CN117487729A - Genetically engineered bacterium for producing sialyllactose and application thereof - Google Patents

Genetically engineered bacterium for producing sialyllactose and application thereof Download PDF

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CN117487729A
CN117487729A CN202311355000.9A CN202311355000A CN117487729A CN 117487729 A CN117487729 A CN 117487729A CN 202311355000 A CN202311355000 A CN 202311355000A CN 117487729 A CN117487729 A CN 117487729A
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gene
encoding
sialyllactose
coli
seq
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张涛
李晨晨
李梦丽
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Jiangnan University
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Abstract

The invention discloses a genetic engineering bacterium for producing sialyllactose and application thereof, belonging to the field of microbial genetic engineering. The invention constructs a metabolic synthesis path of sialyllactose by over-expressing genes, constructs a glutamine circulation system by increasing expression of precursor UDP-acetylglucosamine related synthesis genes, silences expression of side paths in the escherichia coli genome sialyllactose synthesis path, and improves expression level of sialyltransferase by regulating and controlling strength of a promoter and screening fusion protein expression labels, thereby successfully obtaining production strains for efficiently synthesizing sialyllactose, and in shake flask fermentation culture experiments, yields of 3'-SL and 6' -SL respectively reach 8.09g/L and 4.67g/L; in the 3L fermenter, yields of 3'-SL and 6' -SL reached 44.23g/L and 36.29g/L, respectively.

Description

Genetically engineered bacterium for producing sialyllactose and application thereof
Technical Field
The invention relates to a genetic engineering bacterium for producing sialyllactose and application thereof, belonging to the field of microbial genetic engineering.
Background
Human milk oligosaccharides (human milk oligosaccharides, HMOs) are complex mixtures of more than 200 structurally complex, nondigestible, non-nutritive carbohydrates, the content of which in breast milk is next to lactose and lipids. Studies have shown that the probability of developing gastroenteritis, acute otitis media, and other immune diseases in neonates fed with human milk is significantly reduced compared to non-breast feeding, and even dominates the development of future wisdom, the probability of developing obesity and diabetes is also reduced, which is closely related to HMOs in human milk. HMOs are important immunological active ingredients in breast milk, play a vital role in infant health and growth and development, and more animals and clinical experiments prove that the HMOs have beneficial characteristics, such as multiple functional activities of maintaining intestinal ecological balance, resisting adhesion of pathogenic bacteria, regulating immunity, promoting development and repair of nervous system and the like as prebiotics. HMOs can be largely divided into three major categories: (1) fucosylated neutral HMOs; (2) sialylated acidic HMOs; (3) nonfucosylated neutral HMOs. Sialylated HMOs account for about 13% of total HMOs, with sialyllactose (3 (6) '-sialylactose, 3 (6)' -SL) being a representative and simplest compound, with a content of about 2%.3 (6)' -SL is structurally denoted Neu5Ac alpha 2,3 (6) Gal beta 1,4Glc, consisting of N-acetylneuraminic acid (Neu 5 Ac) and lactose units. Has been widely demonstrated to have good prebiotic effect, anti-adhesion antibacterial, antiviral activity, prevention of Necrotizing Enterocolitis (NEC), immunomodulation, regulation of intestinal epithelial cell response, promotion of brain development, and improvement of cognition. The use of 3 '-sialyllactose sodium salt and 6' -sialyllactose sodium salt as novel foods in infant milk powder and food supplements has been approved by the European Union.
The synthesis method of 3 (6)' -SL mainly comprises chemical synthesis, enzymatic synthesis and biological fermentation synthesis. The chemical synthesis of 3 (6)' -SL requires numerous cumbersome protection and deprotection steps. Enzymatic synthesis the cost of the synthesized 3 (6)' -SL is high due to the relatively expensive and low yield of the donor substrate nucleotide sugar. Compared with the enzymatic synthesis, the microbial fermentation method is used for synthesizing the 3 (6)' -SL with higher efficiency and safety. In 2002, priem et al reported for the first time biosynthesis of 3' -SL by fermentation with a metabolic engineering strain, using E.coli JM107 as a host. In 2008 Eric samin et al produced 25.5g/L of 3' -SL by silencing nanT, nanA, nanK, nanE, over-expressing neuB, neuA, neuC and α -2,3 sialyltransferase; in 2010, the subject group constructed a 6' -SL pathway in the same manner, and only 3' -SL was modified from the α -2,3 sialyltransferase in the de novo synthesis pathway to α -2,6 sialyltransferase, and 34g/L of 6' -SL was obtained by fermentation as the highest yield. Furthermore, zhang et al obtained an extracellular yield of 23.1g/L in fed-batch fermentation by inactivating lacZ, nanA and nanK, heterologously over-expressing the 3' -SL pathway genes. However, the currently reported methods for microbial production and the production of sialyllactose still cannot meet the requirements of industrial production. Thus, the search for inexpensive and high-yielding sialyllactose de pathway from the head to solve the bottleneck of current microbial production, and the creation of more efficient production strains is a current urgent need to solve.
Disclosure of Invention
Aiming at the problems and difficulties in the prior art, the invention provides an escherichia coli engineering bacterium for efficiently producing sialyllactose and a construction method thereof.
The invention also provides a chassis cell which takes escherichia coli as a host, and knocks out a beta-galactosidase coding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase coding gene pfkA, an N-acetylmannosamine kinase gene nanK and a mannose-6-phosphate isomerase gene manA.
In one embodiment of the present invention, the chassis cell uses any one of E.coli MG1655, E.coli DH 5. Alpha., E.coli BL21 (DE 3), E.coli JM109, or E.coli HB101 as an expression host
The invention provides a genetic engineering bacterium for preparing 3' -SL, wherein the recombinant escherichia coli comprises the following components: deleting a beta-galactosidase encoding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase encoding gene pfkA, an N-acetylmannosamine kinase gene nanK and a mannose-6-phosphate isomerase gene manA on the genome of escherichia coli, and heterologously expressing a neuB gene encoding acetylneuraminic acid synthase, a neuC gene encoding N-acetylglucosamine isomerase, a neuA gene encoding CMP-sialic acid synthase, and a sialyltransferase gene lst; lactose transferase encoding gene lacY, glutamine synthetase encoding gene glnA and UDP-acetylglucosamine synthesis pathway gene are also over-expressed;
the UDP-acetylglucosamine synthesis pathway is glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU.
In one embodiment of the invention, the genetically engineered bacterium is used for regulating and controlling the protein translation strength of a target gene sialyllactose transferase gene by using the RBS described in any one of SEQ ID NO. 2-4.
In one embodiment of the invention, MBP protein, infB protein and pelB protein with nucleotide sequences shown in SEQ ID NO. 5-7 are adopted as fusion protein tags; MBP protein, infB protein or pelB protein is fused to N end of sialyllactose to raise its soluble expression.
In one embodiment of the invention, the nucleotide sequence of the coding gene lacZ of the beta-galactosidase is shown as SEQ ID NO.8, the nucleotide sequence of the nucleotide aldolase gene nanA is shown as SEQ ID NO.9, the nucleotide sequence of the nucleotide gene pfkA of the 6-phosphofructokinase is shown as SEQ ID NO.10, the nucleotide sequence of the nucleotide gene nanK of the N-acetylmannosamine kinase is shown as SEQ ID NO.11, the nucleotide sequence of the mann-6-phosphate isomerase is shown as SEQ ID NO.12, the nucleotide sequence of the nucleotide gene neuB of the heterologous expression coding acetylneuraminidase is shown as SEQ ID NO.13, the nucleotide sequence of the nucleotide gene neuC of the coding N-acetylglucosamine isomerase is shown as SEQ ID NO.14, the nucleotide sequence of the nucleotide gene neuA of the coding N-sialyltransferase is shown as SEQ ID NO.15, the nucleotide sequence of the nucleotide gene lst of the coding sialyltransferase is shown as SEQ ID NO.16, the nucleotide sequence of the nucleotide gene of the coding N-acetylglucosamine isomerase is shown as SEQ ID NO.16, the nucleotide sequence of the nucleotide gene coding N-acetylglucosamine isomerase is shown as SEQ ID NO.22, and the nucleotide sequence of the nucleotide sequence coding nucleotide sequence of the nucleotide gene coding N-acetylglucosamine isomerase is shown as SEQ ID NO. 18.
In one embodiment of the invention, the deletion refers to knockout or non-expression.
In one embodiment of the invention, pETDuet-1, pRSFDuet-1 and pCDFDuet-1 plasmids are used for overexpression.
In one embodiment of the present invention, the genetically engineered bacterium is an expression host selected from E.coli MG1655, DH 5. Alpha., BL21 (DE 3), JM109 and HB 101.
The invention provides a genetic engineering bacterium for preparing 6' -SL, which comprises the following components: deleting a beta-galactosidase encoding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase encoding gene pfkA, an N-acetylmannosamine kinase gene nanK and a mannose-6-phosphoisomerase gene manA on the genome of escherichia coli, and heterologously expressing a neuB gene encoding acetylneuraminic acid synthase, a neuC gene encoding N-acetylglucosamine isomerase, a neuA gene encoding CMP-sialic acid synthase and a sialyllactose gene pst6-224; lactose transferase encoding gene lacY, glutamine synthetase encoding gene glnA and UDP-acetylglucosamine synthesis pathway gene are also over-expressed;
in one embodiment of the invention, the genetically engineered bacterium is used for regulating and controlling the protein translation strength of a target gene sialyllactose transferase gene by using the RBS described in any one of SEQ ID NO. 2-4.
In one embodiment of the invention, MBP protein, infB protein and pelB protein with nucleotide sequences shown in SEQ ID NO. 5-7 are adopted as fusion protein tags; MBP protein, infB protein or pelB protein is fused to N end of sialyllactose to raise its soluble expression.
The UDP-acetylglucosamine synthesis pathway is glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU.
The nucleotide sequence of the coding gene lacZ of the beta-galactosidase is shown as SEQ ID NO.8, the nucleotide sequence of the nanA gene of the sialic acid aldolase is shown as SEQ ID NO.9, the nucleotide sequence of the pfkA gene of the 6-phosphofructokinase is shown as SEQ ID NO.10, the nucleotide sequence of the nanK gene of the N-acetylmannosamine kinase is shown as SEQ ID NO.11, the nucleotide sequence of the manA gene of the mannose-6-phosphate isomerase is shown as SEQ ID NO.12, the nucleotide sequence of the neuB gene of the heterologous expression coding acetylneuraminidase is shown as SEQ ID NO.13, the nucleotide sequence of the neuC gene of the N-acetylglucosamine isomerase is shown as SEQ ID NO.14, the nucleotide sequence of the neuA gene of the CMP-sialyltransferase is shown as SEQ ID NO.15, the nucleotide sequence of the PST6-224 gene is shown as SEQ ID NO.17, the nucleotide sequence of the nucleotide transferase gene of the nucleotide sequence of the N-acetylmannosamine kinase is shown as SEQ ID NO.17, the nucleotide sequence of the nucleotide transferase is shown as SEQ ID NO.22, and the nucleotide sequence of the nucleotide of the coded N-acetylglucosamine isomerase is shown as SEQ ID NO. 14.
In one embodiment of the invention, the deletion refers to knockout or non-expression.
In one embodiment of the invention, pETDuet-1, pRSFDuet-1 and pCDFDuet-1 plasmids are used for overexpression.
In one embodiment of the present invention, the E.coli is any one of E.coli MG1655, E.coli DH 5. Alpha., E.coli BL21 (DE 3), E.coli JM109, or E.coli HB 101.
In one embodiment of the present invention, the acetylneuraminic acid synthase gene neuB, the N-acetylglucosamine isomerase gene neuC and the CMP-sialic acid synthase gene neuA are derived from Campylobacter jejuni Campylobacter jejuni.
In one embodiment of the present invention, the glucosamine-6-phosphate synthase gene glmS, the glucosamine synthase gene glmM, the UDP-acetylglucosamine pyrophosphorylase gene glmU, the lactose transferase encoding gene lacY and the glutamine synthase gene glnA are derived from E.coli K-12.
In one embodiment of the invention, the sialyltransferase gene is an alpha-2, 3 sialyltransferase gene or an alpha-2, 6 sialyltransferase gene.
In one embodiment of the invention, the gene encoding α -2,3 sialyltransferase is derived from neisseria meningitidis Neisseria meningitidis gene lst; the encoding alpha-2, 6 sialyltransferase gene is selected from the group consisting of the gene pst6-224 derived from Photobacterium sp.
In one embodiment of the invention, the E.coli is E.coli BL21 (DE 3).
In one embodiment of the present invention, the genetically engineered bacterium comprises vectors pETDuet-1, pRSFDuet-1 and pCDFDuet-1, wherein the vectors comprise a neuB gene encoding acetylneuraminic acid synthase, a neuC gene encoding N-acetylglucosamine isomerase, a neuA gene encoding CMP-sialyl synthase, a sialyl lactose transferase gene lst or pst6-224, a lactose transferase gene lacY, a glutamine synthetase gene glnA and a UDP-acetylglucosamine synthesis pathway gene; the UDP-acetylglucosamine synthesis pathway is glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU.
In one embodiment of the present invention, the genetically engineered bacterium expresses gene neuB, neuC, neuA and glnA using pETDuet-1 plasmid.
In one embodiment of the present invention, the genetically engineered bacterium expresses the α -2,3 sialyltransferase gene lst or the α -2,6 sialyltransferase gene pst6-224, as well as the lactose transferase gene lacY, using pRSFDuet-1 plasmid.
In one embodiment of the present invention, the genetically engineered bacterium expresses genes glmM, glmS and glmU using the pCDFDuet-1 plasmid.
In one embodiment of the invention, the recombinant E.coli contains a strongly regulated RBS for regulating the overexpression of the gene of interest.
In one embodiment of the present invention, the ribosome binding site on vector pRSFDuet-1 is replaced with one of RBS T7, RBS 34, RBS 64 and RBS 80.
In one embodiment of the invention, the nucleotide sequences of the ribosome binding sites RBS T7, RBS 34, RBS 64 and RBS 80 are SEQ ID NO. 1-4 in sequence.
In one embodiment of the invention, the recombinant E.coli contains a protein tag for increasing the soluble expression of sialyltransferase.
In one embodiment of the invention, the fusion protein tag InfB is added to the N-terminus of the sialyltransferase.
In one embodiment of the invention, the nucleotide sequences of the fusion protein tags MBP, infB and pelB are shown in SEQ ID NO. 5-7.
The invention provides a method for improving the fermentation production of sialyllactose by escherichia coli, which is characterized in that a beta-galactosidase coding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase coding gene pfkA, an N-acetylmannosamine kinase gene nanK and a mannose-6-phosphate isomerase gene manA on the genome of the escherichia coli are deleted, and a neuB gene encoding acetylneuraminic acid synthetase, a neuC gene encoding N-acetylglucosamine isomerase, a neuA gene encoding CMP-sialyltransferase, a sialyltransferase gene lst or pst6-224 are expressed in a heterologous manner; lactose transferase encoding gene lacY, glutamine synthetase encoding gene glnA and UDP-acetylglucosamine synthesis pathway gene are also over-expressed;
the UDP-acetylglucosamine synthesis pathway is glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU.
The gene engineering bacteria adopts any one of SEQ ID NO. 2-4 to regulate and control the protein translation strength of a target gene sialyllactose transferase gene; preferably, MBP protein, infB protein and pelB protein with nucleotide sequences shown in SEQ ID NO. 5-7 are adopted as fusion protein tags; MBP protein, infB protein or pelB protein is fused to N end of sialyllactose to raise its soluble expression.
In one embodiment of the present invention, the E.coli is any one of E.coli MG1655, E.coli DH 5. Alpha., E.coli BL21 (DE 3), E.coli JM109, or E.coli HB 101.
In one embodiment of the invention, the sialyllactose comprises 3'-SL and 6' -SL.
The invention also provides a method for producing sialyllactose, which is characterized in that glycerol is used as a carbon source, lactose is used as a substrate, and the genetic engineering bacteria are used as fermentation strains for fermentation to produce the sialyllactose.
In one embodiment of the present invention, the amount of glycerol added in the reaction system is: 15-25 g/L.
In one embodiment of the present invention, the lactose is added in an amount of: 5-15 g/L.
In one embodiment of the present invention, the amount of IPTG added by the genetically engineered bacterium is: 0.1 to 0.8mmol/L.
In one embodiment of the invention, the conditions of the fermentation are: 55-65 h.
The invention also provides the genetically engineered bacterium, or the method for improving the yield of sialyllactose or the application of the preparation method in producing sialyllactose and products containing sialyllactose.
Advantageous effects
(1) The invention forms a metabolic pathway of sialyllactose by expressing acetylneuraminic acid synthase gene neuB, N-acetylglucosamine isomerase gene neuC, CMP-sialyl synthase gene neuA, alpha-2, 3 sialyl transferase gene lst and/or alpha-2, 6 sialyl transferase gene pst6-224; and through the overexpression of lactose transferase encoding gene lacY, glutamine synthetase encoding gene glnA and glucosamine-6-phosphate synthetase encoding gene glmS, glucosamine synthetase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU, the balance and accumulation of precursor substances are further regulated and controlled, and constant flow sialyllactose is targeted; silencing a beta-galactosidase encoding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase encoding gene pfkA, an N-acetylmannosamine kinase gene nanK and mannose-6-phosphoisomerase manA, and blocking shunt metabolism of a bypass pathway; finally, the soluble expression of sialyltransferase is further improved by RBS replacement and screening of different fusion proteins, and the purpose of improving sialyllactose is achieved by a series of operations.
(2) The capacity of the engineering bacteria constructed by the invention for producing sialyllactose through fermentation culture: 3'-SL from initial 2.68g/L to 8.09g/L,6' -SL from initial 0.51g/L to 4.67g/L; in a 3L fermentation tank, the yields of 3'-SL and 6' -SL respectively reach 44.23g/L and 36.29g/L, and the production capacity lays a foundation for the industrial production of sialyllactose.
Drawings
FIG. 1 is a diagram of sialyllactose metabolic pathway.
FIG. 2 is a graph showing sialyllactose content.
FIG. 3 is a diagram of sialyllactose fed-batch fermentation.
Detailed Description
The present invention will be further described with reference to examples and drawings, wherein plasmids, PCR reagents, restriction enzymes, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercially available, and the specific operations are carried out according to the kit instructions. Embodiments of the invention are not limited thereto, and other unspecified experimental operations and process parameters are conducted in accordance with conventional techniques. Sequencing of plasmid and DNA products was performed by the Biotechnology Co., ltd. Preparation of E.coli competence: kit for biological engineering (Shanghai) Co., ltd. pETDuet-1, pRSFDuet-1, pCDFDuet-1 referred to in the examples below were purchased from Novagen.
The following examples relate to the following media:
LB liquid medium (g/L): yeast extract 5, tryptone 10, sodium chloride 10.
LB solid medium (g/L): yeast extract 5, tryptone 10, sodium chloride 10, agar powder 20.
Fermentation medium (g/L): glycerin 20, citric acid 1.7, monopotassium phosphate 13.5, diammonium phosphate 4, magnesium sulfate heptahydrate 1.4, trace metal solution 10mL/L (ferric citrate 10g/L, zinc sulfate heptahydrate 2.25g/L, cupric sulfate pentahydrate 1.0g/L, manganese sulfate monohydrate 0.35g/L, borax 0.23g/L, ammonium heptamolybdate 0.11g/L, calcium chloride dihydrate 2.0 g/L), and pH 6.80.
The detection method involved in the following examples is as follows:
the sialyllactose described in the embodiment of the present invention is determined by using High Performance Liquid Chromatography (HPLC), specifically:
the fermentation broth was boiled at 100deg.C for 10min to break the cells, centrifuged at 12000r/min for 10min, and the supernatant was filtered through 0.22 μm membrane and detected by HPLC.
HPLC detection conditions: a differential refractive detector; the chromatographic column is Rezex ROA-organic acid (Phenomenex, USA) with the column temperature of 50 ℃; h with mobile phase of 0.005mmol/L 2 SO 4 The flow rate of the aqueous solution is 0.6mL/min; the sample loading was 10. Mu.L.
The following examples relate to sialyllactose fermentation culture methods:
engineering bacteria colonies cultured overnight on the LB solid medium are selected and inoculated into 5mL of LB liquid medium, and cultured for 12 hours at 37 ℃ and 200r/min to be used as seed liquid. Transferring seed liquid according to 1% (v/v) inoculum size to liquid filling sizeCulturing in 50mL fermentation medium at 37deg.C and 200r/min to bacterial OD 600 The value is 0.6-0.8, IPTG is added to make the final concentration of the mixture be 0.2mmol/L, and lactose is added to make the final concentration of lactose be 10g/L, and the mixture is induced and cultured for 50h under the conditions of 25 ℃ and 200 r/min.
Example 1: silencing of E.coli BL21 (DE 3) genomic gene lacZ, nanA, pfkA, nanK and manA
The lacZ, nanA, pfkA, nanK and manA genes in the escherichia coli BL21 (DE 3) genome are silenced by using a CRISPR-Cas9 gene silencing system, and the specific steps are as follows (the related primer sequences are shown in table 1):
(1) Using E.coli BL21 (DE 3) genomic DNA as a template, homologous upstream and downstream fragments of lacZ, nanA, pfkA, nanK and manA were amplified by PCR using primer pairs lacZ-up-F/R and lacZ-down-F/R, nanA-up-F/R and nanA-down-F/R and pfkA-down-F/R, nanK-up-F/R and nanK-down-F/R, manA-up-F/R and manA-down-F/R, respectively. After the product is purified and recovered, homologous upstream and downstream fragments of lacZ, nanA, pfkA, nanK and manA are respectively used as templates, and the homologous repair arms downZ, downr-nanA, downr-pfkA, downr-nanK and downr-manA are obtained by amplifying and connecting the upstream and downstream fragments through SOE-PCR technology by adopting lacZ-up-F/lacZ-down-R, nanA-up-F/nanA-down-R, pfkA-up-F/pfkA-down-R, nanK-up-F/nanK-down-R and manA-up-F/manA-down-R primers.
(2) The pTargetF plasmid (Addgene: # 62226) was used as a template, lacZ-sg-F/R, nanA-sg-F/R, pfkA-sg-F/R, nanK-sg-F/R and manA-sg-F/R were used as primers, N20 sequences complementary to lacZ, nanA, pfkA, nanK and manA sequences were introduced into the pTargetF plasmid by PCR amplification to obtain pTargetF plasmids with targeting lacZ, nanA, pfkA, nanK and manA (i.e., targeting plasmids pTargetF-lacZ, pTargetF-nanA, pTargetF-pfkA, pTargetF-nanK and pTargetF-manA with lacZ, nanA, pfkA, nanK and manA specific N20 sequences, respectively), the PCR amplified products were transformed into E.coli DH 5. Alpha. Competent cells, coated on LB plates (containing spectinomycin), extracted by overnight culture at 37℃and sequencing.
(3) E.coli BL21 (DE 3) competent cells were thawed on ice for 10min, 5. Mu.L of pCas plasmid (Addgene: # 60847) was added to 100. Mu.L of competent cells and gently mixed. Ice bath for 30min, heat shock at 42 ℃ for 90s, and immediately placing on ice for 2-3 min. 1mL of fresh LB liquid medium is added, after 1h of culture at 30 ℃ and 200r/min, centrifugation is carried out at 3500r/min for 5min, the supernatant is discarded, the thalli are coated on an LB plate containing kanamycin, and the thalli are placed in a constant temperature incubator at 30 ℃ for overnight culture until single colonies of E.coli BL21 (DE 3)/pCas are grown.
(4) Picking up E.coli BL21 (DE 3)/pCas single colony overnight culture seed liquid, transferring LB liquid culture medium according to 1% volume ratio, and culturing OD at 30deg.C 600 To 0.2, arabinose with a final concentration of 30mmol/L was added to induce pCas-lambda-red system expression, and the culture was continued until the logarithmic growth phase was reached to prepare E.coli BL21 (DE 3)/pCas competence.
(5) E.coli BL21 (DE 3)/pCas competence was electrotransformed with pTargetF-lacZ plasmid (total amount 500 ng) and gene homology repair arm donor-lacZ (total amount 1 μg), spread on LB plates (kanamycin and spectinomycin), incubated overnight at 30℃and single colonies on plates were picked for PCR validation, positive transformants were screened and sent to Ten-Sida Biotechnology Co.Ltd for sequencing.
(6) Culturing the positive clone colony which is verified to be successfully silenced by sequencing in the step (5) in LB liquid medium containing kanamycin until OD 600 The value reaches 0.2, IPTG with the final concentration of 0.5mmol/L is added, the mixture is cultured for 12 to 16 hours at 30 ℃ to remove pTargetF plasmid, then the mixture is cultured for 12 hours at 42 ℃, pCas plasmid is removed, and E.coll BL21 (DE 3) delta lacZ of the genome silencing lacZ gene is obtained.
(7) Gene nanA, pfkA, nanK and manA were sequentially silenced with E.coli BL21 (DE 3) ΔlacZ as a host strain, and silencing was performed with reference to silencing of the above gene lacZ to obtain corresponding BL21 (DE 3) ΔlacZ ΔnanA, ΔpfkA ΔnanK and BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA ΔnanK ΔmanA strains.
Table 1: sgRNA and silencing primers
Example 2: construction of sialyllactose recombinant bacteria
The construction of recombinant bacteria comprises the following specific steps (the related primer sequences are shown in Table 2):
(1) neuB, neuC, neuA, lst and pst6-224 Gene fragment acquisition:
the sequence of neuB, neuC, neuA gene derived from Campylobacter jejuni Campylobacter jejuni (the nucleotide sequence of the heterologous expression of the neuB gene encoding acetylneuraminic acid synthetase is shown as SEQ ID NO.13, the nucleotide sequence of the neuC gene encoding N-acetylglucosamine isomerase is shown as SEQ ID NO.14, the nucleotide sequence of the neuA gene encoding CMP-sialyl synthetase is shown as SEQ ID NO. 15), the sequence of lst gene derived from Neisseria meningitidis Neisseria meningitidis (the nucleotide sequence of sialyllactose gene lst is shown as SEQ ID NO. 16), the sequence of pst6-224 gene derived from Photobacterium sp (the nucleotide sequence of sialyllactose gene pst6-224 is shown as SEQ ID NO. 17).
(2) Amplifying a neuB gene fragment by taking the synthesized neuB gene fragment as a template and a neuB-F/neuB-R as a primer, purifying and recovering a DNA fragment, and connecting the recovered gene fragment neuB to NcoI/BamHI cleavage sites of a vector pETDuet-1 through a seamless cloning kit (Nanjinouzan life technologies Co., ltd.) to obtain a plasmid pET-neuB;
amplifying a neuC gene fragment by taking the synthesized neuC gene fragment as a template and a neuC-F/neuC-R as a primer, purifying and recovering a DNA fragment, and connecting the recovered neuC gene fragment between PstI/HindIII enzyme cutting sites of a vector plasmid pET-neuB to obtain a plasmid pET-BC;
the synthesized neuA gene fragment is used as a template, the neuA-F/neuA-R is used as a primer to amplify the neuA gene fragment, the DNA fragment is purified and recovered, and the recovered neuA gene fragment is connected between NdeI/XhoI cleavage sites of a vector pET-BC to obtain a plasmid pET-BCA.
(3) The synthesized lst and pst6-224 gene fragments are used as templates, lst-F/lst-R, pst6-224-F/pst6-224-R are used as primers to amplify the lst and pst6-224 gene fragments, DNA fragments are purified and recovered, and the recovered lst and pst6-224 gene fragments are respectively connected between NcoI/BamHI cleavage sites of a vector pRSFDuet-1 to respectively obtain plasmids pRS-lst and pRS-pst6.
(4) Obtaining of lacY Gene fragment: the genome of Escherichia coli K-12 is used as a template, lacY-F/lacY-R is used as a primer to amplify a lacY gene fragment, DNA fragments are purified and recovered, the recovered lacY gene fragment is respectively connected between NdeI/XhoI cleavage sites of plasmids pRS-lst and pRS-pst6 through a seamless cloning kit (Nanjinopran life technologies Co., ltd.) to finally obtain plasmids pRS-LY and pRS-PY.
TABLE 2 plasmid construction primers
(4) Transferring the plasmids pET-BCA and pRS-LY obtained in the steps into E.coli BL21 (DE 3) DeltalacZ obtained in the example 1 according to key genes in a sialyllactose metabolic synthesis path to obtain engineering bacteria M1; the plasmids pET-BCA and pRS-PY were transferred into E.coli BL21 (DE 3) ΔlacZ to obtain engineering bacterium F1. After fermentation culture of these two strains, the product was confirmed to be sialyllactose by HPLC and LC-MS identification, and the yields were 2.68g/L of 3'-SL and 0.51g/L of 6' -SL, respectively (see Table 3). The sialyllactose metabolic pathway diagrams of strains M1 and F1 are shown in FIG. 1.
Example 3: influence of overexpression of precursor substances of the de novo synthetic pathway on sialyllactose
(1) Overexpression of UDP-acetylglucosamine
The availability of acetylmannosamine and sialic acid has a certain impact on the efficiency of sialyllactose synthesis. UDP-acetylglucosamine is a precursor of acetylmannosamine and sialic acid in the sialyllactose biosynthetic pathway. Improving intracellular flux to the precursor substance UDP-acetylglucosamine to enhance the production of acetylmannosamine and sialic acid, and then targeting the flux to sialyllactose. Three genes, including glmS (encoding glucosamine-6-phosphate synthase), glmM (encoding glucosamine synthase) and glmU (encoding UDP-acetylglucosamine pyrophosphorylase), are critical in the production of UDP-acetylglucosamine. For this, the gene fragments of glmS, glmM and glmU were amplified with primers glmS-F/R, glmM-F/R and glmU-F/R (primer sequences are shown in Table 2) respectively, and the DNA fragments were recovered by purification, and the recovered glmS, glmM and glmU gene fragments were sequentially ligated between NcoI/BamHI cleavage sites of vector pCDFDuet-1 by using the construction method of example 2, using a seamless cloning kit (Nannunozan Life technologies Co., ltd.) to obtain plasmid pCD-SMU.
Based on the strains M1 and F1 in example 2, the plasmids pCD-SMU were expressed, respectively, to obtain 2 different engineering bacteria, which were designated as M2 (E.coli BL21 (DE 3) ΔlacZ/pET-BCA/pRS-LY/pCD-SMU) and F2 E.coli BL21 (DE 3) ΔlacZ/pET-BCA/pRS-PY/pCD-SMU, respectively.
The engineering bacteria M2 is fermented and cultured by shaking bottles, so that the yield of 3' -SL is 3.94g/L; the yield of the engineering bacteria F2 obtained is 1.05g/L. The results indicate that the appropriate expression of UDP-acetylglucosamine can increase the production of sialyllactose.
(2) Construction and expression of glutamine circulation System
Fructose-6-phosphate and glucosamine-6-phosphate are important precursors for the de novo synthesis pathway of 3 (6)' -SL. The cyclic utilization of L-glutamine + fructose-6-phosphate, L-glutamic acid + glucosamine-6-phosphate, can be achieved by constructing a glutamine circulating system (figure 1). Two genes, glmS (encoding glucosamine-6-phosphate synthase) and glnA (encoding glutamine synthase), catalyze this cycle. For this, the glnA gene fragment was amplified with the E.coli K-12 genome as a template and with the primers glnA-F/R (primer sequences shown in Table 2), and the DNA fragment was recovered by purification, and the recovered glnA gene fragment was connected between the SlaI/PacI cleavage sites of pET-BCA by a seamless cloning kit (Nannonvirginal Life technologies Co., ltd.) using the construction method of example 2, to obtain plasmid pET-BCA.
Strains M3 and F3 (Table 3) were formed by expressing the glnA gene using E.coli BL21 (DE 3) DeltalacZ as a host.
TABLE 3 detailed information on engineering bacteria
As a result, it was found that 4.23g/L and 1.29g/L of 3'-SL and 6' -SL were obtained by shake flask fermentation culture. This suggests that overexpression of the glnA gene promotes sialyllactose production.
Example 4: effects of silencing genes nanA, pfkA, nanK and manA on sialyllactose production
To increase the synthesis efficiency of sialyllactose, the silencing strains BL21 (DE 3) ΔlacZ ΔnanA, BL21 (DE 3) ΔlacZ ΔpnakA ΔpfkA ΔnanK and BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA ΔnanK ΔmannA were obtained by blocking other metabolic pathway losses of the precursor substances by silencing the sialylaldehyde aldolase gene nanA, the N-acetylmannosamine kinase gene nanK and the mannose-6-phosphate isomerase manA using the CRISPR/Cas9 system using E.coli BL21 (DE 3) ΔlacZ as the starting strain as described in example 1.
Plasmid combination 1 (pET-BCAA, pRS-LY, PCD-SMU) and plasmid combination 2 (pET-BCAA, pRS-PY, PCD-SMU) were transformed into the silencing strains BL21 (DE 3) ΔlacZ ΔnanA, BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA ΔnanK and BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA ΔnanK ΔmanA, respectively, yielding strains M4 to M7 and F4 to F7, and the highest 3' -SL yields of 6.98g/L and 3.56g/L were obtained by shake flask fermentation culture (see Table 4).
TABLE 4 engineering bacteria detailed information
The results showed that the highest yields of strains M4-M7 and F4-F7 were increased 1.67-fold and 2.76-fold, respectively, over M3 and F3, indicating that blocking the bypass path of sialyllactose helped to increase the conversion of 3'-SL and 6' -SL.
Example 5: increasing soluble expression of sialyltransferases
(1) Substitution of the original ribosome binding site on the expression plasmid
In addition to the ribosome binding site (RBS T7) of the expression plasmid itself, 3 RBSs (RBS 34, RBS 64 and RBS 80) with different intensities are selected (different RBS sequences are shown in Table 5) to regulate the protein translation intensity of the target gene.
TABLE 5RBS sequence
Using the plasmids pRS-LY and pRS-PY constructed in example 2 as templates, corresponding fragments were obtained by using the primers 34lstF/R,64lstF/R,80lstF/R,34pst6F/R,64pst6F/R and 80pst6F/R (primer sequences shown in Table 2), and RBS T7 was assembled using a seamless cloning kit (Nannonuzan Life technologies Co., ltd.) to replace the original vector to obtain the corresponding recombinant plasmids pRS- (34) -LY, pRS- (64) -LY, pRS- (80) -LY, pRS- (34) -PY, pRS- (64) -PY and pRS- (80) -PY.
The obtained different plasmid combinations pRS- (34) -LY, pRS- (64) -LY and pRS- (80) -LY are respectively transformed into BL21 (DE 3) DeltalacZ DeltananA DeltapfkA DeltananK DeltamanA containing plasmids pET-BCAA and pCD-SMU to obtain strains M8-M10;
the different plasmid combinations pRS- (34) -PY, pRS- (64) -PY and pRS- (80) -PY obtained are respectively transformed into BL21 (DE 3) delta lacZ delta nanA delta pfkA delta nanK delta manA containing plasmids pET-BCAA and pCD-SMU to obtain strains F8-F10;
the highest 3'-SL yield of 7.58g/L and the highest 6' -SL yield of 3.89g/L (see Table 6) can be obtained by shake flask fermentation culture of M8-M10 and F8-F10, and the yields are respectively improved by 1.09 times and 1.10 times compared with M3 and F3.
(2) Fusion protein tag addition to promote soluble expression
The fusion protein expression tag can increase the solubility of the fusion protein that is overexpressed in bacteria. According to the invention, 3 protein tags with different sequences, namely MBP protein, infB protein and pelB protein (the nucleotide sequences of fusion protein tags MBP, infB and pelB are shown as SEQ ID NO. 5-7) are selected according to literature reports, and the fusion protein tags are fused at the N end of target protein to improve the soluble expression.
Obtaining gene fragments encoding MBP protein, infB protein and pelB protein: the principal biological engineering (Shanghai) stock company. The synthesized MBP, infB, pelB gene fragment was used as a template, and the MBP-F/MBP-R, infB-F/InfB-R, pelB-F/pelB-R was used as a primer to amplify a MBP, infB, pelB gene fragment (primer sequences shown in Table 2), and the DNA fragment was recovered by purification, and the recovered gene fragment MBP, infB, pelB was ligated to N-terminal of the desired gene lst or pst6-226 in plasmids pRS- (80) -LY and pRS- (80) -PY, respectively, by means of a seamless cloning kit (Nannonuzan life technologies Co., ltd.) to obtain plasmids pRS-MBP- (80) -LY, pRS-InfB- (80) -LY, pRS-pelB- (80) -LY, pRS-MBP- (80) -PY, pRS-InfB- (80) -PY.
The different plasmid combinations pRS-MBP- (80) -LY, pRS-InfB- (80) -LY and pRS-pelB- (80) -LY obtained are respectively transformed into silent strains BL21 (DE 3) DeltalacZ DeltananA DeltapfkA DeltananK DeltamanA containing plasmids pET-BCAA and pCD-SMU to obtain strains M11-M13;
the different plasmid combinations pRS-MBP- (80) -PY, pRS-InfB- (80) -PY and pRS-pelB- (80) -PY obtained were transformed into silent strains BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA ΔnanK ΔmanA containing plasmids pET-BCAA and pCD-SMU, respectively, to give strains F11 to F13. The sialyllactose content of each strain is shown in FIG. 2.
TABLE 6 engineering bacteria detailed information
Strains M11-M13 and F11-F13, the highest 3'-SL yield of 8.09g/L and the highest 6' -SL yield of 4.67g/L (see Table 6) can be obtained by shake flask fermentation culture, and the yields are respectively improved by 1.91 times and 3.62 times compared with M3 and F3, which shows that partial fusion protein tags can promote the transformation of sialyllactose.
Example 6: production of sialyllactose by batch feeding of fermentation tank
Respectively inoculating the genetically engineered bacteria M12 and F12 constructed in the embodiment 5 into 100mL of LB liquid medium, culturing at 37 ℃ for 12h in a shaking flask at 200r/min to obtain seed liquid; the seed solution was inoculated into a fermentation medium having a working volume of 1L at a fermentation temperature of 37℃and a stirring speed of 800r/min at an inoculum size of 10% by volume, and was aerated at 1vvm at pH 6.80 (automatic control of additional ammonia). Fermentation for 18h (OD) 600 About 15), and IPTG was added at a final concentration of 20g/L lactose and 0.5 mmol/L. To maintain the growth of the cells and the synthesis of sialyllactose, 750g/L of glycerol (20 g/L of MgSO) was fed after the initial carbon source was consumed 4 ·7H 2 O) to supplement carbon source, 200g/L lactose is added after the initial lactose is consumed, and the concentration of lactose in the system is maintained to be about 10g/L, and fermentation culture is carried out for 55h. During the whole culture process, the bacterial OD of 3'-SL and 6' -SL 600 Yields of 121 and 97,3'-SL and 6' -SL, respectively, reach up to 44.23g/L and 36.29g/L (FIG. 3).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A chassis cell is characterized in that escherichia coli is taken as a host, and a beta-galactosidase coding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase coding gene pfkA, an N-acetylmannosamine kinase gene nanK and a mannose-6-phosphate isomerase gene manA are knocked out; preferably, the chassis cell uses any one of E.coli MG1655, E.coli DH 5. Alpha., E.coli BL21 (DE 3), E.coli JM109 or E.coli HB101 as an expression host.
2. A genetically engineered bacterium for producing sialyllactose, characterized in that the chassis cell of claim 1 is used as a host, and the neuB gene encoding acetylneuraminic acid synthetase, the neuC gene encoding N-acetylglucosamine isomerase, the neuA gene encoding CMP-sialyl synthetase, the gene encoding sialyllactosyltransferase lst or pst6-224 are expressed in a heterologous manner; meanwhile, the genes glnA and UDP-acetylglucosamine synthesis pathway genes encoding glutamine synthetase are also overexpressed;
the UDP-acetylglucosamine synthesis pathway genes are glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU.
3. The genetically engineered bacterium of claim 2, wherein the genetically engineered bacterium modulates the protein translation strength of a sialyllactose gene of a target gene using any one of SEQ ID nos. 2 to 4; preferably, MBP protein, infB protein and pelB protein with nucleotide sequences shown in SEQ ID NO. 5-7 are used as fusion protein tags, and the expression of sialyllactose transferase is improved by fusing the MBP protein, the InfB protein or the pelB protein at the N end of the sialyllactose transferase; preferably, the genetically engineered bacterium is overexpressed using pETDuet-1, pRSFDuet-1 and pCDFDuet-1 plasmids.
4. The chassis cell according to claim 1 or the genetically engineered bacterium according to claim 2 or 3, wherein the nucleotide sequence of the gene lacZ encoding the β -galactosidase is shown in SEQ ID NO.8, the nucleotide sequence of the sialylaldehyde aldolase gene nanA is shown in SEQ ID NO.9, the nucleotide sequence of the gene pfkA encoding the 6-phosphofructokinase is shown in SEQ ID NO.10, the nucleotide sequence of the gene nanK encoding the N-acetylmannosamine kinase is shown in SEQ ID NO.11, the nucleotide sequence of the gene manA encoding the mannose-6-phosphoisomerase is shown in SEQ ID NO.12, the nucleotide sequence of the gene neuB encoding the acetylneuraminic acid synthetase is shown in SEQ ID NO.13, the nucleotide sequence of the neuC gene of the N-acetylglucosamine isomerase is shown as SEQ ID NO.14, the nucleotide sequence of the neuA gene of the CMP-sialyl synthetase is shown as SEQ ID NO.15, the nucleotide sequence of the sialyl lactose transferase gene lst is shown as SEQ ID NO.16, the nucleotide sequence of the sialyl lactose transferase gene pst6-224 is shown as SEQ ID NO.17, the nucleotide sequence of the glutamine synthetase encoding gene glnA is shown as SEQ ID NO.18, the nucleotide sequence of the glucosamine-6-phosphate synthetase encoding gene glmS is shown as SEQ ID NO.19, the nucleotide sequence of the glucosamine synthetase encoding gene glmM is shown as SEQ ID NO.20, and the nucleotide sequence of the UDP-acetylglucosamine pyrophosphorylase encoding gene glmU is shown as SEQ ID NO. 21.
5. The genetically engineered bacterium of any one of claims 2 to 4, wherein the genetically engineered bacterium is an expression host comprising any one of β -galactosidase encoding gene lacZ, sialylaldehyde aldolase gene nanA, 6-phosphofructokinase encoding gene pfkA, N-acetylmannosamine kinase gene nanK, and mannose-6-phosphate isomerase gene manA knocked out of e.coli MG1655, e.coli dh5α, e.coli BL21 (DE 3), e.coli JM109, or e.coli HB 101.
6. A method for improving the fermentation production of sialyllactose by escherichia coli, which is characterized in that the method is that a beta-galactosidase coding gene lacZ, a sialylaldehyde aldolase gene nanA, a 6-phosphofructokinase coding gene pfkA, an N-acetylmannosamine kinase gene nanK and a mannose-6-phosphate isomerase gene manA on the genome of the escherichia coli are deleted, and a neuB gene encoding acetylneuraminic acid synthase, a neuC gene encoding N-acetylglucosamine isomerase, a neuA gene encoding CMP-sialyltransferase, a sialyltransferase gene lst or pst6-224 are expressed in a heterologous manner; meanwhile, the glutamine synthetase coding genes glnA and UDP-acetylglucosamine synthesis pathway genes are also overexpressed;
the UDP-acetylglucosamine synthesis pathway genes are glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and UDP-acetylglucosamine pyrophosphorylase encoding gene glmU;
the gene engineering bacteria adopts any one of SEQ ID NO. 2-4 to regulate and control the protein translation strength of a target gene sialyllactose transferase gene; preferably, MBP protein, infB protein and pelB protein with nucleotide sequences shown in SEQ ID NO. 5-7 are used as fusion protein tags, and the expression of sialyllactose is improved by fusing the MBP protein, the InfB protein or the pelB protein at the N end of the sialyllactose.
7. The method of claim 6, wherein the E.coli is any one of E.coli MG1655, E.coli DH 5. Alpha., E.coli BL21 (DE 3), E.coli JM109, or E.coli HB 101.
8. A method for preparing sialyllactose, which is characterized in that glycerol is used as a carbon source, lactose is used as a substrate, and the genetic engineering bacteria of any one of claims 2-5 are used as fermentation strains to produce the sialyllactose by fermentation.
9. The method according to claim 8, wherein the glycerol is added in the reaction system in an amount of: 15-25 g/L; preferably, the lactose is added in the following amount: 5-15 g/L.
10. Use of a genetically engineered bacterium according to any one of claims 2 to 5 or a method according to any one of claims 6 to 9 for the preparation of sialyllactose or a product containing sialyllactose.
CN202311355000.9A 2023-10-18 2023-10-18 Genetically engineered bacterium for producing sialyllactose and application thereof Pending CN117487729A (en)

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