Method for increasing yield of 2 '-fucosyllactose through synergistic effect of multiple alpha-1, 2-fucosyltransferases

Abstract

The invention relates to the field of microbial metabolism engineering, and discloses a method for increasing the yield of 2 '-fucosyllactose (2'-FL) through the synergistic effect of various alpha-1, 2-fucosyltransferases. According to the method, two or more alpha-1, 2-fucosyltransferases with different sources are co-expressed in microbial cells, and the complementary enzymatic characteristics of the alpha-1, 2-fucosyltransferases are utilized, so that the synthesis efficiency of the 2 '-FL is remarkably improved, and the yield of the 2'-FL is effectively improved. The microbial cell is escherichia coli C43 (DE3) (E. coli C43 lacZ waaF with lacZ and waaF genes knocked out, the serial number is SL), a pETDuet-1 plasmid is constructed, and genes manB, manC, gmd, wcaG and zwf are overexpressed on a pETDuet-1 vector, so that the microbial cell can be used for preparing the microbial cell. In a 5L fermentation tank, after the final strain SL07 is used for final fermentation for 68 hours, the yield of 2 '-FL reaches 148g / L, and the production intensity reaches 2.18 g / L / h. The embodiment shows that the engineering strain for co-expressing the alpha-1, 2-fucosyltransferase from different sources can obviously improve the yield of the 2 '-FL.

Description

Technical Field

[0001] This invention relates to the field of microbial metabolic engineering, and specifically to a method for synergistically increasing the yield of 2′-fucosylated lactose (2′-FL) by co-expressing multiple α-1,2-fucosyltransferases (α-1,2-FT) from different sources. Background Technology

[0002] 2′-Fucosyllactose (2′-FL), an oligosaccharide abundant in human breast milk, plays a crucial role in infant health, effectively promoting gut health, preventing infant diarrhea, and is an important component for ensuring normal growth and development in infants. Currently, the synthesis of 2′-FL using microbial cell factories has become the main production method, and α-1,2-fucosyltransferase, as a key enzyme catalyzing the synthesis of 2′-FL from GDP-fucose and lactose, directly determines the yield of 2′-FL.

[0003] However, existing technologies mostly employ single-source α-1,2-FT enzymes (such as WbgL, SAMT, FutCB, NsFutC, etc.), which have several shortcomings. In terms of catalytic efficiency, their reaction rates are relatively low, making it difficult to complete large-scale synthetic transformations per unit time, thus limiting the rapid and large-scale production of 2′-FL. Regarding expression stability, their expression levels in microbial cells are prone to fluctuations, resulting in a lack of a stable enzyme supply for the entire synthesis process, thereby affecting the stable increase in 2′-FL yield. Furthermore, single-source enzymes often only exhibit good catalytic activity under relatively fixed conditions, with narrow tolerance to reaction conditions such as temperature and pH. Even slight changes in external environmental conditions can significantly affect their catalytic activity, making it difficult to sustain efficient 2′-FL synthesis and ultimately hindering further breakthroughs in 2′-FL yield. Therefore, there is an urgent need to develop a novel enzyme combination strategy to overcome the bottlenecks of existing technologies. Summary of the Invention

[0004] This invention aims to overcome the many shortcomings of existing technologies and provide a highly efficient synthesis method based on a space synthetic biology strategy, specifically a method to increase 2′-FL yield through the synergistic action of multiple α-1,2-fucosyltransferases. Specifically, this invention co-expresses two or more α-1,2-FTs from different sources, fully utilizing their complementary enzymatic properties. This allows enzymes from different sources to function in their respective strengths, cooperating and synergistically, thereby significantly improving the synthesis efficiency of 2′-FL and ultimately achieving a substantial increase in 2′-FL yield. Detailed Implementation

[0005] Example 1: Chassis Construction

[0006] In this embodiment, Escherichia coli C43(DE3) is used as an example to construct a starting strain for the metabolic synthesis of 2′-FL.

[0007] Specifically, the lacZ and waaF genes were sequentially knocked out in Escherichia coli C43(DE3) (purchased from the China Industrial Microbial Culture Collection Center). The resulting chassis strain, E. coli C43△lacZ△waaF, was designated SL.

[0008] During gene manipulation, plasmids pGRB and pRedCas9 were used to express sgRNA and Cas9 protein.

[0009] The specific construction process includes:

[0010] 1. Knockout of the lacZ gene

[0011] Using the E. coli C43(DE3) genome as a template, upstream homologous arm primers (UP-lacZ-S, Up-lacZ-A) and downstream homologous arm primers (DN-lacZ-S, DN-lacZ-A) were designed based on the upstream and downstream sequences of the lacZ gene. The upstream and downstream homologous arm fragments were amplified by PCR. These fragments were then fused using overlap PCR to obtain the lacZ gene knockout fragment (lacZ-up-down). An N20 sequence targeting lacZ was designed online, and a short DNA fragment was prepared by annealing with primers gRNA-lacZ-S and gRNA-lacZ-A. This N20 short DNA fragment was ligated into pGRB using a seamless cloning kit (Novizan one-step cloning kit) to obtain the pGRB-lacZ plasmid. Subsequently, competent cells of E. coli C43(DE3) / pCas were prepared (IPTG was added during the preparation of competent cells for induction). The plasmid pGRB-lacZ and the fragment "lacz-up-down" were electroporated into C43 / pCas basal cells containing the pCas plasmid. After verification and identification, the strain E. coli C43(DE3)△lacZ with lacZ knocked out was obtained.

[0012] 2. Knockout of the waaF gene

[0013] Using the E. coli C43(DE3) genome as a template, upstream homologous arm primers (UP-waaF-S, Up-waaF-A) and downstream homologous arm primers (DN-waaF-S, DN-waaF-A) were designed based on the upstream and downstream sequences of the waaF gene. The upstream and downstream homologous arm fragments were amplified by PCR. These fragments were then fused using overlap PCR to obtain the waaF gene knockout fragment (waaF-up-down). An N20 sequence targeting waaF was designed online, and a short DNA fragment was prepared by annealing with primers gRNA-waaF-S and gRNA-waaF-A. This N20 short DNA fragment was ligated into pGRB using a seamless cloning kit (Novizan one-step cloning kit) to obtain the pGRB-waaF plasmid. Subsequently, competent cells of E. coli C43(DE3)△lacZ / pCas were prepared (IPTG was added during the preparation of competent cells for induction). The plasmid pGRB-waaF and the fragment "waaF-up-down" were electroporated into C43△lacZ / pCas basal cells containing the pCas plasmid. After verification and identification, the strain E. coli C43(DE3)△lacZ△waaF with waaF knocked out was obtained.

[0014] Example 2: Construction of expression vector

[0015] Using gene synthesis and molecular cloning techniques, the genes manB, manC, gmd, wcaG, zwf, fbp, gsk, rcsB, and lacY were overexpressed on plasmids, and at least two of the following α-1,2-FTs were transformed into the sclerotium bacteria:

[0016] NsFutC (derived from the genus Neisseria sp.)

[0017] SAMT (derived from Azospirillum lipoferum)

[0018] WbgL (derived from E. coli O126)

[0019] FutCB (derived from Bacillus cereus) Strain number Combination SL01 FutCB SL02 SAMT SL03 WbgL SL04 NsFutC SL05 FutCB + SAMT SL06 FutCB + WbgL SL07 FutCB + NsFutC SL08 SAMT + WbgL SL09 SAMT + NsFutC SL10 WbgL + NsFutC

[0020] The specific construction process includes:

[0021] 1. Construction of pETDuet-BCGWZ

[0022] Genes manB, manC, gmd, wcaG, and zwf were overexpressed on the pETDuet-1 vector.

[0023] PCR amplification was performed using primer pair ET-zwf-F / ET-zwf-R with pETDuet-1 as a template, yielding a linearized vector containing homologous arms at both ends. PCR amplification was then performed using primer pair zwf-F / zwf-R with the wild-type E. coli C43 genome as a template, yielding the zwf target gene fragment containing homologous arms at both ends. A seamless cloning reaction system (using the Novizan one-step cloning kit) was prepared according to the kit instructions, using the linearized vector and target gene fragment. After incubation at 37°C for 30 min, 10 µL of the product was transformed into 100 µL of DH5α chemicompetent cells. After verification by colony PCR and sequencing, the recombinant plasmid pETDuet-zwf was obtained.

[0024] PCR amplification was performed using primer pair new-all-ET-F / all-ET-R with pETDuet-zwf as a template, yielding a linearized vector containing homologous arms at both ends. The target gene cpsG-cpsB fragment was amplified by PCR using primer pair cpsG-F / cpsB-R-1 with the wild-type E. coli C43 genome as a template; the target gene gmd-fcl fragment was amplified by PCR using primer pair gmd-F / fcl-R with the E. coli C43 genome as a template. A second round of PCR was performed using the target gene fragments as templates to introduce the RBS site and promoter sequence. The linearized vector, the target gene fragment cpsG-cpsB, and the target gene fragment gmd-fcl were seamlessly cloned. After verification by colony PCR and sequencing, the recombinant plasmid pETDuet-BCGWZ was obtained.

[0025] 2. Construction of pAC series plasmids

[0026] Using pACYCDuet-1 as a vector, the fbp, gsk, rcsB, and lacY genes were overexpressed, and NsFutC (derived from Neisseria sp.), SAMT (derived from Azospirillum lipoferum), WbgL (derived from E. coli O126), FutCB (derived from Bacillus cereus), or different combinations thereof, were overexpressed, resulting in the construction of a total of 10 pAC series plasmids.

[0027] First, pAC-01 was constructed by overexpressing the FutCB, fbp, gsk, rcsB, and lacY genes using pACYCDuet-1 as a vector.

[0028] PCR amplification was performed using primer pair AC-kanR-F / AC-kanR-R with pACYCDuet-1 as a template, yielding a linearized vector containing homologous arms at both ends. PCR amplification was then performed using primer pair kanR-F / kanR-R with pET28a as a template, yielding the kanR target gene fragment containing homologous arms at both ends. Seamless cloning was performed using the linearized vector and target gene fragment according to the kit instructions. After verification by colony PCR and sequencing, the plasmid pACYCDuet-kanR was obtained.

[0029] PCR amplification was performed using primer pair AC-lacY-F / AC-lacY-R with pACYCDuet-kanR as a template, yielding a linearized vector containing homologous arms at both ends. PCR amplification was then performed using primer pair lacY-F / lacY-R with the wild-type E. coli C43(DE3) genome as a template, yielding the lacY target gene fragment containing homologous arms at both ends. Seamless cloning was performed using the linearized vector and the target gene fragment. After verification by colony PCR and sequencing, the recombinant plasmid pACYCDuet-lacY was obtained.

[0030] The target gene futCB fragment was amplified by PCR using primer pair futCB-F / futCB-R with the synthesized futCB gene as a template.

[0031] The target gene rcsB fragment was amplified by PCR using primer pair rcsB-F / rcsB-R with E. coli MG1655 genome as template.

[0032] PCR amplification was performed using primer pair all-AC-F / all-AC-R-1 with pACYCDuet-lacY as a template. A linearized vector containing partial homologous arms at both ends was recovered. The PCR product was then used as a template for a second round of PCR with primer pair all-AC-F / all-AC-R-2 to introduce the linearized vector containing complete homologous arm sequences. The linearized vector, the target gene fragment futCB, and the target gene fragment rcsB were seamlessly cloned. After verification by colony PCR and sequencing, the recombinant plasmid pACYCDuet-FFGRL (numbered pAC-01) was obtained.

[0033] Subsequently, reverse PCR was performed using primer pair pAC01-F / R with pAC-01 plasmid as template (amplifying the remaining sequences on the vector except for the FutcB gene sequence, to be used as a universal linearization vector to ligate genes such as NsFutC, SAMT, and WbgL). The PCR product was recovered as a linearization vector (pAC-FGRL fragment). Using seamless cloning technology, several genes encoding α-1,2-FT from different sources were ligated into plasmid vectors respectively.

[0034] Using primer pair SAMT-F / R and the synthesized SAMT gene as a template, the target gene SAMT fragment containing homologous arms at both ends was amplified by PCR. This fragment was then seamlessly cloned with the "pAC-FGRL fragment". After colony identification and plasmid sequencing, plasmid pAC-02 was obtained.

[0035] Using the primer pair WbgL-F / R and the synthesized WbgL gene as a template, the target gene WbgL fragment containing homologous arms at both ends was amplified by PCR. This fragment was then seamlessly cloned with the "pAC-FGRL fragment". After colony identification and plasmid sequencing, plasmid pAC-03 was obtained.

[0036] Using the primer pair NsFutC-F / R and the synthesized WbgL gene as a template, the target gene NsFutC fragment containing homologous arms at both ends was amplified by PCR. This fragment was then seamlessly cloned with the "pAC-FGRL fragment". After colony identification and plasmid sequencing, plasmid pAC-04 was obtained.

[0037] Subsequently, NsFutC, SAMT, WbgL, and FutCB were combined and expressed on the vector to construct the vector:

[0038] Using primer pair 2-pAC-F / R, reverse PCR was performed with pAC-01 plasmid as a template. The PCR product was recovered and used as a linearized vector (pAC-01-fragment). The genes SAMT, WbgL, and NsFutC, each containing homologous arms at both ends, were amplified using primer pairs 2-SAMT-F / R, 2-WbgL-F / R, and 2-NsFutC-F / R, respectively. These were then seamlessly cloned with the "pAC-01-fragment." Colony identification and plasmid sequencing yielded plasmids pAC-05, pAC-06, and pAC-07, respectively.

[0039] Using primer pair 2-pAC-F / R, reverse PCR was performed with pAC-02 plasmid as a template. The PCR product was recovered and used as a linearized vector (pAC-02-fragment). Genes WbgL and NsFutC, containing corresponding homologous arms at both ends, were amplified using primer pairs 2-WbgL-F / R and 2-NsFutC-F / R, respectively. These were then seamlessly cloned with the "pAC-02-fragment." Colony identification and plasmid sequencing yielded plasmids pAC-08 and pAC-09, respectively.

[0040] Using primer pair 2-pAC-F / R, reverse PCR was performed with pAC-03 plasmid as a template. The PCR product was recovered and used as a linearized vector (pAC-03 fragment). The gene NsFutC, containing corresponding homologous arms at both ends, was amplified using primer pair 2-NsFutC-F / R. This gene was then seamlessly cloned with the "pAC-03 fragment." After colony identification and plasmid sequencing, plasmid pAC-10 was obtained.

[0041] Example 3 Construction of recombinant engineered bacteria

[0042] The plasmid pETDuet-BCGWZ described in Example 2 was electroporated into the chassis bacteria SL (E. coli C43△lacZ△waaF) described in Example 1 to obtain strain SL0 (E. coli C43△lacZ△waaF / pETDuet-BCGWZ).

[0043] SL0 competent cells were prepared, and plasmids pAC01~10 described in Example 2 were transformed into cells to obtain 10 recombinant engineered bacteria SL01~SL10.

[0044] Strain number Plasmid 1 Plasmid 2 SL01 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-FutCB-fbp-J23107-gsk-rcsB-lacIP-lacY SL02 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-SAMT-fbp-J23107-gsk-rcsB-lacIP-lacY SL03 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-WbgL-fbp-J23107-gsk-rcsB-lacIP-lacY SL04 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-NsFutC-fbp-J23107-gsk-rcsB-lacIP-lacY SL05 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-FutCB-fbp-J23111-SAMT-J23107-gsk-rcsB-lacIP-lacY SL06 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-FutCB-fbp-J23111-WbgL-J23107-gsk-rcsB-lacIP-lacY SL07 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-FutCB-fbp-J23111-NsFutC-J23107-gsk-rcsB-lacIP-lacY SL08 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-SAMT-fbp-J23111-WbgL-J23107-gsk-rcsB-lacIP-lacY SL09 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-SAMT-fbp-J23111-NsFutC-J23107-gsk-rcsB-lacIP-lacY SL10 pET-J23118-manB-manC-J23104-gmd-wcaG-lacIP-zwf pAC-J23111-WbgL-fbp-J23111-NsFutC-J23107-gsk-rcsB-lacIP-lacY

[0045] Example 4: Shake-flask fermentation verification

[0046] Strains S01–S10 were cultured overnight in 50 mL LB medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin at 37°C and 220 rpm. Then, 10% (v / v) of the seed culture was inoculated into the fermentation medium for shake-flask culture.

[0047] The fermentation medium consists of: lactose 15 g / L, glycerol 30 g / L, yeast extract (or yeast powder) 5.0 g / L, tryptone (or corn steep liquor powder) 10.0 g / L, Na2HPO4·12H2O 17.1 g / L, (NH4)2HPO4 1.0 g / L, KH2PO4 3.0 g / L, NH4Cl 2.0 g / L, trisodium citrate 2.0 g / L, magnesium sulfate·7H2O 1.4 g / L, thiamine (VB1) 10.0 mg / L, and trace elements 1‰. The composition of the trace salt stock solution is as follows: FeCl3·6H2O 25.0 g / L, CaCl2·2H2O 2.3 g / L, ZnCl2 2.6 g / L, CuSO4·5H2O 2.0 g / L, MnSO4·H2O 2.5 g / L, Na2MoO4·2H2O 2.6 g / L, and H3BO3 0.7 g / L.

[0048] The cells were cultured at 37°C and 220 rpm for the first 5 hours, and then cooled to 34°C for 72 hours.

[0049] During fermentation, 25 g / L of glycerol and 10 g / L of lactose were added to the fermentation broth after 24 h of fermentation; and 10 g / L of glycerol and 10 g / L of lactose were added to the fermentation broth after 48 h of fermentation.

[0050] Samples were taken after fermentation, and the 2'-FL content was detected by HPLC-MS. The results showed that SL07 had the best fermentation effect, with a yield of 33.7 g / L.

[0051] strain number OD Production g / L SL01 25.15 16.3 SL02 22.75 16.5 SL03 19 31.3 SL04 29.7 15.1 SL05 21.2 30.1 SL06 21.6 22.4 SL07 21.25 33.7 SL08 19 20.8 SL09 19.45 24 SL10 14.55 33

[0052] Example 5: Feed-in batch fermentation in a 5L fermenter

[0053] The SL07 strain was fermented in a 5L fermenter. Seed culture: The culture was carried out overnight in fresh LB medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin at 37°C and 220 rpm. 10% (v / v) of the seed culture was inoculated into the fermentation medium (same as the shake-flask fermentation medium). During fermentation, the pH was adjusted with dilute ammonia to maintain it between 6.7 and 6.9. The initial culture temperature was 37°C, and the temperature was slowly lowered to 34°C from 11 to 15 hours. Glycerol and lactose were added after 12-13 hours of fermentation.

[0054] After 68 hours of final fermentation, the yield of 2′-FL reached 148 g / L, and the production intensity reached 2.18 g / L / h, demonstrating excellent potential for industrial production.

Claims

1. A method for increasing the yield of 2′-fucosyllactose (2′-FL), characterized in that, Includes the following steps: (1) Two or more α-1,2-fucosyltransferases (α-1,2-FT) from different sources are co-expressed in microbial cells, wherein the α-1,2-FT is selected from Neisseria sp., Azospirillumlipoferum, Escherichia coli O126 and Bacillus cereus; (2) The microbial cells are cultured under suitable culture conditions to enable the microbial cells to synthesize 2′-FL.

2. The method according to claim 1, characterized in that: The microbial cells were Escherichia coli C43(DE3) (E. coli C43(DE3)).

3. The method according to claim 1, characterized in that: The microbial cells were E. coli C43(DE3) (E. coli C43△lacZ△waaF, numbered SL) with the lacZ and waaF genes knocked out.

4. The method according to claim 1, characterized in that, The pETDuet-BCGWZ plasmid contained in SL overexpresses the genes manB, manC, gmd, wcaG, and zwf in the pETDuet-1 vector.

5. The method according to claim 1, characterized in that, SL contains the pACYCDuet-1 vector, which integrates four genes: fbp, gsk, rcsB, and lacY.

6. The method according to claim 1, characterized in that: The two or more α-1,2-FTs from different sources include α-1,2-FTs from Neisseria and α-1,2-FTs from Escherichia coli O126.

7. The method according to claim 1, characterized in that: The two or more α-1,2-FTs from different sources include α-1,2-FTs from Azotobacter lipogenicus and α-1,2-FTs from Bacillus cereus.

8. The method according to claim 1, characterized in that: The culture conditions include a culture temperature of 25℃-37℃ and a culture time of 12-72 hours.

9. The method according to claim 1, characterized in that: In a 5L fermenter, using strain SL07 (E. coli C43 △lacZ△waaF / pETDuet-BCGWZ / pAC-J23111-FutCB-fbp-J23111-NsFutC-J23107-gsk-rcsB-lacIP-lacY), after 68 hours of final fermentation, the yield of 2′-FL reached 148 g / L, and the production intensity reached 2.18 g / L / h.