon source is added to meet cell growth. The pH is maintained at 6.8 ± 0.2 throughout the process, and the foam is controlled by adding a defoamer. The dissolved oxygen is controlled by adjusting the stirring speed (100-900 rpm) and the ventilation volume (2-8 vvm). After the fermentation is completed, the contents of L-fucose, 2-FL, lactose, and glycerol in the fermentation broth are measured. The results are shown in Figure 5 .

[0122] The results are as follows Figure 5 As shown in Figure 2, after 65 h of fermentation, the L-fucose production reached 61.91 g / L, and the OD 600 The highest value reached 118, and high yield could be achieved without the addition of antibiotics, indicating its production potential in large-scale industrial applications.

[0123] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. An engineered Escherichia coli producing L-fucose, characterized in that: Using Escherichia coli as the starting strain, the following transformations of AF items were performed to obtain the result; A, knockout of β-galactosidase gene lacZ and UDP-glucose lipid carrier transferase gene wcaJ; B. Using a strong constitutive promoter P J23119 Promote the expression of phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB C. Using a strong constitutive promoter P J23119 Promote expression of the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG; D. Integrate the α-1,2-fucosyltransferase gene BKHT at the recA site, arsB site, and ldhA site. E, knockout of the fucose isomerase-L-fucokinase gene cluster fucI-fucK; F. Integration of α-L-fucosidase gene afcA.

2. The L-fucose-producing engineered Escherichia coli according to claim 1, characterized in that: The α-L-fucosidase gene afcA was integrated into the fucIK locus.

3. The L-fucose-producing engineered Escherichia coli according to claim 1, characterized in that: The Escherichia coli was also transformed with the following items G and F: G. Plasmid pRSFDuet-1 was used to express the phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB and the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-wcaG; H. Use plasmid pETDuet-1 to express the positive transcriptional regulatory factors rcsA and rcsB and the α-1,2-fucosyltransferase gene BKHT.

4. The L-fucose-producing engineered Escherichia coli according to claim 2, characterized in that: The Escherichia coli has also undergone the following transformation: I: Using the plasmid pETDuet-1, one or more of the phosphomannose mutase gene manC, the mannose-1-phosphate guanyl transferase gene manB, the GDP-mannose-6-dehydrogenase gene gmd, and the GDP-fucose synthase gene wcaG are enhanced in expression, preferably, the mannose-1-phosphate guanyl transferase gene manB is enhanced in expression.

5. An engineered Escherichia coli producing L-fucose according to claim 3 or 4, characterized in that: The Escherichia coli has also undergone the following transformations J and K: J: inserting the hok / sok system between the CDF ori and lacI sites of the plasmid pRSFDuet-1; K: knocking out the thioadenosine kinase gene cysC in the E. coli, and cloning the thioadenosine kinase gene cysC into the plasmid pETDuet-1.

6. The L-fucose-producing engineered Escherichia coli according to claim 5, characterized in that: The nucleotide sequence of the hok / sok system is as shown in SEQ ID NO.2 or has at least 98% identity with SEQ ID NO.2; The nucleotide sequence of the thioadenosine kinase gene cysC is shown in SEQ ID NO.4 or has at least 98% identity with SEQ ID NO.

4.

7. The L-fucose-producing engineered Escherichia coli according to claim 1, characterized in that: The Escherichia coli includes one or more of E. coli BL21 (DE3), E. coli K-12, and E. coli Top10.

8. Use of the L-fucose-producing engineered Escherichia coli according to claim 1 in producing L-fucose.

9. A method for producing L-fucose, characterized in that: The L-fucose-producing engineered Escherichia coli according to any one of claims 1 to 7 is fermented and cultured to produce L-fucose.

10. The method for producing L-fucose according to claim 9, characterized in that: The fermentation culture uses glycerol as a carbon source and lactose as a substrate.

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