CN116042671A - Nucleic acid molecule, expression vector and microorganism for preparing breast milk oligosaccharide - Google Patents

Nucleic acid molecule, expression vector and microorganism for preparing breast milk oligosaccharide Download PDF

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CN116042671A
CN116042671A CN202310120743.1A CN202310120743A CN116042671A CN 116042671 A CN116042671 A CN 116042671A CN 202310120743 A CN202310120743 A CN 202310120743A CN 116042671 A CN116042671 A CN 116042671A
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fucosyllactose
breast milk
milk oligosaccharide
bkht
nucleic acid
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沐万孟
王浩
朱莺莺
陈柔霖
王芬
陈逸晗
刘超平
王瑞妍
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Beijing Huaxi Rongxi Biotechnology Research Co ltd
Jiangnan University
Bloomage Biotech Co Ltd
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Beijing Huaxi Rongxi Biotechnology Research Co ltd
Jiangnan University
Bloomage Biotech Co Ltd
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Abstract

The invention provides a nucleic acid molecule for encoding BKHT protein, which comprises a sequence shown as SEQ ID NO.2 or a nucleotide sequence with at least 98% sequence identity with SEQ ID NO. 2. The BKHT protein coded by the nucleic acid molecule is derived from Helicobacter pylori sp.13S00401-1, the amino acid sequence of the BKHT protein is shown as SEQ ID NO.1, the BKHT protein has excellent alpha-1, 2-fucosyltransferase catalytic activity and strict substrate specificity, when the BKHT protein is used for catalyzing GDP-fucose and lactose to synthesize 2' -fucosyllactose, the yield of the 2' -fucosyllactose can reach 94.68g/L, the lactose conversion rate can reach 0.98mol2' -FL/mol lactose, and especially, the BKHT protein has good industrialization prospect because of no generation of byproduct of the double fucosyllactose.

Description

Nucleic acid molecule, expression vector and microorganism for preparing breast milk oligosaccharide
Technical Field
The application relates to the technical field of bioengineering, in particular to a nucleic acid molecule, an expression vector and a microorganism for preparing breast milk oligosaccharide.
Background
Breast milk oligosaccharides (HMOs) are the 3 rd largest solid substance next to lactose and fat in breast milk, and are an indispensable component for infant development. Of the more than 200 identified HMOs, 2 '-fucosyllactose (2' -FL), the most abundant oligosaccharide in HMOs, is also one of the earliest HMOs approved by the us FDA and european union for addition to infant milk powder, plain foods, dietary supplements and/or medical foods, and has received widespread attention. However, the current production cost of 2' -FL is high and the production method has certain limitations.
Currently, the production modes of 2' -fucosyllactose mainly comprise three modes of chemical synthesis, enzyme catalytic synthesis and microbial fermentation. The cost of raw materials required by the chemical synthesis is high, and protective groups are required to be introduced, so that the steps are tedious. In contrast, the enzyme catalysis method and the biological fermentation method have mild and green reaction conditions, and are the most competitive production modes.
The core of the enzyme catalysis method or the biological fermentation method for producing the 2' -fucosyllactose is to screen alpha-1, 2-fucosyltransferase with high activity and high substrate specificity. At present, alpha-1, 2-fucosyltransferase for synthesizing 2' -FL is mainly FucT2 from helicobacter pylori and WbgL from escherichia coli O126, but the two enzymes still have the problems of low catalytic activity, poor substrate specificity and the like, and the synthesis of 2' -FL is accompanied by the synthesis of byproduct of disaccharide tetrasaccharide DFL, so that the cost of downstream separation and purification is greatly increased, the conversion rate of lactose to 2' -FL is reduced, and the material input cost is increased intangibly.
Disclosure of Invention
The object of the present invention is to provide a nucleic acid molecule, an expression vector and a microorganism for the preparation of breast milk oligosaccharides. Firstly, the BKHT protein with alpha-1, 2-fucosyltransferase function is obtained through screening, and can specifically catalyze the synthesis of 2' -fucosyllactose; secondly, the BKHT protein is subjected to codon optimization to obtain a coding gene sequence shown as SEQ ID NO.2, so that a BKHT expression vector and a microorganism containing the BKHT coding gene and the BKHT expression vector are constructed, and an engineering bacterium which has good adaptability, high yield and no byproduct generation is provided for breast milk oligosaccharide synthesis; third, the present application provides a method for preparing breast milk oligosaccharides, in particular 2' -fucosyllactose, using the above-described nucleic acid molecules, expression vectors and microorganisms.
In one aspect, the present application provides a nucleic acid molecule encoding a BKHT protein, said nucleic acid molecule comprising a sequence as shown in SEQ ID No.2 or a nucleotide sequence having at least 98% sequence identity to SEQ ID No. 2.
Preferably, the nucleic acid molecule comprises a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID No. 2.
More preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.
Preferably, the amino acid sequence of the BKHT protein comprises the sequence shown as SEQ ID NO.1 or an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 1.
More preferably, the amino acid sequence of the BKHT protein is shown as SEQ ID NO. 1.
In another aspect, the present application also provides an expression vector comprising the nucleic acid molecule described above.
Preferably, the expression vector is a pET expression vector and/or a pRSF expression vector.
In a preferred embodiment, the pET expression vector is pET-AB-BKHT, and the construction method of the pET-AB-BKHT is as follows: the plasmid pET-ABW is used as a template, a BKHT (with a sequence shown as SEQ ID NO. 2) is used as a target fragment to replace the wbgL gene in the pET-ABW, and finally the expression vector pET-AB-BKHT is obtained.
The plasmid pET-ABW can be seen from Chinese patent document with publication number CN114874964A, and the construction method and application of recombinant escherichia coli with high yield of 2' -fucosyllactose are recorded.
In another aspect, the present application also provides a microorganism comprising the above nucleic acid molecule and/or the above expression vector.
Preferably, the microorganism contains the above nucleic acid molecule and the above expression vector.
In a preferred embodiment, the microorganism is BYBB, which is obtained from the co-transfer of plasmids pRSF-CBGW and pET-AB-BKHT into BWLBC strain (integrated with BKHT coding sequence).
Plasmid pRSF-CBGW can be seen from Chinese patent document with publication number CN112342176A, 2' -fucosyllactose gene engineering bacteria and application thereof.
Further, the microorganism is one or more of corynebacterium glutamicum, bacillus subtilis and escherichia coli; preferably, the microorganism is E.coli.
Preferably, the strong promoter is PJ23119; more preferably, the nucleotide sequence of the PJ23119 is shown as SEQ ID NO. 7.
In a preferred embodiment, the strain BWLWC is obtained after engineering of E.coli BL21 (DE 3). The nucleic acid molecule (SEQ ID NO. 2) encoding the BKHT protein is used for replacing the wbgL gene controlled by a strong promoter on the genome of the host strain BWLWC, so that the strain BWLBC is obtained. The target sequence of the nucleic acid molecule integrated sgRNA is shown as SEQ ID NO. 8.
The specific obtaining method of the escherichia coli BWLWC can be seen from Chinese patent document with publication number of CN114874964A, and the construction method and application of the recombinant escherichia coli with high yield of 2' -fucosyllactose are recorded.
It will be appreciated by those skilled in the art that the above-described modifications may be made using known gene editing methods, such as using a CRISPR-Cpf1 gene editing system, a CRISPR-Cas9 gene editing system, RNA interference (RNAi) mediated gene knockout mode, and the like.
In a preferred embodiment, gene editing is performed using a CRISPR-Cpf1 gene editing system.
In another aspect, the present application also provides a whole cell catalyst comprising the microorganism described above.
In another aspect, the application also provides the application of the nucleic acid molecule, the expression vector, the microorganism or the whole cell catalyst in preparing breast milk oligosaccharide; preferably, the breast milk oligosaccharide is 2 '-fucosyllactose and/or 3' -fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
In another aspect, the present application also provides a method for producing a milk oligosaccharide, the method comprising producing a milk oligosaccharide using the above nucleic acid molecule, or the above expression vector, or the above microorganism, or the above whole cell catalyst; preferably, the breast milk oligosaccharide is 2 '-fucosyllactose and/or 3' -fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
Preferably, the method for producing breast milk oligosaccharide specifically comprises the following steps: lactose is used as a substrate, glycerol is used as a carbon source, and the microorganism is utilized for fermentation to prepare the 2' -fucosyllactose.
More preferably, lactose and/or glycerol are fed in the production process; more preferably, the lactose concentration is 3-15g/L; more preferably, the concentration of glycerol is 3-15g/L.
More preferably, the lactose concentration is 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L or 15g/L.
More preferably, the lactose concentration is 8g/L.
More preferably, the glycerol concentration is 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L or 15g/L.
In a preferred embodiment, a method of producing breast milk oligosaccharides comprises the steps of: culturing the microorganism in LB culture medium at 37deg.C and 200rpm for 8-12 hr to obtain seed solution, transferring to initial fermentation culture medium with inoculum size of 2%, and growing to OD 600 After =0.6-0.8, IPTG was added at a final concentration of 0.1mM while 8g/L lactose was added, and induction culture was continued at 25 ℃ at 200rpm for 72 hours.
The yield of the 2 '-fucosyllactose can reach 94.68g/L, the lactose conversion rate can reach 0.98mol2' -FL/mol lactose, the theoretical conversion value is close, and the byproduct of the disaccharide fucose tetraose is not present.
On the other hand, the application also provides the breast milk oligosaccharide prepared by the method; preferably, the breast milk oligosaccharide is 2 '-fucosyllactose and/or 3' -fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
In another aspect, the present application also provides a composition comprising the breast milk oligosaccharide described above; preferably, the breast milk oligosaccharide is 2' -fucosyllactose and/or 3-fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
In another aspect, the application also provides application of the breast milk oligosaccharide or the composition in preparing medicines, foods or cosmetics containing the breast milk oligosaccharide; preferably, the breast milk oligosaccharide is 2' -fucosyllactose and/or 3-fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
The invention has the following beneficial effects:
1. the alpha-1, 2-fucosyltransferase BKHT obtained by screening has excellent catalytic activity and strict substrate specificity, and provides a new engineering enzyme for the preparation of breast milk oligosaccharide;
2. the BKHT protein is subjected to codon optimization to obtain the coding gene sequence shown as SEQ ID NO.2, so that a BKHT expression vector and a microorganism containing the BKHT coding gene and the BKHT expression vector are constructed, and an engineering bacterium which has good adaptability, high yield and no byproduct generation is provided for breast milk oligosaccharide synthesis;
3. the novel engineering bacteria provided by the application are utilized to prepare the 2' -fucosyllactose, the yield of the 2' -fucosyllactose reaches 94.68g/L, the lactose conversion rate reaches 0.98mol2' -FL/mol lactose, and especially the generation of byproduct-free bisfucosyllactose is avoided, the downstream separation and purification cost is obviously reduced, and the method has good industrialization prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a diagram of engineering bacterium modification principle and 2' -FL synthetic route;
FIG. 2 is a diagram showing an alignment of a novel enzyme BKHT derived from Helicobacter pylori sp.13S00401-1 and specifically encoding alpha-1, 2-glycosyltransferase with other E.coli O126 sources, H.pyri UA802 sources and B.fragilis NCTC 9343 sources and sequences encoding alpha-1, 2-glycosyltransferase;
FIG. 3 is a graph (A) showing HPAEC-PAD results and a graph (B) showing the comparison of productivity of fermentation products of engineering bacteria for synthesizing 2' -fucosyllactose;
FIG. 4 is a graph (A) showing the results of 2 '-fucosyllactose HPAEC-PAD and a graph (B) showing the comparison of the capacity of 2' -fucosyllactose with different lactose concentrations;
FIG. 5 is a diagram showing fermentation results of engineering bacteria for producing 2' -fucosyllactose in a 5L fermenter in an enlarged scale.
Detailed Description
In order to more clearly illustrate the general concepts of the present application, the following detailed description is made by way of example with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer.
The plasmids, endonucleases, PCR enzymes, column type DNA extraction kits, DNA gel recovery kits and the like used in the following examples are commercially available products, and specific operations are performed according to the kit instructions.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention are all performed according to Molecular Cloning: ALaboratory Manual (fourths Edition) using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and conventional techniques in the relevant arts, which are conventional in the art.
Wherein, the initial seed liquid culture medium uses LB liquid culture medium, and the specific composition of the initial seed liquid culture medium is peptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L.
The initial fermentation medium (namely the glycerol quantification medium) is used for glycerol quantification of 2' -fucosyllactose production, and comprises the following specific components: 20g/L glycerin, 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate, 4.5mg/L thiamine and 10ml/L trace elements (trace element mother liquor: 10g/L ferrous sulfate heptahydrate, 2.2g/L zinc sulfate heptahydrate, 1.0g/L copper sulfate pentahydrate, 0.38g/L manganese sulfate monohydrate, 0.02g/L sodium borate decahydrate, 0.1g/L ammonium molybdate, 2.0g/L anhydrous calcium chloride), the pH of the initial fermentation medium was 6.8.
The glycerol supplementing system is as follows: 600g/L glycerin, 20g/L magnesium sulfate heptahydrate, 0.2g/L thiamine.
The lactose supplement is: 200g/L lactose.
HPLC configuration and detection parameters:
chromatographic column: carbohydrate Analysis (Rezex ROA-organic acid h+ (8%)); a detector: a differential detector; mobile phase: 0.5mmol/L, H 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 0.6mL/min; column temperature: 60 ℃; sample injection amount: 10 mu L.
HPAEC-PAD configuration and detection parameters:
chromatographic column: dionex CarboPac PA10 column (4 mm. Times.250 mm); a detector: a pulse current detector; mobile phase: three eluents were used for gradient elution, including A: ultrapure water, B:1M sodium acetate, and C:250mM NaOH. Elution procedure was performed with the following gradient: 0-15min,40% A0% B60% C to 31% A9% B60% C;15-15.1min,31% A9% B60% C to 40% A0% B60% C;15.1-25min,40% A, 0% B, 60% C; all are V/V. The flow rate was set to 1.0mL/min.
In the application, the escherichia coli BWLWC is used as a template, a CRISPR-Cpf1 gene editing system is utilized to target and replace a wbgL gene controlled by a strong promoter on the escherichia coli BWLWC genome, so that the strain BWLBC is obtained, and the specific transformation principle is shown in figure 1.
Wherein, the construction of plasmid pET-ABW comprises:
the rcsA and rcsB genes were amplified from E.coli K-12MG1655 chromosome with rcsA-F/R and rcsB-F/R, respectively, and the vector fragment was amplified from pET-W (ligation of the wbgl gene fragment to pETDuet-1 vector) with rcsAB-V-F/R, and the DNA fragment was recovered by gel. The amplified rcsA and rcsB gene fragments and vector fragments are connected through a Jeep kit (NEB reagent company in the United states) to obtain a plasmid pET-ABW, and the expression of the pathway enzyme genes is regulated by using the rcsA and rcsB positive transcription regulation factors.
The construction method of pET-AB-BKHT in the application comprises the following steps: the plasmid pET-ABW is used as a template, and BKHT (with a sequence shown as SEQ ID NO. 2) is used as a target fragment to replace the wbgL gene in pET-ABW.
The plasmid pET-ABW and a host (E.coli) BWLWC can be found in Chinese patent document with publication number CN114874964A, and the construction method and application of recombinant E.coli with high yield of 2' -fucosyllactose are recorded.
Plasmid pRSF-CBGW can be seen from Chinese patent document with publication number CN112342176A, genetic engineering bacteria for producing 2' -fucosyllactose and application thereof.
Example 1 selection of enzymes with Fusarium fucosyltransferase Functions and construction of expression vectors therefor
In this example, based on NCBI database (https:// www.ncbi.nlm.nih.gov /), classical fucT2 and wbgL in glycosyltransferase family are used as templates, BLAST modules on NCBI website are screened one by one, and alignment is performed according to key conserved sites and similarity, so as to obtain a novel enzyme derived from Helicobacter sp.13S00401-1 of Helicobacter pylori, named BKHT (GenBank: PAF 50342.1), the amino acid sequence of which is shown in SEQ ID NO. 1.
The amino acid sequence alignment of the enzyme BKHT derived from Helicobacter sp.13S00401-1 of the genus Helicobacter with the enzyme wcfB derived from other E.coli O126-derived enzymes, the enzyme fucT2 derived from H.pyri UA802 and the enzyme wcfB derived from B.fragilis NCTC 9343 is shown in FIG. 2. As can be seen from the results of FIG. 2, the amino acid sequence of BKHT has only 31.05% similarity with the presently disclosed alpha-1, 2-fucosyltransferase fucT2 (GenBank: AAC 99764.1) derived from helicobacter pylori; the amino acid sequence similarity with the alpha-1, 2-fucosyltransferase wbgL (GenBank: ADN 43847.1) derived from the escherichia coli O126 disclosed at present is only 30.58%; the similarity with the presently disclosed alpha-1, 2-fucosyltransferase wcfB from Bacteroides fragilis (GenBank: CAH 06753.1) is only 32.85%, so BKHT belongs to a brand new alpha-1, 2-fucosyltransferase.
In this example, the plasmid pET-ABW was used as a template, and the BKHT was used as a target fragment, to construct a pET-AB-BKHT expression vector. Wherein, the specific sequence of the target fragment after codon optimization is shown as SEQ ID NO. 2. Meanwhile, a pET-AB-BKHT-1 expression vector is also constructed, wherein the target fragment is not subjected to codon optimization, and the specific sequence is shown as SEQ ID NO.3 (GenBank: MLAR 01000006.1).
In addition, three other sources of alpha-1, 2-fucosyltransferases, named wbgL, fucT2, wcfB, were annotated based on the NCBI database in this example, and specific sequences after codon optimization were shown as SEQ ID No.4, SEQ ID No.5, and SEQ ID No. 6. And constructing pET-AB-fucT2 and pET-AB-wcfB expression vectors by taking the pET-ABW plasmid as a template (namely pET-AB-wbgL).
In this example, the E.coli BL21 (DE 3) DeltalacZ DeltawcaJ was co-transformed with the plasmid pRSF-CBGW and the pET expression vectors constructed as described above (pET expression vectors respectively represent pET-AB-wbgL, pET-AB-fucT2, pET-AB-wcfB, pET-AB-BKHT or pET-AB-BKHT-1) to obtain strains BY01, BY02, BY03, BY04 and BY05, respectively, for shake flask fermentation verification.
The specific method for shake flask fermentation verification comprises the following steps: strains BY01, BY02, BY03, BY04 or BY05 were grown on a resistant agar plate overnight and inoculated into 4mL of LB medium with the corresponding antibiotics as an initial seed solution, and cultured for 8-12h at 37 ℃ and 200 rpm; inoculating 2mL of initial seed solution into glycerol quantitative culture medium (100 mL volume) with corresponding antibiotics, and continuing to grow to OD under the same culture parameters 600 =0.6-0.8 followed by addition of IPTG at final concentration of 0.1mM while 8g/L lactose, 25 ℃,200rpm continued induction culture for 72h. 1mL of the fermentation broth was centrifuged at 10000rpm for 10min, and the supernatant was used for HPAEC-PAD measurement, and the results of shake flask fermentation verification are shown in Table 1.
Plasmid pET-ABW and a preparation method thereof can be seen from Chinese patent document with publication number CN114874964A, and a construction method and application of recombinant escherichia coli with high yield of 2' -fucosyllactose are recorded; the plasmid pRSF-CBGW can be described in the genetic engineering bacterium of 2' -fucosyllactose produced by Chinese patent document with publication number CN112342176A and application thereof.
TABLE 1 engineering Strain Productivity information for use of BL21 (DE 3) DeltalacZ DeltawcaJ for 2' -fucosyllactose production
Figure BDA0004079896600000091
Figure BDA0004079896600000101
Results As shown in Table 1 and FIG. 3, 2 '-fucosyllactose (2' -FL) was produced extracellularly in cultures of engineered E.coli expressing WbgL, fucT2, wcfB, BKHT and BKHT-1, respectively, at 4.15, 3.16, 3.67, 7.90 and 1.77 g/L. BKHT-expressing strain BY04 has a higher advantage than BY01, BY02 and BY03 in terms of 2' -FL production, increased BY more than 1.90 times; in addition, the BKHT-expressing strain BY04 after codon optimization has higher advantage than BY05 without codon optimization, and the yield is increased BY more than 4.46 times; the heterologous protein expression is subjected to codon optimization, and synonymous codons corresponding to high-abundance tRNA are selected, so that the protein expression is promoted, and the protein has higher production potential. Also, the lactose conversion rate of strain BY04 is highest, 0.95mol 2' -FL/mol lactose, 1.20, 1.27 and 1.17 times higher than that of strains BY01, BY02 and BY 03; in addition, strain BY01 (expressing wbgL) produced 0.24g/L of disaccharide tetrasaccharide and 0.06g/L of 3-fucosyllactose, whereas BY02 (expressing fucT 2) produced 0.65g/L of disaccharide tetrasaccharide, no production of disaccharide tetrasaccharide and 3-fucosyllactose was observed in strains BY03 and BY04 expressing WcfB and BKHT, respectively. It was demonstrated that the efficient activity and enzyme specificity of alpha-1, 2-fucosyltransferase are one of the key limiting factors for the large-scale production of 2'-FL, whereas the enzyme BKHT derived from Helicobacter pylori sp.13S00401-1 newly screened in example 1 has high 2' -FL productivity as alpha-1, 2-fucosyltransferase without producing by-products, and has a strong production potential.
EXAMPLE 2 construction and validation experiments of recombinant strains for 2' -fucosyllactose production
In this embodiment, host BWLWC is used as a template, a CRISPR-Cpf1 gene editing system is used for integrating a target new enzyme BKHT, targeting the wbgL in the genome of E.coli BWLWC, and a constitutive promoter PJ23119 (nucleotide sequence is shown as SEQ ID NO. 7) is used for replacing the original promoter to control the expression of BKHT, and the specific operation is as follows:
using the E.coli BWLWC genome as an amplification template of target fragments UP and Down, amplifying an upstream sequence fragment of a recA locus by using a recA-UP-F/recA-UP-R primer, and amplifying a downstream sequence fragment by using the recA-Down-F/recA-Down-R primer in the same way; the PJ23119-BKHT-T7 terminator fragment is arranged between the upstream fragment and the downstream fragment, and forms a homology arm with the upstream fragment and the downstream fragment during amplification, the three-fragment amplified product is recovered and purified through gel, and the three-fragment amplified product is amplified and connected through overlapping PCR, and then the target donor DNA template ZH-BKHT is obtained; the original pEcgRNA-N23 plasmid is used as a template, and a target sequence N23 (a nucleotide sequence is shown as SEQ ID NO. 8) of crRNA is searched on wbgL in a escherichia coli BWLWC genome to replace the original sequence for positioning, cutting and recombination of Cpf 1; the correctly sequenced pEcgRNA-wbgL-N23 (200 ng) was electroporated with the ZH-BKHT (800 ng) fragment into the target strain with Cpf1 electrotransformation competence, the electrotransformation was set to 2.5kv (2 mm electrotransformation cup), 900. Mu.L LB pre-chilled on ice was added immediately after electrotransformation, incubated at 37℃for 1h and then plated with kanamycin (Kan) and spectinomycin (Spc) plates, and incubated overnight in an oven at 37 ℃. And obtaining the BWLBC strain integrating BKHT after the subsequent sequencing verification is successful.
Wherein, the nucleotide sequence of the target donor DNA template ZH-BKHT is as follows: TCTGTGCGGTATT TCACACCGCATATGCTGGATCCTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTGATCAGACCTTTGTTTAACTTTAAAGGAGGTGATAAAAATGAAAAACAAAGTGCAGATTAAAATGCAAGGCCGCCTGGGCAATCAGTTTTTTATTTATGCGTTTGCGAAAAGTTTACTGCACAAACTGCAGAGCAGCGATAACCTGGATATTAAAAAAACCAAAGTGATTCTGCTGTGCGATGAATATGAAAACCTGGCGAGCGCGGATATTTTTAAATATAACATTGATCTGAGCCTGTTTGATCTGAAATTTTATGGCAGCCGCATTAGCAGTAGCAACTTTTTTCTGCGCGCGACCCGCAAGGCGATTTATCTGAGCACCGGCACCGTGATTCGCATTGGCATTAAAGATACCGCGAAAAAGAAAAGCTTTTATATTCAGCTGCCGATTCTGACCGAAACCTATACCCAAGGCGAATTTGATGAAAACGTGATTAAAGATT GCCTGAACAAATGCCTGCTGGGTTATTTTCAGAGCGAACTGTATTTTGAAGATATTAAAGAAAGCCTGAAAAAAGATTTTGTGCTGAAAGCGCCGCTGAAAAGCAAAACGCAGAAAGCGCTGACCAAAATTCGCCAAGATAGCAAAGCGATCTTTGTGCATATTCGCCGCGGCGATTATCTGGCGCCGGCGAACATTACCCTGTATGCGAGCCTGGGCGCGAACTACTATAAAAGTGCGATTAAACTGGCGCTGGAAAAAATTCCGGATGCGAAATTTTATATTTTTAGCGATGATATTGCGTGGGTGAAAGAAGAAGGCAAAAGCCTGCTGGGCTTTGAAGGCCTGGAATGCGAATTTATGGATCTGCATGGCGAAAACGAAGGCTATCTGGATCTGGAACTGATGCGCGCGTGCAAAGGCGCGATTACCGCGAACAGCAGCTTTAGCTGGTGGGGCGCGTATCTGATTGAAGAACCGAAAGTGGTGATTGCGCCGTTTCCGTTTCATTTTATTCATAGCAACGTGGATCTGTTACTGGATAGCTGGATTGTGCTGGATTATAAGAGTGGCGATGTGCTGAAAAAAGAAGATTATCTGTATGAAACCCTGAAAACCAAAAACAAACAAGAAAGCAACAGCAAAAGCACGAGCAGCTATTTTCGCTTTCATAACAGCAGCGGCGGTGGCGGTGGCAACACCTTTCGCTATGGTCAGTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG;
the nucleotide sequence of the plasmid pEcgRNA-wbgL-N23 is: TCGAGTTCATGTGCAGCTCC ATCAGCAAAAGGGGATGATAAGTTTATCACCACCGACTATTTGCAACAGTGCCGTTGATCGTGCTATGATCGACTGATGTCATCAGCGGTGGAGTGCAATGTCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGC GGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAAGATGCCGCTCGCCAGTCGATTGGCTGAGCTCATGAAGTTCCTATTCCGAAGTTCCGCGAACGCGTAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGCTGGATCCTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTAGAGGTAGAGACGCGAGGTCTAAGAACTTTAAATAATTTCTACTGTTGTAGATGAATCACAGTCTTCACTTAGCATAATTTCTACTGTTGTAGATCGTCTCTG AACTGATTCAAGCAAGCTTAAACCCAGCTCAATGAGCTGGGTTTTTTGTTTGTTTTTTCAAACTTAGTTAGCTTGGCCTTATTAACGTTGATATAATTTAAATTTTATTTGACAAAAATGGGCTCGTGTTGTACAATAAATGTAGTGATAGCGGTACCCTCGAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAATAGAGGGTAAGTAAAAGAAAGGAGGTTTATTTTATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAACGAACGGCAGATCAGAATTTTGTAATAAAAAAAGAGCCTGCTCATTACACTGCGGGCTCTTTTTCATGGTCAGAAGACGGGTAACCAAGATAACAAAGCTTAGATCTATTACCCTGTTATCCCTAC.
Removal of pEcgRNA-wbgL-N23 and Cpf1 from recombinant strain BWLBC: inoculating the bacteria with successful sequencing verification into a 4mL LB test tube (10 mM rhamnose and 2 μL kanamycin with final concentration are added simultaneously during inoculation) for 10-12h at 37 ℃, streaking on a kanamycin (Kan) plate after finishing, and culturing at 37 ℃; after single colony is grown on the plate, scribing lines are respectively carried out on the plate (Kan plate and Kan+Spc plate), and culturing is carried out at 37 ℃, wherein bacteria which grow on the Kan plate but do not grow on the Kan plate are the pEcgRNA-wbgL-N23 which is successfully removed; the single colonies were then further inoculated into a liquid LB medium containing glucose (5 g/L), and cultured at 200rpm at 37℃for 10-12 hours. Then, about 10. Mu.L of the bacterial liquid was streaked on a plate containing 5g/L glucose and 10g/L sucrose, and cultured at 37℃for 10-12 hours. And randomly selecting single colonies, respectively streaking on the non-resistant plate and the Kan-containing plate, and obtaining the host with the plasmid removed finally and successfully by the bacteria which grow on the non-resistant plate and do not grow on the Kan plate.
The plasmids pRSF-CBGW and pET-AB-BKHT of example 1 were transferred into the target engineering bacterium BWLBC to obtain the final recombinant BYBB.
Plasmid pRSF-CBGW can be seen from Chinese patent document with publication number CN112342176A, 2' -fucosyllactose gene engineering bacteria and application thereof; the BWLWC as host is disclosed in Chinese patent document No. CN114874964A, and the construction process and application of recombinant colibacillus with high yield of 2' -fucosyllactose are described.
Shake flask fermentation verification is carried out on the recombinant strain BYBB, lactose of 5g/L, lactose of 8g/L, lactose of 10g/L and lactose of 15g/L are added during induction, and the specific shake flask fermentation method is the same as in example 1, and the results are shown in Table 2.
TABLE 2 engineering Strain Productivity information of BYBB for the production of 2' -fucosyllactose
Figure BDA0004079896600000151
The results as shown in Table 2 and FIG. 4 show that the yield of 2' -fucosyllactose can be achievedTo 11.13g/L,8g/L lactose consumption is complete, lactose conversion rate reaches 0.98mol2' -FL/mol lactose, OD 600 10.6.
When the lactose concentration is less than 10g/L, the yield of the product 2' -FL increases with increasing lactose concentration. When lactose was added at a concentration of 15g/L, the yield and lactose conversion of 2' -FL decreased slightly, probably because high lactose concentration was detrimental to the growth of the strain or the activity of alpha-1, 2-fucosyltransferase. Overall, the addition of 5 and 8g/L lactose was consumed at the end of the fermentation, and the yield of 2' -FL and lactose conversion reached almost theoretical maximum. The yield of 2'-FL in lactose produced by BYBB strain reaches 0.98mol2' -FL/mol lactose, which is close to the theoretical conversion value.
Example 3 fed batch fermentation experiments
To further verify the effect of the recombinant strain BYBB described in example 2 on the scale-up production, a fed-batch fermentation experiment was performed in this example.
The batch fed-batch fermentation experiment is specifically as follows: transferring the overnight grown flat-plate strain BYBB into 4mL LB liquid culture medium with corresponding antibiotics as primary initial seed liquid, shake culturing at 37deg.C for about 12 hr, and OD 600 About 2.5; then the first seed liquid is inoculated into a second glycerol quantitative culture medium (with corresponding antibiotics and thiamine as plasmid resistance screening and strain growth) according to the initial inoculum size of 2% (v/v), and the culture is continued for about 10 hours to OD under the same temperature and rotation speed condition 600 Between 1.8 and 2.2; the second-level seed solution is inoculated into a 5L fermentation tank containing 1.5L of glycerol quantitative culture medium (30 g/L of glycerol) at an inoculation proportion of 10% (v/v), dissolved oxygen is set to be 30%, the culture temperature is 37 ℃, the rotation speed is related to the dissolved oxygen, the stirring speed is 200-800rpm, the aeration rate is 2-8vvm, and the pH is 6.75.
Culture OD 600 About 15-18, 0.1mM IPTG and 8g/L lactose were then added thereto, and the culture temperature was changed to 25 ℃. The pH in the growth process is regulated and controlled by ammonia water to maintain the pH stability of the whole fermentation. Lactose and glycerol consumption were measured periodically during fermentation, keeping lactose and glycerol at a concentration of 3-15g/L as much as possible to promote empirically high productivity.After the fermentation, 1mL of the fermentation broth was centrifuged at 12000rpm for 2-3min at room temperature, and the supernatant was filtered with a water-based filter head (0.22 μm), followed by HPLC detection, and qualitative and quantitative detection based on standard information.
As shown in FIG. 5, the biomass of the thallus in the earlier stage of fermentation and the accumulation amount of 2' -FL show a positive correlation trend, and the strain gradually enters the stationary phase after fermentation for about 30 hours, so that the accumulation rate of the product starts to be obviously slowed down. Feeding materials in the fermentation process, and controlling the final concentration of lactose and glycerol at 3-15g/L. The whole fermentation time is not less than 80 hours, and the final bacterial OD 600 The yield of 2 '-fucosyllactose reaches 94.68g/L and the lactose conversion rate reaches 0.98mol2' -FL/mol lactose.
The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.

Claims (10)

1. A nucleic acid molecule encoding a BKHT protein, said nucleic acid molecule comprising a sequence as shown in SEQ ID No.2 or a nucleotide sequence having at least 98% sequence identity to SEQ ID No. 2.
2. An expression vector comprising the nucleic acid molecule of claim 1.
3. A microorganism, characterized in that it comprises the nucleic acid molecule according to claim 1 and/or the expression vector according to claim 2.
4. A microorganism according to claim 3, wherein the microorganism is one or more of corynebacterium glutamicum, bacillus subtilis, escherichia coli; preferably, the microorganism is E.coli.
5. A whole cell catalyst, characterized in that it comprises the microorganism of claim 3 or 4.
6. Use of the nucleic acid molecule of claim 1, or the expression vector of claim 2, or the microorganism of claim 3 or 4, or the whole cell catalyst of claim 5, for the preparation of a breast milk oligosaccharide; preferably, the breast milk oligosaccharide is 2 '-fucosyllactose and/or 3' -fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
7. A method for producing a milk oligosaccharide, comprising producing a milk oligosaccharide using the nucleic acid molecule of claim 1, or the expression vector of claim 2, or the microorganism of claim 3 or 4, or the whole cell catalyst of claim 5; preferably, the breast milk oligosaccharide is 2 '-fucosyllactose and/or 3' -fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
8. A breast milk oligosaccharide produced by the method of claim 7; preferably, the breast milk oligosaccharide is 2 '-fucosyllactose and/or 3' -fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
9. A composition comprising the breast milk oligosaccharide of claim 8; preferably, the breast milk oligosaccharide is 2' -fucosyllactose and/or 3-fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
10. Use of the breast milk oligosaccharide of claim 8 or the composition of claim 9 in the preparation of a medicament, food or cosmetic product comprising a breast milk oligosaccharide; preferably, the breast milk oligosaccharide is 2' -fucosyllactose and/or 3-fucosyllactose; more preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
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