CN117487728A - Application of escherichia coli engineering strain for efficiently producing 2' -fucosyllactose - Google Patents

Application of escherichia coli engineering strain for efficiently producing 2' -fucosyllactose Download PDF

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CN117487728A
CN117487728A CN202311327641.3A CN202311327641A CN117487728A CN 117487728 A CN117487728 A CN 117487728A CN 202311327641 A CN202311327641 A CN 202311327641A CN 117487728 A CN117487728 A CN 117487728A
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fucosyllactose
recombinant strain
seq
gene
nucleotide sequence
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张康
吴敬
王璐瑶
高圣琦
刘同乐
蔡博涵
张梦薇
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Jiangnan University
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Jiangnan University
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Abstract

The invention discloses application of an escherichia coli engineering strain for efficiently producing 2' -fucosyllactose, belonging to the fields of synthetic biology, microbial metabolism engineering technology, fermentation engineering technology and the like. The invention provides a regulation strategy of the expression level of key enzymes in the de-novo synthesis way of 2'-fucosyllactose, knocks out a series of genes related to substrate degradation and intermediate product split in escherichia coli, simultaneously respectively carries out regulation and control of genome overexpression or plasmid overexpression on different enzymes, optimizes RBS, greatly improves the efficiency of metabolic pathways, and utilizes recombinant strain constructed by the related strategy to enable the obtained escherichia coli engineering strain to produce 125g/L of 2' -FL by taking glycerol as a carbon source and 115g/L of 2'-FL by taking glucose as a carbon source under the condition of a 3-L fermentation tank, thereby having important significance for industrialized production of 2' -FL.

Description

Application of escherichia coli engineering strain for efficiently producing 2' -fucosyllactose
Technical Field
The invention relates to application of an escherichia coli engineering strain for efficiently producing 2' -fucosyllactose, belonging to the fields of synthetic biology, microbial metabolism engineering technology, fermentation engineering technology, microbial genetic engineering and the like.
Background
Human Milk Oligosaccharides (HMOs) are a complex class of structurally diverse oligosaccharides that are critical in neonatal health and development. In terms of structural composition, 2'-fucosyllactose (2' -FL) is a lactose linked at the reducing end and a fucose (Fuc) linked at the non-reducing end to galactose (Gal) in the lactose structure via an alpha-1, 2 linkage. It has the highest ratio in HMOs compared to other substances. Studies have shown that infant formula supplemented with 2' -FL has nutritional ingredients and efficacy that more closely approximate breast milk. Many studies have shown that 2' -FL is capable of modulating the balance of intestinal flora in infants, resisting pathogen attachment and immunomodulation, promoting brain and neurological development in infants. Currently, 2' -FL has entered the commercial production stage, but its industrialization progress is limited by factors such as its yield, cost, quality and safety.
Early scientific studies have isolated 2' -FL directly from human milk. Currently, there are three main methods for synthesizing 2' -FL: chemical synthesis, enzymatic synthesis and whole cell biosynthesis. The production process of chemical and enzymatic synthesis of 2' -FL is complicated and inefficient, which hinders mass production thereof. In contrast, whole cell biosynthesis methods can be used to achieve large-scale production using engineered bacteria, and are ideal alternatives to the above-described methods. Intracellular synthesis of 2' -FL involves two pathways. The first is called the salvage pathway, in which exogenous L-fucose is converted to GDP-L-fucose by the catalysis of an L-fucokinase/GDP-fucose pyrophosphorylase (L-fucokinase/GDP-fucose pyrophosphorylase, FKP) enzyme. Then, the GDP-L-fucose produced by the catalytic action of the alpha-1, 2-fucosyltransferase reacts with lactose to form 2' -FL. Another route is the de novo synthesis route. The host synthesizes GDP-L-fucose through a range of metabolic pathways using inexpensive raw materials such as glucose, sucrose, or glycerol as carbon sources. Then, GDP-L-fucose is reacted with lactose under the catalysis of alpha-1, 2-fucosyltransferase to produce 2' -FL. The de novo synthetic pathway is more economical than the salvage pathway and thus increasingly dominates the industrial manufacture of 2' -FL.
Currently, reported production methods generally use glycerol as a fermentation carbon source to obtain high density fermentation and synthesis to obtain high 2' -FL yields. However, glycerol is inconvenient to determine its content in real time during fermentation and residual glycerol is also difficult to completely separate from the product. The production process using glucose as a carbon source is more convenient and the cost is lower than that of glycerol.
In summary, the prior art is limited in that when glucose is used as a carbon source in 2'-FL synthesis, a large amount of acetic acid, an intermediate metabolite, is accumulated, ultimately resulting in lower yields of 2' -FL. Resulting in too low a yield of fucosylated human milk oligosaccharides to meet the demands of industrial production.
Disclosure of Invention
[ technical problem ]
The invention aims to provide an escherichia coli engineering strain capable of efficiently producing 2' -fucosyllactose from two carbon sources, namely glucose and glycerol.
Technical scheme
In order to solve the technical problems, the invention regulates and controls different expression levels of key enzymes of the de novo synthesis pathway of 2'-fucosyllactose so as to improve the pathway efficiency and the production level of 2' -fucosyllactose.
The invention provides a recombinant strain for producing 2'-fucosyllactose, which takes escherichia coli as a host to regulate and control the expression of 2' -fucosyllactose from a de novo synthesis pathway enzyme, and comprises free expression genes: the gene cluster of ManB-ManC, gmd-WcaG, guanosine kinase Gsk, 2' -fucosyllactose FutC with fusion protein tag at N end, DNA binding transcription activators rcsA and rcsB, beta-galactoside permease LacY, sugar efflux transporter setA, NADP (+) -dependent glucose-6-phosphate dehydrogenase zwf are overexpressed; knocking out the genome gene: GDP-mannose hydrolase NudD (NCBI accession number Gene ID: 946559), beta-galactosidase LacZ (NCBI accession number Gene ID: 945006), GDP-mannose hydrolase NudK (NCBI accession number Gene ID 947072), glucose-1-phosphate transferase undecanoate WcaJ (NCBI accession number Gene ID 946583), ATPase component ClpY of HslVU protease (NCBI accession number Gene ID 948430), peptidase component ClpQ of HslVU protease (NCBI accession number Gene ID 948429), lon protease Lon (NCBI accession number Gene ID 945085), galactosido-acetyltransferase lacA (NCBI accession number Gene ID 945674), isocitrate lyase regulator iclR (NCBI accession number Gene ID 948524), phosphoacetyltransferase ptA (NCBI accession number Gene ID 946778), transcriptional dual regulator arcA (NCBI accession number Gene ID 948874), oxidase poxB (NCBI accession number 946132), formate lyase lacA (NCBI accession number Gene ID 945674), phosphoacetyltransferase regulator icR (NCBI accession number Gene ID 5487), and pyruvate dehydrogenase (NCBI accession number NCBI ID 37G.
In one embodiment of the invention, the fusion protein tag is ubiquitin-like modification molecule SUMO.
In one embodiment of the invention, the recombinant strain expresses the gene episomally using plasmids pRSF-Duet-1, pET-Duet-1 and/or pCDF-Duet-1.
In one embodiment of the invention, the expression of the gene is initiated by a double T7 promoter.
In one embodiment of the invention, the recombinant strain expresses the ManB-ManC gene cluster and the Gmd-WcaG gene cluster using pRSF-Duet-1 plasmid.
In one embodiment of the invention, the recombinant strain expresses genes Gsk and FutC using pET-durt-1 plasmid.
In one embodiment of the invention, the recombinant strain expresses genes LacY, setA, rcsA, rcsB and Zwf using the pCDF-durt-1 plasmid.
In one embodiment of the invention, the recombinant strain uses RBS to regulate the expression intensity of the ManB-ManC gene cluster and the Gmd-WcaG gene cluster.
In one embodiment of the invention, the recombinant strain modulates the ManB-ManC gene cluster using RBS29 and the Gmd-WcaG gene cluster using RBS 34.
In one embodiment of the invention, the host cell comprises E.coli BL21star (DE 3), E.coli K12 MG1655 or E.coli JM109.
In one embodiment of the invention, the host cell is E.coli BL21star (DE 3).
In one embodiment of the invention, the nucleotide sequences of the encoding genes of mannose-1-guanyl phosphate transferase ManC and mannosyl phosphate mutase ManB are shown as SEQ ID NO. 1;
in one embodiment of the invention, the nucleotide sequences of the coding genes of the GDP-mannose-4, 6-dehydratase Gmd and GDP-fucose synthase WcaG are shown in SEQ ID NO. 2;
in one embodiment of the invention, the nucleotide sequences of the coding genes of the DNA binding transcriptional activators rcsA and rcsB are shown in SEQ ID NO.3 and SEQ ID NO. 4;
in one embodiment of the invention, the nucleotide sequence of the coding gene of the 2' -fucosyllactose FutC is shown in SEQ ID NO. 5;
in one embodiment of the invention, the nucleotide sequence of the coding gene of the beta-galactoside permease LacY is shown in SEQ ID NO. 6;
in one embodiment of the invention, the nucleotide sequence of the coding gene of the sugar efflux transporter SetA is shown in SEQ ID NO. 7;
in one embodiment of the invention, the nucleotide sequence of the encoding gene of the guanosine kinase Gsk is shown as SEQ ID NO. 8;
in one embodiment of the present invention, the nucleotide sequence of the coding gene of NADP (+) -dependent glucose-6-phosphate dehydrogenase Zwf is shown in SEQ ID NO. 9;
in one embodiment of the invention, the coding sequence of the ubiquitin-like modifier molecule SUMO is shown in SEQ ID NO. 10.
In one embodiment of the invention, the nucleotide sequence of RBS29 is set forth in SEQ ID NO. 11; the nucleotide sequence of RBS34 is shown as SEQ ID NO. 12.
The invention provides a method for producing 2'-fucosyllactose, which is to take the recombinant strain as a fermentation strain to produce and prepare the 2' -fucosyllactose.
In one embodiment of the invention, the method uses glycerol or glucose as a single carbon source and lactose as a substrate.
In one implementation method of the invention, the shake flask fermentation method comprises the following steps: inoculating the constructed engineering bacteria into an LB liquid culture medium, culturing for 10-16 hours at 35-38 ℃ and 180-220 rpm in a shaking bottle to obtain seed liquid; inoculating the seed liquid into fermentation medium at 0.5-2% of inoculation amount, shaking flask culture to OD at 35-38 deg.c and 180-220 rpm 600 0.5-2 mM IPTG with final concentration of 0.1-0.2 mM is added, and lactose with final concentration of 8-12 g/L is added for induction culture at 20-DEG C and 200 rpm.
In one embodiment of the invention, the 3-L fermenter fermentation process is: the recombinant strain is inoculated in a fermentation culture medium, cultured until the OD600 is 15-20, and added with lactose with the final concentration of 5g/L and IPTG with the final concentration of 0.1mM.
In one embodiment of the present invention, glycerol or glucose is fed so as to have a concentration of not less than 3g/L, and lactose is fed so as to have a concentration of not less than 3 g/L.
In one embodiment of the invention, the fermentation conditions of the 3-L fermentation tank are that the culture temperature is 25-37 ℃, the stirring rotation speed is 300-800 r/min, the ventilation rate is 0.8-1.2 vvm, the pH is 6.5-7.0, and the fermentation time is 48-72 h.
In one embodiment of the invention, the fermentation medium contains 3.375g/L KH 2 PO 4 ，0.7g/L MgSO 4 ·7H 2 O，2.0g/L(NH 4 ) 2 HPO 4 ，1.7g/L C 6 H 8 O 7 ·H 2 O,5g/L industrial yeast powder, 2.5g/L porcine bone peptone, 8g/L glycerol and 10mL of microelement solution.
In one embodiment of the invention, the trace element solution takes 5mol/L HCl as mother solution and 2.0g/L CaCl 2 ，0.1g/L(NH 4 ) 6 Mo 7 O 24 ，2.25g/L ZnSO 4 ·7H 2 O，0.5g/L MnSO 4 ·4H 2 O，10.0g/L FeSO 4 ·7H 2 O，3.0g/L CuSO 4 ·5H 2 O，0.23g/L Na 2 B 4 O 7 ·10H 2 O，0.18g/L CoCl 2 ·6H 2 O。
The invention also provides application of the recombinant strain in producing 2'-fucosyllactose or a product containing 2' -fucosyllactose.
[ advantageous effects ]
The invention regulates and controls the expression obtained by mannose-1-guanyl phosphate transferase ManC, mannomutase ManB, GDP-mannose-4, 6-dehydratase Gmd, GDP-fucose synthetase WcaG, beta-galactoside permease LacY, DNA combined transcription activator rcsA and rcsB, sugar efflux transporter SetA, guanosine kinase Gsk and NADP (+) -dependent glucose-6-phosphate dehydrogenase Zwf in combination and knocks out GDP-mannose hydrolase NudD and GDP-mannose hydrolase NudK in the escherichia coli host 2'-fucosyl lactose synthesis pathway, beta-galactosidase LacZ, undecalactoglucose-1-phosphotransferase WcaJ, the ATPase component ClpY of HslVU protease, the peptidase component ClpQ of HslVU protease, the Lon protease Lon, the galactoside O-acetyltransferase lacA, the isocitrate lyase regulator iclR expresses phosphoacetyl transferase ptA, the transcriptional dual regulator arcA, pyruvate oxidase poxB, pyruvate formate lyase pflB, lactate dehydrogenase ldhA, glucose phosphotransporter ptsG, and the transcriptional level is trimmed using plasmid copy number and RBS strength to achieve the goal of increasing 2' -fucosyllactose yield (FIG. 1).
The fermentation production verification of the fucosylation oligosaccharide (taking 2' -fucosyl lactose as an example and glycerol or glucose as a substrate) in the 3-L tank is carried out by adopting the metabolic engineering bacterium provided by the invention, and the result shows that the production level of the 2' -fucosyl lactose (2 ' -FL) taking glycerol as a single carbon source can reach 125g/L at the highest, and the production level of the 2' -fucosyl lactose (2 ' -FL) taking glucose as a single carbon source can reach 115g/L at the highest.
Drawings
Fig. 1: de novo synthetic pathway for the production of 2' -fucosyllactose.
Wherein, manB, phosphomannose mutase; manC, mannose-1-phosphate guanyl transferase; gmd GDP-mannose-4, 6-dehydratase; wcaG, GDP-fucose synthase; futC,2' -fucosyllactose; setA, sugar efflux transporter; lacZ, beta-galactosidase; lacY, beta-galactosidase; rcsA and RcsB, DNA binding transcriptional activators; zwf, guanosine kinase Gsk and NADP (+) -dependent glucose-6-phosphate dehydrogenase.
Fig. 2: schematic representation of recombinant plasmid pRSF-Duet-1-ManC-ManB-Gmd-WcaG.
Fig. 3: schematic representation of recombinant plasmid pET-Duet-1-Gsk-SUMO-FutC.
Fig. 4: schematic representation of recombinant plasmid pCDF-Duet-1-LacY-SetA-RcsA-RcsB-Zwf.
Fig. 5: schematic of liquid phase detection of standard 2' -fucosyllactose samples, standard lactose samples, standard glycerol samples and standard glucose samples.
Fig. 6: summary of final yields of 2' -fucosyllactose produced in 3-L fermentors using glycerol as carbon source.
Fig. 7: summary of final yields of 2' -fucosyllactose produced in a 3-L fermenter using glucose as carbon source.
Fig. 8: the 3-L fermentation tank takes glycerol as a carbon source to produce each component of the 2' -fucosyllactose.
Fig. 9: the 3-L fermentation tank uses glucose as a carbon source to produce each component of 2' -fucosyllactose.
Detailed Description
The present invention will be further described with reference to examples and drawings, wherein plasmids, PCR reagents, restriction enzymes, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercially available, and the specific operations are performed according to the kit instructions.
Embodiments of the invention are not limited thereto, and other unspecified experimental operations and process parameters are conducted in accordance with conventional techniques. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
Definition:
ManB-ManC Gene Cluster: consists of mannose-1-guanyl phosphate transferase ManC and phosphomannose mutase ManB, wherein the ManC and the ManB are continuous gene fragments on the genome of escherichia coli, and the nucleotide sequence is shown as SEQ ID NO. 1.
Gmd-WcaG Gene Cluster: consists of GDP-mannose-4, 6-dehydratase Gmd and GDP-fucose synthetase WcaG, gmd and WcaG are continuous gene fragments on the genome of escherichia coli, and the nucleotide sequence is shown as SEQ ID NO. 2.
The method for measuring the 2' -fucosyllactose in the embodiment of the invention is an HPLC method, and specifically comprises the following steps:
chromatographic column: aminex HPX-87H; a detector: a differential detector; mobile phase: 5mM sulfuric acid; flow rate: 0.6mL/min; column temperature: 35 ℃; sample injection amount: 10 mu L.
The main culture medium adopted by the embodiment of the invention is as follows:
LB medium: 10g/L tryptone, 5g/L yeast extract, 5g/LNaCl.
Fermentation medium: 3.375g/L KH 2 PO 4 ，0.7g/L MgSO 4 ·7H 2 O，2.0g/L(NH 4 ) 2 HPO 4 ，1.7g/L C 6 H 8 O 7 ·H 2 O,5g/L industrial yeast powder, 2.5g/L porcine bone peptone, 8g/L glycerol, 10mL of microelement solution and the balance of water. The pH was adjusted to about 7.2.
The microelement solution comprises the following components: taking 5mol/L HCl as mother liquor, 2.0g/L CaCl 2 ，0.1g/L(NH 4 ) 6 Mo 7 O 24 ，2.25g/L ZnSO 4 ·7H 2 O，0.5g/L MnSO 4 ·4H 2 O，10.0g/L FeSO 4 ·7H 2 O，3.0g/L CuSO 4 ·5H 2 O，0.23g/LNa 2 B 4 O 7 ·10H 2 O，0.18g/L CoCl 2 ·6H 2 O。
And (3) material supplementing liquid: carbon source: glycerol or glucose 600g/L; a substrate: lactose 200g/L.
pH regulation: 50% ammonia (v/v).
Concentration of antibiotic mother liquor: ampicillin 100mg/L, kanamycin 30mg/L, and spectinomycin 50mg/L.
Concentration of inducer: isopropyl-beta-D-thiogalactopyranoside (IPTG) was added at a concentration of 0.1mM.
TABLE 1 knockout Gene primer sequences
TABLE 2 overexpression vector primer sequences
The invention is further illustrated by the following examples.
Example 1: knockout of the E.coli BL21star (DE 3) genomic gene NudD, lacZ, nudK, wcaJ, clpYQ, lon, lacA, iclR, ptA, arcA, poxB, pflB, ldhA and ptsG.
The NudD, lacZ, nudK, wcaJ, clpYQ, lon, lacA, iclR, ptA, arcA, poxB, pflB, ldhA and ptsG genes in the escherichia coli BL21star (DE 3) genome are knocked out by using a CRISPR-Cas9 gene knockout system, and the specific steps are as follows (the related primer sequences are shown in Table 1):
(1) Using the E.coli BL21star (DE 3) genome as a template, primer pairs NudD-US-F/R and NudD-DS-F/R, lacZ-US-F/R and LacZ-DS-F/R, nudK-US-F/R and NudK-DS-F/R, wcaJ-US-F/R and WcaJ-DS-F/R, clpYQ-US-F/R and ClpYQ-DS-F/R, lon-US-F/R and Lon-DS-F/R, lacA-US-F/R and lacA-DS-F/R, iclR-US-F/R and iclR-DS-F/R, ptA-US-F/R and ptA-DS-F/R, arcA-F/R and arcA-F/R, poxB-US-F/R and poxB-DS-F/R, and pordB-DS-F/R, and fldG-DS-F/R, and sshA-DS-F/R were amplified by PCR, respectively, and the amplification of the supernatant was performed. The template was then used for the upstream and downstream fragments of NudD, lacZ, nudK, wcaJ, clpYQ, lon, lacA, iclR, ptA, arcA, poxB, pflB, ldhA and ptsG, respectively, using NudD-US-F/NudD-DS-R, lacZ-US-F/LacZ-DS-R, nudK-US-F/NudK-DS-R, wcaJ-US-F/WcaJ-DS-R, clpYQ-US-F/ClpYQ-DS-R, lon-US-F/Lon-DS-R, lacA-US-F/lacA-DS-R, iclR-US-F/iclR-DS-R, ptA-US-F/ptA-DS-R, arcA-US-F/arcA-DS-/R, poxB-US-F/poxB-DS-R, pflB-US-F/pflB-DS-R, ldhA-US-F/ldhA-DS-R, ptsG-US-F/ptsG-DS-R, and the primer was used to recover the complete fragments by PCR.
(2) Using the original pTargetF plasmid (Addgene: # 62226) as template, nudD-sg-F/R, lacZ-sg-F/R, nudK-sg-F/R, wcaJ-sg-F/R, clpYQ-sg-F/R, lon-sg-F/R, lacA-sg-F/R, iclR-sg-F/R, ptA-sg-F/R, arcA-sg-F/R, poxB-sg-F/R, pflB-sg-F/R, ldhA-sg-F/R and ptsG-sg-F/R as primers, the N20 sequences on the original plasmid were replaced with N20 sequences complementary to NudD, lacZ, nudK, wcaJ, clpYQ, lon, lacA, iclR, ptA, arcA, poxB, pflB, ldhA and ptsG sequences, respectively, to obtain the various pTargetF plasmids with targeted NudD, lacZ, nudK, wcaJ, clpYQ, lon, lacA, iclR, ptA, arcA, poxB, pflB, ldhA and ptsG (i.e., targeted plasmids with NudD, lacZ, nudK, wcaJ, clpYQ, lon, lacA, iclR, ptA, arcA, poxB, pflB, ldhA and ptsG specific N20 sequences, respectively). Coli JM109 was transformed competent, coated with LB plate (containing spectinomycin), and the plasmid was extracted by amplification culture at 37℃and sequenced.
(3) Taking pCas plasmid (Addgene: # 60847) and escherichia coli BL21star (DE 3) to be competent, placing on ice for 5min until competent melting, transferring competent cells into a precooled electrode cup, lightly knocking to enable the competent cells not to be bubble, and cooling in ice water for 5-10min; placing the electrode cup on an electrotransport device, adjusting electric shock parameters to 200 omega, 25 mu F and 2.5kV, hearing beeping after electric shock, checking the time to be 3ms-5ms, taking out the electrode cup, immediately adding 900 mu L of LB liquid culture medium into a sterile operation table, quickly and gently resuspending thalli cells, and transferring the thalli cells into a sterilized centrifuge tube; placing the centrifuge tube in a shaking table at 30 ℃ for shaking gently for 60min for resuscitation, centrifuging at low speed for 1min, discarding the supernatant medium, leaving about 150 mu L of medium to suspend and mix the thalli, and coating the thalli in LB solid medium with corresponding resistance; and (3) culturing the bacillus coli in an inverted incubator at the constant temperature of 30 ℃ overnight, picking a monoclonal colony for colony PCR verification, and adding a culture medium for culturing after the colony is verified to be correct to obtain the competent escherichia coli BL21star (DE 3)/pCas.
(4) 200ng of targeting plasmid pTargetF with iclR specific N20 sequence constructed in the step (2) and 1000ng of donor DNA fragment (namely the complete iclR template obtained in the step (1)) are electrically transferred to the E.coli BL21star (DE 3)/pCas competence prepared in the step (3), coated on LB plate (kanamycin and spectinomycin) and cultured for 24 hours at 30 ℃, positive colonies on the plate are picked up and cultured for 10 hours in LB, and sequencing verification is carried out by Jin Weizhi Biotech Co.
(5) And (3) picking the positive clone colony which is successfully knocked out by sequencing and verifying in the step (4) to a 4mL LB liquid test tube, adding IPTG with the final concentration of 1mM and 30mg/L kanamycin, culturing at 30 ℃ for 8-16h to remove pTargetF plasmid, culturing at 42 ℃ for 12h to remove pCas plasmid, and obtaining escherichia coli BL21star (DE 3) delta iclR with genome knocked-out iclR gene, wherein escherichia coli BL21star (DE 3) delta iclR is used as host bacteria.
(6) By the same method, the ptA gene of the escherichia coli BL21star (DE 3) Δiclr genome was knocked out to obtain the corresponding escherichia coli BL21star (DE 3) Δiclr Δpta by combining the targeting plasmid pTargetF with the ptA specific N20 sequence obtained in step (2) and 1000ng of the donor DNA fragment (i.e., the complete ptA template obtained in step (1)).
(7) And (3) taking the escherichia coli engineering bacteria BL21star (DE 3) delta iclR delta ptA as an original strain, and finally constructing and obtaining the engineering escherichia coli BL21star (DE 3) delta NudDeltaLacZ delta NudK delta WcaJ delta ClpYQ delta Lon delta lacA delta iclR delta ptA delta arcA delta poxB delta pfLdsA delta ldhA delta ptsG by referring to the operations of the steps (2) to (5).
Example 2: construction of recombinant bacterium overexpression genes of the de novo synthesis path of 2' -fucosyllactose.
The construction of the recombinant bacterium over-expression gene comprises the following specific steps (the related primer sequences are shown in table 2):
(1) Obtaining ManB, manC, gmd, wcaG, rcsA, rcsB, lacY, setA, gsk, zwf gene fragment: using BL21star (DE 3) genome as a template, using RcsA-F/RcsA-R, rcsB-F/RcsB-R, lacY-F/LacY-R, setA-F/SetA-R, gsk-F/Gsk-R, zwf-F/Zwf-R to amplify RcsA, rcsB, lacY, setA, gsk and Zwf gene fragments, respectively, since ManB-ManC and Gmd-WcaG were consecutive gene fragments on E.coli genome, and BC-F/BC-R and GW-F/GW-R to amplify ManB-ManC and Gmd-WcaG gene fragments, respectively, the recovered Gmd-WcaG and ManB-ManC gene fragments were ligated to vector SF-Duet-1 by a seamless cloning kit (Nannuo pran life sciences Co., ltd.) to obtain pRSF-Duet-1-CBGW (FIG. 2). The gene fragment of recovered Gsk was ligated to pET-Duet-1 of a plasmid, pET-Duet-1-Gsk, by the same ligation method. The recovered LacY, setA, rcsA, rcsB and Zwf gene fragments were ligated in sequence to pCDF-Duet-1 of the plasmid, which was pCDF-Duet-1-RSZL, using the same ligation method (FIG. 4).
(2) Obtaining FutC gene fragment: the 2' -fucosyllactose FutC gene sequence derived from helicobacter pylori was synthesized by the company limited to intelligent biotechnology, suluzhou, and the synthesized FutC gene fragment was connected to the vector pET-Duet-1-Gsk by a seamless cloning kit (the company limited to intelligent biotechnology, suluzhan) to obtain the plasmid pET-Duet-1-Gsk-FutC.
(3) Construction of fusion protein plasmid pET-Duet-1-Gsk-SUMO-FutC
Obtaining a gene fragment SUMO of ubiquitin-like protein modified molecule (SUMO) through total gene synthesis; the expression plasmid pET-Duet-1-Gsk-FutC was amplified by inverse PCR linearization using the correctly sequenced recombinant plasmid pET-Duet-1-Gsk-FutC in step (2) above as a template, followed by digestion of the template plasmid with DpnI enzyme, and purification and recovery of the pET-Duet-1-Gsk-FutC linearized vector fragment. The SUMO gene fragment and linearization vector pET-Duet-1-Gsk-FutC were then recombinantly ligated using a seamless cloning kit. Wherein the gene fragment of SUMO is ligated to the N-terminus of the FutC gene fragment, (GGGGS) 2 The gene sequence of the flexible Linker is designed at the junction of the C end of SUMO and the N end of FutC to construct a fusion protein sequence SUMO-FutC. The recombinant reaction system is reacted for 30min at 37 ℃, then transformed into competent cells of Escherichia coli JM109, subjected to heat shock for 90s at 42 ℃, subjected to ice bath for 2min, recovered at 37 ℃ for 1h, coated with an ampicillin-resistant LB plate with a final concentration of 0.1mM, and cultured at 37 ℃ for 10-12h. Finally, single colonies on the ampicillin resistance plate are selected for colony PCR verification, and whether the fusion protein SUMO-FutC is constructed successfully is confirmed by sequencing. Finally, the fusion protein plasmid pET-Duet-1-Gsk-SUMO-FutC was obtained (FIG. 3).
(4) Construction of expression plasmid pRSF-Duet-1-CBGW for optimizing RBS
The translational strength of selected RBS was obtained from MIT biological standard part registry, and RBS29 (nucleotide sequence shown as SEQ ID NO. 11) and RBS34 (nucleotide sequence shown as SEQ ID NO. 12) were replaced before the gene clusters of ManB-ManC and Gmd-WcaG in pRSF-Duet-1-CBGW, respectively.
(5) The plasmids pCDF-Duet-1-RSZL, pET-Duet-1-Gsk-SUMO-FutC and pRSF-Duet-1-CBGW obtained in steps (1), (2) and (4) were transferred into example 1 according to the key genes in the 2'-fucosyllactose synthesis pathway to obtain E.coli BL21star (DE 3) ΔNudΔLacZ ΔNudK ΔWcaJ ΔClpYQ Δlon ΔlacA ΔiclR Δpta ΔarcA ΔpoxB ΔpflB ΔldhA ΔptsG, and the engineering strain was fermented and cultured to confirm that the product was 2' -fucosyllactose by HPLC identification.
Example 3 Synthesis of 2' -fucose lactose by fermentation Using E.coli with glycerol as a carbon source (shake flask fermentation)
(1) Inoculating engineering bacteria F2 constructed in the embodiment 2 into LB culture medium containing corresponding antibiotics, culturing at 37 ℃ and 200rpm in a shaking flask for 12 hours to obtain seed liquid; inoculating the seed solution into 50mL of fermentation medium with an inoculum size of 1% (v/v), shaking and culturing at 37 ℃ and 200rpm until OD600 is 08; IPTG was added at a final concentration of 0.1mM, and lactose was added simultaneously to a lactose concentration of 10g/L at 25℃and 200rpm for induction culture for 96 hours.
(2) After induction fermentation, samples were taken at a fixed time, and the amount of 2' -fucosyllactose produced by fermentation of the engineered E.coli of the present invention was measured by HPLC.
(3) In the shake flask fermentation process, the RBS before Gmd-WcaG gene cluster on pRSF-dure-1 is replaced by RBS34, so that compared with the original strain, the glycerol is a carbon source, and the yield is improved to 12g/L from 11 g/L.
Example 4 Synthesis of 2' -fucose lactose by fermentation Using E.coli with glucose as carbon Source (shake flask fermentation)
(1) Inoculating engineering bacteria F2 constructed in the embodiment 2 into LB culture medium containing corresponding antibiotics, culturing at 37 ℃ and 200rpm in a shaking flask for 12 hours to obtain seed liquid; inoculating the seed solution into 50mL fermentation medium at 1% (v/v) inoculum size, shaking flask culture at 37deg.C and 200rpm to OD 600 1.5; IPTG was added at a final concentration of 0.1mM, and lactose was added simultaneously to a lactose concentration of 10g/L at 25℃and 200rpm for induction culture for 96 hours.
(2) After induction fermentation, samples were taken at a fixed time, and the amount of 2' -fucosyllactose produced by fermentation of the engineered E.coli of the present invention was measured by HPLC.
(3) In the shake flask fermentation process, the RBS before Gmd-WcaG gene cluster on pRSF-dure-1 is replaced by RBS34, so that compared with the original strain, the yield of glucose serving as a carbon source is improved to a small extent from 6g/L to 7g/L.
EXAMPLE 5 Synthesis of 2' -fucose lactose (3-L tank) by fermentation Using Escherichia coli Using Glycerol as a carbon Source
(1) Inoculating engineering bacteria F2 constructed in the example 2 into LB culture medium containing corresponding antibiotics, culturing for 12h at 37 ℃ at 200rpm/min, transferring into fermentation culture medium, culturing at 37 ℃ at 300-750 rpm until OD 600 Adding IPTG (0.1 mM) and primary lactose 5g/L to 15-20, adding feed liquid in a uniform feeding mode, feeding glycerol at a feeding speed of 4.5-11.0 g/L/h and feeding lactose at a feeding speed of 1.5-3.0 g/L/h, and transferring to 25 ℃ for continuous fermentation. The pH is regulated and controlled to be 6.5 by adopting 50% ammonia water in the whole fermentation process.
(2) After induction fermentation, samples were taken at a fixed time, and the amount of 2' -fucosyllactose produced by fermentation of the engineered E.coli of the present invention and the amounts of the individual component standard samples were measured by HPLC, and the measurement results are shown in FIG. 5. The detection results and the final yields of the components are shown in FIG. 6 and FIG. 8, the final yield of the 2' -fucosyllactose can reach 125g/L, the yield is 1.6g/L/h, and the lactose conversion rate is 1.17mol/mol.
EXAMPLE 6 Synthesis of 2' -fucose lactose (3-L tank) by fermentation Using E.coli with glucose as carbon Source
(1) Inoculating engineering bacteria F2 constructed in the example 2 into LB culture medium containing corresponding antibiotics, culturing for 12h at 37 ℃ at 200rpm/min, transferring into fermentation culture medium, and culturing at 37 ℃ at 300-800 rpm until OD 600 Adding IPTG (0.1 mM) and 5g/L of primary lactose until reaching 10, simultaneously adopting a uniform feeding mode to add feeding liquid, controlling the feeding speed of glucose to be 6-15 g/L/h and the feeding speed of lactose to be 1.5-5.0 g/L/h, and cooling to 25 ℃ to continue fermentation. The pH is regulated and controlled to be 6.8 by adopting 50% ammonia water in the whole fermentation process.
(2) After induction fermentation, sampling is carried out at a fixed time, and the amount of 2' -fucosyllactose produced by fermentation of the engineering escherichia coli and the standard samples of each component are detected by using an HPLC instrument. The detection results and the final yields of the components are shown in FIG. 7 and FIG. 9, the highest yield of 2' -fucosyllactose can reach 115g/L, the yield is 1.35g/L/h, and the lactose conversion rate is 0.91mol/mol.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A recombinant strain for producing 2'-fucosyllactose, characterized in that the expression of 2' -fucosyllactose de novo synthesis pathway enzyme is regulated and controlled by using escherichia coli as a host, comprising, knocking out genome genes: knocking out the E.coli BL21 (DE 3) strain of GDP-mannitohydrolase NudD, beta-galactosidase LacZ, GDP-mannitohydrolase NudK, ATPase component ClpY of undecanoglucose-1-phosphotransferase WcaJ, hslVU protease, peptidase component ClpQ of HslVU protease, lon protease Lon, galactoside O-acetyltransferase lacA, knocking out isocitrate lyase regulator iclR, phosphoacetyltransferase ptA, transcriptional double regulation factor arcA, pyruvate oxidase poxB, pyruvate formate lyase pflB, lactate dehydrogenase ldhA and glucose phosphate transporter ptsG; episomally expressed genes: the gene cluster of ManB-ManC, the gene cluster of Gmd-WcaG, DNA binding transcriptional activators rcsA and rcsB, 2' -fucosyllactose synthase FutC with fusion protein tag at the N-terminal, beta-galactoside permease LacY, sugar efflux transporter SetA, guanosine kinase Gsk and NADP (+) -dependent glucose-6-phosphate dehydrogenase Zwf are overexpressed; the fusion protein tag is ubiquitin-like protein modified molecule SUMO.
2. A recombinant strain for the production of 2' -fucosyllactose according to claim 1, wherein the recombinant strain expresses genes episomally using multicopy expression plasmids.
3. A recombinant strain for the production of 2' -fucosyllactose according to claim 2, wherein the recombinant strain expresses genes episomally using plasmids pRSF-Duet-1, pET-Duet-1 and/or pCDF-Duet-1.
4. A recombinant strain producing 2' -fucosyllactose according to claim 2 or 3, wherein the recombinant strain expresses the ManB-ManC gene cluster and the Gmd-WcaG gene cluster using the pRSF-durt-1 plasmid; genes Gsk and FutC were expressed using pET-durt-1 plasmid; genes LacY, setA, rcsA, rcsB and Zwf were expressed using the pCDF-Duet-1 plasmid.
5. A recombinant strain for producing 2' -fucosyllactose according to any one of claims 1 to 4, wherein the recombinant strain modulates the ManB-ManC gene cluster by RBS29 and modulates the Gmd-WcaG gene cluster by RBS 34.
6. A recombinant strain for the production of 2' -fucosyllactose according to any one of claims 1 to 5, wherein the host cell comprises e.coli BL21star (DE 3), e.coli K12 MG1655 or e.coli JM109.
7. A recombinant strain for the production of 2' -fucosyllactose according to claim 1, wherein the nucleotide sequence of the ManB-ManC gene cluster is shown in SEQ ID No. 1; the nucleotide sequence of the Gmd-WcaG gene cluster is shown as SEQ ID NO. 2; the nucleotide sequences of coding genes of the DNA binding transcription activating factors rcsA and rcsB are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequence of the coding gene of the 2' -fucosyllactose synthase FutC is shown as SEQ ID NO. 5; the nucleotide sequence of the coding gene of the beta-galactoside permease LacY is shown as SEQ ID NO. 6; the nucleotide sequence of the coding gene of the sugar efflux transporter SetA is shown as SEQ ID NO. 7; the nucleotide sequence of the encoding gene of the guanosine kinase Gsk is shown as SEQ ID NO. 8; the nucleotide sequence of the coding gene of the NADP (+) -dependent glucose-6-phosphate dehydrogenase Zwf is shown in SEQ ID NO. 9; the protein tag is ubiquitin-like protein modified molecule SUMO, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 10.
8. A method for producing 2'-fucosyllactose, characterized in that 2' -fucosyllactose is produced by using the recombinant strain according to any one of claims 1 to 7 as a fermentation strain.
9. The method of claim 8, wherein the method uses glycerol or glucose as a single carbon source and lactose as a substrate.
10. Use of a recombinant strain according to any one of claims 1 to 7, or of a method according to claim 8 or 9, for the production of 2'-fucosyllactose or of a product comprising 2' -fucosyllactose.
CN202311327641.3A 2023-10-13 2023-10-13 Application of escherichia coli engineering strain for efficiently producing 2' -fucosyllactose Pending CN117487728A (en)

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