CN116426452A

CN116426452A - Recombinant escherichia coli strain for synthesizing 2' -fucosyllactose and application thereof

Info

Publication number: CN116426452A
Application number: CN202310420876.0A
Authority: CN
Inventors: 马一榕; 黄兵; 蒋发现; 马志国; 姚萌
Original assignee: Guoqiang Biotechnology Nanjing Co ltd
Current assignee: Guoqiang Biotechnology Nanjing Co ltd
Priority date: 2023-04-19
Filing date: 2023-04-19
Publication date: 2023-07-14

Abstract

The invention discloses a method for synthesizing 2'-fucosyllactose by using a recombinant escherichia coli strain, wherein the recombinant escherichia coli strain takes escherichia coli as an initial strain, a phosphofructokinase I coding gene pfkA is knocked out, a phosphofructokinase II coding gene pfkB natural promoter is replaced by a pR promoter, and a CI857 repressor coding gene is integrated on a chromosome, and the escherichia coli can balance the growth of thalli and the production of 2' -fucosyllactose through a temperature regulation glycolysis path; in the fermentation process, glucose is used as a carbon source, lactose is added as a co-substrate to ferment and produce 2'-fucosyllactose, the concentration of the 2' -fucosyllactose can reach 130 g/L, and the lactose conversion rate can reach 98% in a 5L fermentation tank for 72 h.

Description

Recombinant escherichia coli strain for synthesizing 2' -fucosyllactose and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and mainly relates to construction and application of recombinant escherichia coli for producing 2' -fucosyllactose.

Background

Breast milk is generally considered to be the most important nutritional source for infants, and Human Milk Oligosaccharides (HMOs) as the third largest solid component next to lactose and fat in breast milk (content of about 5-15 g/L) play a key role in promoting the development of infant intestinal flora and preventing pathogen adhesion to epithelial cells. Human milk oligosaccharides include 2'-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 6 '-sialyllactose (6' -SL) and the like, which promote the growth of bifidobacteria and produce receptor analogues, protecting infants from infection by intestinal pathogens. Among them, 2'-fucosyllactose (2' -FL) is the highest content human milk oligosaccharide in secreted breast milk, which is about 30% of the total amount of human milk oligosaccharide, and has important nutritional and medical values. Currently, the U.S. and european union food safety authorities have approved the addition of 2'-fucosyllactose as a nutritional supplement to infant formulas, and china has also claimed a new variety of 2' -fucosyllactose as a food additive since 2021. Therefore, the 2' -fucosyllactose has wide prospect in the future infant milk powder market as a high-value nutritional supplement.

Over the past decade, various methods have been tried to produce 2' -fucosyllactose, including breast milk isolation, chemical synthesis, enzymatic conversion, microbial fermentation, and the like. The chemical synthesis of 2'-fucosyllactose requires that L-fucose is used as a substrate to undergo a four-step reaction to synthesize fucosyl donor, lactose is used as a substrate to undergo a two-step reaction to generate lactose acceptor, and the donor and the acceptor undergo a four-step reaction to finally generate the product 2' -fucosyllactose. In the method, L-fucose with high cost is required to be used as a substrate, and the method is subjected to multi-step reaction, so that the cost is high, and the steps are complicated, so that the method is not suitable for large-scale application and production. Compared with chemical methods, biological methods are currently mainly synthesized by adopting an enzymatic method and a microbial fermentation method. Enzymatic synthesis conditions are mild, but most methods require GDP-L-fucose as a substrate and are quite costly. The microbial fermentation method has become the main method of the current industrial production because the microorganisms can be quickly propagated and are easy to culture, and the low-cost carbon sources such as glucose or glycerol can be utilized to realize the production of high-value 2' -fucosyllactose. Chinese patent CN115216500A, mannose-6-phosphate is synthesized by taking glucose as a substrate enzyme, then mannose-6-phosphate is taken as a substrate, and lactose, cofactors NADpH, GTP and four enzymes of GDP-L-fucose synthesis paths and alpha-1, 2-fucosyltransferase are added, and the 2' -fucosyllactose is obtained by enzyme catalytic reaction. The method needs to carry out enzyme catalytic reaction by a two-step method, the reaction involves 8 enzymes, and GDP-L-fucose needs to be additionally added, so that the catalytic step is complex, and the economic cost is high. In Chinese patent CN114874964A, the escherichia coli takes glycerol as a carbon source and lactose as a substrate to synthesize the 2'-fucosyllactose from the head, the method avoids the addition of L-fucose and reduces the production cost, but the glycerol is taken as a byproduct in the diesel oil production process and is not suitable for the production of food grade 2' -fucosyllactose. Chinese patent CN114672448A, in escherichia coli, uses glucose and lactose as mixed carbon sources to ferment and enhance the supply of cofactors, the extracellular yield of 2' -fucosyllactose reaches 5.21 g/L, but the yield is far from sufficient to meet the industrialization requirements.

Disclosure of Invention

In view of the above technical background, the invention aims to obtain a strain Wen Minda enterobacteria by genetic engineering means, which can highly produce recombinant bacteria of 2'-fucosyllactose, and the escherichia coli can balance the growth of the bacteria and the production of the 2' -fucosyllactose by regulating and controlling the glycolysis path through temperature.

In order to achieve the above object, the present invention adopts a scheme that a recombinant E.coli strain for synthesizing 2' -fucosyllactose takes E.coli as an original strain, a phosphofructokinase I encoding gene pfkA is knocked out, a phosphofructokinase II encoding gene pfkB natural promoter is replaced by pR promoter, and a CI857 repressor protein encoding gene is integrated on a chromosome.

According to the invention, after the phosphofructokinase I encoding gene pfkA is knocked out, the natural promoter of the phosphofructokinase II encoding gene pfkB is replaced by a temperature sensitive pR promoter, and meanwhile, the repressor protein CI857 encoding gene is integrated on a chromosome, so that the expression of CI857 is started by an inducible promoter or a constitutive promoter pL. The CI857 protein has activity under the condition of 30 ℃ and can inhibit pR promoter; and at 37 ℃, the CI857 activity is extremely low and is close to inactivation, and the pR promoter loses the inhibition effect of the repressor protein and has the capacity of promoting gene transcription. In view of the above, the temperature is controlled, so that the recombinant bacteria can normally grow in the early growth stage, and the density of the bacteria is increased; in the middle and late growth stages, the glycolytic pathway is inhibited, so that fructose-6-phosphate is more diverted to the synthesis of GDP-L-fucose, and the precursor supply capacity of 2' -fucosyllactose is improved.

Specifically, the escherichia coli is selected from escherichia coli BL21 (DE 3) or MG1655; the CI857 repressor encoding gene is expressed in an inducible promoter or a constitutive promoter. Further preferably, the CI857 repressor encoding gene is inducible for expression with the inducible promoters Ptrc or PT7 or Ptac; or the CI857 repressor encoding gene is constitutively expressed as a constitutive promoter, pL or J23100.

Preferably, the recombinant E.coli strain has been knocked out of the gene encoding the beta-galactosidase lacZ and the gene encoding the UDP-glucose lipid carrier transferase wcaJ. The knock-out of beta-galactosidase coding gene lacZ in escherichia coli blocks lactose degradation pathway, the knock-out of UDP-glucose lipid carrier transferase coding gene wcaJ blocks GDP-L-fucose degradation, thus realizing accumulation of glycosyl donor GDP-L-fucose and glycosyl acceptor lactose.

Preferably, the ability of lactose to enter the cell is enhanced by over-expressing the lactose permease encoding gene lacY, facilitating the supply of glycosyl receptors. Specifically, the lactose osmotic enzyme coding gene lacY of the recombinant escherichia coli strain is subjected to over-expression modification, wherein the over-expression modification mode comprises free expression or integration expression on a chromosome through a plasmid; further preferably, lacY is expressed by inducible promoter Ptrc or PT7 or Ptac, or constitutive promoter J23119, etc.

Preferably, the supply of glycosyl donors is enhanced by over-expression of a GDP-L-fucose synthesis pathway encoding gene. The GDP-L-fucose synthesis pathway coding gene manC, manB, gmd, wcaG of the recombinant escherichia coli strain is subjected to over-expression modification, wherein the over-expression modification mode comprises free expression or integration on a chromosome through a plasmid; further preferably, the gene expressed by integrating the GDP-L-fucose synthesis pathway encoding gene on chromosome is expressed using a constitutive promoter J23119.

Specifically, the genes encoding 4 enzymes in the mannose-6-phosphate to GDP-L-fucose synthesis pathway are integrated on chromosome or expressed episomally by plasmids, including the mannose-phosphate mutase encoding gene manB, the mannose-1-guanylate transferase encoding gene manC, the GDP-D-mannose-4, the 6-dehydratase encoding gene gmd, the GDP-L-fucose synthase encoding gene wcaG, and the genes in the pathway are expressed by inducible promoters Ptrc or PT7 or Ptac in an inducible manner or constitutive promoters PJ23119 and the like.

Preferably, the recombinant E.coli strain has been over-expressed with the 2' -fucosyltransferase encoding gene wbgL, the over-expression modification comprising episomal expression by a plasmid or expression integrated on the chromosome. It is further preferred that the wbgL is expressed by induction with an inducible promoter Ptrc or PT7 or Ptac or constitutive promoter J23119.

Preferably, the gene expressed freely is used as a vector by one or two of plasmids pETDuet, pRSFDuet, pCDFDuet, pACYCDuet, pCOLADuet, pET a, pTrc99a, pBbA1a, pBbE1k and the like; the gene integration site is a pseudogene site or a site which does not affect the expression of the GDP-fucose synthesis pathway gene.

The invention also provides a method for synthesizing 2'-fucosyllactose, which takes the recombinant escherichia coli strain as a fermentation strain to produce the 2' -fucosyllactose, preferably, the recombinant escherichia coli strain takes grape as the fermentation strainSugar is used as a carbon source, lactose is added as a co-substrate for fermentation to produce 2' -fucosyllactose, the early temperature is controlled to be 35-38 ℃ in the fermentation process, and the fermentation temperature is controlled to be equal to the OD of thalli ₆₀₀ When the fermentation temperature reaches more than 25 ℃, the fermentation temperature is slowly reduced to 28-32 ℃.

The effective effects are as follows:

1. the recombinant bacterium disclosed by the invention can balance the growth of the bacterium and the production of 2' -fucosyllactose by regulating and controlling the glycolysis path through temperature. Specifically, expression of CI857 is initiated by knocking out the phosphofructokinase I encoding gene pfkA, by replacing the native promoter of the phosphofructokinase II encoding gene pfkB with a temperature sensitive pR promoter, and integrating the repressor protein CI857 encoding gene into the chromosome, with an inducible promoter or constitutive promoter pL. The CI857 protein has activity under the condition of 30 ℃ and can inhibit pR promoter; and at 37 ℃, the CI857 activity is extremely low and is close to inactivation, and the pR promoter loses the inhibition effect of the repressor protein and has the capacity of promoting gene transcription. In view of the above, the temperature is controlled, so that the recombinant bacteria can normally grow in the early growth stage, and the density of the bacteria is increased; in the middle and late growth stages, the glycolytic pathway is inhibited, so that fructose-6-phosphate is more diverted to the synthesis of GDP-L-fucose, and the precursor supply capacity of 2' -fucosyllactose is improved.

2. In the fermentation process of the recombinant bacterium, glucose is used as a carbon source, lactose is added as a co-substrate for fermentation to produce 2'-fucosyllactose, the concentration of the 2' -fucosyllactose can reach 130 g/L, and the lactose conversion rate can reach 98% in a 5L fermentation tank for fermentation for 72 h.

Drawings

FIG. 1 is a diagram of the metabolic pathway of 2' -fucosyllactose in recombinant bacteria;

FIG. 2 is a graph showing the accumulation of 2' -fucosyllactose by recombinant bacteria in a 5L fermenter;

FIG. 3 is a liquid chromatogram of 2' -fucosyllactose and lactose standard;

FIG. 4 is a liquid chromatogram of a sample of 2' -fucosyllactose broth;

FIG. 5 is a liquid chromatogram of 2' -fucosyllactose fermentation medium.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

In the invention, the amino acid sequence of 2' -fucosyltransferase from Escherichia coli O126 strain is shown as SEQ ID NO.1, and we entrust general purpose organism (Anhui) Co., ltd.

All gene sequencing services to which the present invention relates were commissioned by general biological (Anhui) Inc.

The present invention will be further illustrated by the following specific examples.

Example 1

Construction of recombinant plasmids

The recombinant bacterium of the invention knocks out the beta-galactosidase encoding gene lacZ and the UDP-glucose lipid carrier transferase encoding gene wcaJ to block lactose metabolism and improve the accumulation of the precursor GDP-L-fucose.

The knockdown construction uses the approach of Crispr-Cas9 gene editing, and this example is described in detail with the knockdown development of lacZ.

Firstly, competent preparation of escherichia coli BL21 (DE 3) strain is carried out, and the specific operation is as follows:

(1) Coli BL21 (DE 3) was inoculated and cultured overnight at 37℃and 200 rpm in a tube containing 5 mL of LB medium.

(2) Inoculating into 50 mL LB culture medium at 1% inoculum size, culturing at 37deg.C to OD ₆₀₀ About 0.6 (about 2 h).

(3) The bacterial liquid was transferred to a 50 mL precooled centrifuge tube and placed on ice for 5 min at 4℃and 4000 rpm.

(4) The supernatant was discarded, and a 10% glycerol solution, 15. 15 mL pre-chilled, was added to suspend the cells, and the cells were centrifuged at 4000 rpm at 4℃for 5 min.

(5) Repeating (4) twice.

(6) The supernatant was discarded, and 500 ul pre-chilled 10% glycerol solution was added to gently suspend the cells, which were then sub-packaged into sterile 1.5 ml centrifuge tubes at 100 ul/tube and stored in a-80 ℃ refrigerator for use.

The recombinant escherichia coli BL21 (DE 3) strain containing the pCas9 plasmid is prepared by adopting an electrotransformation method:

(1) Mu.l of pCas9 plasmid was added to 100 ul of competent bacteria of BL21 (DE 3), gently mixed and ice-bathed for 20 min.

(2) The pre-chilled 0.2. 0.2 cm cuvette sides were wiped clean and competent cells were all added to the cuvette for electrotransformation at 2.5 KV voltage.

(3) Immediately after the electric shock, 700 ul LB culture solution without antibiotics is added, and the culture is carried out at 30 ℃ for 1h so as to revive the thalli.

(4) The cells were spread uniformly on LB plates containing kanamycin resistance, and cultured in an incubator at 30℃for 16 h.

(5) Single colonies were picked for colony PCR and used for subsequent knockout construction after verification of correct positive clones.

This example mainly teaches the construction of pCDFDuet-CBWGWY, which is the insertion of six gene fragments manC, manB, gmd, wcaG, lacY and WbgL on the pCDFDuet plasmid, all genes on the plasmid being expressed by the T7 promoter.

The amino acid sequence SEQ ID NO.1 of the 2' -fucosyltransferase involved in the construction process of the pCDFDuet-CBWGWY plasmid is obtained by searching NCBI data, and has the sequence number of QTF37967.1, and the base sequence of the gene corresponding to the amino acid sequence is entrusted to be synthesized by general biological (Anhui) Co. The construction of the essence grain also relates to a phosphomannomutase (manB) amino acid sequence SEQ ID NO.2, a mannose 1-guanylate transferase (manC) amino acid sequence SEQ ID NO.3, a GDP-D-mannose-4, a 6-dehydratase (gmd) amino acid sequence SEQ ID NO.4, a GDP-L-fucose synthase (wcaG) amino acid sequence SEQ ID NO.5 and a lactose permease (lacY) amino acid sequence SEQ ID NO. 6, and gene fragments corresponding to the amino acid sequences are obtained from a PCR amplification of an escherichia coli BL21 (DE 3) strain.

The plasmid was constructed by one-step cloning. The construction of the plasmid is completed by two steps, wherein the first step is the construction of pCDFDuet-CBW, wherein three gene fragments of manC, manB and wbgL are inserted into the pCDFDuet plasmid, and the pCDFDuet-CBW is constructed by using a one-step cloning kit, which is purchased from Tiangen Biochemical technology (Beijing) Co., ltd, and the construction flow is referred to the one-step cloning kit specification.

First pass through pCDFDuet plasmidEcoAfter single cleavage with R I restriction enzyme, linearized plasmids were recovered. The manC and manB fragments are respectively amplified by PCR from the genome of escherichia coli BL21 (DE 3) by taking manC-F/manC-R and manB-F/manB-R as primers, the wbgL fragment is amplified by PCR from the synthesized wbgL gene fragment by taking wbgL-F/wbgL-R as primers, the fragments are recovered by glue, and the enzyme digestion and glue recovery reagent use modes are operated according to the reagent instruction.

The pCDFDuet-CBW plasmid is sequenced after construction, so that the accuracy of the inserted sequence is ensured.

The second step was to construct pCDFDuet-CBWGWY, pCDFDuet-CBWGWY as three gene fragments of gmd, wcaG and lacY inserted on pCDFDuet-CBW plasmid using a one-step cloning kit purchased from Tiangen Biochemical technologies (Beijing) Co., ltd, and the construction procedure was referred to the one-step cloning kit specification.

Specifically, the pCDFDuet-CBW plasmid was digested with Mfe I restriction enzyme, and then the linearized plasmid was recovered. The gmd, wcaG, lacY fragment is amplified from the genome of the escherichia coli BL21 (DE 3) by using gmd-F/gmd-R, wcaG-F/wcaG-R and lacY-F/lacY-R as primers, and the fragment is recovered by using the enzyme digestion and the gel recovery reagent according to the use mode of the reagent specification.

The pCDFDuet-CBWGWY plasmid is sequenced after construction, so that the accuracy of the inserted sequence is ensured.

Table 1 shows the primer sequences involved in this example.

TABLE 1

Example 2

Knock-out construction of recombinant strains

(5) Repeating (4) twice.

The pTarget-lacZ plasmid and the donor fragment for knocking out the lacZ gene were constructed by the following specific procedures:

PCR amplification is carried out by taking the original pTarget plasmid as a template and pTarget-lacZ-F/pTarget-R as a primer, the N20 sequence on the original pTarget plasmid is replaced by an N20 sequence which can target the lacZ gene, and fragments are recovered by glue after the amplification is finished. The recovered fragment was demethylated with Dpn I restriction endonuclease to remove the original plasmid template, then introduced into Top10 competence, placed on ice for 20 min, heat-shocked at 42℃for 90 s, placed on ice for 5 min, added with LB medium without resistance 700 ul, incubated at 37℃for 45 min, and plated with LB plate containing spectinomycin resistance.

The Donor fragment contains the upstream and downstream homology arms of the lacZ gene for repair after cleavage of the lacZ gene by Cas 9. The construction method is that BL21 (DE 3) genome sequence is used as a template, and lacZ-up-F/lacZ-up-R is used as a primer to amplify the upper homology arm lacZ-up; the lacZ-dm-F/lacZ-dm-R is used as a homology arm lacZ-dm under primer amplification, and the products are recovered by glue. Overlapping PCR is carried out by taking two fragments of lacZ-up and lacZ-dm as templates and lacZ-up-F/lacZ-dm-R as primers, splicing upstream and downstream homology arms, and recovering fragments by glue, wherein the fragments are used as Donor for knocking out lacZ genes.

Coli BL21 (DE 3) containing pCas9 plasmid was prepared as follows:

(1) Coli BL21 (DE 3) containing pCas9 plasmid was inoculated and cultured overnight at 30℃and 200 rpm in LB tubes to which kanamycin resistance was added.

(2) Inoculating into 50 mL LB culture medium at 1% inoculum size, culturing at 30deg.C to OD ₆₀₀ About 0.2. Mu.l of 1M arabinose solution was addedCulturing is continued until OD ₆₀₀ About 0.6.

(5) Repeating (4) twice.

The pTarget-lacZ plasmid of 2 ul and the donor fragment of 400 ng were added to 100 E.coli BL21 (DE 3) competent with pCas9 plasmid ul, gently mixed and placed on ice for 20 min. Electrotransformation was performed under the condition of 2.5 and KV, after the electrotransformation was completed, 700 ul antibiotic-free LB culture solution was added, after incubation at 30℃for 1h, LB plates containing both kanamycin and spectinomycin resistance were spread, and placed in an incubator at 30℃for 16 h.

Monoclonal on the plate was picked for colony PCR validation and sent to sequencing, positive clones sequenced correctly were inoculated in LB tubes containing 0.2 mM IPTG and kanamycin resistance, and incubated overnight at 30 ℃ to remove the pTarget plasmid; the monoclonal with the pTarget plasmid removed was inoculated into a non-resistant LB tube, and cultured overnight at 42℃to remove the pCas9 plasmid, thereby finally obtaining a recombinant strain of E.coli with the lacZ gene knocked out in BL21 (DE 3).

wcaJ was knocked out in BL21 (DE 3) using Crispr-Cas9 gene editing techniques, specific procedures were referenced to the knockdown of lacZ, and primer sequences involved in the knockdown procedure are shown in table 2. The recombinant E.coli after knockdown of both lacZ and wcaJ was designated as SFL-1.

TABLE 2

Example 3

Construction of temperature-sensitive control system strain

Recombinant strains for controlling the expression of phosphofructokinase II in a glycolytic pathway by CI857 proteins are constructed on the basis of SFL-1 strains. In Escherichia coli, phosphofructokinase I and phosphofructokinase II realize phosphorylation of fructose-6-phosphate, and in order to balance bacterial growth and product accumulation, expression of phosphofructokinase is regulated and controlled by a temperature-sensitive promoter, so that recombinant bacteria can normally grow in the early stage, and phosphorylation of fructose-6-phosphate is inhibited in the middle and late stages, so that fructose-6-phosphate flows to GDP-L-fucose synthesis more. Therefore, the construction idea of the recombinant strain is to knock out the phosphofructokinase I coding gene pfkA on the basis of the SFL-1 strain, so that the phosphofructokinase II can only be used for catalytic conversion in the step of phosphorylation. The native promoter of the phosphofructokinase II encoding gene pfkB was replaced with pR promoter which was repressed by the CI857 repressor protein, while the CI857 protein encoding gene, which in this example was selected for its constitutive promoter pL to promote expression, was integrated on the genome.

In this example, the construction of recombinant strain was performed in two steps, the first step being to knock out the pfkA gene in SFL-1 strain using the Crispr-Cas9 gene editing technique, and specific procedures were as described in example 2 for the lacZ knockout, with the primer sequences involved in the pfkA knockout procedure being shown in table 3.

After the pfkA knockout was successful, the CI857 protein encoding gene and pR promoter were substituted for the natural promoter of pfkB. Wherein the amino acid sequence of CI857 protein SEQ ID NO.7, which sequence, after codon optimization in E.coli, delegated the synthesis of gene fragments by general organism (Anhui) Co.Ltd. The synthesized CI857 gene fragment is amplified by taking CI857-F/CI857-R as a primer, a constitutive promoter pL is added in a primer sequence in the amplification process, pfkB-up and pfkB-dm are respectively amplified by taking pfkB-up-F/pfkB-up-R and pfkB-dm-F/pfkB-dm-R as primers, BL21 (DE 3) genome is taken as a template, and CI857, pfkB-up and pfkB-dm are obtained by glue recovery, wherein pR promoter sequences are added in the primer in the amplification process. Using overlap PCR, donor sequences with up-CI857-pR-dm elements were obtained.

The construction method of pTarget-pfkB plasmid is described in example 2. The pTarget-pfkB plasmid and the Donor fragment having the up-CI857-pR-dm element were added to competent cells from which the pfkA gene had been knocked out for electrotransformation. Specific procedures for electrotransformation and subsequent loss of the pTarget plasmid and the pCas9 plasmid are described in example 2.

The recombinant strain of E.coli containing the temperature-sensitive control system constructed in this example was designated SFL-2.

TABLE 3 Table 3

Example 4

Use of recombinant strains containing plasmids

The fermentation strain in this example was obtained by introducing the pCDFDuet-CBWGWY plasmid constructed in example 1 into recombinant strain SFL-2. Preparation method and electrotransformation method for competence referring to example 2 above, SFL-2 strain after plasmid introduction was designated SFL-3.

In this example, SFL-3 recombinant strain was used as the fermentation strain, and 5L fermenter was used as the fermentation vessel. The fermentation medium is selected from optimized M9 medium, namely 12.8 g/L disodium hydrogen phosphate heptahydrate, 3 g/L potassium dihydrogen phosphate, 2 g/L ammonium chloride, 0.5 g/L sodium chloride, 0.25 g/L magnesium sulfate heptahydrate, 15 mg/L calcium chloride dihydrate, 2 g/L yeast extract and 10 ml/L microelement mixture. The trace element mixture contained 5 g/L EDTA,0.83 g/L ferric chloride hexahydrate, 84 mg/L zinc chloride, 0.13 mg/L cupric chloride dihydrate, 10 mg/L cobalt chloride dihydrate, 10 mg/L boric acid, 1.6 mg/L manganese chloride tetrahydrate, and the initial glucose was 20 g/L.

The specific fermentation process is as follows: SFL-3 was inoculated into LB liquid medium containing spectinomycin and cultured overnight at 37 ℃. 1% of the inoculum size is transferred into 200 ml fresh LB medium to be used as secondary seed culture, and when OD ₆₀₀ When the temperature reaches about 4, the secondary seed solution is inoculated into a fermentation tank for expansion culture at the temperature of 37 ℃ according to the inoculation amount of 10 percent. The dissolved oxygen concentration is kept between 20 and 40 percent in the fermentation process, the pH is controlled between 6.8 and 7.2, ammonia water is adopted as a pH regulator, the aeration ratio is 1 vvm, and the bacterial OD ₆₀₀ When the temperature reaches 25 ℃, slowly cooling to 30 ℃, adding 0.1 mMIPTG was used as inducer and 10 g/L lactose. Lactose is fed in as a substrate in the fermentation process, and the lactose concentration is maintained to be 5-10 g/L.

FIG. 2 is a graph showing the fermentation yield of 2'-fucosyllactose of recombinant bacteria in a 5L fermenter, wherein the production rate of 2' -fucosyllactose can reach 4 g/L/h 15-35 h after inoculation, and the production rate is in a mass accumulation stage of the product. The bacterial cells grow to 20 h and OD ₆₀₀ The growth plateau is reached at 150 f, at which time the product accumulates in large quantities. The yield of the 2' -fucosyllactose can reach 130 g/L after 72 h fermentation, and the lactose conversion rate is 98%.

Example 5

Construction and application of recombinant strain without plasmid

In this example, recombinant strain SFL-4 with integrated genome wbgL, manC, manB, gmd, wcaG and lacY was constructed on the basis of strain SFL-2, and the genes integrated and expressed in this strain were expressed by using constitutive promoter J23119. Wherein, the WbgL and lacY integration sites are flhd and the integration site of manC, manB, gmd, wcaG is motA. The gene integration process adopts Crispr-Cas9 gene editing technology, and the embodiment uses the gene integration development of motA site for detail.

Firstly, competent preparation of the escherichia coli SFL-2 strain is carried out, and the specific operation is as follows:

(1) Coli SFL-2 was inoculated in a tube containing 5 mL of LB medium and cultured overnight at 37℃and 200 rpm.

(5) Repeating (4) twice.

The recombinant escherichia coli SFL-2 strain containing the pCas9 plasmid is prepared by adopting an electrotransformation method:

(1) Mu.l of pCas9 plasmid was added to 100 ul of SFL-2 competent bacteria, and the mixture was gently mixed and ice-bathed for 20 min.

(3) Immediately after the electric shock is finished, 700 ul LB culture solution without antibiotics is added, and the culture is carried out for 1h at 30 ℃ to revive the thalli.

(5) Single colonies were picked for colony PCR and used for subsequent integration construction after verification of correct positive clones.

The pTarget-motA plasmid and the donor fragment were constructed for the integrated motA site, specifically as follows:

PCR amplification is carried out by taking the original pTarget plasmid as a template and pTarget-motA-F/pTarget-R as a primer, the N20 sequence on the original pTarget plasmid is replaced by an N20 sequence which can target motA genes, and fragments are recovered by glue after amplification is completed. The recovered fragment was demethylated with Dpn I restriction endonuclease to remove the original plasmid template, then introduced into Top10 competence, placed on ice for 20 min, heat-shocked at 42℃for 90 s, placed on ice for 5 min, added with LB medium without resistance 700 ul, incubated at 37℃for 45 min, and plated with LB plate containing spectinomycin resistance.

The Donor fragment contains the upstream and downstream homology arms of the motA gene, the tandem manC and manB gene expression cassette, which is driven by the J23119 promoter, and the tandem gmd and wcaG gene expression cassette, which is driven by the J23119 promoter, and the upstream and downstream homology arm sequences are used for repair of the motA gene after it has been cleaved by Cas 9. The construction method comprises the steps of taking BL21 (DE 3) genome sequence as a template and motA-up-F/motA-up-R as a primer to amplify the upper homologous arm motA-up; the motA-dm-F/motA-dm-R is the homology arm motA-dm under primer amplification; amplifying a manC gene fragment containing a J23119 promoter by manC-F/manC-R; amplifying the manB gene fragment by manB-F/manB-R; amplifying the gmd fragment containing the J23119 promoter by using the gmd-F/gmd-R; the wcaG-F/wcaG-R gene fragment was amplified and the product was recovered by gel. Respectively taking three fragments of motA-up, manC and manB as templates, taking motA-up-F/manB-R as primers for overlapping PCR, splicing homologous arm, manC and manB genes, and recycling fragment 1 by glue; overlapping PCR is carried out by taking three fragments of gmd, wcaG and motA-dm as templates and gmd-F/motA-dm-R as primers, splicing the gmd, wcaG genes and homologous arms under motA, and recycling fragment 2 by glue. Finally, overlapping PCR is carried out by taking the gel recovery fragment 1 and the gel recovery fragment 2 as templates and taking the motA-up-F/motA-dm-R as primers, and splicing the upper homologous arm of the motA, the manC, manB, gmd, wcaG and the lower homologous arm of the motA, and the gel recovery fragment is inserted into the Donor of the manC, manB, gmd, wcaG gene expression cassette started by the J23119 promoter as a motA gene locus for standby.

The preparation method of the escherichia coli SFL-2 competence containing pCas9 plasmid comprises the following specific steps:

(1) Coli SFL-2 containing pCas9 plasmid was inoculated and cultured overnight at 30℃and 200 rpm in LB tube to which kanamycin resistance was added.

(2) Inoculating into 50 mL LB culture medium at 1% inoculum size, culturing at 30deg.C to OD ₆₀₀ About 0.2. Mu.l of 1M arabinose solution was added and the culture continued until OD ₆₀₀ About 0.6.

(5) Repeating (4) twice.

The pTarget-motA plasmid of 2 ul and the donor fragment of 400 ng were added to 100. 100 ul E.coli SFL-2 competent with pCas9 plasmid, gently mixed and left on ice for 20 min. Electrotransformation was performed under the condition of 2.5 and KV, after the electrotransformation was completed, 700 ul antibiotic-free LB culture solution was added, after incubation at 30℃for 1h, LB plates containing both kanamycin and spectinomycin resistance were spread, and placed in an incubator at 30℃for 16 h.

Monoclonal on the plate was picked for colony PCR validation and sent to sequencing, positive clones sequenced correctly were inoculated in LB tubes containing 0.2 mM IPTG and kanamycin resistance, and incubated overnight at 30 ℃ to remove the pTarget plasmid; the single clone from which pTarget plasmid was removed was inoculated into a non-resistant LB tube, and cultured overnight at 42℃to remove pCas9 plasmid, and finally, an E.coli recombinant strain in which a gene expression cassette for expressing manC, manB, gmd and wcaG by the J23119 promoter was integrated in the SFL-2 strain was obtained.

The process of integrating the two WbgL and lacY genes at flhd site was referred to above for the gene integration process at motA site, and the primer sequences involved in the gene integration process of this example are shown in Table 4.

The recombinant E.coli strain with genomic integration wbgL, manC, manB, gmd, wcaG and lacY constructed in this example was designated SFL-4.

In this example, SFL-4 recombinant strain was used as the fermentation strain, and 5L fermenter was used as the fermentation vessel. Fermentation medium reference was made to the M9 medium optimized in example 4.

The specific fermentation process is as follows: SFL-4 was inoculated into LB liquid medium containing no antibody and cultured overnight at 37 ℃. 1% of the inoculum size is transferred into 200 ml fresh LB medium to be used as secondary seed culture, and when OD ₆₀₀ When the temperature reaches about 4, the secondary seed solution is inoculated into a fermentation tank for expansion culture at the temperature of 37 ℃ according to the inoculation amount of 10 percent. The dissolved oxygen concentration is kept between 20 and 40 percent in the fermentation process, the pH is controlled between 6.8 and 7.2, ammonia water is used as a pH regulator, and the ventilation ratio is 1 vvm. When OD is ₆₀₀ When 25 is reached, the temperature is slowly reduced to 30 ℃ and 10 g/L lactose is added. Lactose is fed in as a substrate in the fermentation process, and the lactose concentration is maintained to be 5-10 g/L.

The SFL-4 recombinant strain is fermented for 72 hours, the yield of 2' -fucosyllactose can reach 48 g/L, and the lactose conversion rate is 98%.

TABLE 4 Table 4

Example 6

Construction and application of recombinant strain of escherichia coli MG1655 without plasmid

In this example, using E.coli MG1655 as the starting strain, lacZ, wcaJ and pfkA were knocked out, the knockdown method was as described in example 2 and example 3, the native promoter of pfkB was replaced with pR promoter controlled by CI857 temperature sensitive protein, and the procedure was as described in example 3. Six genes wbgL, manC, manB, gmd, wcaG and lacY were integrated on the genome of E.coli MG1655, and the gene to be integrated and expressed in this example was expressed using the constitutive promoter J23119. Wherein WbgL and lacY integration sites are flhd and manC, manB, gmd, wcaG integration sites are motA, and the method of gene integration is described in example 5. The plasmid-free recombinant strain constructed by taking Escherichia coli MG1655 as an initial strain is named SFL-5.

In this example, SFL-5 recombinant strain was used as the fermentation strain, and 5L fermenter was used as the fermentation vessel. Fermentation medium reference was made to the optimized M9 medium of example 4 and fermentation process reference was made to example 5.

The SFL-4 recombinant strain is fermented for 72 hours, the yield of 2' -fucosyllactose can reach 62 g/L, and the lactose conversion rate is 97%.

Example 7

Determination of 2' -fucosyllactose

The 2' -fucosyllactose was detected by High Performance Liquid Chromatography (HPLC). Before on-machine detection, pretreatment is needed to be carried out on a fermentation liquor sample, and the specific treatment method comprises the following steps: the resulting fermentation broth was centrifuged at 12000 rpm for 2 min. Taking supernatant, diluting with water to a concentration range of 25 mg/L-500 mg/L, uniformly mixing, filtering with a 0.22 um water-based film, and transferring the filtered sample to a sample bottle for upper machine analysis.

Agilent 1200 was used as a high performance liquid chromatograph, rezex ROA Organic Acid column (250×4.6mm×2.7 μm) was used as column, a differential detector was used, the column temperature was set to 50deg.C, the mobile phase was 100% aqueous sulfuric acid solution of 2.5 mM, the flow rate was set to 0.4 ml/min, and the amount of sample introduced per time was 5 ul.

As can be seen from fig. 3, the peak time of the 2' -fucosyllactose standard was 9.9 min, the peak time of the lactose standard was 10.7 min, and under the same detection conditions, as shown in fig. 4, the fermentation broth sample also detected 2' -fucosyllactose as a product, which was confirmed to be 2' -fucosyllactose by comparison with the blank medium of fig. 5.

The results of the above examples show that we succeeded in constructing recombinant E.coli strains capable of producing 2' -fucosyllactose with high efficiency. The strain takes glucose as a carbon source and lactose as a common substrate, avoids the addition of high-value L-fucose by optimizing a GDP-L-fucose synthesis way, and reduces the production cost. Meanwhile, by introducing a temperature-sensitive control system, the central carbon metabolic pathway is dynamically regulated, and finally the recombinant strain is fermented for 72 hours, the yield of the 2' -fucosyllactose can reach 130 g/L, and the lactose conversion rate is 98% and is the highest level reported so far.

While the invention has been described in terms of its general and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Therefore, any simple modification, variation and equivalent structural transformation made to the above embodiments according to the technical substance of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A recombinant escherichia coli strain for synthesizing 2' -fucosyllactose is characterized in that escherichia coli is taken as an original strain, a phosphofructokinase I coding gene pfkA is knocked out, a phosphofructokinase II coding gene pfkB natural promoter is replaced by a pR promoter, and a CI857 repressor protein coding gene is integrated on a chromosome.

2. The recombinant escherichia coli strain for synthesizing 2' -fucosyllactose according to claim 1, wherein the escherichia coli is escherichia coli BL21 (DE 3) or MG1655.

3. A recombinant escherichia coli strain for the synthesis of 2' -fucosyllactose according to claim 1, wherein the CI857 repressor encoding gene is expressed as an inducible promoter or a constitutive promoter.

4. The recombinant escherichia coli strain for synthesizing 2' -fucosyllactose according to claim 1, wherein the beta-galactosidase encoding gene lacZ and the UDP-glucose lipid carrier transferase encoding gene wcaJ of the recombinant escherichia coli strain have been knocked out.

5. A recombinant escherichia coli strain for synthesizing 2' -fucosyllactose according to claim 1, wherein a lactose permease encoding gene lacY of the recombinant escherichia coli strain has been subjected to an overexpression modification, wherein the overexpression modification comprises episomal expression by a plasmid or expression by integration on a chromosome.

6. The recombinant escherichia coli strain for synthesizing 2' -fucosyllactose according to claim 1, wherein the GDP-L-fucose synthesis pathway encoding gene manC, manB, gmd, wcaG of the recombinant escherichia coli strain has been subjected to an overexpression modification, wherein the overexpression modification comprises episomal expression by a plasmid or expression by integration on a chromosome.

7. A recombinant escherichia coli strain for the synthesis of 2'-fucosyllactose according to claim 1, wherein the 2' -fucosyltransferase encoding gene wbgL of the recombinant escherichia coli strain has been subjected to an over-expression modification comprising episomal expression by a plasmid or expression integrated on a chromosome.

8. A recombinant escherichia coli strain for the synthesis of 2' -fucosyllactose according to any one of claims 5-7, wherein all freely expressed genes are carried by one or two of the pETDuet, pRSFDuet, pCDFDuet, pACYCDuet, pCOLADuet, pET a, pTrc99a, pBbA1a, pBbE1k plasmids.

9. A method for synthesizing 2'-fucosyllactose, wherein 2' -fucosyllactose is produced by fermentation using the recombinant escherichia coli strain as defined in any one of claims 1 to 7 as a fermentation strain.

10. The method for synthesizing 2'-fucosyllactose according to claim 9, wherein said recombinant E.coli strain is fermented to produce 2' -fucosyllactose by using glucose as a carbon source and lactose as a co-substrate, and the pre-stage temperature is controlled to 35-38deg.C when the OD of the cells is ₆₀₀ When the fermentation temperature reaches more than 25 ℃, the fermentation temperature is slowly reduced to 28-32 ℃.