CN118028129A - Construction method and application of recombinant Escherichia coli for synthesizing lactoyl-N-neotetraose - Google Patents
Construction method and application of recombinant Escherichia coli for synthesizing lactoyl-N-neotetraose Download PDFInfo
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- CN118028129A CN118028129A CN202410213996.8A CN202410213996A CN118028129A CN 118028129 A CN118028129 A CN 118028129A CN 202410213996 A CN202410213996 A CN 202410213996A CN 118028129 A CN118028129 A CN 118028129A
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- fermentation
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- 241000588724 Escherichia coli Species 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title abstract description 9
- 230000002194 synthesizing effect Effects 0.000 title abstract description 4
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Classifications
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Abstract
The invention discloses a construction method and application of recombinant escherichia coli for synthesizing lactoyl-N-neotetraose, belonging to the field of microbial genetic engineering. The invention takes engineering strain MGC02 as an original strain, optimizes the enzyme expression level by introducing beta-1, 3-N-acetylglucosaminyl transferase LgtA and beta-1, 4-galactosyl transferase CpsIaJ and using various promoters, improves precursor supply, introduces lux type quorum sensing system and optimizes QS promoter, and finally, the extracellular titer of LNnT is 20.33g/L, the productivity is 0.41g/L/h, thereby having good industrialization prospect.
Description
Technical Field
The invention relates to a construction method and application of recombinant escherichia coli for synthesizing lactoyl-N-neotetraose, belonging to the field of microbial genetic engineering.
Background
As a group of structurally complex and diverse glycans, human Milk Oligosaccharides (HMOs) are present in large amounts in human milk, in amounts between 5 and 15g/L, being the third largest solid component next to lactose and lipids. lacto-N-neotetraose (LNnT) is an extremely important neutral core oligosaccharide and has been approved by the us FDA and EU as a nutritional supplement for addition to infant formulas. Numerous studies have shown that LNnT has a variety of health benefits to infants, including prebiotic effects, modulating immunity, antiviral, and anti-adhesion and antibacterial activity. Various methods have been used to synthesize LNnT effectively, and among them, microbial synthesis methods can use inexpensive glycerol or glucose as a carbon source, which reduces production costs, and such methods have little influence on the environment, and thus have received much attention. Using lactose as a substrate, endogenous UDP-N-acetylglucosamine (UDP-GlcNAc) as a donor, beta-1, 3-N-acetylglucosamine transferase (EC 2.4.1.149) catalyzes the first step of transglycosylation to produce lacto-N-trisaccharide II (LNTri II), an essential precursor for LNnT biosynthesis. The precursor LNTri II was then converted to the final product LNnT under catalysis of beta-1, 4-galactosyltransferase (EC 2.4.1.22) using UDP-galactose (UDP-Gal) as a glycoside donor.
For the production of LNnT by microbiological synthesis, patent CN113652385B discloses a method for increasing the yield of lacto-N-neotetraose by screening for beta-1, 4-galactosyltransferase, in which the yield of recombinant lacto-N-neotetraose is 12.14g/L. The existing microbial synthesis method still has the problem of low yield in LNnT production, and the lower LNnT cannot meet the industrial production requirement. In addition, there is still a need for the use of the inducer IPTG in the production of LNnT by existing microbiological synthesis methods, on the one hand, there is a potential toxicity of IPTG and a potential safety risk in food production, and on the other hand, the use of IPTG can greatly increase the cost of industrialization.
Disclosure of Invention
[ Technical problem ]
The existing microbial synthesis method for producing LNnT has the problems of low yield, safety risk brought by the use of IPTG and high cost.
Technical scheme
Aiming at the technical problems, the invention provides a recombinant strain which can efficiently and constitutively produce LNnT through a quorum sensing system.
The first object of the invention is to provide a recombinant strain, which takes escherichia coli as an initial strain, and performs the following genetic engineering operations on the initial strain:
(1) Knocking out a beta-galactosidase coding gene lacZ and a UDP-glucose lipid carrier transferase coding gene wcaJ on the genome of the original strain;
(2) Replacing the promoter of the gene lacY encoding the beta-galactosidase on the genome with promoter P J23119;
(3) Beta-1, 3-N-acetylglucosaminyl transferase coding gene lgtA is expressed by a promoter P QS03, and beta-1, 4-galactosyltransferase coding gene cpsIaJ is expressed by an expression element luxR-P QS33 -luxI.
In one embodiment, the E.coli comprises E.coli MG1655.
In one embodiment, the Gene IDs of the Gene lacZ, the Gene wcaJ, and the Gene lacY are respectively: 945006, 946583 and 949083.
In one embodiment, the nucleotide sequence of the promoter P J23119 is shown in SEQ ID No. 3.
In one embodiment, the lgtA gene is derived from neisseria meningitidis (NEISSERIA MENINGITIDIS) and has the nucleotide sequence shown in SEQ ID No. 2.
In one embodiment, the cpsIaJ gene is derived from Streptococcus agalactiae (Streptococcus agalactiae) and has the nucleotide sequence shown in SEQ ID NO. 1.
In one embodiment, the nucleotide sequence of the promoter P QS03 is shown in SEQ ID NO.5, and the nucleotide sequence of the expression element luxR-P QS33 -luxI is shown in SEQ ID NO. 10.
It is a second object of the present invention to provide a method for producing lacto-N-neotetraose by fermentation using the above recombinant bacterium.
In one embodiment, the method comprises: inoculating the recombinant bacteria to a seed culture medium, culturing at 30-40 ℃ for 8-14h to obtain seed liquid, transferring the seed liquid to a fermentation culture medium, culturing until OD 600 is 0.6-0.8, adding lactose, reducing the fermentation temperature to 20-28 ℃, and fermenting to produce lactoyl-N-neotetraose.
In one embodiment, the method comprises:
(1) Inoculating the recombinant bacteria to a seed culture medium, and culturing for 8-14h at 30-40 ℃ to obtain primary seed liquid;
(2) Transferring the primary seed liquid obtained in the step (1) into a fermentation medium according to the transfer amount of 0.5% -2%, and culturing for 3-6h at 30-40 ℃ to obtain a secondary seed liquid;
(3) Inoculating the secondary seed liquid obtained in the step (2) into a fermentation culture medium according to the inoculum size of 5% -15%, culturing until the OD 600 is 30-50, adding lactose, and fermenting at 25-35 ℃ to produce lactoyl-N-neotetraose.
In one embodiment, the seed medium formulation comprises: 1-10g/L of yeast extract, 5-20g/L of peptone and 5-20g/L of sodium chloride. Optionally, the formula of the seed culture medium comprises: 5g/L of yeast extract, 10g/L of peptone and 10g/L of sodium chloride.
In one embodiment, the fermentation medium is formulated to include: 10-30g/L glycerol, 1-10g/L yeast extract, 5-20g/L potassium dihydrogen phosphate, 1-10g/L diammonium hydrogen phosphate, 0.5-3g/L citric acid, 0.5-3g/L magnesium sulfate heptahydrate, 1-20ml/L trace metal element and 1-10mg/L thiamine. Optionally, the formulation of the fermentation medium comprises: 20g/L glycerol, 5g/L yeast extract, 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate and 10ml/L trace metal elements, 4.5mg/L thiamine.
In one embodiment, the trace metal formulation comprises: 5-20g/L ferrous sulfate, 1-5g/L zinc sulfate heptahydrate, 0.5-2g/L anhydrous copper sulfate, 0.1-1g/L manganese sulfate monohydrate, 0.1-1g/L sodium borate decahydrate, 0.1-1g/L ammonium molybdate and 0.5-5g/L calcium chloride dihydrate. Optionally, the trace metal element formulation includes: 10g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 0.35g/L manganese sulfate monohydrate, 0.23g/L sodium borate decahydrate, 0.11g/L ammonium molybdate, and 2.0g/L calcium chloride dihydrate.
In one embodiment, the lactose concentration in the fermentation system is maintained at 2-5g/L by feeding.
In one embodiment, the glycerol concentration in the fermentation system is maintained at 3-10g/L by feeding.
In one embodiment, the fermentation time is not less than 50 hours.
It is a third object of the present invention to provide the use of the recombinant bacterium described above or the method described above in the medical, food or chemical field.
In one embodiment, the use comprises producing lacto-N-neotetraose or a lacto-N-neotetraose containing product using the recombinant bacteria described above or the method.
Compared with the prior art, the invention has the following beneficial effects:
(1) The recombinant strain constructed by the invention does not need IPTG for induction, and can produce LNnT in a constitutive mode. The use of IPTG can greatly increase the cost of industrial production, and has a certain safety risk, and the strain does not need IPTG induction, so that the production cost can be greatly saved, the risk is reduced, and the strain is more suitable for industrial popularization and application.
(2) The strain provided by the invention has the advantages that the yield of LNnT (low-density polyethylene) fermented for 50h reaches 20.33g/L in a 5L fermentation tank, and the strain has a good industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of the metabolic pathway of lacto-N-neotetraose biosynthesis in recombinant E.coli.
FIG. 2 is a graph showing comparison of the yield of recombinant E.coli after supply of the strengthening precondition.
FIG. 3 is a schematic diagram of quorum sensing (A) and a comparison graph of yield after quorum sensing (B) was introduced.
Detailed Description
The present invention will be further described in more detail by way of specific examples.
Commercial products such as plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the examples below were prepared according to the kit instructions. The conventional methods of colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electrotransformation, competent cell preparation, and bacterial genome extraction and preservation were carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition). Sequencing of plasmid and DNA products was done by Jin Weizhi Biotechnology Inc. in Suzhou.
The medium involved in the fermentation of LNnT is as follows:
(1) LB liquid medium: 5g/L of yeast extract, 10g/L of peptone and 10g/L of sodium chloride.
(2) LB solid medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar powder.
(3) Fermentation medium: 20g/L glycerol, 5g/L yeast extract, 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate and 10ml/L trace metal elements (10 g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 0.35g/L manganese sulfate monohydrate, 0.23g/L sodium borate decahydrate, 0.11g/L ammonium molybdate, 2.0g/L calcium chloride dihydrate), adjusting the pH of the culture medium to 6.8 with sodium hydroxide, sterilizing at 115 ℃ for 30min, and adding 4.5mg/L thiamine before use.
Antibiotic concentration: ampicillin 100mg/L (liquid medium), ampicillin 200mg/L (solid medium), kanamycin 50mg/L, streptomycin 50mg/L, chloramphenicol 34mg/L.
Fed-batch fermentation feed liquid: glycerin 600g/L, magnesium sulfate heptahydrate 20g/L, thiamine 0.2g/L, ampicillin 100mg/L, and streptomycin 50mg/L. pH regulation: 50% ammonia (v/v).
Shake flask fermentation production of LNnT: the engineering strain is inoculated into 4mL LB liquid medium containing ampicillin and streptomycin, the engineering strain is cultivated for 10 to 12 hours under the conditions of 37 ℃ and 200rpm, seed solution is inoculated into 20mL fermentation medium according to the inoculum size of 2 percent, the engineering strain is cultivated at 37 ℃ and 200rpm, lactose with the final concentration of 5g/L is added when OD 600 is increased to 0.6 to 0.8, IPTG is optionally added (the addition of IPTG is not needed when the target gene is expressed by a conventional trc/tac/J23119 promoter, the addition of IPTG is not needed when LNnT is produced by a quorum sensing system), the final concentration is 0.5mM,25 ℃ and the induction cultivation is continued for 96 hours at 200 rpm.
Fed-batch fermentative production of LNnT: 2L of fermentation medium was added to a 5L bioreactor and sterilized at 115℃for 30 minutes. After sterilization, the temperature of the bioreactor was gradually reduced to 30 ℃ using a condenser. Before inoculation, the pH of the DM medium was adjusted to about 6.8 using 50% (v/v) NH3.H2O. The dissolved oxygen level in the bioreactor is maintained between 25% and 35%. The prepared seed solution is inoculated into a bioreactor with 10 percent of inoculum size, after 18.5 hours of culture, OD 600 reaches about 40, lactose feed is started to be fed into the culture medium, sampling is carried out at intervals, the concentration of the residual carbon source and the concentration of lactose are monitored, and the concentration of glycerol is kept at 3-10g/L and the concentration of lactose is kept at 2-5g/L through the feed.
Detection of the content of the substances (LNnT, LNTri II, lactose and glycerol) in the fermentation broth: 1mL of the fermentation broth was centrifuged at 10,000rpm for 10min, and the supernatant was filtered through a 0.22 μm aqueous filter and the content was measured by HPLC.
HPLC detection conditions: high Performance Liquid Chromatography (HPLC) system (Waters e 2695); chromatographic column: rezex ROA-organic acid H+ (8%); a detector: waters 2414RIDetector differential detector; mobile phase: 5mM H 2SO4; flow rate: 0.6mL/min; column temperature: 60 ℃; sample injection amount: 10 mu L.
Example 1: construction of recombinant plasmid vector
The primers used for plasmid construction are listed in Table 1.
TABLE 1 construction primers for recombinant plasmid vectors
The method comprises the following specific steps:
The plasmid pTrc99a-lgtA is obtained by amplifying a pCO-lgtA plasmid vector from Chinese patent document with publication number CN113684164A as a template, pTrc-lgtA-F/R as a primer to obtain lgtA gene fragment, pTrc99a as a template, a pair of primers pTrc-lgtA-V-F/R to obtain vector fragment, respectively recovering the two fragments by gel, and connecting the two fragments by Gibson kit (NEB reagent Co. U.S.A.). Then, the whole plasmid PCR was performed on pTrc99a-lgT A plasmid using primer pairs pTrc-tac-F/R and pTrc-J23119-F/R, and the promoters on plasmid pTrc99a-lgT A were replaced with promoters tac and J23119, respectively, to obtain plasmids pTac-lgT A and pJ23119-lgT A. the nucleotide sequences of tac and J23119 promoters are shown as SEQ ID NO.4 and SEQ ID NO.3 respectively.
The nucleotide sequence of β -1, 4-galactosyltransferase CpsIaJ is shown as SEQ ID NO.1, which is synthesized by GENEWIZ Biotech (Suzhou, china) and inserted into the first multiple cloning site of the pCDFDuet-1 plasmid vector to give plasmid pCD-CpsIaJ. And (3) carrying out full plasmid PCR by using pCD-CpsIaJ as a template and using primer pairs tac-F/R and J23119-F/R respectively, and replacing the T7 promoter on the plasmid pCD-CpsIaJ with the promoter tac and J23119 respectively to obtain plasmids pCD-TCP and pCD-JCP.
The plasmid pZE-luxR-P QS33 -luxI-eGFP is used as a template, a primer pair LuxRI-F/R is used for amplification to obtain a luxR-P QS33 -luxI sequence, pTrc99a-lgtA and pCD-CpsIaJ plasmid vectors are used as templates, luxRI-V-F/R and LuxRI-CPS-V-F/R are used as primers for amplification to obtain corresponding vector fragments, and then the two fragments are connected to obtain plasmids pTrc99a-luxR-P QS33 -luxI-lgtA and pCD-luxR-P QS33 -luxI-CP. With these two constructed plasmids as templates, whole plasmid PCR was performed using primer pairs HP-PQS03-F/R, HP-PQS13-F/R, HP-PQS23-F/R and HP-PQS43-F/R, respectively, and P QS33 was replaced with promoters P QS03,PQS13,PQS23 and P QS43. The nucleotide sequences of the promoters P QS03,PQS13,PQS23,PQS33 and P QS43 are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9. Further, whole plasmid PCR was performed on pTrc99a series plasmids using primers HP-QuluxR-F and HP-QuluxR-R (Trc) to obtain plasmids from which luxR was removed, followed by whole plasmid PCR using primer pair HP-QuluxI-F/R to finally obtain pTrc99a series plasmids from which luxR and luxI were removed; plasmid information constructed by performing whole plasmid PCR on pCD series plasmids using primers HP-QuluxR-F and HP-QuluxR-R (CPS) to obtain plasmid with luxR removed, followed by whole plasmid PCR using primers HP-QuluxI-F (CPS) and HP-QuluxI-R to finally obtain pCD series plasmids with luxR and luxI removed is shown in Table 2 below.
Table 2 constructed plasmids
Plasmid pZE-luxR-P QS33 -luxI-eGFP is disclosed in the literature Ge,C.;Yu,Z.;Sheng,H.;Shen,X.;Sun,X.;Zhang,Y.;Yan,Y.;Wang,J.;Yuan,Q.Redesigning regulatory components of quorum-sensing system for diverse metabolic control.Nat Commun 2022,13(1),2182.
Example 2: knockout of host bypass genes and promoter fortification of key enzymes
TABLE 3 construction primers for host engineering
The method comprises the following specific steps:
the method pEcCpf/pcrEG was used to knock out the ugd gene in E.coli and to replace the original promoter sequence of the galE gene.
Knock-out of the ugd gene:
(1) pEcCpf1 was plasmid-transferred to MG 1655. DELTA.wcaJ. DELTA.lacZ.DELTA.P lacY::PJ23119 (MGC 02, a strain disclosed in document Chen,R.,Zhu,Y.,Wang,H.,Liu,Y.,Meng,J.,Chen,Y.,Mu,W.,2023.Engineering Escherichia coli MG1655 for highly efficient biosynthesis of 2'-fucosyllactose by de novo GDP-fucose pathway.J.Agric.Food Chem.71(40),14678-14686.),, after single colonies were grown on plates, and inoculated into a test tube containing 4mL of LB medium containing kanamycin resistance, and cultured for 12 hours, to prepare electrotransfer competent cells.
(2) Long primers Ugd-F and Ugd-R for homologous recombination were synthesized from GENEWIZ Biotech (Suzhou, china) at a final concentration of 100. Mu.M.
(3) The pcrEG plasmid was PCR amplified using primers ugd-N23-F/R and the resulting linear DNA was circularized by single-fragment one-step cloning to a pcrEG-Deltaugd plasmid targeting ugd.
(4) The prepared electrotransformation competence containing pEcCpf < 1 > plasmid is taken on ice, pcrEG-Deltaugd plasmid and long primer Ugd-F or Ugd-R are added after thawing, and 1mL of LB culture medium which is fully precooled is added immediately after electrotransformation. After gently pipetting and mixing and transferring to a sterile 1.5mL ep tube, culturing was carried out for 2h at 37℃and 200 r/min. Then, the whole cells were plated on a double antibody plate (final concentration: 50. Mu.g/L kanamycin and 50. Mu.g/L spectinomycin), and cultured in an incubator at 37℃for 12 to 15 hours in an inverted manner.
(5) Single colonies grown were verified by colony PCR. If the external validation band is shorter than the negative control band, the gene knockout may be successful. And (3) performing bacteria P verification by using an external verification primer delta ugd-YZ-F/R, and sending a sample with a short external verification result band to Suzhou gold intelligent biotechnology limited company for sequencing, wherein a colony matched with the theoretical sequence is a colony with successful knockout ugd.
(6) Inoculating the above-verified bacteria, culturing in a 4mL test tube for 12 hours (50. Mu.g/L kanamycin and rhamnose with a final concentration of 10mM were added at the time of inoculation) to remove pcrEG-Deltaugd plasmid, further inoculating the target bacteria into a liquid LB medium containing 5g/L glucose and further streaking the bacterial liquid onto a plate containing 5g/L glucose and 15g/L sucrose to remove pEcCpf plasmid, and finally obtaining the strain MG1655 DeltawcaJ DeltalacZ DeltaP lacY::PJ23119 Deltaugd designated as MGC12.
Replacement of the original promoter sequence of the galE gene:
The original promoters of the galE gene were replaced with promoters P J23119 and P tac, respectively, using MGC02 as the starting strain. The construction method is identical to the knockout ugd, and is different in that the homologous recombination templates containing the tac promoter fragment are obtained by overlap extension PCR by using 1-tac-F, 2-tac-R and 3-tac-R as primers, and the homologous recombination templates containing the J23119 promoter fragment are obtained by overlap extension PCR by using 1-J23119-F, 2-J23119-R and 3-J23119-R as primers. The hosts finally obtained, in which the original galE gene promoter was replaced with P tac and P J23119, were designated MGC02T and MGC02J, respectively.
Example 3: lactoyl-N-neotetraose produced by recombinant strain fermentation
(1) Production of lactoyl-N-neotetraose by shake flask fermentation
The plasmids pTrc99a-lgT A, pTac-lgT A, pJ23119-lgT A and pCD-JCP, pCD-TCP obtained in example 1 were transferred into E.coli recombinant strain MGC02 in the manner shown in Table 4, respectively, to obtain strains MGC02-nA, MGC02-nB, MGC02-nC, MGC02-nD, MGC02-nE, MGC02-nF, and the yields of lactoyl-N-neotetraose were verified by fermentation.
The fermentation method comprises the following steps: the recombinant strains are respectively grown on a resistant agar LB plate overnight, inoculated into 4mL LB culture medium with corresponding antibiotics as initial seed liquid, and cultured for 8-12 h at 37 ℃ and 200 rpm. And (3) inoculating 400 mu L of seed solution into 20mL of fermentation medium, culturing at 37 ℃ and 200rpm until the OD 600 is 0.6-0.8, adding 0.5mM IPTG with the final concentration, and continuously performing induction culture at 25 ℃ and 200rpm for 96 hours. 1mL of the fermentation broth was centrifuged at 12,000rpm for 10min, and the supernatant was used for HPLC measurement.
As a result, as shown in Table 4, the yield of lacto-N-neotetraose was highest in strain MGC02-nB, 1.31g/L.
TABLE 4 engineering strain shake flask fermentation information for LNnT production
(2) Improving precursor supply
To increase precursor supply to further increase LNnT production, plasmids pTrc99a-lgtA and pCD-JCP were introduced into the constructed key precursor-enhanced hosts MGC12, MGC02T, MGC J, and fermentation validated LNnT production. The enhancement of either the knockout bypass gene ugd or galE is to increase the supply of UDP-Gal, which is a galactosyl donor from precursor LNTri II to the final synthesis of LNnT.
As a result of the fermentation conditions in the step (1), as shown in Table 4, knocking out the bypass gene ugd did not result in an increase in LNnT yield, and after strengthening galE to increase UDP-Gal supply, LNnT yield was further increased to 1.77g/L, and the group of enhancement of galE with P J23119 was superior to P tac.
Example 4: introduction of quorum sensing systems into host engineering bacteria to produce LNnT
(1) Fermentation production of LNnT by introducing quorum sensing system
The QS system was introduced into the engineered strain to evaluate its potential to improve LNnT synthesis. The recombinant strains MGC02-A33, MGC02-B33 and MGC02-C33 were obtained by introducing the plasmids pTrc99a-luxR-PQS 33-luxI-lgTAs, pTrc99a-PQS 33-lgTAs, pCD-luxR-PQS33-luxI-CP and pCD-PQS33-CP constructed in example 2 into MGC 02. The LNnT production of the recombinant strain was verified by fermentation under substantially the same conditions as in example 3, the only difference being that there was no need to add IPTG to initiate gene expression, and the results are shown in table 5.
After the lux type quorum sensing system is introduced into host engineering bacteria, the LNnT yields of the obtained strains MGC02-A33, MGC02-B33 and MGC02-C33 are 1.32 g/L, 3.05g/L and 1.04g/L respectively. This suggests that the implementation of the lux-type QS system is a very efficient method of increasing LNnT production. By autonomously and dynamically regulating the expression of lgtA and cpsIaJ, the production of LNnT was increased to 3.05g/L, 2.33 times that of the control MGC 02-nB. Furthermore, the results of the study also show that the location of luxR-luxI on the plasmid is very important under the quorum sensing system under the control of the PQS33 promoter, which shows a significant advantage over the pTrc99a vector (expression lgtA) when it is located on the pCD vector (expression cpsIaJ), whereas the effectiveness is greatly reduced when the luxR-luxI is present on both vectors.
TABLE 5 engineering strain shake flask fermentation information for the production of LNnT by introducing a quorum sensing system
(2) Promoter optimization
Promoters PQS03, PQS13, PQS23, PQS33 and PQS43 have different fluorescence peaks and trigger ODs, and the fluorescence peaks gradually rise and the trigger ODs gradually decrease from PQS03 to PQS 43. To further optimize the QS system, PQS33 in MGC02-B33 and MGC02-C33 were replaced with PQS03, PQS13, PQS23 and PQS43, respectively, to obtain two LNnT-producing libraries each containing 25 host engineering bacteria. In the first library, luxR-luxI was present on the pCD vector only, while in the other library it was present on both the pTrc99a vector and the pCD vector.
As shown in table 5, these strains exhibited different LNnT production levels. The best production strain in the first library was MGC02-B03 with an LNnT yield of 3.56g/L, a 1.7 fold increase compared to the strain MGC02-nB prior to introduction into the quorum sensing system. In the other pool, the highest yield was 3.33g/L, 1.54 fold increase compared to the pre-optimized level. The best producer strain in the first library contained pTrc99a-PQS03-lgT A and pCD-luxR-PQS33-luxI-CP plasmids, while the best producer strain in the other library contained pTrc99a-luxR-PQS23-luxI-lgT A and pCD-luxR-PQS43-luxI-CP plasmids.
The results indicated that strain MGC02-B03, which had been expressed by promoter PQS03 to control LgtA and by promoter PQS33 to control CpsIaJ, gave the highest yield of LNnT, indicating that the timing of expression of the lgtA and cpsIaJ genes was a key factor when LNnT was expressed using the QS system. Driving LgtA with QS promoters at higher trigger OD and driving expression of CpsIaJ at lower trigger OD resulted in higher LNnT titers. By fine tuning the timing of LgtA and CpsIaJ expression, the LNnT yield was further increased and the yield of the best producing strain reached 3.56g/L.
Example 5: fed-batch fermentation production of LNnT
In order to further verify the amplification effect of the high-yield LNnT strain, an engineering strain MGC02-B03 is selected for carrying out a fed-batch fermentation experiment of a 5L fermentation tank.
After the recombinant strain MGC02-B03 grows on an ampicillin and streptomycin-containing agar LB plate overnight, single colony is selected from the plate and inoculated into 4mL of LB culture medium containing ampicillin and streptomycin antibiotics for overnight culture, and the single colony is used as primary seed liquid; 1mL of the primary seed solution is inoculated into 100mL of fermentation medium and is subjected to expansion culture for 3-6h at 37 ℃ and 200rpm, so as to obtain secondary seed solution. The prepared secondary seed liquid was then inoculated into a 5L fermenter containing 2L of fermentation medium at an inoculum size of 10%. The fermentation culture temperature was controlled at 30℃throughout, and lactose addition was started when OD 600 reached about 40. When the concentration of the glycerol is lower than 3g/L, adding a glycerol feed supplement liquid to maintain the concentration of the glycerol in the fermentation system at 3-10g/L, when the concentration of the lactose is lower than 2g/L, adding a lactose feed supplement liquid to maintain the concentration of the lactose in the fermentation system at 2-5g/L, and regulating and controlling the pH by ammonia water to maintain the pH of the whole fermentation to be stable between 6.6 and 6.8.
Samples taken when the fermentation had proceeded to 50 hours and examined for LNnT production, which showed that the maximum production of LNnT in the supernatant was 20.33g/L and the productivity was 0.41g/L, which was 5.71 times that of the shake flask fermentation.
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. The recombinant bacterium is characterized in that the recombinant bacterium takes escherichia coli as an initial strain, and the initial strain is subjected to the following genetic engineering operation:
(1) Knocking out a beta-galactosidase coding gene lacZ and a UDP-glucose lipid carrier transferase coding gene wcaJ on the genome of the original strain;
(2) Replacing the promoter of the gene lacY encoding the beta-galactosidase on the genome with promoter P J23119;
(3) Expressing a beta-1, 3-N-acetylglucosaminyl transferase coding gene lgtA by using a promoter P QS03, and expressing a beta-1, 4-galactosyltransferase coding gene cpsIaJ by using an expression element luxR-P QS33 -luxI;
The Gene IDs of the Gene lacZ, the Gene wcaJ and the Gene lacY are respectively: 945006, 946583 and 949083; the nucleotide sequence of the promoter P J23119 is shown in SEQ ID NO. 3; the nucleotide sequence of the lgtA gene is shown as SEQ ID NO. 2; the nucleotide sequence of cpsIaJ gene is shown as SEQ ID NO. 1; the nucleotide sequence of the promoter P QS03 is shown in SEQ ID NO. 5; the nucleotide sequence of the expression element luxR-P QS33 -luxI is shown as SEQ ID NO. 10.
2. The recombinant bacterium according to claim 1, wherein the escherichia coli comprises escherichia coli MG1655.
3. A method for producing lacto-N-neotetraose, characterized in that the recombinant bacterium according to claim 1 or 2 is used for fermentation production of lacto-N-neotetraose.
4. A method according to claim 3, characterized in that the method comprises: inoculating the recombinant bacteria to a seed culture medium, culturing at 30-40 ℃ for 8-14h to obtain seed liquid, transferring the seed liquid to a fermentation culture medium, culturing until OD 600 is 0.6-0.8, adding lactose, reducing the fermentation temperature to 20-28 ℃, and fermenting to produce lactoyl-N-neotetraose.
5. A method according to claim 3, characterized in that the method comprises the steps of:
(1) Inoculating the recombinant bacteria to a seed culture medium, and culturing for 8-14h at 30-40 ℃ to obtain primary seed liquid;
(2) Transferring the primary seed liquid obtained in the step (1) into a fermentation medium according to the transfer amount of 0.5% -2%, and culturing for 3-6h at 30-40 ℃ to obtain a secondary seed liquid;
(3) Inoculating the secondary seed liquid obtained in the step (2) into a fermentation culture medium according to the inoculum size of 5% -15%, culturing until the OD 600 is 30-50, adding lactose, and fermenting to produce lactoyl-N-neotetraose.
6. The method according to claim 5, wherein the fermentation production temperature in step (3) is 25 to 35 ℃.
7. The method according to claim 6, wherein the concentration of glycerol in the fermentation system is maintained at 3-10g/L and the concentration of lactose is maintained at 2-5g/L by feeding.
8. The method of claim 7, wherein the fermentation time is not less than 50 hours.
9. Use of the recombinant bacterium of any one of claims 1-2, or the method of any one of claims 2-8, in the medical, food or chemical field.
10. The use according to claim 9, characterized in that the use comprises the production of lacto-N-neotetraose or a lacto-N-neotetraose containing product using the recombinant bacterium or the method.
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