CN115806890A - Genetically engineered bacterium for high yield of 3-fucosyllactose and construction method and application thereof - Google Patents
Genetically engineered bacterium for high yield of 3-fucosyllactose and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to a genetic engineering bacterium for high yield of 3-fucosyllactose, a construction method and application thereof. The genetic engineering bacteria simultaneously express lactose permease, GDP-mannose dehydrogenase, GDP-fucose synthetase and alpha-1, 3-fucosyltransferase. The alpha-1, 3-fucosyltransferase is alpha-1, 3-fucosyltransferase from Neobacillus cucumis, the coding gene of the alpha-1, 3-fucosyltransferase is Fut3Bc, and the nucleotide sequence is shown in SEQ ID NO. 1. According to the genetic engineering bacteria constructed by the invention, as alpha-1, 3-fucosyltransferase Fut3Bc is an optimized codon, the efficiency of synthesizing 3-FL is higher, and the genetic engineering bacteria are more suitable for expression in Saccharomyces cerevisiae, when the engineering bacteria are used for producing 3-FL by fermenting and taking lactose and galactose as substrates, the yield of 3-FL is greatly improved, and can reach 0.93g/L, which is 5.4 times of the reported alpha-1, 3-FT futA strain for synthesizing 3-FL in common use, and important reference and guidance are provided for industrial synthesis of 3-FL.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a genetic engineering bacterium for high yield of 3-fucosyllactose, and a construction method and application thereof.
Background
3-fucosyllactose (3-FL) is an important component in breast milk oligosaccharide, 3-FL is an important component of breast milk oligosaccharide (HMOs), and accounts for 25-55% of the total amount of HMOs in breast milk of 18-month infants. 3-FL in promoting human health plays an important role, for example, 3-fucosyllactose specifically inhibits pathogenic Escherichia coli, norovirus and corresponding host receptor binding and thus avoid infection. The 3-fucosyllactose can inhibit the interaction of Pseudomonas aeruginosa with local epithelial cells, thereby preventing infection of gastrointestinal tract, urethra and respiratory system by Pseudomonas aeruginosa. The 3-fucosyllactose can also specifically proliferate specific beneficial bacteria in intestinal tract to regulate the structure and function of intestinal flora. In addition, 3-fucosyllactose promotes the growth of beneficial microorganisms such as bifidobacteria in the gut by up-regulating the expression of gut goblet cell secretion-associated genes. The construction of the high-yield 3-FL strain has important significance for further research on the function of 3-FL and industrial synthesis of 3-FL.
Biosynthesis of 3-FL is a well-established method. The biosynthesis of 3-FL is carried out by binding a donor molecule of GDP-fucose to an acceptor lactose by alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase, alpha-1, 3-FT) to produce a molecule of 3-FL. The construction of recombinant strains of E.coli with high 3-FL yield has been studied. However, escherichia coli may produce endotoxin, and is susceptible to phage contamination during fermentation, so that industrial synthesis of 3-FL by Escherichia coli is limited.
Saccharomyces cerevisiae is a microorganism generally regarded as safe, and no 3-FL Saccharomyces cerevisiae synthesis has been reported. However, the structural analog 2'-FL of 3-FL has been successfully synthesized in Saccharomyces cerevisiae, whereas 3-FL requires the same substrate as 2' -FL synthesis, whereas the glycosyltransferase catalyzing 3-FL synthesis is alpha-1, 3-FT.
Therefore, the technical personnel in the field need to solve the problems that the alpha-1, 3-FT with high activity is searched and expressed in the saccharomyces cerevisiae, the recombinant saccharomyces cerevisiae for synthesizing the 3-fucosyllactose is constructed, the fermentation and synthesis of the 3-fucosyllactose are effectively promoted, and a solid foundation is laid for the future large-scale production.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a genetic engineering bacterium for high yield of 3-fucosyllactose, and a construction method and application thereof. According to the invention, the saccharomyces cerevisiae can absorb lactose and synthesize GDP-fucose by taking GDP-mannose as a precursor in cells through metabolic engineering modification, and the high-yield alpha-1, 3-FT of 3-FL is found when the saccharomyces cerevisiae is expressed by heterologously expressing alpha-1, 3-fucosyltransferase from various biological sources. Thereby improving the yield of the 3-FL and providing an important reference for industrial production of the 3-FL.
The technical scheme of the invention is as follows:
a genetic engineering bacterium for high yield of 3-fucosyllactose simultaneously expresses lactose permease, GDP-mannose dehydrogenase, GDP-fucose synthetase and alpha-1, 3-fucosyltransferase.
According to the optimization of the invention, the genetic engineering bacteria are recombinant saccharomyces cerevisiae, and the saccharomyces cerevisiae is saccharomyces cerevisiae W303-1a; the genotype of the saccharomyces cerevisiae W303-1a is MATa ade2-1 can1-100 ura3-1 leu2-3,112his3-11,15.
According to the invention, the lactose permease is lactose permease from Kluyveromyces lactis, and the coding gene of the lactose permease is Lac12, and Genbank accession number is X06997.1.
Preferably, the GDP-mannose dehydrogenase is GDP-mannose dehydrogenase from Escherichia coli K12, and the coding gene of the GDP-mannose dehydrogenase is Gmd and the Genbank accession number is WP-182915037.1.
Preferably, the GDP-fucose synthetase is GDP-fucose synthetase from Escherichia coli K12, and the coding gene of the GDP-fucose synthetase is Wcag and Genbank accession number is WP-000043654.1.
According to the invention, the alpha-1, 3-fucosyltransferase is alpha-1, 3-fucosyltransferase from Neobacillus cumis, the coding gene of the alpha-1, 3-fucosyltransferase is Fut3Bc, and the nucleotide sequence is shown in SEQ ID NO. 1. The codon of the enzyme is optimized and designed, so that the enzyme is more suitable for saccharomyces cerevisiae.
The construction method of the genetic engineering bacteria comprises the following steps:
(1) Inserting lactose permease gene Lac12 of Kluyveromyces lactis into vector plasmid pRS304 to construct expression vector pRS304-Lac12;
(2) Inserting GDP-mannose dehydrogenase gene Gmd and GDP-fucose synthetase gene Wcag of escherichia coli K12 into a vector plasmid pRS306 together to construct an expression vector pRS306-Gmd-Wcag;
(3) Inserting an alpha-1, 3-fucosyltransferase gene Fut3Bc of Neobasilus cucumis into a vector plasmid pRS305 to construct an expression vector pRS305-Fut3Bc;
(4) And sequentially transforming the expression vector pRS304-Lac12, pRS306-Gmd-Wcag and the expression vector pRS305-Fut3Bc into Saccharomyces cerevisiae W303-1a, and selecting a positive recon to obtain the genetic engineering bacterium FL303.
According to the optimization of the invention, the construction method of the genetic engineering bacteria specifically comprises the following steps:
(1) Performing PCR amplification by using a Kluyveromyces genome as a template to obtain the lactose permease Lac12, wherein the PCR primer sequence is as follows:
gal1-LAC12-F：5′-GAAAAAACCCATGGCAGATCA TTCG-3′，
LAC12-cyc1-R：5′-AATTACATGATTAAACAGATTCTGCCTC-3′；
then through fusion PCR, galactose-inducible promoter P is used gal1 And a terminator T cyc1 Construction of the expression cassette Pgal1-lac12-T cyc1 Finally, the expression cassette Pgal1-lac12-T was fused seamlessly cyc1 Connecting with pRS304 to obtain pRS304-Lac12 plasmid;
(2) Taking Escherichia coli K12 genome as a template to carry out PCR amplification to obtain GDP-mannose dehydrogenase Gmd and GDP-fucose synthetase Wcag, wherein the PCR primer sequences are as follows:
10-Gmd-F：5′-ACCTCATGTCAAAAGTCGCTCTCATCAC-3′，
Gmd-cyc-R：5′-CGGATTTATGACTCCAGCGCGATC-3′；
adh-WcaG-F：5′-CTCTGGCTTACCCCCGAAAGCGGTCTT-3′，
WcaG-10-R：5′-CGACGATAAGATGAGTAAACAACGAGTTTTTATTGCTG-3′；
then fusion PCR is carried out to obtain the galactose bidirectional inducible promoter P gal1,10 Constructing a bidirectional expression cassette, and finally connecting the bidirectional expression cassette with pRS306 through seamless fusion to obtain pRS306-Gmd-Wcag plasmid;
(3) Artificially synthesizing an alpha-1, 3-fucosyltransferase gene Fut3Bc with codon optimized design, and then carrying out PCR amplification by taking the gene as a template to obtain the alpha-1, 3-fucosyltransferase Fut3Bc, wherein the PCR primer sequence is as follows:
Fut3Bc-F：5′-GTCAAGGAGAAAAAACCCATGAAACCAAAAATTAAAATT-3′，
Fut3Bc-R：5′-TGACATAACTAATTACATGATTATCTTCTATCAATTTTTC-3′；
then fusion PCR is carried out to induce the promoter P with galactose gal1 And a terminator T cyc1 Construction of expression cassette Pgal1-fut3Bc-T cyc1 Finally, the expression cassette Pgal1-fut3Bc-T is fused seamlessly cyc1 Connecting with pRS305 to obtain pRS305-Fut3Bc plasmid;
(4) The pRS304-Lac12 plasmid obtained in the step (1) is transformed into Saccharomyces cerevisiae W303-1a to obtain a strain FL01; then, the pRS306-Gmd-Wcag plasmid obtained in the step (2) is transformed into a strain FL01 to obtain a strain FL03; and finally, the pRS305-Fut3Bc plasmid is transformed into a strain FL03, and a positive recon is selected to obtain a genetic engineering strain FL303.
The application of the genetic engineering bacteria in the production of 3-fucosyllactose.
According to the invention, the preferable application is to produce the 3-fucosyllactose by fermenting lactose and galactose as substrates, and the specific production process is as follows:
activating the genetically engineered bacteria on an LB solid culture medium, selecting an activated single colony, inoculating the activated single colony into an YNB liquid culture medium for culturing for 24 hours, transferring the culture solution into an YPAD liquid culture medium, adjusting the initial inoculation amount to ensure that the initial OD of the fermentation solution is 0.25-0.35, culturing for 24 hours at 30 ℃ and 200r/min, supplementing 25-35 g/L of galactose and 1-3 g/L of lactose, continuing to ferment, and producing the 3-fucosyllactose.
Wherein the YNB liquid medium comprises the following components: 5g/L (NH) 4 ) 2 SO 4 1.7g/L Yeast Nitrogen Base (from Biotech), 20g/L glucose.
The YPAD liquid culture medium comprises the following components: 20g/L peptone, 10g/L yeast extract 20g/L glucose, 0.06g/L adenine sulfate.
The invention has the beneficial effects that:
according to the invention, lactose permease, GDP-mannose dehydrogenase, GDP-fucose synthetase and alpha-1, 3-fucose transferase are simultaneously expressed in saccharomyces cerevisiae, and a new genetic engineering bacterium is constructed, because the alpha-1, 3-fucose transferase Fut3Bc is an optimized codon, the efficiency of synthesizing 3-FL is higher, and the genetic engineering bacterium is more suitable for expression in saccharomyces cerevisiae, therefore, when the engineering bacterium is used for fermenting and producing 3-FL by taking lactose and galactose as substrates, the yield of 3-FL is greatly improved, can reach 0.93g/L, is 5.4 times of the reported commonly used alpha-1, 3-FT futA strain for synthesizing 3-FL, and provides important reference and guidance for industrial synthesis of 3-FL.
Drawings
Fig. 1 is a graph of alignment analysis and amino acid sequence alignment of α -1, 3-fucosyltransferases FutA, fut3 and Fut3Bc (a) a graph of evolutionary tree analysis of α -1,3-fucosyltransferase (B), with α -1, 2-fucosyltransferase FutC from h.pyloi 26695 as a control.
FIG. 2 shows PCR verification of genetically engineered bacteria expressing alpha-1, 3-fucosyltransferases from different biological sources.
FIG. 3 shows the yield of 3-FL in genetically engineered bacteria expressing α -1, 3-fucosyltransferases from different biological sources.
FIG. 4 is a LC-MS analysis chart of strain FL303 fermentation product.
FIG. 5 shows the fermentation product of strain FL303 1 H-NMR chart.
Detailed Description
The technical solution of the present invention is further described below with reference to the following examples and drawings, but the scope of the present invention is not limited thereto. Reagents and medicines involved in the examples are common commercial products unless otherwise specified; the experimental procedures referred to in the examples are those conventional in the art unless otherwise specified.
The Saccharomyces cerevisiae W303-1a used in the present invention is a commonly commercially available strain, and is available from microorganism collection centers or strain sales companies.
Example 1: construction of Strain FL01
1. Amplifying by taking saccharomyces cerevisiae genome DNA as a template to obtain galactose-inducible promoter P gal1 (BamHI-GAL 1-F/GAL1-LAC 12-R) and terminator T cyc1 The sequence of (lac 12-CYC1-F/CYC 1-XhoI-R), the PCR primer sequence is specifically as follows:
BamHI-GAL1-F：5′-GCTCTAGAACTAGTGCGCGGATCCAGTACGGATTA GAAG-3′，
GAL1-LAC12-R：5′-GATCTGCCATGGGTTTTTTCTCCTT-3′；
lac12-CYC1-F：5′-ATCTGTTTAATCATGTAATTAGTTATGTCACG-3′，
CYC1-XhoI-R：5′-GTACCGGGCCCCCCCCCGCTCGAGGCAAATTAAAGCCTT-3′。
PCR amplification was performed using the high Fidelity DNA Polymerase Phanta Super-Fidelity DNA Polymerase available from Vazyme, and the PCR amplification system was formulated according to the kit instructions.
PCR amplification procedure: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, renaturation at 55 deg.C for 15s, extension at 72 deg.C for 1min/kb for 30 cycles, post-extension at 72 deg.C for 5min, and storage at 12 deg.C.
The promoter P gal1 The nucleotide sequence of (A) is shown as SEQ ID NO.4, and the terminator T cyc1 The nucleotide sequence of (A) is shown in SEQ ID NO. 5.
2. Performing PCR amplification by taking a Kluyveromyces genome as a template to obtain a lactose permease Lac12 sequence, wherein the PCR primer sequence is as follows:
gal1-LAC12-F：5′-GAAAAAACCCATGGCAGATCA TTCG-3′，
LAC12-cyc1-R：5′-AATTACATGATTAAACAGATTCTGCCTC-3′；
PCR amplification was performed using the high Fidelity DNA Polymerase Phanta Super-Fidelity DNA Polymerase from Vazyme, and the PCR amplification system was formulated according to the kit instructions.
PCR amplification procedure: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, renaturation at 55 deg.C for 15s, extension at 72 deg.C for 1min/kb,30 cycles, post-extension at 72 deg.C for 5min, and storage at 12 deg.C
3. Based on a Vazyme high-Fidelity DNA polymerase Phanta Super-Fidelity DNA polymerasePCR kit, the promoter P gal1 Lac12 sequence and terminator T cyc1 The method comprises the following steps of 1:3:1 is added into a PCR system to be used as a template for fusion PCR, and a fusion fragment 1 is obtained. Then fusion PCR was performed to fuse fragment 1 and promoter P gal1 And a terminator T cyc1 Construction of the expression cassette Pgal1-lac12-T cyc1 . Finally, the expression cassette Pgal1-lac12-T is fused through a seamless fusion method based on T5 exonuclease cyc1 And the pRS304 is connected to obtain pRS304-Lac12 plasmid.
The seamless fusion method can be referred to as: xia Y, li K, li J, wang T, gu L, xun L.T5 exonuclease-dependent assembly of bacteria a low-cost method for influencing binding and site-directed mutagenesis, nucleic acids research.2018;47, e15-e15.
After the pRS304-Lac12 plasmid was linearized with restriction enzyme Bsu36I, transferred into Saccharomyces cerevisiae W303-1a by lithium acetate transformation, screened by tryptophan-deficient YNB solid medium to obtain positive clones, and further verified by PCR, the correct transformant obtained was named FL01.
The strain FL01 obtained in this example can absorb lactose added extracellularly.
Example 2: construction of Strain FL03
PCR amplification is carried out by taking Saccharomyces cerevisiae W303-1a genome DNA as a template to obtain ADH1t sequence (Sa)cI-ADH1-F/ADH 1-wcAG-R), galactose bidirectional inducible promoter P gal1,10 (wcag-10-F/10-gmd-R) and CYC1t sequence; performing PCR amplification by taking an escherichia coli K12 genome as a template, and performing PCR amplification on a GDP-mannose dehydrogenase Gmd sequence (10-Gmd-F/Gmd-cyc-R) and a GDP-fucose synthetase Wcag sequence (adh-Wcag-F/Wcag-10-R). The PCR primer sequences are specifically shown in Table 1.
TABLE 1
PCR amplification was performed using the high Fidelity DNA Polymerase Phanta Super-Fidelity DNA Polymerase from Vazyme, and the PCR amplification system was formulated according to the kit instructions.
PCR amplification procedure: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, renaturation at 55 deg.C for 15s, extension at 72 deg.C for 1min/kb for 30 cycles, post-extension at 72 deg.C for 5min, and storage at 12 deg.C.
The promoter P gal，10 The nucleotide sequence of (A) is shown as SEQ ID NO. 6.
The Gmd sequence, promoter P gal，10 The Wcag sequence is as follows 1:3:1 is added into a PCR system to be used as a template for fusion PCR, and a fusion fragment 2 is obtained. And then the ADH1t sequence, the fusion fragment 2 and the CYC1t sequence are expressed in a proportion of 1:3:1 is added into a PCR system to be used as a template to carry out fusion PCR to obtain a fusion fragment P gal1，10 -gmd-wcaG. Then fusion PCR was performed to fuse fragment P gal1，10 -gmd-wcaG and galactose bidirectional inducible promoter P gal1,10 Construct a bidirectional expression cassette. And finally, connecting the bidirectional expression cassette with pRS304 by a seamless fusion method based on T5 exonuclease to obtain pRS306-Gmd-Wcag plasmid.
The pRS306-Gmd-Wcag plasmid was linearized with restriction enzyme NcoI, transferred into the strain FL01 by the lithium acetate transformation method, screened by a tryptophan-deficient YNB solid medium to obtain positive clones, and verified by PCR to obtain the correct transformant designated as FL03.
The strain FL03 obtained in this example can absorb extracellular lactose, and can synthesize GDP-fucose by using intracellular GDP-mannose as a substrate.
Example 3: construction of genetically engineered bacterium FL303
1. The inventor compares the alpha-1, 3-fucosyltransferase gene Fut3 derived from human with the alpha-1, 3-fucosyltransferase gene FutA template derived from helicobacter pylori in NCBI database, and finds the unreported gene Fut3Bc derived from Neobasilus cucumis. The results of NCBI Blast analysis showed that the three belong to the GT10 superfamily, and the bioinformatics predicted that the gene Fut3Bc has the activity of alpha-1, 3-fucosyltransferase. Fut3Bc has only 18.53% amino acid sequence identity to Fut3 of human origin, while it has 36.43% amino acid sequence identity to FutA of h. The DNA sequence of the protein is subjected to codon optimization and synthesized by Jinzhi corporation, and the nucleotide sequence is shown as SEQ ID NO. 4. The codon of the enzyme is optimized and designed, so that the enzyme is more suitable for saccharomyces cerevisiae.
Then respectively carrying out PCR amplification by taking the human alpha-1, 3-fucosyltransferase gene Fut3 (SEQ ID NO. 2), the helicobacter pylori alpha-1, 3-fucosyltransferase gene futA (SEQ ID NO. 3) and the codon-optimized gene Fut3Bc (SEQ ID NO. 1) as templates to respectively obtain a lactose permease Fut3 sequence (Fut 3 human-F/R), a futA sequence (futA Hp-F/R) and a Fut3Bc sequence (Fut 3 Bc-F/R); taking Saccharomyces cerevisiae genome DNA as a template for amplification to obtain a promoter P gal1 (the primer is pRS305-XbaI-gal1p-F/gal1 p-R) and the terminator T cyc1 Sequence (primer is Cyc1t-F/pRS305-HindIII-Cyc1 t-R); specific PCR primers are shown in Table 2.
PCR amplification was performed using the high Fidelity DNA polymerase Phanta Super-Fidelity DNApolymerase available from Vazyme, and the PCR amplification system was prepared according to the kit instructions
PCR amplification procedure: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, renaturation at 55 deg.C for 15s, extension at 72 deg.C for 1min/kb,30 cycles, post-extension at 72 deg.C for 5min, and storage at 12 deg.C
The promoter P gal1 The corresponding alpha-1, 3-glycosyltransferase (Fut 3, futA and Fut3 BC) and the terminator T cyc1 Mixing the raw materials in a ratio of 1:3:1 is added into a PCR system to carry out fusion PCR, fusion fragments 4, 5 and 6 were obtained, respectively. Then fusion PCR is carried out to fuse the fragments 4, 5 and 6 and the promoter P gal1 And a terminator T cyc1 Construction of expression cassettes P separately gal1 -fut3-T cyc1 、P gal1 -futA-T cyc1 And P gal1 -fut3Bc-T cyc1 Finally, the expression cassette P is expressed by a seamless fusion method based on T5 exonuclease gal1 -fut3-T cyc1 、P gal1 -futA-T cyc1 And P gal1 -fut3Bc-T cyc1 Respectively connected with pRS304 to obtain pRS305-P gal1 -fut3-T cyc1 、pRS305-P gal1 -futA-T cyc1 And pRS305-P gal1 -fut3Bc-T cyc1 A plasmid.
TABLE 2
(ii) pRS305-P gal1 -fut3-T cyc1 、pRS305-P gal1 -futA-T cyc1 And pRS305-P gal1 -fut3Bc-T cyc1 The plasmid is transferred into a strain FL03 to obtain genetically engineered bacteria FL301 (expression Fut 3), FL302 (expression FutA) and FL303 (expression Fut3 Bc), and the specific operation method is as follows:
pRS305-P gal1 -fut3-T cyc1 、pRS305-P gal1 -futA-T cyc1 And pRS305-P gal1 -fut3Bc-T cyc1 The plasmid was digested with the restriction enzyme BspTI and the linearized vector was obtained by gel recovery. The strain FL03 is cultured in a tryptophan and uracil deficient yeast SC liquid culture medium overnight until OD is 0.6-0.8, the thallus is collected, a linearized fucosyltransferase carrier is respectively converted into the FL03 by a lithium acetate conversion method, and the FL03 is coated on the tryptophan, uracil and leucine deficient yeast SC solid culture medium. After transformants grow on the plate, selecting the transformants to be in a yeast SC liquid culture medium corresponding to auxotrophy, after the strain grows for 24 hours, extracting genome DNA for PCR verification, verifying primers pRS305-XbaI-gal1p-F and pRS305-HindIII-Cyc1t-R, and preserving the strains verified to be positive (figure 2)Recombinant strains FL301, FL302 and FL303 were obtained.
Example 4: detection of 3-fucosyllactose synthesizing capability of recombinant strains FL301, FL302 and FL303
1. Fermentative production of 3-fucosyllactose
Fermentation: the deposited strains FL301, FL302 and FL303 were inoculated into a test tube and transferred to a 100mL Erlenmeyer flask with 20mL YPAD broth (YP medium, 2% glucose, 0.06g/L adenine sulfate), and the initial inoculum size was adjusted to give an initial OD of 0.2 in the fermentation broth. The shake flask was incubated in a shaker at 30 ℃ with shaking (200 rpm). After fermenting for 24 hours, supplementing 30g/L galactose and 2g/L lactose, continuing to ferment for 96 hours, wherein 10g/L galactose is added in 48 hours and 72 hours respectively, and sampling for detection.
And (3) treatment of a fermentation sample: collecting 1mL fermentation liquid, centrifuging at 6000 Xg for 8min, and filtering the supernatant with 0.22 μ M microporous membrane to obtain extracellular sample. After washing the remaining cells with 1mL of distilled water 2 times, glass beads and 1mL of distilled water were added in an amount equivalent to the cells, and the cells were disrupted by circulating the mixture in a homogenizer at 6000rpm for 45s for 5 times. The cell disruption solution (13000 Xg) is centrifuged for 10min, and the supernatant is taken and filtered by a 0.22 mu M microporous filter membrane to obtain an intracellular sample.
Detecting a fermentation sample: detection was performed using Shimadzu (Shimadzu) liquid chromatograph. The intracellular and extracellular samples to be tested were transferred to a liquid phase vial, and the sample size for analysis was 15. Mu.L. Rezex ROA-Organic Acid H was used + (8%) column (Phenomenex, torrance, CA, USA) chromatography, at 50 deg.C, mobile phase is 0.005 NH 2 SO 4 The flow rate was 0.6mL/min. Under these conditions, the 3-FL retention time is about 7.8min.
Yield analysis: the peak area of the 3-FL standard is taken as the basis to calculate the yield of the 3-FL in different strains, and the result is shown in figure 3. As can be seen from FIG. 3, 3-FL synthesis was not detected in the strain FL301 expressing the humanized α -1,3-fucosyltransferase gene Fut3, and a small amount of 3-FL was synthesized in the strain FL302 expressing the α -1,3-fucosyltransferase gene FutA, with an intracellular and extracellular total yield of about 0.17g/L, wherein the intracellular 3-FL was 0.12g/L and accounted for 71% of the total yield; the total yield of 3-FL in the strain FL303 expressing the Fut3Bc gene of the invention is about 0.93g/L, wherein the yield of intracellular 3-FL is about 0.52g/L and accounts for 56% of the total yield. The 3-FL yield in FL303 is 5.4 times that of FL 302.
2. Identification of gene engineering bacterium FL303 fermentation product
Although the yield of 3-FL was high in the strain FL303 expressing the gene Fut3Bc, fut3Bc was an unreported alpha-1, 3-fucosyltransferase, and the retention time of the catalytic product in Saccharomyces cerevisiae was consistent with that in HPLC, the following experiment further verified the structure of the product detected in the strain FL303, confirming that the catalytic product of Fut3Bc was 3-fucosyllactose.
Liquid chromatography-mass spectrometry analysis: the resulting 3-FL fermentation extracellular sample was analyzed in a liquid chromatograph-mass spectrometer (LTQ-Orbitrap velos pro ETD) using a Rezex ROA-Organic Acid H + (8%) column chromatography with column temperature of 50 deg.C, mobile phase of 0.005M formic acid, and flow rate of 0.6mL/min. At 7.8min, consistent with 3-FL retention time, [ M + Na ] consistent with 3-FL molecular weight was present] + =511.1629 (fig. 4), which preliminarily verified that expression of Fut3Bc catalyzed results in 3-FL.
1 H-nuclear magnetic resonance analysis: firstly, a pure product presumed to be 3-FL is extracted from a FL303 fermentation sample. Nine times the volume of precooled ethanol is added into the fermentation liquor, and the mixture is continuously stirred for 2 hours to fully precipitate sugar in the solution. The mixture was then centrifuged, the resulting precipitate was sufficiently suspended in 3mL of distilled water, and the supernatant was collected after centrifugation to obtain a mixed sugar solution. The mixed sugar solution was then added to the molecular sieve at 10mM NH 4 HCO 3 Eluting at a flow rate of about 0.6mL/min, collecting with an automatic collector after 1.5h, collecting one tube every 5min, determining the number of distribution tubes of 3-FL by TLC detection, combining the solutions only containing 3-FL tubes, and lyophilizing to obtain pure product of 3-FL. Dissolving the lyophilized 2' -FL pure product in 600 μ L of heavy water D 2 In O, freeze-drying, dissolving repeatedly and freeze-drying for 3 times, dissolving in 600 μ L D 2 O, 0.1 μ L acetone was added as a chemical shift internal reference and the solution was transferred to a nuclear magnetic tube for nuclear magnetic resonance analysis. The hydrogen spectrum of the product is shown in FIG. 5. From the figure5, the chemical shift of the characteristic peak is consistent with that of the 3-FL standard, and the catalytic product of FutBC in the saccharomyces cerevisiae is proved to be 3-FL.
Claims (10)
1. The genetic engineering strain for high yield of 3-fucosyllactose is characterized by simultaneously expressing lactose permease, GDP-mannose dehydrogenase, GDP-fucose synthetase and alpha-1, 3-fucosyltransferase.
2. The genetically engineered bacterium producing 3-fucosyllactose in high yield according to claim 1, wherein the genetically engineered bacterium is recombinant saccharomyces cerevisiae, and the saccharomyces cerevisiae is saccharomyces cerevisiae W303-1a.
3. The genetically engineered bacterium producing 3-fucosyllactose in high yield according to claim 1, wherein the lactose permease is lactose permease from Kluyveromyces lactis, the coding gene of which is Lac12, and Genbank accession number is X06997.1.
4. The genetically engineered bacterium producing 3-fucosyllactose in high yield according to claim 1, wherein the GDP-mannose dehydrogenase is GDP-mannose dehydrogenase from escherichia coli K12, and the coding gene of the GDP-mannose dehydrogenase is Gmd, and the Genbank accession number is WP-182915037.1.
5. The genetically engineered bacterium producing 3-fucosyllactose in high yield according to claim 1, wherein the GDP-fucose synthetase is a GDP-fucose synthetase from escherichia coli K12, the coding gene of the GDP-fucose synthetase is WcaG, and the Genbank accession number is WP _000043654.1.
6. The genetically engineered bacterium capable of highly producing 3-fucosyllactose according to claim 1, wherein the α -1,3-fucosyltransferase is α -1,3-fucosyltransferase from Neobasilus cuumis, the encoding gene of the α -1,3-fucosyltransferase is Fut3Bc, and the nucleotide sequence of the α -1,3-fucosyltransferase is shown in SEQ ID No. 1.
7. The method for constructing a genetically engineered bacterium according to claim 1, comprising the steps of:
(1) Inserting lactose permease gene Lac12 of Kluyveromyces lactis into vector plasmid pRS304 to construct expression vector pRS304-Lac12;
(2) Inserting GDP-mannose dehydrogenase gene Gmd and GDP-fucose synthetase gene Wcag of Escherichia coli K12 into a vector plasmid pRS306 together to construct an expression vector pRS306-Gmd-Wcag;
(3) Inserting an alpha-1, 3-fucosyltransferase gene Fut3Bc of Neobasilus cucumis into a vector plasmid pRS305 to construct an expression vector pRS305-Fut3Bc;
(4) And sequentially transforming the expression vector pRS304-Lac12, pRS306-Gmd-Wcag and the expression vector pRS305-Fut3Bc into saccharomyces cerevisiae W303-1a, and selecting a positive recon to obtain the genetically engineered bacterium FL303.
8. The method for constructing the genetically engineered bacteria of claim 7, which comprises the following steps:
(1) Performing PCR amplification by using a Kluyveromyces genome as a template to obtain the lactose permease Lac12, wherein the PCR primer sequence is as follows:
gal1-LAC12-F：5′-GAAAAAACCCATGGCAGATCATTCG-3′，
LAC12-cyc1-R：5′-AATTACATGATTAAACAGATTCTGCCTC-3′；
then through fusion PCR, galactose-inducible promoter P is used gal1 And a terminator T cyc1 Construction of the expression cassette Pgal1-lac12-T cyc1 Finally, the expression cassette Pgal1-lac12-T was fused seamlessly cyc1 Connecting with pRS304 to obtain pRS304-Lac12 plasmid;
(2) Taking Escherichia coli K12 genome as a template to carry out PCR amplification to obtain GDP-mannose dehydrogenase Gmd and GDP-fucose synthetase Wcag, wherein the PCR primer sequences are as follows:
10-Gmd-F：5′-ACCTCATGTCAAAAGTCGCTCTCATCAC-3′，
Gmd-cyc-R：5′-CGGATTTATGACTCCAGCGCGATC-3′；
adh-WcaG-F：5′-CTCTGGCTTACCCCCGAAAGCGGTCTT-3′，
WcaG-10-R：5′-CGACGATAAGATGAGTAAACAACGAGTTTTTATTGCTG-3′；
then fusion PCR is carried out to obtain the galactose bidirectional inducible promoter P gal1,10 Constructing a bidirectional expression cassette, and finally connecting the bidirectional expression cassette with pRS306 through seamless fusion to obtain pRS306-Gmd-Wcag plasmid;
(3) Artificially synthesizing an alpha-1, 3-fucosyltransferase gene Fut3Bc with codon optimized design, and then carrying out PCR amplification by taking the gene as a template to obtain the alpha-1, 3-fucosyltransferase Fut3Bc, wherein the PCR primer sequence is as follows:
Fut3Bc-F：5′-GTCAAGGAGAAAAAACCCATGAAACCAAAAATTAAAATT-3′，
Fut3Bc-R：5′-TGACATAACTAATTACATGATTATCTTCTATCAATTTTTC-3′；
then through fusion PCR, galactose-inducible promoter P is used gal1 And a terminator T cyc1 Construction of the expression cassette Pgal1-fut3Bc-T cyc1 Finally, the expression cassette Pgal1-fut3Bc-T is fused seamlessly cyc1 Connecting with pRS305 to obtain pRS305-Fut3Bc plasmid;
(4) The pRS304-Lac12 plasmid obtained in the step (1) is transformed into Saccharomyces cerevisiae W303-1a to obtain a strain FL01; then, the pRS306-Gmd-Wcag plasmid obtained in the step (2) is transformed into a strain FL01 to obtain a strain FL03; and finally, the pRS305-Fut3Bc plasmid is transformed into a strain FL03, and a positive recon is selected to obtain the genetically engineered bacterium FL303.
9. The use of the genetically engineered bacterium of claim 1 in the production of 3-fucosyllactose.
10. The use according to claim 9, wherein the use is for the fermentative production of 3-fucosyllactose using lactose and galactose as substrates, and the specific production process is as follows:
activating the genetically engineered bacteria of claim 1 on an LB solid culture medium, selecting an activated single colony, inoculating the activated single colony into an YNB liquid culture medium for culturing for 24 hours, then transferring the culture solution into an YPAD liquid culture medium, adjusting the initial inoculation amount to ensure that the initial OD of a fermentation liquid is 0.25-0.35, culturing for 24 hours at 30 ℃ and 200r/min, supplementing 25-35 g/L galactose and 1-3 g/L lactose, and continuing to ferment to produce 3-fucosyllactose;
wherein the YNB liquid medium comprises the following components: 5g/L (NH) 4 ) 2 SO 4 1.7g/L Yeast Nitrogen Base (from Biolabs), 20g/L glucose;
the YPAD liquid culture medium comprises the following components: 20g/L peptone, 10g/L yeast extract 20g/L glucose, 0.06g/L adenine sulfate.
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