CN117487726A - Genetically engineered bacterium with endogenous hyaluronic acid coating, construction method and application thereof - Google Patents
Genetically engineered bacterium with endogenous hyaluronic acid coating, construction method and application thereof Download PDFInfo
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- CN117487726A CN117487726A CN202210919679.9A CN202210919679A CN117487726A CN 117487726 A CN117487726 A CN 117487726A CN 202210919679 A CN202210919679 A CN 202210919679A CN 117487726 A CN117487726 A CN 117487726A
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- hyaluronic acid
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- engineered bacterium
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Abstract
The invention relates to a genetically engineered bacterium with an endogenous hyaluronic acid coating, and a construction method and application thereof. The genetically engineered bacterium is constructed by taking probiotics as an initial strain, over-expressing hyaluronic acid synthase, uridine diphosphate-glucose-6-dehydrogenase, glucose-1-phosphate uridylate transferase and L-glutamine-D-fructose-6-phosphate aminotransferase, knocking out epsA genes, and down-regulating the expression level of lactic acid dehydrogenase. The invention provides a construction method of the genetically engineered bacterium, the constructed genetically engineered bacterium can form a hyaluronic acid coating outside a bacterium body, has higher survival ability and longer colonization in vivo, can continuously generate hyaluronic acid in intestinal tracts after colonization, and can further supplement the hyaluronic acid in vivo. The genetically engineered bacterium provided by the invention can be used for producing hyaluronic acid, can be used for preparing probiotics for enhancing intestinal barriers and regulating intestinal flora, and a composition of the probiotics and prebiotics, and has good application value.
Description
Technical Field
The invention relates to a genetically engineered bacterium with an endogenous hyaluronic acid coating, and a construction method and application thereof, and belongs to the technical field of biology.
Background
Probiotics are "living microorganisms" that are beneficial to host health and are effective in treating gastrointestinal disorders and food allergies, improving brain function and inflammation in the body. In recent years, bacterial biotherapy has been widely focused by researchers by engineering microorganisms to perform specific functions in vivo. With the development of synthetic biology and genetic manipulation tools, traditional probiotics can play a more positive role after being reasonably engineered.
However, the digestive process of the gastrointestinal tract impairs oral probiotic activity, for which various probiotic delivery systems have been developed, wherein coating techniques perform well on oral probiotic delivery, e.g. after Escherichia coli Nissle (EcN) 1917 has been coated with Tannic Acid (TA) and mucin (mucin) in sequence, the gastrointestinal fluid tolerance is significantly improved, survival and colonisation in the body is improved, and at the same time play an important role in restoring the intestinal barrier and regulating intestinal homeostasis. Bifidobacteria encapsulated with a lipid bilayer coating may significantly increase intestinal colonisation, produce a constant concentration of gamma-aminobutyric acid in the intestine, and maintain a longer half-life for the treatment of parkinson's disease. In natural environments, certain microorganisms can protect against the environment by producing their own biofilms or other types of extracellular matrix. In light of the above, it is suggested that the biofilm-coated bacillus subtilis can be obtained by culturing bacillus subtilis to obtain a self-coating of the biofilm, and the bacillus subtilis coated with the biofilm has 125 times of high oral utilization rate and 17 times of high colonization rate, and has better treatment effect in a mouse model for treating staphylococcus aureus infection. Thus, enhancing self-protection by stimulating the ability of the strain itself is also an effective way of oral probiotic delivery. Compared with an exogenous coating, the protective coating of the microorganism provides more durable and more powerful protection for the thalli, and the thalli only needs to be cultured during preparation, so that the operation process is simple.
Hyaluronic Acid (HA) is a glycosaminoglycan that can be synthesized by microorganisms, itself as an active ingredient of the extracellular matrix providing a protective layer for bacteria. In the process of producing hyaluronic acid by corynebacterium glutamicum, the morphological observation of the thallus shows that the wrapping layer formed by the hyaluronic acid outside the thallus limits the thallus to absorb nutrient substances, and simultaneously, the thallus can be well isolated from the external environment. In particular, hyaluronic acid has been widely used for modification and delivery of drugs based on its good biocompatibility and physiological functions, for example, bilirubin can effectively treat inflammatory bowel disease after being coated with hyaluronic acid, and has a better therapeutic effect on bacterial enteritis after lactobacillus rhamnosus is coated with a reduction-responsive hyaluronic acid hydrogel. These studies above have largely inspired that we designed a hyaluronic acid self-coating for oral probiotic delivery. Meanwhile, the prebiotic effect of the hyaluronic acid is expected to be fully exerted, so that good combination of probiotics and prebiotics is realized. However, there are few strains in nature that have a protective shell of natural hyaluronic acid, and thus it is a primary problem to confer the ability to endogenously produce hyaluronic acid to probiotic strains and to form a protective layer outside the cell.
Streptococcus thermophilus Streptococcus thermophilus (S.thermophilus) is a common lactic acid bacterium and is widely used in daily fermented foods such as yogurt, cheese, etc. Currently, several studies suggest that s.thermophilus has many beneficial functions in inhibiting colorectal cancer, alleviating lactose intolerance and treating antibiotic-associated diarrhea. However, the oral delivery efficiency of s.thermophilus has yet to be improved to allow better therapeutic action in vivo. At present, no study has been reported on the enhancement of the oral delivery effect of probiotics by engineering them to produce a hyaluronic acid coating. Based on the above, the invention stimulates the S.thermophilus to form the hyaluronic acid self-protective layer through strain transformation, thereby enhancing the tolerance of the strain in the gastrointestinal tract and generating better probiotics in vivo.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a genetically engineered bacterium with an endogenous hyaluronic acid coating, and a construction method and application thereof.
The technical scheme of the invention is as follows:
the genetically engineered bacterium with the endogenous hyaluronic acid coating is constructed by taking probiotics as an original strain, performing genetic engineering operation to overexpress hyaluronic acid synthase, uridine diphosphate-glucose-6-dehydrogenase, glucose-1-phosphate uridylate transferase and L-glutamine-D-fructose-6-phosphate aminotransferase, knocking out epsA genes, and down-regulating the expression level of lactate dehydrogenase.
Preferably according to the invention, the probiotics are those in the form of generally accepted safety additives, including streptococcus thermophilus, lactococcus lactis, lactobacillus bulgaricus and bifidobacteria;
further preferably, the probiotic is streptococcus thermophilus.
According to the invention, preferably, the hyaluronic acid synthase is derived from streptococcus pyogenes (Streptococcus pyogenes), the coding gene is sphasA, and the nucleotide sequence after codon optimization is shown as SEQ ID NO. 1; the uridine diphosphate-glucose-6-dehydrogenase and glucose-1-phosphate uridylic acid transferase are derived from streptococcus zooepidemicus (Streptococcus zooepidemicus), the coding genes of which are hasB and hasC respectively, and the IDs of GenBank are CP001129, sez _0200 and CP001129, sez _0201 respectively; the L-glutamine-D-fructose-6-phosphate aminotransferase is from streptococcus thermophilus (S.thermophilus), the coding gene is glmS, the ID of GenBank is CP000419, STER_0903; the hasB and hasC are two contiguous gene sequences; the GenBank ID of the epsA gene nucleotide sequence is CP000419, STER_1071.
According to a preferred aspect of the present invention, the down-regulating of the expression level of lactic acid in streptococcus thermophilus is specifically: mutating the aspartic acid residue at position 9 of a streptococcus thermophilus CRISPR-related protein (CRISPR-associated protein, cas 9) to an alanine residue, mutating the histidine residue at position 599 to an alanine residue, making it a Cas9 (read Cas9, dCas 9) which loses DNA cleavage activity; the aim of down-regulating the expression level of lactic acid in streptococcus thermophilus is achieved by respectively expressing dcas9 and guide RNA (sgRNA) of targeted lactic acid dehydrogenase by using a strong promoter P11, wherein the ID of GenBank of the protein related to the streptococcus thermophilus CRISPR is CP000419 and STER_0709; the sgRNA sequence is shown as SEQ ID NO. 2.
The construction method of the genetically engineered bacterium with the endogenous hyaluronic acid coating comprises the following steps:
(1) Constructing an overexpression cassette HP11-SP comprising a strong promoter P11 and sphasA of Streptococcus pyogenes; constructing an overexpression cassette HP32-ZBC containing a strong promoter P32 and hasB and hasC of streptococcus zooepidemicus; constructing an overexpression cassette HP32-S comprising a strong promoter P32 and glmS of Streptococcus thermophilus;
(2) Constructing a knockout box HDE containing upper and lower homology arm sequences of the epsA gene;
(3) Constructing a mutation box HD1 comprising a mutation of the aspartic acid residue at position 9 of Cas9 to an alanine residue; constructing a mutation box HH2 comprising a mutation of the histidine residue at position 599 of Cas9 to an alanine residue; constructing an insertion cassette HP11 comprising a strong promoter P11; constructing an insertion box HP11-sgRNA1 containing a strong promoter P11 and sgRNA1;
(4) Sequentially converting the over-expression cassettes HP11-SP, HP32-ZBC and HP32-S into streptococcus thermophilus LMD-9, and selecting positive recombinants to obtain genetically engineered bacteria P32-ZBCS;
(5) Converting the knockout box HDE into genetic engineering bacteria P32-ZBCS, and selecting positive recombinants to obtain knockout strain DE;
(6) The mutant box HD1, the mutant box HH2, the insert box HP11 and the insert box HP11-sgRNA1 are transformed into a knockout strain DE, positive recombinants are selected, and the genetically engineered bacterium L1 with endogenous hyaluronic acid coating is obtained.
The genetically engineered bacterium with the endogenous hyaluronic acid coating is applied to the production of hyaluronic acid, has the characteristics of safety and high efficiency in the fermentation production of hyaluronic acid, and is suitable for the production and preparation of food-grade hyaluronic acid.
The genetically engineered bacterium with the endogenous hyaluronic acid coating is applied to the production of fermented milk.
The application of the genetically engineered bacterium with the endogenous hyaluronic acid coating in increasing survival and colonization of probiotics;
the method for increasing the survival and the colonization of the probiotics comprises the following steps: the genetically engineered bacterium with the endogenous hyaluronic acid coating can form the hyaluronic acid coating outside the bacterium body, so that the protection of the bacterial strain is enhanced, the gastric juice resistance is good, and the bacterial strain has higher survival ability and longer colonization in vivo.
The genetically engineered bacterium with the endogenous hyaluronic acid coating is applied to supplementing hyaluronic acid in vivo;
the hyaluronic acid in the body is supplemented specifically as follows: the hyaluronic acid contained in the culture of the genetically engineered bacterium with the endogenous hyaluronic acid coating realizes exogenous supplementation of the hyaluronic acid in vivo, and the genetically engineered bacterium generates the hyaluronic acid in the intestinal tract so as to realize continuous supplementation of the hyaluronic acid in the intestinal tract.
A probiotic composition for enhancing intestinal barrier and regulating intestinal flora, comprising the genetically engineered bacterium with endogenous hyaluronic acid coating.
A probiotic and prebiotic composition for enhancing the intestinal barrier and regulating the intestinal flora, comprising a genetically engineered bacterium having an endogenous hyaluronic acid coating as described above or a culture of a genetically engineered bacterium as described in claim 1.
According to a preferred aspect of the present invention, the enhancing intestinal barrier and regulating intestinal flora specifically comprises: increasing goblet cell and mucus secretion in the gut to enhance the gut barrier makes the gut flora more biased towards short chain fatty acid synthesis.
Preferably according to the invention, the probiotic composition, probiotic and prebiotic composition comprise dairy products, food products, health products and medicaments.
Drawings
Fig. 1: transmission electron microscope pictures of wild streptococcus thermophilus LMD-9 and genetically engineered bacterium L1.
Fig. 2: the yield and molecular weight results of the wild streptococcus thermophilus LMD-9, the genetically engineered bacterium P32-ZBCS, the knockout strain DE and the genetically engineered bacterium L1 in the SM17 culture medium are shown.
Fig. 3: the yield and molecular weight of hyaluronic acid in fermented milk of genetically engineered bacterium L1 are shown.
Fig. 4: the results of feeding wild Streptococcus thermophilus LMD-9 and the number of Streptococcus thermophilus in the mouse feces of the genetically engineered bacterium L1 fermented milk are shown.
Fig. 5: results of the content of hyaluronic acid in serum (A) and feces (B) of mice fed with wild Streptococcus thermophilus LMD-9 and genetically engineered bacterium L1 fermented milk.
Fig. 6: the expression result diagram of mucin Muc2 in intestinal tissues of mice fed with wild streptococcus thermophilus LMD-9 and genetically engineered bacterium L1 fermented milk.
Fig. 7: results of intestinal barrier of mice fed with wild Streptococcus thermophilus LMD-9 and genetically engineered bacterium L1 fermented milk are shown.
Fig. 8: results of intestinal flora of mice fed with fermented milk of wild Streptococcus thermophilus LMD-9 and genetically engineered bacterium L1 are shown.
Detailed Description
The invention is described below by means of specific embodiments. The technical means employed in the present invention are methods well known to those skilled in the art unless specifically stated. The following examples are intended to further illustrate the present invention and are not intended to limit the scope of the invention.
The standard PCR amplification system described in the examples below is: 2X Phanta Max Buffer. Mu.L, dNTP Mix (10 mM) 1. Mu.L, primer-F2. Mu.L, primer-R2. Mu.L, template DNA 100ng,Phanta Max Super-Fidelity DNA Polymerase 1. Mu.L, ddH 2 O was added to a total volume of 50. Mu.L.
The standard PCR procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 72℃for 15s, and elongation at Tm-2℃for 1kb/min for 30 cycles; extending at 72 ℃ for 5min, and preserving at 4 ℃.
The standard overlay PCR system is: 2X Phanta Max Buffer. Mu.L, dNTP Mix (10 mM) 1. Mu.L, primer-F2. Mu.L, primer-R2. Mu.L, template DNA fragments (molar ratio 1) 100ng,Phanta Max Super-Fidelity DNA Polymerase. Mu.L, ddH 2 O was added to a total volume of 50. Mu.L.
The standard overlay PCR procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 72℃for 30s, and elongation at Tm-2℃for 1kb/min (total length according to long fragment after fusion) for 30 cycles; extending at 72 ℃ for 5min, and preserving at 4 ℃.
The content of hyaluronic acid was determined by the method of sulfuric acid-carbazole: taking 500 mu L of a sample, adding the sample into 3mL of sodium tetraborate sulfuric acid solution, heating for 10min in a boiling water bath, adding 100 mu L of carbazole reagent, and heating for 15min. The absorbance at 530nm was measured. The uronic acid content and the hyaluronic acid content of the sample were calculated according to the standard curve.
Determination of hyaluronic acid molecular weight using a multi-angle laser scatterometer: the chromatographic column was OHPak SB-806M HQ, the mobile phase was 0.2mol/L sodium nitrate (containing 0.02% sodium azide) and the flow rate was 0.5mL/min.
EXAMPLE 1 overexpression of key enzymes in the hyaluronan synthetic pathway
1. And (3) respectively taking the synthesized strong promoter P11 fragment and the codon optimized hyaluronate synthase sphasA gene as templates by adopting a standard PCR amplification system and a standard PCR amplification program, and respectively carrying out PCR amplification by using primers P11-SP-F4/R4 and P11-SP-F5/R5 to obtain strong promoter P11 and hyaluronate synthase sphasA sequences, wherein the primer sequences are as follows:
P11-SP-F4：5′-tcggactcgcgaattcattgcgtcgtagatgtgcaaggtg-3′；
P11-SP-R4：5′-accaattacctccaaaaatgttatttagattagc-3′；
P11-SP-F5：5′-ctaaataacatttttggaggtaattggtatgccaattttcaagaagacattg-3′；
P11-SP-R5：5′-catgtaatcactccttcttaattacaaattacttaaaaattgtaacttttttacgtgta-3′。
2. the upstream homology arm segment of the over-expression cassette HP11-SP is obtained by PCR amplification by using a standard PCR amplification system and a standard PCR amplification program and using a streptococcus thermophilus LMD-9 genome (GenBank: CP 000419.1) as a template and using P11-SP-F1/R1 as a primer; the P11-SP-F6/R6 is used as a primer, and the downstream homology arm fragment of the overexpression box HP11-SP is obtained through PCR amplification, wherein the primer sequence is as follows:
P11-SP-F1：5′-ggctgtccttttggaagagtcg-3′；
P11-SP-R1：5′-tcaacacactcttaagtttgcaatgaattcgcgagtccgac-3′；
P11-SP-F6：5′-gaaggagtgattacatgttaggtcaacatgaagatgtg-3′；
P11-SP-R6：5′-ttaatctctcacaaatacatcgtgagtgtg-3′。
3. adopting a standard PCR amplification system and a standard PCR amplification program, taking a SacB-Cat box (shown as SEQ ID NO. 3) as a template, taking P11-SP-F2/R2 and Cat-F/SP-R2 as primers, and obtaining a double-screening mark of the over-expression box HP11-SP and a chloramphenicol resistance fragment through PCR amplification; the directional repeated segment of the overexpression box HP11-SP is obtained by PCR amplification by taking the streptococcus thermophilus LMD-9 genome as a template and taking P11-SP-F3/R3 as a primer, wherein the primer sequence is as follows:
Cat-F:5′-cagttaacaaataattacagtaatattgacttttaaaaaaggattg-3′；
P11-SP-F2：5′-caaacttaagagtgtgttgatagtgcagtatc-3′；
P11-SP-R2：5′-gtaatataaaaaccttcttcaactaacggggcag-3′；
P11-SP-F3：5′-gaagaaggtttttatattacaagttcttcctaatgatggtttggttg-3′；
P11-SP-R3：5′-caatgaattcgcgagtccgac-3′。
4. using standard overlay PCR system and program, fusing the upstream homology arm segment and double screening mark with P11-SP-F1/R2 as primer to medium length segment 1, fusing chloramphenicol resistance segment, targeting repetitive segment and strong promoter P11 with Cat-F/P11-SP-R4 as primer to medium length segment 2, fusing strong promoter P11, sphasA gene and downstream homology arm segment with primer P11-SP-F4/R6 to medium length segment 3; the above 3 middle-length fragments were fused to sphasA sequence using P11-SP-F1/R6 as primer to obtain the overexpression cassette HP11-SP (shown as SEQ ID NO. 4).
Wherein the upstream homology arm fragment is 1-1000 bp, the double-screening mark fragment is 1001-3367 bp, the directional repeated fragment is 3368-3538 bp, the strong promoter P11 fragment is 3539-3819 bp, the hyaluronic acid synthase sphasA is 3820-5105 bp, and the downstream homology arm fragment is 5106-6223 bp.
5. According to the method described in 1-4, the upstream homology arm fragment of the over-expression cassette HP32-ZBC is obtained by PCR amplification with the codon optimized hyaluronate synthase sphasA gene as a template and the primer P11-SP-F5/P32-ZBC-R1; the synthesized strong promoter P32 fragment and streptococcus zooepidemicus genome (GenBank: CP 001129) are used as templates, and P32-ZBC-F2/R2 and P32-ZBC-F3/R3 are used as primers, and the strong promoter P32, hasB and hasC sequences are obtained through PCR amplification; taking streptococcus zooepidemicus genome as a template and P32-ZBC-F5/R5 as a primer, and performing PCR amplification to obtain a directional repeated segment of the overexpression cassette HP32-ZBC; the primer P32-ZBC-F6/P11-SP-R6 and the streptococcus thermophilus LMD-9 genome are used as templates, and the downstream homology arm fragment of the overexpression box HP32-ZBC is obtained through PCR amplification; the upstream homology arm fragment and the strong promoter P32 are fused into a medium-length fragment 4, hasB, hasC sequences and double-screening markers are fused into a medium-length fragment 5 by adopting a standard overlay PCR system and a standard overlay PCR program, and chloramphenicol fragments, oriented repeated fragments and downstream homology arm fragments are fused into a medium-length fragment 6; the above 3 medium-length fragments were fused to obtain an overexpression cassette HP32-ZBC (shown as SEQ ID NO. 5).
Wherein the upstream homology arm fragment is 1-1260 bp, the strong promoter P32 is 1261-1445 bp, hasB and hasC sequences are 1446-3603 bp, the double-screening mark fragment is 3604-5970 bp, the oriented repeated fragment is 5971-6173 bp, and the downstream homology arm fragment is 6174-7292 bp.
The primer sequences are as follows:
P32-ZBC-R1:ttacttaaaaattgtaacttttttacgtgta；
P32-ZBC-F2：5′-gtaaaaaagttacaatttttaagtaaagatcttcgaattcggtcctc-3′；
P32-ZBC-R2：5′-cacttcaaaattcctccgaatatttttttac-3′；
P32-ZBC-F3：5′-cggaggaattttgaagtgaaaatttctgtagcaggctc-3′；
P32-ZBC-R3：5′-caacacactcttaagtttgctatttcttgacgtccttgttc-3′；
P32-ZBC-F5：5′-gttgaagaaggtttttatattactgaccgatgcgattgac-3′；
P32-ZBC-R5：5′-cttcatgttgacctaacatctatttcttgacgtccttgttc-3′；
P32-ZBC-F6：5′-atgttaggtcaacatgaagatg-3′。
6. according to the method of 1-4, the upstream homology arm and glmS sequence of the over-expression cassette HP32-S are obtained by PCR amplification by taking the streptococcus thermophilus LMD-9 genome as a template and taking P32-ZBCS-F1/R1 and P32-ZBCS-F5/R5 as primers; the synthesized strong promoter P32 is used as a template, P32-ZBCS-F2/R2 and P32-ZBCS-F4/R4 are used as primers, and the directional repeated fragments of the strong promoter P32 and the overexpression box HP32-S are respectively obtained through PCR amplification; the upstream homology arm fragment, the strong promoter P32 and the double screening marker are fused into a medium-length fragment 7 by adopting a standard overlay PCR system and a standard overlay PCR program, and the chloramphenicol resistance fragment, the targeting repeat fragment and the downstream homology arm fragment are fused into a medium-length fragment 8; the above 2 intermediate fragments and glmS sequences were fused to give the overexpression cassette HP32-S (shown as SEQ ID NO. 6).
Wherein the upstream homology arm fragment is 1-1116 bp, the strong promoter P32 fragment is 1117-1301 bp, the double-screening mark fragment is 1302-3668 bp, the directional repeated fragment is 3669-3853 bp, and the glmS fragment is 3854-4936 bp.
The primer sequences are as follows:
P32-ZBCS-F1：5′-ttagttatcaaaagtgacctccttaac-3′；
P32-ZBCS-R1：5′-aggaccgaattcgaagatctatttttaataataacctcaagtcaaagagg-3′；
P32-ZBCS-F2：5′-agatcttcgaattcggtcct-3′；
P32-ZBCS-R2：5′-caacacactcttaagtttgttcaaaattcctccgaatatttttttac-3′；
P32-ZBCS-F4：5′-gaagaaggtttttatattacagatcttcgaattcggtcct-3′；
P32-ZBCS-R4：5′-caccaacaattccacacatttcaaaattcctccgaatatttttttac-3′；
P32-ZBCS-F5：5′-atgtgtggaattgttggtg-3′；
P32-ZBCS-R5：5′-gttagctttaacaagaacctg-3′。
7. conversion: wild streptococcus thermophilus LMD-9 is activated overnight in 1mL of LM17 liquid culture medium, inoculated in 1mL of LM17 liquid culture medium according to the volume ratio of 1:100, and subjected to activation culture for 6 hours at 37 ℃ and 100r/min, and activated bacterial liquid is subjected to activation culture for 1:60 volume ratio (OD 600 =0.05) was again transferred to 6mL LM17 liquid medium containing 1 μmol/L of the inducing peptide ComS to grow OD 600 Reaching 0.2 to 0.3 to obtain the streptococcus thermophilus LMD-9 competent cells. 300 mu L of competent cells are added into 25 mu L of overexpression cassette HP11-SP, incubation is continued for 1.5h at 37 ℃, centrifugation and concentration are carried out until 50 mu L,3 mu g/mL of chloramphenicol resistant LM17 solid medium is coated, culture is carried out for 24h at 37 ℃, then streaking is carried out again on 3 mu g/mL of chloramphenicol resistant LM17 solid medium, single colony is selected, 1 mu L of non-resistant medium containing 10% sucrose is coated after relaxation culture is carried out for 24h at 37 ℃ through non-resistant LM17 liquid mediumOn SM17 solid culture medium, culturing at 37 ℃ for 24 hours for screening, randomly picking a plurality of single colonies, and carrying out bacterial liquid PCR verification to obtain positive strains. Based on the above, the over-expression cassettes HP32-ZBC and HP32-S are transformed according to the transformation method, and the obtained positive strain is the genetic engineering bacteria P32-ZBCS.
The amino acid residue sequence of the induction peptide ComS is as follows: LPYFAGCL.
EXAMPLE 2 knockout of the Positive regulatory factor epsA in the extracellular polysaccharide Synthesis Gene Cluster
Adopting a standard PCR amplification system and a standard PCR amplification program, taking a streptococcus thermophilus LMD-9 genome as a template, taking DE-F1/R1, DE-F3/R3 and DE-F4/R4 as primers, and respectively obtaining an upstream homology arm fragment, a directional repeated fragment and a downstream homology arm fragment of the knockout box through PCR amplification; the upstream homology arm fragment and the double screening marker are fused into a medium length fragment 9, and the chloramphenicol fragment, the oriented repeated fragment and the downstream homology arm fragment are fused into a medium length fragment 10 by adopting a standard overlay PCR system and program; the above 2 mid-length fragments were fused to a knockout cassette HDE (shown as SEQ ID No. 7).
Wherein the upstream homology arm fragment is 1-1051 bp, the double-screening marker fragment is 1052-3418 bp, the directed repeated fragment is 3419-3626 bp, and the downstream homology arm fragment is 3627-4477 bp.
The primer sequences are as follows:
DE-F1：5′-atgtcaattcatatttctgccaaac-3′；
DE-R1：5′-caacacactcttaagtttgataaattgctcctaaaaattaaaattaagt-3′；
DE-F3：5′-gaagaaggtttttatattacggtaaatatctgacaatttaaagtttaac-3′；
DE-R3：5′-tccaaagaaatataaattgctcctaaaaattaaaattaagt-3′；
DE-F4：5′-ggagcaatttatatttctttggataccattaatgctttg-3′；
DE-R4：5′-gcatctttcataaatggtggtctc-3′。
the knockout cassette HDE was transformed into strain P32-ZBCS according to the transformation method described in example 1, and the knockout strain DE was obtained after screening and verification.
EXAMPLE 3 Down-regulating lactic acid Synthesis construction of genetically engineered bacterium L1 with endogenous hyaluronic acid coating
1. Adopting a standard PCR amplification system and a standard PCR amplification program, taking a streptococcus thermophilus LMD-9 genome as a template, taking D-F1/R1, D-F2/R2 and DE-F4/R4 as primers, and carrying out PCR amplification to obtain an upstream homology arm fragment, a directed repeated fragment and a downstream homology arm fragment of the mutant box HD1; the upstream homology arm fragment, the targeting repeat fragment and the double screening marker are fused into a medium length fragment 11, and the chloramphenicol resistance fragment and the downstream homology arm fragment are fused into a medium length fragment 12 by using a standard overlay PCR system and procedure; the above 2 intermediate fragments were fused to obtain a mutant cassette HD1 (shown in SEQ ID NO. 8).
Wherein the upstream homology arm fragment is 1-1027 bp, the directional repeated fragment is 1028-1189 bp, the double-screening marker fragment is 1190-3556 bp, and the downstream homology arm fragment is 3557-4575 bp.
The primer sequences are as follows:
D-F1：5′-aagtctatgaagctcttgaaccctacc-3′；
D-R1：5′-caacagaacctataccgatagcaagtcctaaaactaagtcactcat-3′；
D-F2：5′-atcggtataggttctgttggtg-3′；
D-R2：5′-caacacactcttaagtttgtctacgatgttttttacgtcgtg-3′；
D-F4：5′-agttgaagaaggtttttatattacatcggtataggttctgttggtg-3′；
D-R4：5′-gtatgaatctcagccttacctg-3′。
2. according to the method in 1, the upstream homology arm fragment, the oriented repeated fragment and the downstream homology arm fragment of the mutant box HH2 are obtained by PCR amplification by taking the streptococcus thermophilus LMD-9 genome as a template and taking H-F1/R1, H-F2/R2 and H-F4/R4 as primers; the upstream homology arm fragment, the targeting repeat fragment and the double screening marker are fused into a medium length fragment 13, and the chloramphenicol resistance fragment and the downstream homology arm fragment are fused into a medium length fragment 14 by using a standard overlay PCR system and procedure; the above 2 fragments were fused using standard overlay PCR system and procedure to give the mutant cassette HH2 (shown as SEQ ID NO. 9).
Wherein the upstream homology arm fragment is 1-1011 bp, the directional repeated fragment is 1012-1172 bp, the double-screening mark fragment is 1173-3539 bp, and the downstream homology arm fragment is 3540-4628 bp.
The primer sequences are as follows:
H-F1：5′-ccagacgagtttagagcagcaaaag-3′；
H-R1：5′-gatagaaagaggtaaaatagcatctacttcaaactgattagaattatttatc-3′；
H-F2：5′-attttacctctttctatcacattcg-3′；
H-R2：5′-caacacactcttaagtttggactcacgtacaaaagcttttaattc-3′；
H-F4：5′-gttgaagaaggtttttatattacattttacctctttctatcacattcg-3′；
H-R4：5′-ctgactgtaagacaactttattgttac-3′。
3. the upstream homology arm segment inserted into the box HP11 is obtained by adopting a standard PCR amplification system and a standard PCR amplification program, taking the streptococcus thermophilus LMD-9 genome as a template and taking D-F1/P11-R1 as a primer and performing PCR amplification; taking the synthesized promoter P11 as a template, taking D-F2/R2 as a primer, and obtaining a strong promoter P11 through PCR amplification; then, a standard overlay PCR system and a program are adopted, a strong promoter P11, a directional repeated segment in a mutation box HD1 and a double-screening marker are fused into a medium-length segment 15, and a chloramphenicol segment and a downstream homology arm in the mutation box HD1 are fused into a medium-length segment 16; the fusion of the upstream homology arm fragment with the above 2 mid-length fragments was continued to give the insert HP11 (shown as SEQ ID NO. 10).
Wherein the upstream homology arm fragment is 1-1000 bp, the strong promoter P11 is 1001-1308 bp, the directional repeated fragment 1309-1449 bp in the mutation box HD1, the double-screening mark 1450-3816 bp, and the downstream homology arm fragment 3817-4835 bp.
The primer sequences are as follows:
P11-R1：5′-ttaataatccccttatgcttttttcta-3′；
D-F2：5′-atcggtataggttctgttggtg-3′；
D-R2：5′-caacacactcttaagtttgtctacgatgttttttacgtcgtg-3′。
4. adopting a standard PCR amplification system and a standard PCR amplification program, taking a streptococcus thermophilus LMD-9 genome as a template, taking P11-sgRNA-F1/R1 and P11-sgRNA-F6/R6 as primers, and obtaining an upstream homology arm fragment and a downstream homology arm fragment inserted into the box HP11-sgRNA1 through PCR amplification; the synthetic promoter P11 is used as a template, and the P11-sgRNA-F2/R2 is used as a primer, and the strong promoter P11 is obtained through PCR amplification; the synthesized sgRNA is used as a template, and P11-sgRNA-F3/R3 is used as a primer, and the sgRNA1 is obtained through PCR amplification; taking a streptococcus thermophilus LMD-9 genome as a template, taking P11-sgRNA-F4/R4 as a primer, and carrying out PCR amplification to obtain a directional repeated segment inserted into a box; then, adopting a standard overlay PCR system and a program to fuse an upstream fragment, a strong promoter P11 and a fusion sgRNA1 to obtain a medium-length fragment 17, and fusing a directional repeated fragment and a double-screening marker to obtain a medium-length fragment 18 and fusing a chloramphenicol fragment and a downstream homology arm fragment to obtain a medium-length fragment 19; fragment 17 and fragment 18 are fused as fragment 20; fragment 19 was fused to 20 to give insert HP11-sgRNA1 (shown as SEQ ID NO. 11).
Wherein the upstream homology arm fragment is 1-1182 bp, the strong promoter P11 fragment is 1183-1463 bp, the directional repeated fragment is 1464-1651 bp, the sgRNA1 sequence is 1653-1834 bp, the double-screening mark fragment is 1835-4201 bp, and the downstream homology arm fragment is 4202-5274 bp.
The primer sequences are as follows:
P11-sgRNA-F1：5′-atgacaaatcgtaaagacgatcacat-3′；
P11-sgRNA-R1：5′-accttgcacatctacgacgtcaggctaaaacagttcccca-3′；
P11-sgRNA-F6：5′-gttgaagaaggtttttatattacgcctagaaattaaaaagtgaaggggctc-3′；
P11-sgRNA-R6：5′-agctagggcaacacggtcgttaac-3′；
P11-sgRNA-F2：5′-cgtcgtagatgtgcaaggt-3′；
P11-sgRNA-R2：5′-tggtgacgggttcctacagtaccaattacctccaaaaatgttatttagattag-3′；
P11-sgRNA-F3：5′-actgtaggaacccgtcaccagtttttgtactctcaagatttaagtaac-3′；
P11-sgRNA-R3：5′-catggtgagtgcctcctt-3′；
P11-sgRNA-F4：5′-aaggaggcactcaccatggcctagaaattaaaaagtgaaggggct-3′；
P11-sgRNA-R4：5′-ctatcaacacactcttaagtttgagggcctctgattatctataatc-3′。
5. the knock-out cassette HD1 was transformed into strain DE according to the transformation method described in example 1, resulting in strain D1; transforming mutant cassette HH2 into strain D1, resulting in strain D91; converting the insert cassette HP11 into strain D91, resulting in strain D92; and (3) converting the inserted cassette HP11-sgRNA1 into a strain D92, and screening and verifying to obtain the genetically engineered bacterium L1 with the endogenous hyaluronic acid coating.
The result of transmission electron microscope observation of the genetically engineered bacterium L1 constructed in the example is shown in FIG. 1. As can be seen from fig. 1, a protective layer of hyaluronic acid was attached around the genetically engineered bacterium L1.
EXAMPLE 4 preparation of hyaluronic acid
1. Inoculating the genetically engineered bacterium P32-ZBCS into SM17 liquid culture medium containing 2% of sucrose, and fermenting and culturing at 37 ℃ and 100r/min for 48 hours to obtain fermentation bacterial liquid.
The specific method for extracting hyaluronic acid from fermentation broth comprises the following steps:
adding 20mL of fermentation broth into 20mL of 0.1% SDS, standing at room temperature for 10min, centrifuging at 4 ℃ for 5min at 5000 Xg, repeating for one time, adding sodium chloride with the final concentration of 0.1mol/L into the supernatant, adding 3 times volume of absolute ethyl alcohol after complete dissolution, and precipitating with ethanol at 4 ℃ overnight; dissolving the precipitate after centrifugation by using 4mL of 0.1mol/L sodium chloride solution, adding 1% of cetylpyridinium chloride, complexing and precipitating for 2-3 h, dissociating the complex for 5-6 h by using 0.4mol/L sodium chloride solution, and precipitating the dissociated solution by using 3 times of volume of alcohol for 2-3 h to obtain hyaluronic acid.
According to the above method, hyaluronic acid was prepared using wild Streptococcus thermophilus LMD-9, knockout strain DE and genetically engineered bacterium L1 as production strains. And then the yield and the relative molecular mass of the hyaluronic acid prepared by the wild streptococcus thermophilus LMD-9, the genetically engineered bacterium P32-ZBCS, the knockout strain DE and the genetically engineered bacterium L1 are respectively measured by a sulfuric acid-carbazole method or a multi-angle laser scatterometer, and the result is shown in figure 2.
As shown in FIG. 2, the yield of hyaluronic acid of the wild streptococcus thermophilus LMD-9 is 3mg/L, the yield of hyaluronic acid of the genetically engineered bacterium P32-ZBCS is 50-60 mg/L, the yield of hyaluronic acid of the knockout strain DE is 60-65 mg/L, and the yield of hyaluronic acid of the genetically engineered bacterium L1 is about 75-80 mg/L.
Example 5 preparation of fermented milk
The genetic engineering bacteria L1 are firstly subjected to the scratch culture on an LM17 solid culture medium, and single bacterial colony is selected and activated for 12 hours in the LM17 liquid culture medium. Inoculating 1% of inoculum size into 6mL of SM17 liquid culture medium, and growing for 6-8 h to obtain seed liquid; inoculating the seed solution into 100mL 10% skim milk powder containing 2% sucrose according to the proportion of 2% -4%, and fermenting and culturing for 24-48 h at 37 ℃ and 100r/min to obtain fermented milk.
Wherein, the pH of the fermentation liquid is adjusted to 7.0 by adopting sodium carbonate solution with the concentration of 3mol/L in 4-6 h and 16-18 h of fermentation.
The fermented milk prepared in this example was subjected to live bacteria detection and hyaluronic acid content measurement, and the results are shown in fig. 3. As can be seen from FIG. 3, the content of the genetically engineered bacterium L1 in the genetically engineered bacterium L1 fermented milk is more than 1.0X10 8 CFUs/mL, the content of hyaluronic acid is 180-200 mg/L, and the relative molecular mass of hyaluronic acid is 500-1000 kDa.
The fermented milk of the wild streptococcus thermophilus LMD-9 is prepared according to the same method, the content of the wild streptococcus thermophilus LMD-9 is not significantly different from that of the genetically engineered bacterium L1, and the content of hyaluronic acid is only 8mg/L.
EXAMPLE 6 in vivo Effect of genetically engineered bacterium L1 fermented milk
The C57BL/6J mice of 7 weeks old were fed 200. Mu.L of the genetically engineered bacterium L1 fermented milk of example 5 each day, and the fermented milk of the wild Streptococcus thermophilus LMD-9 was fed continuously for 14 days with the same volume as the control. Mice were provided with sufficient food and water during the feeding period. Mice of each group were bled and faeces collected by tail-breaking before gavage, during gavage at days 1, 4, 7, 14 and at days 1, 4, 7, 14 after the end of gavage, respectively. Wherein the sampling time during the lavage is 6 hours after the lavage.
The hyaluronic acid content of the blood samples was measured using an ELISA kit (available from Wohan cloud cloning Co.). Standing at room temperature for 2h, centrifuging at 2000r/min for 15min to obtain serum, and diluting with PBS for 3 times; after 50. Mu.L of sample is added, 50. Mu.L of detection solution A is immediately added, and the mixture is incubated for 1h at 37 ℃; spin-drying, and washing the plate for 3 times; adding 100 mu L of detection solution B, and incubating for 30min at 37 ℃; washing the plate for 5 times; the TMB substrate was added at 90. Mu.L and incubated at 37℃for 10min, stop solution was added at 50. Mu.L, and the read at 450nm was immediately performed, and the results are shown in FIG. 4.
Quantification of s.thermophilus in feces: 180mg of feces was taken in TE Buffer (20 mmol/L Tris, pH8.0;2mmol/L Na) 2 -EDTA;1.2% Triton X-100) 300. Mu.L, and after mixing with a high-pressure homogenizer, adding 20mg/mL lysozyme 400. Mu.L at 37deg.C for 2 hours, extracting genomic DNA from feces according to the procedure of feces/soil genomic DNA extraction kit (available from double Warewrites medical science). The amount of the engineering strain in the feces was determined by absolute quantification using primers A-F/R according to standard PCR amplification system and procedure, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the serum of mice fed with the genetically engineered bacterium L1 fermented milk had an increased content of hyaluronic acid. In the mouse feces fed with the genetically engineered bacterium L1 for fermentation, the survival rate of the genetically engineered bacterium L1 is maintained at 10 7 The CFUs/g feces are higher than that of wild streptococcus thermophilus by 2.0-3.8X10 6 CFUs/g feces. In particular, on the first gastric lavage day, wild Streptococcus thermophilus only has a concentration of 3.4X10 5 Survival rate of CFUs/g faeces. After the gastric lavage is finished, the genetically engineered bacterium L1 has longer colonization time than the wild streptococcus thermophilus LMD-9, the wild streptococcus thermophilus LMD-9 is recovered to the initial level in the first week, but the genetically engineered bacterium L1 can still keep 2.8X10 5 Survival of CFUs/g feces.
The primer sequences are as follows:
A-F：5′-cagccataatggtgaaatcagt-3′；
A-R：5′-ttgagcattgcgaacttgtga-3′。
determination of hyaluronic acid content in feces: another 100mg of feces was added to 900. Mu.L of PBS buffer and the mixture was ground for 1min. The assay was performed using a radioimmunokit (sold by Shanghai Sail Co.), specifically: standard and sample (50 μl) were labeled and incubated for 1h at 37deg.C, hyaluronic acid binding protein antibody 37℃for 40min, secondary antibody 20min, and centrifugation at 3800r/min for 15min. The radioactivity of the precipitate was counted by a counter, and the result is shown in fig. 5.
As is clear from FIG. 5, the feces of mice fed with the fermented milk of the genetically engineered bacterium L1 during the stomach irrigation period had a very high content of hyaluronic acid, whereas the feces of mice fed with the fermented milk of the wild-type Streptococcus thermophilus had almost no content of hyaluronic acid detected, and in particular, the genetically engineered bacterium L1 after colonization still produced a certain level of hyaluronic acid during the recovery period, and 10 to 30ng/mL of hyaluronic acid was detected in the feces.
Intestinal barrier analysis in mice: the expression level of mucin Muc2 in intestinal tissues was relatively quantitatively analyzed by a standard PCR amplification system using the primer MUC2-F/R, and the results are shown in FIG. 6. The intestinal tissues of mice were analyzed by PAS and AB staining, and the results are shown in FIG. 7. As can be seen from fig. 6 and 7, although there was no significant difference between the two groups in the expression level of mucin Muc2, it was found that the cup cell and mucus secretion in the intestinal tract of mice fed with the genetically engineered bacterium L1 fermented milk was higher than that of the wild-type streptococcus thermophilus fermented milk, i.e., the genetically engineered bacterium L1 had a more positive effect in enhancing the barrier.
The primer sequences are as follows:
MUC2-F：5′-cctgaagactgtcgtgctgt-3′；
MUC2-R：5′-gggtagggtcacctccatct-3′。
the diversity of the intestinal flora of mice was analyzed by measuring the total length of 16S, and the result is shown in FIG. 8, the intestinal flora of mice fed with the fermented milk of the genetically engineered bacterium L1 was significantly changed relative to the fermented milk of the wild type Streptococcus thermophilus, and the intestinal flora was also a factor of the enhancement of intestinal barrier, which is specifically shown by the increase of the type and abundance of the flora concerning the synthesis of short chain fatty acids.
Claims (10)
1. The genetically engineered bacterium with the endogenous hyaluronic acid coating is characterized by taking probiotics as an original strain, and is constructed by over-expressing hyaluronic acid synthase, uridine diphosphate-glucose-6-dehydrogenase, glucose-1-phosphate uridylate transferase and L-glutamine-D-fructose-6-phosphate aminotransferase through genetic engineering operation, knocking out epsA genes, and down-regulating the expression level of lactic acid dehydrogenase.
2. The genetically engineered bacterium with an endogenous hyaluronic acid coating of claim 1, wherein the probiotic is a probiotic in a safe additive state, including streptococcus thermophilus, lactococcus lactis, lactobacillus bulgaricus and bifidobacteria;
further preferably, the probiotic is streptococcus thermophilus.
3. The use of the genetically engineered bacterium with an endogenous hyaluronic acid coating of claim 1 in the production of hyaluronic acid; the genetically engineered bacterium has the characteristics of safety and high efficiency in the fermentation production of hyaluronic acid, and is suitable for the production and preparation of food-grade and pharmaceutical-grade hyaluronic acid.
4. The use of the genetically engineered bacterium with an endogenous hyaluronic acid coating of claim 1 in the production of fermented milk.
5. Use of the genetically engineered bacterium with an endogenous hyaluronic acid coating of claim 1 to increase probiotic survival and colonization;
the method for increasing the survival and the colonization of the probiotics comprises the following steps: the genetically engineered bacterium with an endogenous hyaluronic acid coating of claim 1 can form a hyaluronic acid coating outside the bacterium body, thereby enhancing the protection of the strain itself, having good gastrointestinal fluid resistance, higher survival ability in vivo and longer colonization.
6. The use of the genetically engineered bacterium with an endogenous hyaluronic acid coating of claim 1 for supplementing hyaluronic acid in vivo;
the hyaluronic acid in the body is supplemented specifically as follows: the method for continuously supplementing hyaluronic acid in intestinal tracts, which comprises the step of preparing hyaluronic acid in the intestinal tracts by using the genetically engineered bacterium with the endogenous hyaluronic acid coating, wherein the hyaluronic acid contained in the culture of the genetically engineered bacterium with the endogenous hyaluronic acid coating realizes exogenous supplementation of the hyaluronic acid in vivo.
7. A probiotic composition for enhancing the intestinal barrier and regulating the intestinal flora, characterized in that the probiotic composition comprises the genetically engineered bacterium of claim 1.
8. A probiotic and prebiotic composition for enhancing the intestinal barrier and regulating the intestinal flora, characterized in that it comprises the genetically engineered bacterium of claim 1 or a culture of the genetically engineered bacterium of claim 1.
9. The probiotic composition according to claim 7 or the probiotic and prebiotic composition according to claim 8, characterized in that said enhancing the intestinal barrier, regulating the intestinal flora, are in particular: increasing goblet cell and mucus secretion in the gut to enhance the gut barrier makes the gut flora more biased towards short chain fatty acid synthesis.
10. The probiotic composition according to claim 7 or the probiotic and prebiotic composition according to claim 8, wherein said probiotic composition, probiotic and prebiotic composition comprise dairy products, food products, health products and pharmaceuticals.
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| CN202210919679.9A CN117487726A (en) | 2022-08-02 | 2022-08-02 | Genetically engineered bacterium with endogenous hyaluronic acid coating, construction method and application thereof |
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