CN116676243A

CN116676243A - Construction method and application of recombinant escherichia coli producing 2' -fucosyllactose

Info

Publication number: CN116676243A
Application number: CN202211037018.XA
Authority: CN
Inventors: 江正强; 梁山泉; 杨绍青; 何滋; 闫巧娟
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2023-09-01

Abstract

The invention discloses recombinant escherichia coli for producing 2' -fucosyllactose, a construction method and application thereof. The recombinant escherichia coli is any one of the following: 1) Comprises an alpha-1, 2-fucosyltransferase and an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene; 2) The gene of 1) does not contain at least one of a beta-galactosidase gene, a UDP-glucose lipid carrier transferase gene, a fucose isomerase gene, a fucokinase gene, an arabinose isomerase gene and a rhamnose isomerase gene; 3) In addition to the features of 2), a fucose transporter gene and/or a lactose permease gene are contained; 4) In addition to having the characteristics of 3), does not contain genes related to outer membrane lipid synthesis and modification; 5) Besides the features of 4), no genes related to outer membrane lipopolysaccharide synthesis, modification and/or transport are contained. The recombinant escherichia coli can be used for industrially producing 2' -fucosyllactose.

Description

Construction method and application of recombinant escherichia coli producing 2' -fucosyllactose

Technical Field

The invention belongs to the technical field of synthetic biology, and particularly relates to a construction method and application of recombinant escherichia coli producing 2' -fucosyllactose.

Background

Breast milk has an irreplaceable nutritional value as an optimal food for infants, both as formula milk and as cow milk, the biggest difference being the kind and content of oligosaccharides in breast milk. Human Milk Oligosaccharides (HMOs) are the third largest solid component next to lactose and fat in breast milk, at about 5-15g/L. Of the HMOs, 2'-fucosyllactose (2' -FL) content is highest, up to 31%. There is growing evidence that 2' -FL has many functional activities beneficial to infants, including prebiotic activity, prevention of pathogen adhesion, modulators of intestinal epithelial cell response, immunomodulators, prevention of necrotizing enterocolitis, and promotion of brain development, etc. It is the physiological functions of 2'-FL, and the 2' -FL is approved as a new food material in the United states, european Union, canada, australia, new Zealand and other countries and regions.

The main methods for obtaining 2' -FL are extraction from breast milk, chemical synthesis, enzymatic synthesis and microbial synthesis. Compared with the other three methods, the microbial synthesis method has more economical efficiency and high efficiency. The microbial synthesis of 2' -FL is the transfer of fucosyl from GDP-L-fucose to lactose by the action of an alpha-1, 2-fucosyltransferase. GDP-L-fucose is used as a donor for the synthesis of 2' -FL, and there are two different synthetic routes: de novo synthesis pathway and salvage synthesis pathway. The de novo synthesis pathway involves overexpression of phosphomannose mutase ManB, mannose-1-phosphoguanyl transferase ManC, GDP-mannose-6-dehydrogenase Gmd and GDP-L-fucose synthase WcaG. Overexpression of these enzyme genes will break the metabolic balance of the host, affect bacterial growth, and limit further increases in GDP-L-fucose and 2' -FL production. Glucose or glycerol is less costly as a substrate, but the longer catalytic steps of the de novo synthesis pathway result in loss of metabolic flux and intermediates and a relatively low final yield. The salvage synthesis approach only needs to overexpress L-fucose-kinase/GDP-L-fucose pyrophosphorylase Fkp, and the metabolic influence of taking L-fucose as an initial substrate on thalli is low.

Hosts for producing 2' -FL have been developed in the present day, such as E.coli, saccharomyces cerevisiae, bacillus subtilis, and Corynebacterium glutamicum. The escherichia coli has the characteristics of clear background, complete carrier receptor system, rapid growth, simple culture and the like, and is the most studied host for synthesizing 2' -FL at present. In E.coli, the beta-galactosidase gene LacZ, UDP-glucose lipid carrier transferase gene WcaJ and the fucose isomerase/fucokinase gene FucI-FucK gene cluster were knocked out, the expression intensities of ManB, manC, gmd, wacG, fkp and FucT2 were regulated and the fermentation conditions were optimized, and the 2' -FL yield at the shake flask level was 3.81g/L (e Mo Meng et al, CN 110804577A). Under low pH culture conditions, the synthesis of the colibacillus S17-3 kola acid is promoted, GDP-L-fucose is a key precursor substance for synthesizing the kola acid, and the synthesis of the kola acid is blocked by knocking out WcaJ, so that the accumulation of intracellular GDP-L-fucose is improved. On this basis, the recombinant bacterium S17-3 produced 0.62g/L of 2'-FL (Sun Junsong et al, CN111218488A; chen Q, et al (2020) "Engineering a colanic acid biosynthesis pathway in E.coli for manufacturing 2' -fucesyl transferase". Process Biochem 94:79-85 "), by heterologous expression of the alpha-1, 2-fucosyltransferase. Increasing the soluble expression of fucosyltransferases is also an important aspect of increasing 2' -FL production. 4 different fusion protein tags are fused at the N end of FutC by adopting a flexible linker, so that the solubility and the biological activity of the FutC are improved, and the yield of 2' -FL (shake flask fermentation) is increased from 1.71g/L to 2.94g/L (Liu Long et al, CN 112322565A).

Many efforts have been made to synthesize 2' -FL so far, but the yield of 2' -FL synthesized by E.coli is still not high, the conversion efficiency of the substrates lactose and fucose is low, and the production cost of 2' -FL is high. Therefore, the construction of recombinant microorganisms that efficiently synthesize 2' -FL using synthetic biology means would be of great interest to the art.

Disclosure of Invention

The invention aims to provide a recombinant microorganism for efficiently producing 2' -fucosyllactose, in particular to escherichia coli.

In order to solve the technical problems, the invention firstly provides recombinant microorganisms.

The recombinant escherichia coli is the following A5), A4), A3), A2) or A1):

a1 The recombinant E.coli contains a G1 gene, wherein the G1 gene is an alpha-1, 2-fucosyltransferase gene and an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene;

a2 The recombinant E.coli contains the G1 gene, the recombinant E.coli does not contain the K1 gene, and the K1 gene is 6, 5, 4, 3, 2 or 1 of 6 genes of beta-galactosidase gene, UDP-glucose lipid carrier transferase gene, fucose isomerase gene, fucokinase gene, arabinose isomerase gene and rhamnose isomerase gene;

A3 The recombinant escherichia coli contains the G1 gene and does not contain the K1 gene, the recombinant escherichia coli contains a G2 gene, and the G2 gene is a fucose transporter gene and/or a lactose permease gene;

a4 The recombinant E.coli contains the G1 gene and the G2 gene, and does not contain the K1 gene and genes related to outer membrane lipid synthesis and modification;

a5 The recombinant E.coli contains the G1 gene and the G2 gene, and does not contain the K1 gene, genes related to outer membrane lipid synthesis and modification, and genes related to outer membrane lipopolysaccharide synthesis, modification and/or transport.

The recombinant E.coli can express the alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene.

In the present application, the α -1, 2-fucosyltransferase may have a function of transferring fucose on GDP-fucose to lactose to obtain 2' -fucosyllactose.

In the present application, the L-fucose kinase/GDP-L-fucose pyrophosphorylase may have a function of transferring extracellular fucose into cells and converting intracellular fucose into GDP-fucose.

In the present application, the beta-galactosidase may have the following function to metabolize lactose into galactose and glucose.

In the present application, UDP-glucose lipid carrier transferase may have the following functions, and is a key enzyme for synthesizing colanic acid by GDP-fucose.

In the present application, the fucose isomerase may have a function of converting fucose into fucoidan.

In the present application, fucoidan may have the following functions, a key enzyme for converting fucose.

In the present application, arabinose isomerase may have the following functions to catalyze isomerization of fucose.

In the present application, rhamnose isomerase may have the following function to catalyze isomerization of fucose.

In the present application, the fucose transporter may have a function of transporting extracellular fucose into cells.

In the present application, lactose permease may have the following function to transport extracellular lactose into the cell.

In the present application, the enzyme involved in outer membrane lipid synthesis and modification of gene expression may have the following functions, and is a key enzyme for outer membrane lipid synthesis and modification in E.coli.

In the present application, the enzyme expressed by the genes related to outer membrane lipopolysaccharide synthesis, modification and/or transport may have the following functions, and is a key enzyme for outer membrane lipopolysaccharide synthesis, modification and/or transport in E.coli.

A2 The recombinant E.coli may be any of the following:

B1 The recombinant E.coli does not contain a beta-galactosidase gene;

b2 The recombinant E.coli does not contain the beta-galactosidase gene and UDP-glucose lipid carrier transferase gene;

b3 The recombinant E.coli does not contain the beta-galactosidase gene, the UDP-glucose lipid carrier transferase gene and the fucose isomerase gene;

b4 The recombinant E.coli contains an alpha-1, 2-fucosyltransferase gene and an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, and does not contain a beta-galactosidase gene, a UDP-glucolipid carrier transferase gene, a fucose isomerase gene and a fucokinase gene;

b5 The recombinant E.coli contains an alpha-1, 2-fucosyltransferase gene and an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, and does not contain a beta-galactosidase gene, a UDP-glucolipid carrier transferase gene, a fucose isomerase gene, a fucokinase gene and an arabinose isomerase gene;

b6 The recombinant E.coli contains alpha-1, 2-fucosyltransferase genes and L-fucose kinase/GDP-L-fucose pyrophosphorylase genes, and does not contain beta-galactosidase genes, UDP-glucose lipid carrier transferase genes, fucose isomerase genes, fucokinase genes, and arabinose isomerase genes and rhamnose isomerase genes.

The recombinant E.coli may have the following characteristics:

the alpha-1, 2-fucosyltransferase is derived from helicobacter pylori, escherichia coli, bacteroides fragilis or synechococcus elongatus;

and/or, the L-fucose kinase/GDP-L-fucose pyrophosphorylase is derived from bacteroides fragilis;

and/or, the fucose transporter is derived from escherichia coli;

and/or, the lactose permease is derived from escherichia coli.

In the above, the E.coli may be E.coli K12-MG1655;

in the recombinant E.coli B1) -B6) described in A2) above, the alpha-1, 2-fucosyltransferase gene may in particular be derived from helicobacter pylori.

In the recombinant E.coli B6) described in A2) above, the α -1, 2-fucosyltransferase gene may be derived from E.coli in particular.

In the recombinant E.coli B6) described in A2) above, the α -1, 2-fucosyltransferase gene may be derived from Bacteroides fragilis.

In the recombinant E.coli B6) described in A2) above, the alpha-1, 2-fucosyltransferase gene may in particular originate from Haematococcus elongatus.

In the present application, the α -1, 2-fucosyltransferase gene derived from E.coli may be a WbgL gene.

In the present application, the α -1, 2-fucosyltransferase gene derived from Bacteroides fragilis may be a WcfB gene.

In the present application, the α -1, 2-fucosyltransferase gene derived from Haematococcus elongatus may be Te2FT gene.

In the present application, the fucose kinase/GDP-L-fucose pyrophosphorylase gene may be derived from Bacteroides fragilis.

In the present application, the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene derived from Bacteroides fragilis may be Fkp gene.

The recombinant E.coli may have the following characteristics:

the outer membrane lipid synthesis and modification related gene is at least one of LpxL gene, lpxM gene, lpxP gene and LpxT gene;

and/or, the outer membrane lipopolysaccharide synthesis, modification and/or transport related gene is at least one of a WaaF gene, a WaaL gene, a WaaV gene, a WaaW gene, a WaaY gene, a WaaT gene, a WaaO gene, a WaaP gene, a WaaG gene and a WaaQ gene.

The recombinant E.coli may have the following characteristics:

the beta-galactosidase is any one of the following proteins:

h1 Amino acid sequence is a protein shown in ACT42197.1 (16-FEB-2017) of GenBank;

h2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of the H1) and having more than 80% of identity with the protein of the H1) and beta-galactosidase activity;

H3 Fusion proteins with beta-galactosidase activity obtained by ligating a tag at the N-and/or C-terminus of H1) or H2);

and/or, the UDP-glucose lipid carrier transferase is any one of the following proteins:

j1 Amino acid sequence is a protein shown in ACT43804.1 (16-FEB-2017) of GenBank;

j2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of J1) and having 80% or more identity with the protein shown in J1) and UDP-glucose lipid carrier transferase activity;

j3 Fusion proteins with UDP-glucose lipid carrier transferase activity obtained by ligating a tag at the N-terminal and/or C-terminal of J1) or J2);

and/or, the fucose isomerase is any one of the following proteins:

k1 Amino acid sequence is a protein shown in ACT44469.1 (16-FEB-2017) of GenBank;

k2 Fusion protein which is obtained by substituting and/or deleting and/or adding amino acid residues on the protein of the K1), has more than 80 percent of identity with the protein shown in the K1) and has fucose isomerase activity;

k3 Fusion proteins with the same function obtained by connecting labels at the N end and/or the C end of K1) or K2);

and/or, the fucoidan is any one of the following proteins:

L1) an amino acid sequence is a protein shown in GenBank ACT44470.2 (16-FEB-2017);

l2) a protein having an fucoidan activity and having 80% or more identity to the protein represented by L1) obtained by substitution and/or deletion and/or addition of an amino acid residue to the protein of L1);

l3) a fusion protein having fucoidan activity obtained by ligating a tag to the N-terminus and/or C-terminus of L1) or L2);

and/or, the arabinose isomerase is any one of the following proteins:

m1) the amino acid sequence is a protein shown in GenBank ACT41965.1 (16-FEB-2017);

m2) a protein having an arabinose isomerase activity and having 80% or more identity to the protein represented by M1) obtained by substitution and/or deletion and/or addition of an amino acid residue to the protein of M1);

m3) a fusion protein having arabinose isomerase activity obtained by ligating a tag to the N-terminal and/or C-terminal of M1) or M2);

and/or the rhamnose isomerase is any one of the following proteins:

n1) the amino acid sequence is a protein shown in GenBank ACT45582.2 (16-FEB-2017);

n2) a protein having rhamnose isomerase activity, which is obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of N1) and has 80% or more identity with the protein shown in N);

N3) a fusion protein with rhamnose isomerase activity obtained by connecting a tag at the N-terminal and/or C-terminal of N1) or N2);

and/or the L-fucose kinase/GDP-L-fucose pyrophosphorylase derived from bacteroides fragilis is any one of the following proteins:

o1) the amino acid sequence is a protein shown in the sequence SEQ ID NO. 1;

o2) a protein obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of O1) and having an L-fucose kinase/GDP-L-fucose pyrophosphorylase activity, wherein the protein has an identity of 80% or more to the protein represented by O1);

o3) a fusion protein having L-fucose kinase/GDP-L-fucose pyrophosphorylase activity obtained by ligating a tag to the O-terminus and/or C-terminus of O1) or O2);

and/or the alpha-1, 2-fucosyltransferase from helicobacter pylori is any one of the following proteins:

p1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 2;

p2) a protein having an alpha-1, 2-fucosyltransferase activity, which is obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of P1) and has 80% or more of the identity with the protein represented by P);

p3) a fusion protein having an alpha-1, 2-fucosyltransferase activity obtained by ligating a tag to the P-and/or C-terminal of P1) or P2);

And/or the alpha-1, 2-fucosyltransferase from E.coli is any one of the following proteins:

q1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 3;

q2) a protein having an alpha-1, 2-fucosyltransferase activity, which is obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of Q1) and has 80% or more of the identity with the protein represented by Q);

q3) a fusion protein having an alpha-1, 2-fucosyltransferase activity obtained by ligating a tag to the Q-terminus and/or the C-terminus of Q1) or Q2);

and/or the alpha-1, 2-fucosyltransferase derived from bacteroides fragilis is any one of the following proteins:

r1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 4;

r2) a protein which is obtained by substituting and/or deleting and/or adding an amino acid residue of the protein of R1), has more than 80% of the identity with the protein shown in R), and has alpha-1, 2-fucosyltransferase activity;

r3) a fusion protein with alpha-1, 2-fucosyltransferase activity obtained by linking a tag to the R end and/or the C end of R1) or R2);

and/or the alpha-1, 2-fucosyltransferase from Haematococcus elongatus is any one of the following proteins:

S1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 5;

s2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues on the protein of the S1), has more than 80% of identity with the protein shown in the S) and has the same function;

s3) connecting a label at the S end and/or the C end of the S1) or the S2) to obtain fusion protein with the same function;

and/or lactose permease is any one of the following proteins:

t1) the amino acid sequence is a protein shown in GenBank NP-414877.1 (Mar 8,2022);

t2) a protein which has 80% or more identity with the protein represented by T) and has lactose permease activity, wherein the protein of T1) is obtained by substitution and/or deletion and/or addition of an amino acid residue;

t3) a fusion protein with lactose permease activity obtained by connecting a label at the T end and/or the C end of T1) or T2);

and/or the fucose transporter is any one of the following proteins:

u1) the amino acid sequence is a protein shown in GenBank NP-417281.1 (Mar 8,2022);

u2) a protein having a fucose transporter activity, which is obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of U1) and has 80% or more of the identity with the protein represented by U);

U3) a fusion protein with fucose transporter activity obtained by connecting a tag to the U end and/or the C end of U1) or U2);

and/or, the protein encoded by the LpxL gene is any one of the following:

v1) the amino acid sequence is a protein shown in GenBank ACT42945.1 (16-FEB-2017);

v2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues on the protein of V1), has more than 80% of identity with the protein shown in V), and has activity related to outer membrane lipid synthesis and modification;

v3) a fusion protein with outer membrane lipid synthesis and modification related activities obtained by ligating a tag at the V-terminal and/or C-terminal of V1) or V2);

and/or, the protein encoded by the LpxM gene is any one of the following:

w1) the amino acid sequence is a protein shown in GenBank ACT43680.1 (16-FEB-2017);

w2) a protein obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of W1) and having 80% or more identity with the protein represented by W) and having activity related to outer membrane lipid synthesis and modification;

w3) a fusion protein with outer membrane lipid synthesis and modification related activities obtained by connecting a tag to the W end and/or the C end of W1) or W2);

And/or the protein encoded by the LpxP gene is any one of the following:

x1) the amino acid sequence is a protein shown in GenBank ACT44109.1 (16-FEB-2017);

x2) a protein obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of X1) and having an activity related to outer membrane lipid synthesis and modification, wherein the protein has 80% or more of the identity with the protein represented by X);

x3) a fusion protein with outer membrane lipid synthesis and modification related activities obtained by connecting a tag to the X end and/or the C end of X1) or X2);

and/or, the protein encoded by the LpxT gene is any one of the following:

y1) the amino acid sequence is a protein shown in GenBank ACT43927.1 (16-FEB-2017);

y2) a protein obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of Y1) and having an activity related to outer membrane lipid synthesis and modification, wherein the protein has 80% or more of the identity with the protein represented by Y);

y3) a fusion protein with outer membrane lipid synthesis and modification related activities obtained by linking a tag at the Y-terminus and/or the C-terminus of Y1) or Y2);

and/or, the WaaF gene encodes a protein as follows:

z1) the amino acid sequence is a protein shown in GenBank ACT45276.1 (16-FEB-2017);

Z2) a protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of Z1) and having 80% or more of the same identity with the protein shown in Z) and having activity related to outer membrane lipopolysaccharide synthesis and modification;

z3) a fusion protein with outer membrane lipopolysaccharide synthesis and modification related activity obtained by connecting a label at the Z end and/or the C end of Z1) or Z2);

and/or, the WaaL gene encodes a protein as follows:

z1) the amino acid sequence is a protein shown in GenBank ACT45278.1 (16-FEB-2017);

and/or, the WaaV gene encodes a protein as follows:

t1) the amino acid sequence is a protein shown in GenBank ACT45279.1 (16-FEB-2017);

t2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of the T1), has more than 80% of identity with the protein shown in the T), and has activity related to outer membrane lipopolysaccharide synthesis and modification;

T3) a fusion protein with outer membrane lipopolysaccharide synthesis and modification related activities obtained by connecting a label at the T end and/or the C end of T1) or T2);

and/or, the WaaW gene encodes any one of the following proteins:

n1) the amino acid sequence is a protein shown in GenBank ACT45280.1 (16-FEB-2017);

n2) a protein obtained by substituting and/or deleting and/or adding an amino acid residue in the protein of N1) and having an activity related to outer membrane lipopolysaccharide synthesis and modification, wherein the protein has an identity of 80% or more to the protein represented by N);

n3) a fusion protein with outer membrane lipopolysaccharide synthesis and modification related activities obtained by connecting a label at the N end and/or the C end of N1) or N2);

and/or the protein encoded by the WaaY gene is any one of the following:

o1) the amino acid sequence is a protein shown in GenBank ACT45281.1 (16-FEB-2017);

o2) a protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of O1) and having an activity related to outer membrane lipopolysaccharide synthesis and modification, wherein the protein has an identity of 80% or more with the protein shown in O);

o3) fusion protein with outer membrane lipopolysaccharide synthesis and modification related activities obtained by connecting labels at the O end and/or the O end of O1) or O2);

And/or, the WaaT gene encodes a protein as follows:

l1) the amino acid sequence is a protein shown in GenBank of ACT45282.1 (16-FEB-2017) and GenBank of ACT45283.1 (16-FEB-2017);

l2) a protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of L1), which has more than 80% of the identity with the protein shown in L) and has activity related to outer membrane lipopolysaccharide synthesis and modification;

l3) a fusion protein having an outer membrane lipopolysaccharide synthesis and modification-related activity obtained by linking a tag to the L-terminus and/or C-terminus of L1) or L2);

and/or, the WaaO gene encodes a protein as follows:

r1) is a protein with the amino acid sequence shown in GenBank of ACT45284.1 (16-FEB-2017);

r2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of R1), has more than 80% of identity with the protein shown in R), and has activity related to outer membrane lipopolysaccharide synthesis and modification;

r3) is connected with a label at the R end and/or the C end of R1) or R2) to obtain fusion protein with outer membrane lipopolysaccharide synthesis and modification related activities;

and/or, the WaaP gene encodes a protein as follows:

F1 Amino acid sequence is a protein shown in ACT45285.2 (16-FEB-2017) of GenBank;

f2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of the F1) and having more than 80% of identity with the protein shown in the F) and activity related to outer membrane lipopolysaccharide synthesis and modification;

f3 Fusion proteins with outer membrane lipopolysaccharide synthesis and modification related activities obtained by linking tags at the F-terminal and/or C-terminal of F1) or F2);

and/or, the WaaG gene encodes a protein as follows:

g1 Amino acid sequence is a protein shown in ACT45286.1 (16-FEB-2017) of GenBank;

g2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of the G1), has more than 80 percent of identity with the protein shown in the G), and has activity related to outer membrane lipopolysaccharide synthesis and modification;

g3 Fusion proteins with outer membrane lipopolysaccharide synthesis and modification related activities obtained by linking tags at the G-terminal and/or C-terminal of G1) or G2);

and/or, the WaaQ gene encodes a protein as follows:

h1 Amino acid sequence is a protein shown in ACT45287.1 (16-FEB-2017) of GenBank;

h2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of the H1) and having more than 80% of identity with the protein shown in the H) and activity related to outer membrane lipopolysaccharide synthesis and modification;

H3 Fusion proteins having activities related to outer membrane lipopolysaccharide synthesis and modification obtained by linking a tag to the H-terminus and/or C-terminus of H1) or H2).

In the present application, the alpha-1, 2-fucosyltransferase gene derived from helicobacter pylori may be the FutC gene. The FutC gene sequence may be as shown in sequence SEQ ID No. 6.

In the present application, the α -1, 2-fucosyltransferase gene derived from E.coli may be a WbgL gene. The WbgL gene sequence may be as shown in SEQ ID NO. 7.

In the application, the alpha-1, 2-fucosyltransferase gene WcfB gene derived from Bacteroides fragilis. The WcfB gene sequence can be shown as sequence SEQ ID NO. 8.

In the present application, the α -1, 2-fucosyltransferase gene derived from Haematococcus elongatus may be Te2FT gene. The Te2FT gene sequence can be shown as a sequence SEQ ID NO. 9.

In the present application, the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene may be Fkp gene. The Fkp gene sequence can be represented by the sequence SEQ ID NO. 10.

In the present application, the protein encoded by the LpxL gene has lauroyl carrier protein (ACP) -dependent acyltransferase activity.

In the present application, the protein encoded by the LpxM gene has myristoyl Acyl Carrier Protein (ACP) -dependent acyltransferase activity.

In the present application, the protein encoded by the LpxP gene has palmitoyl Acyl Carrier Protein (ACP) -dependent acyltransferase activity.

In the present application, the protein encoded by the LpxT gene has lipid A1-diphosphate synthase/undecylenic pyrophosphate: lipid A1-phosphotransferase activity.

In the present application, the protein encoded by the WaaF gene has ADP-heptose: LPS-heptyl transferase II activity.

In the present application, the protein encoded by the WaaL gene has lipid a core-surface polymer ligase activity.

In the present application, the WaaV gene encodes a protein having a beta-1, 3-glucosyltransferase activity.

In the present application, the protein encoded by the WaaW gene has UDP-galactose: (galactosyl) LPS alpha-1, 2-galactosyltransferase activity.

In the present application, the protein encoded by the WaaY gene has a phosphorylating activity involved in core oligosaccharide heptose.

In the present application, the protein encoded by the WaaT gene has the IS1 protein lnsa/lnsb activity.

In the present application, the protein encoded by the WaaO gene has UDP-glucose: (glucosyl) LPS alpha-1, 3-glucosyltransferase activity.

In the present application, the protein encoded by the WaaP gene has kinase activity of phosphorylating lipopolysaccharide core heptose.

In the present application, the protein encoded by the WaaG gene has UDP-glucose: (heptosyl) lipopolysaccharide alpha-1, 3-glucosyltransferase/lipopolysaccharide core biosynthesis protein/lipopolysaccharide glucosyltransferase I activity.

In the present application, the protein encoded by the WaaQ gene has lipopolysaccharide core biosynthesis protein activity.

In the present application, the lactose permease gene may be LacY gene. The LacY gene sequence can be represented by the sequence SEQ ID NO. 11.

In the present application, the fucose transporter gene may be a FucP gene. The FucP gene sequence can be shown as a sequence SEQ ID NO. 12.

In the application, the beta-galactosidase gene sequence is nucleotide 332766-335840 of GenBank of CP001509.3 (16-FEB-2017).

In the application, the UDP-glucose lipid carrier transferase gene sequence is nucleotide 2019078-2020472 of GenBank of CP001509.3 (16-FEB-2017).

In the application, the fucose isomerase gene sequence is nucleotide 2773864-2775639 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the sequence of the fucoidan gene is nucleotide 2775748 to 2777166 of GenBank of CP001509.3 (16-FEB-2017).

In the application, the arabinose isomerase gene sequence is nucleotide 69640-71142 of GenBank of CP001509.3 (16-FEB-2017).

In the application, the rhamnose isomerase gene sequence is nucleotide 4002645-4003904 of GenBank CP001509.3 (16-FEB-2017). In the present application, the LpxL gene sequence is nucleotide 1118229 to 1119149 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the LpxM gene sequence is nucleotide 1884946 to 1885917 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the LpxP gene sequence is nucleotide 2373530 to 2374450 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the LpxT gene sequence is nucleotide 2163703 to 2164416 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaF gene sequence is nucleotide 3662908-3663954 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaL gene sequence is nucleotide 3665008-3666261 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaV gene sequence is nucleotide 3666307-3667290 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaW gene sequence is nucleotide 3667375-3668400 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaY gene sequence is nucleotide 3668426-3668974 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaT gene sequence is nucleotide 3669128-3670800 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaO gene sequence is nucleotide 3670916-3671932 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaP gene sequence is nucleotide 3671948-3672745 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaG gene sequence is nucleotide 3672738-3673862 of GenBank of CP001509.3 (16-FEB-2017).

In the present application, the WaaQ gene sequence is nucleotide 3673859-3674917 of GenBank of CP001509.3 (16-FEB-2017).

The recombinant E.coli may have the following characteristics:

the recombinant escherichia coli contains plasmid combinations with different copy numbers, the plasmid combinations are used for regulating and controlling the expression of the alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, and the plasmids are pCDFDu et-1, pETDuet-1 or pRSFDuet-1;

and/or the recombinant escherichia coli contains recombinant vectors in different operon forms, wherein the expression of the alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene is regulated by the recombinant vectors, and the operon forms are operons, pseudooperons or monocistronic.

In the present application, the α -1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene may be expressed in the different operon forms.

The alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene may be organized for expression as an operon;

alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene can be organized into pseudooperon form for expression

Alpha-1, 2-fucosyltransferase genes and L-fucose kinase/GDP-L-fucose pyrophosphorylase genes may be organized but expressed in cistron form

In the present application, in the recombinant E.coli B6) of A2), the source of the α -1, 2-fucosyltransferase gene may be E.coli; the α -1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene may be in any of the following forms:

c1 Alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are expressed in pRSF Duet-1;

c2 Alpha-1, 2-fucosyltransferase gene is expressed in pRSFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pETDuet-1;

c3 Alpha-1, 2-fucosyltransferase gene is expressed in pRSFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pCDFDuet-1;

c4 Alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are expressed in pRSF Duet-1;

C5 Alpha-1, 2-fucosyltransferase gene is expressed in pETDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pETDuet-1; the method comprises the steps of carrying out a first treatment on the surface of the

C6 Alpha-1, 2-fucosyltransferase gene was expressed in pETDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene was expressed in pCDFDuet-1;

c7 Alpha-1, 2-fucosyltransferase gene is expressed in pCDFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pRSFDuet-1;

c8 Alpha-1, 2-fucosyltransferase gene is expressed in pCDFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pETDuet-1;

c9 Alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene were expressed in pCDF Duet-1.

In the present application, in the recombinant E.coli B6) of A3), the source of the α -1, 2-fucosyltransferase gene may be E.coli, the α -1, 2-fucosyltransferase gene is expressed in pRSFDuet-1, and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene is expressed in pCDFDuet-1; wherein the fucose transporter gene and lactose permease gene may be in any of the following forms:

D1 The fucose transporter gene was expressed in pRSFDuet-1, without lactose permease gene;

d2 Lactose permease gene expressed in pRSFDuet-1, without fucose transporter gene;

d3 Fucose transporter gene is expressed in pRSFDuet-1 and lactose permease gene is expressed in pRSFDuet-1.

In the recombinant E.coli B6) of A4), the source of the alpha-1, 2-fucosyltransferase gene may be E.coli, the alpha-1, 2-fucosyltransferase gene is expressed in pRSFDuet-1, and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene is expressed in pCDFDuet-1; wherein the fucose transporter gene and lactose permease gene may be in any of the following forms:

In the above, the recombinant E.coli may be the LpxL gene, the LpxM gene, the LpxP gene and the LpxT gene in addition to the above-described characteristics.

In addition to the above-described characteristics, the outer membrane lipopolysaccharide synthesis, modification and/or transport related gene may be a WaaF gene, a WaaL gene, a WaaV gene, a WaaW gene, a WaaY gene, a WaaT gene, a WaaO gene, a WaaP gene, a WaaG gene and a WaaQ gene.

The recombinant E.coli may have the following characteristics:

the recombinant E.coli was constructed as follows.

The application also provides a method for constructing recombinant escherichia coli to solve the problems.

The method for constructing the recombinant escherichia coli comprises the following steps of carrying out any one of the following operations on the recipient escherichia coli:

c1 Introducing the above-described alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene into a recipient E.coli to obtain the recombinant E.coli of A1) of claims 1-5;

c2 Knocking out the beta-galactosidase gene of any one of claims 1 to 5 of the recombinant escherichia coli of A1) to obtain the recombinant escherichia coli of A2) of claims 1 to 5;

c3 Knocking out the beta-galactosidase gene of the recombinant escherichia coli of A1) and the UDP-glucose lipid carrier transferase gene of any one of claims 1-5 to obtain the recombinant escherichia coli of A2) of claims 1-5;

C4 Knocking out the beta-galactosidase gene, the UDP-glucose lipid carrier transferase gene and the fucose isomerase gene of any one of claims 1-5 of A1) to obtain the recombinant escherichia coli of A2) in claims 1-5;

c5 Knocking out A1) the beta-galactosidase gene, the UDP-glucose lipid carrier transferase gene, the fucose isomerase gene and the fucokinase gene of any one of claims 1-5 of the recombinant escherichia coli to obtain the recombinant escherichia coli of A2) of claims 1-5;

c6 Knocking out the beta-galactosidase gene, the UDP-glucose lipid carrier transferase gene, the fucose isomerase gene, the fucokinase gene and the arabinose isomerase gene of any one of claims 1-5 of A1) of the recombinant escherichia coli to obtain the recombinant escherichia coli of A2) of claims 1-5;

c7 Knocking out the beta-galactosidase gene, UDP-glucose lipid carrier transferase gene, fucose isomerase gene, fucokinase gene, arabinose isomerase gene and rhamnose isomerase gene of any one of claims 1-5 of A1) to obtain recombinant escherichia coli of A2) of claims 1-5;

C8 Introducing the fucose transporter gene and/or lactose permease gene of any one of claims 1-5 into the recombinant escherichia coli of A2) to obtain the recombinant escherichia coli of A3) of claims 1-5;

c9 Knocking out genes related to outer membrane lipid synthesis and modification in any one of claims 1 to 5 of the recombinant escherichia coli of A3) to obtain the recombinant escherichia coli of A4) in claims 1 to 6;

c10 Knocking out genes related to synthesis, modification and/or transportation of outer membrane lipopolysaccharide of any one of claims 1-5 of the recombinant escherichia coli of A4) to obtain the recombinant escherichia coli of A5) of claims 1-5.

In the method, the alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are introduced into the recipient E.coli by a recombinant plasmid, the recombinant plasmid expresses the alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, the recombinant plasmid regulates the expression of the alpha-1, 2-fucose transferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, and the recombinant plasmid is pCDFDuet-1, pETDuet-1 or pRSFDuet-1;

and/or the number of the groups of groups,

The alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are introduced into the recipient E.coli by a recombinant plasmid expressing the alpha-1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, which regulates the expression of the alpha-1, 2-fucose transferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene in the form of different operons, which are operons, pseudooperons or monocistronic.

The recombinant plasmid combination is any one of the following combinations:

d1 Alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are expressed in pRSFDuet-1;

d2 Alpha-1, 2-fucosyltransferase gene is expressed in pRSFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pETDuet-1;

d3 Alpha-1, 2-fucosyltransferase gene is expressed in pRSFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pCDFDuet-1;

d4 Alpha-1, 2-fucosyltransferase gene is expressed in pETDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pRSFDuet-1;

D5 Alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are expressed in pETD uet-1;

d6 Alpha-1, 2-fucosyltransferase gene was expressed in pETDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene was expressed in pCDFDuet-1;

d7 Alpha-1, 2-fucosyltransferase gene is expressed in pCDFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pRSFDuet-1;

d8 Alpha-1, 2-fucosyltransferase gene is expressed in pCDFDuet-1, and L-fucoskinase/GDP-L-fucose pyrophosphorylase gene is expressed in pETDuet-1;

d9 Alpha-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene were expressed in pCDFDuet-1.

The operon form may be any of the following:

d10 A-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are organized into an operon form for expression;

d11 A-1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are organized into a pseudooperon form for expression;

d12 A 1, 2-fucosyltransferase gene and an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are organized into a monocistronic form for expression;

The α -1, 2-fucosyltransferase gene in C1) above may be derived from helicobacter pylori.

The recombinant E.coli obtained in the above, wherein the alpha-1, 2-fucosyltransferase gene introduced into the recombinant E.coli is derived from E.coli, bacteroides fragilis or Haematococcus elongatus.

The recombinant E.coli obtained in the above, C9), the α -1, 2-fucosyltransferase gene was expressed in pRSFDuet-1, and the L-fucokinase/GDP-L-fucose pyrophosphorylase gene was expressed in pCDFDuet-1.

The recombinant E.coli described in C10) above may be the recombinant E.coli obtained in C9).

In the above, C11) the outer membrane lipid synthesis and modification related genes may be LpxL gene, lpxM gene, lpxP gene and LpxT gene.

Above, C12) the outer membrane lipopolysaccharide synthesis, modification and transport related genes are the WaaF gene, the WaaL gene, the WaaV gene, the WaaW gene, the WaaY gene, the WaaT gene, the WaaO gene, the WaaP gene, the WaaG gene and the WaaQ gene.

In order to solve the problems, the application also provides application.

The application is the application of any one of the following in preparing 2 '-fucosyllactose or constructing recombinant escherichia coli producing 2' -fucosyllactose;

D1 Recombinant E.coli as described in any one of the above or a method as described above;

d2 A nucleic acid molecule that is any one of:

d21 A G1 gene as defined in any one of the above;

d22 A G1 gene as defined in any one of the above and a G2 gene as defined in any one of the above;

d3 D2) an expression cassette comprising said nucleic acid molecule;

d4 A recombinant vector comprising D2) said nucleic acid molecule, or a recombinant vector comprising D3) said expression cassette

D5 A recombinant microorganism comprising D2) said nucleic acid molecule, or a recombinant microorganism comprising D3) said expression cassette, or a recombinant microorganism comprising D4) said recombinant vector;

e1 A nucleic acid composition consisting of D2) the nucleic acid molecule and a nucleic acid molecule for knocking out any one of the following genes:

e11 A K1 gene as defined in any one of the above;

e12 the K1 gene and the outer membrane lipid synthesis and modification related genes as described in any one of the above;

e13 Any one of the above K1 gene, the outer membrane lipid synthesis and modification related gene, and the knock-out outer membrane lipopolysaccharide synthesis, modification and transport related gene;

e2 An expression cassette comprising E1) the nucleic acid composition;

e3 A recombinant vector comprising E1) said nucleic acid composition, or a recombinant vector comprising E2) said expression cassette;

E4 A recombinant microorganism comprising the nucleic acid composition of E1), or a recombinant microorganism comprising the expression cassette of E2), or a recombinant microorganism comprising the recombinant vector of E3).

In the present application, the method used for the knockout of the gene can utilize a CRISPR-Cas9 gene knockout system.

In the present application, the sgRNA for knocking out lpxL gene may be 5 '-CCAGAGTGTTCTCCGCCACTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-3'.

In the present application, the sgRNA for knocking out lpxM gene may be 5 '-ACAGATTGATTAACGCACGAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-3'.

In the present application, the sgRNA from which lpxP gene is knocked out may be 5 '-ATCGGCAGAAATAATCTGCGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-3'.

In the present application, the sgRNA for knocking out lpxT gene may be 5 '-ACCAGGAAAGAAACAGCGCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-3'.

In the present application, the sgRNA for knocking out the WaaF gene may be CTGCTGCTCGGTATTCAAAGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-3'.

In the present application, the sgRNA for knocking out the WaaL-Q gene may be 5' -ACCAGGAAAGAAACAGCGCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc. The WaaL-Q gene may be at least one of a WaaL gene, a WaaV gene, a WaaW gene, a WaaY gene, a WaaT gene, a WaaO gene, a WaaP gene, a WaaG gene, and a WaaQ gene.

In order to solve the above problems, the present application also provides a method for producing 2' -fucosyllactose.

The method comprises culturing the recombinant escherichia coli to obtain a fermentation product, and obtaining 2' -fucosyllactose from the fermentation product.

In the present application, the method for producing 2' -fucosyllactose may be: glycerol is used as a carbon source, and lactose and L-fucose are used as substrates, and sodium thiosulfate is used as an auxiliary material to produce 2' -fucosyllactose in a fermentation system.

The E.coli may be BL21 star (DE 3).

The application aims to construct recombinant escherichia coli for efficiently synthesizing 2' -FL based on a salvage pathway by utilizing a synthetic biology means. The CRISPR-Cas9 gene editing system is utilized to weaken bypass metabolism, increase the accumulation of intracellular 2'-FL, screen fucosyltransferase from different sources, optimize the expression intensity of target genes according to plasmid combinations with different copy numbers, and overexpress genes of fucose transport and lactose transport so as to increase the synthesis of 2' -FL. By modifying the cell membrane of the strain, the material transfer capacity of the recombinant bacteria and the substrate utilization rate are increased, and the yield of 2' -FL is further improved; the method successfully constructs an escherichia coli engineering strain with the potential of industrialized production of 2' -FL, the yield of 2' -fucosyllactose in fermentation liquor can reach 7.28g/L, and a foundation is laid for the transformation of the engineering strain for industrialized production of 2' -fucosyllactose.

Drawings

FIG. 1 shows the gene knockout result. Graph a: lacZ gene knockout results; graph B: wcaJ gene knockout results; graph C: the result of knockout of the FucIK gene cluster; graph D: araA gene knockout results; diagram E: a RhaA gene knockout result;

FIG. 2 is a graph showing the effect of gene knockout and different sources of α -1, 2-fucosyltransferase on the yield and biomass of 2' -fucosyllactose in E.coli BL21 star (DE 3). Graph a: effects of knockdown of lactose, fucose and GDP-L-fucose metabolism genes (beta-galactosidase genes LacZ, UDP-glucose lipid carrier transferase genes WcaJ, fucose isomerase/fucokinase genes FucI-FucK gene cluster, arabinose isomerase genes AraA and rhamnose isomerase genes RhaA) on 2' -FL yield of engineering bacteria; graph B: effects of alpha-1, 2-fucosyltransferase FutC, wbgL, wcfB and Te2FT on engineered 2' -FL yield and biomass.

FIG. 3 is the effect of expression of WbgL and Fkp on yield and biomass of engineered E.coli 2' -fucosyllactose in different operon forms and different copy number plasmids. Graph a: the target genes are organized into different operon forms; graph B: yield and biomass of engineered E.coli 2' -fucosyllactose when WbgL and Fkp are expressed in different operon forms; graph C: yield of engineered E.coli 2' -fucosyllactose and biomass when WbgL and Fkp were expressed in different copy number plasmids.

FIG. 4 is the effect of overexpressing the LacY gene for lactose transport and the FucP gene for fucose transport on the yield and biomass of engineered E.coli 2' -fucosyllactose.

FIG. 5 shows the effect of knocking out the genes involved in the synthesis and modification of outer membrane lipids and the yield and biomass of 2' -fucosyllactose relative to engineering E.coli. Graph a: lpxL gene knockout results; graph B: lpxM gene knockout results; graph C: lpxP gene knockout results; graph D: lpxT gene knockout results; diagram E: knocking out the influence of related genes of outer membrane lipid synthesis and modification on the yield and biomass of engineering bacteria 2' -FL.

FIG. 6 shows the effect of knocking out genes involved in lipopolysaccharide synthesis, modification and transport and the yield and biomass of 2' -fucosyllactose relative to E.coli. Graph a: waaF gene knockout results; graph B: waaL-Q gene cluster knockout results; graph C: knocking out the influence of genes related to synthesis, modification and transportation of the outer membrane lipopolysaccharide on the yield and biomass of engineering bacteria 2' -FL.

FIG. 7 results of engineering bacterium FBL27 in a 5L fermenter for production of 2' -fucosyllactose, black arrow indicates 15h of fermentation, and inducer was added to induce expression of the target gene.

FIG. 8 is a graph A showing the results of HPLC and ESI identification of 2' -FL-producing engineering bacterium FBL01 fermentation products: HPLC identification result; graph B: ESI identification results.

Detailed Description

The plasmids (unless otherwise specified), restriction enzymes, PCR reagents, plasmid extraction kits, DNA gel recovery kits and the like used in the examples below are commercially available products, and specific operations are performed according to the kit instructions. Plasmid, DNA synthesis, primer synthesis and DNA sequencing were performed by Beijing, the biological sciences Co.

The raw materials for preparing the LB liquid medium are as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and the balance of water.

The raw materials for preparing the LB agar solid medium are as follows: LB liquid medium and agar 15g/L.

The fermentation medium was prepared from the following raw materials: 2.68g/L of ammonium sulfate, 1g/L of ammonium citrate, 26.42g/L of glycerin, 14.6g/L of dipotassium hydrogen phosphate, 0.241g/L of magnesium sulfate, 2g/L of sodium sulfate, 4g/L of sodium dihydrogen sulfate monohydrate, 0.5g/L of ammonium chloride, 10mg/L of thiamine, 23ml/L of trace salt stock solution A and the balance of water.

Wherein, the pH of trace salt stock solution A is 7.2, and the composition is as follows: 25g/L of ferric trichloride hexahydrate, 2g/L of calcium chloride dihydrate, 2g/L of zinc chloride, 2g/L of disodium molybdate dihydrate, 1.9g/L of copper sulfate pentahydrate, 0.5g/L of boric acid and the balance of water.

The feed medium is prepared from the following raw materials: glycerin 50% (v/v), magnesium sulfate 23.5g/L, sodium thiosulfate 3.2g/L, thiamine 0.6g/L, trace salt stock solution B119 ml/L, and the balance water.

Wherein, the composition of trace salt stock solution B is as follows: 0.5g/L of calcium chloride dihydrate, 16.7g/L of ferric trichloride hexahydrate, 20.1g/L of disodium ethylenediamine tetraacetate, 0.18g/L of zinc sulfate heptahydrate, 0.1g/L of magnesium sulfate monohydrate, 0.16g/L of copper sulfate pentahydrate, 0.18g/L of cobalt chloride hexahydrate and the balance of water.

Determination method of lactose, fucose, 2' -fucosyllactose and glycerol: HPLC assay. The fermentation broth was centrifuged at 8000rpm for 10min in a boiling water bath for 10min, and the supernatant was filtered through a 0.22 μm filter and analyzed quantitatively by HPLC. Quantitative analysis was performed using a standard curve method (external standard method). HPLC assay conditions: differential reflectance detector (RID), detector temperature 35 ℃; the column was Rezex ROA-Organic Acid h+ (8%) column (Phenomenex inc., CA, USA) column temperature 60 ℃; mobile phase: 5mM H ₂ SO ₄ The flow rate of the solution is 0.6mL/min; the sample volume was 10. Mu.L.

Example 1: influence of different sources of fucosyltransferases on the Synthesis of 2' -fucosyllactose by E.coli engineering bacteria

In this example, plasmids were constructed by means of cleavage ligation, and the primer sequences involved are shown in Table 1.

1. Construction of recombinant E.coli FBL06-1

1.1 construction of recombinant E.coli BL 21. Delta. LacZ

The LacZ gene (nucleotide sequence (CDS) is nucleotide 332766-335840 of GenBank: CP001509.3 (16-FEB-2017)) in Escherichia coli BL21 star (DE 3) (Invitrogen Co., ltd.: C60003) was knocked out by using CRISPR-Cas9 gene knockout system, and the encoded amino acid sequence was protein beta-D-galactosidase of GenBank: ACT42197.1, to obtain recombinant Escherichia coli BL 21. DELTA. LacZ deleted in gene LacZ. The primer sequences involved are shown in Table 1.

1.1.1 construction of the pTargetF plasmid containing sgRNA. PCR amplification was performed using the original pTargetF plasmid (Wuhan vast Biotechnology Co., ltd., cat# P0743) as a template and LacZ-sg-F and LacZ-sg-R as primers (Table 1) to obtain PCR products, removing template DNA by using DpnI, then transforming E.coli DH 5. Alpha. Competent cells, coating LB plates (containing 50. Mu.g/mL spectinomycin), culturing at 37℃and extracting the plasmid and sequencing to obtain the plasmid pTargetF-LacZ.

pTargetF-LacZ is an expression vector for expressing sgRNA targeting LacZ gene, which replaces 5 '-CATCGCCGCAGCGGTTTCAG-3' of pTargetF (Wuhan vast Ling Biotechnology Co., ltd., cat. No. P0743) with N20 sequence (5 '-CGGGTGAACTGATCGCGCAG-3') complementary to LacZ sequence, and the other nucleotide sequence of pTargetF is kept unchanged.

1.1.2 preparation of donor DNA fragments. The upstream homology arm of the LacZ gene was PCR amplified using the E.coli BL21 star (DE 3) genome as a template, lacZ-up-F (5 '-TTGGCAACCGTGGCAGAAG-3') and LacZ-up-R (5 '-GACTGGGAAAACCCTGGCCGGTCGCTACCATTACCAGTTG-3'), and the downstream homology arm of the LacZ gene was PCR amplified using LacZ-down-F (5 '-CAACTGGTAATGGTAGCGACCGGCCAGGGTTTTCCCAGTC-3') and LacZ-down-R (5 '-GGTAGTGGGATACGACGATACC-3'), and the above fragments were recovered. And then, using an upstream homology arm of the LacZ gene and a downstream homology arm of the LacZ gene as templates, and adopting LacZ-up-F and LacZ-down-R primers to obtain a complete donor DNA fragment for carrying out homologous recombination with the LacZ gene through overlap PCR, and recovering the DNA fragment by glue.

1.1.3 The pCas plasmid (Whan vast, biotechnology Co., ltd.) was introduced into E.coli BL21 star (DE 3) to obtain pCas-containing recombinant E.coli BL21 star (DE 3)/pCas.

1.1.4 100ng of the pTargetF-LacZ plasmid of step 1.1.1 and 400ng of the donor DNA fragment for homologous recombination with LacZ gene of step 1.1.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24h, and the LacZ knockout effect was confirmed by PCR (primers LacZ-F and LacZ-R). The result shows that the positive cloning bacteria obtain 1000bp PCR product and the wild type escherichia coli BL21 star (DE 3) obtains 3398bp PCR product.

1.1.5 Removal of the pTargetF-LacZ and pCas plasmids gave E.coli BL21 (DE 3) ΔLacZ. The positive clone colonies were picked up to a 5ml LB liquid tube, IPTG and 50mg/L kanamycin were added to a final concentration of 0.5mM, and cultured at 30℃for 8-16 hours to obtain pTargetF-LacZ-removed strains. The strain was then incubated at 37℃for 12h for removal of the pCas plasmid. E.coli BL21 (DE 3) ΔLacZ (recombinant E.coli BL21 ΔLacZ or strain BL21 ΔLacZ for short) was obtained after sequencing verification. Coli BL21 (DE 3) ΔLacZ was deleted from the LacZ gene by nucleotides 91 to 2488 (position 332766 of GenBank CP001509.3 was designated as position 1 of LacZ gene) as compared with wild-type E.coli BL21 star (DE 3), and the encoded protein was deleted from amino acids 31 to 831 of GenBank ACT42197.1, whereby the LacZ gene was knocked out.

1.2 construction of the Strain BL 21. Delta. LacZ. Delta. WcaJ

The method of reference 1.1 knocks out the WcaJ gene (nucleotide sequence (CDS) is nucleotide 2019078 to 2020472 of GenBank: CP001509.3 (16-FEB-2017), the encoded amino acid sequence is protein (UDP-glucose lipid carrier transferase gene) of GenBank: ACT 43804.1) in recombinant escherichia coli bl21 Δlacz Δwcaj deleted from WcaJ gene using CRISPR-Cas9 gene knockout system. The primer sequences involved are shown in Table 1.

1.2.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-TTTATCAATGTGCTGACCGG-3'), resulting in a pTargetF plasmid with sgRNA targeting the WcaJ gene (nucleotide sequence (CDS) is nucleotide 2019078-2020472 of GenBank: CP001509.3 (16-FEB-2017), the encoded amino acid sequence is protein of GenBank: ACT43804.1 (UDP-glucose lipid carrier transferase gene)), named pTargetF-WcaJ. LacZ-sg-F and LacZ-sg-R were replaced with WcaJ-sg-F and WcaJ-sg-R (Table 1), and the rest was 1.1.1.

1.2.2 preparation of donor DNA fragments. The upstream homology arm of the WcaJ gene was amplified by PCR using the E.coli BL21 star (DE 3) genome as a template, using WcaJ-up-F (5 '-GGTTTCGATCATGCCGATTT-3') and WcaJ-up-R (5 '-CGGACTATGGCTGGTTTGCCGGTGTTCAAAGGTTTCGTTAAC-3'), and the downstream homology arm of the WcaJ gene was amplified by PCR using WcaJ-down-F (5 '-GTTAACGAAACCTTTGAACACCGGCAAACCAGCCATAGTCCG-3') and WcaJ-down-R (5 '-TTACTTCCGTGATTTCGCTTACT-3'), and the above fragments were recovered by gel. And then, by taking an upstream homology arm of the WcaJ gene and a downstream homology arm of the WcaJ gene as templates and adopting WcaJ-up-F and WcaJ-down-R, obtaining a complete donor DNA fragment for carrying out homologous recombination with the WcaJ gene through overlap PCR, and recycling the DNA fragment by glue.

1.2.3 is the same as 1.1.3.

1.2.4 100ng of the pTargetF-WcaJ plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24 hours, and PCR (primers WcaJ-F and WcaJ-R) was performed to verify the WcaJ gene knockout effect. The result shows that the positive clone bacteria obtain 1000bp PCR product, the wild type escherichia coli BL21 star (DE 3) obtains 2135bp PCR product, and the strain BL21 delta LacZ obtains 2135bp PCR product.

1.2.5 pTargetF-WcaJ plasmid and pCas plasmid in positive clone were removed according to the method of 1.1.5 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ (recombinant E.coli ΔLacZ ΔWcaJ or strain BL21 ΔLacZ ΔWcaJ for short). The WcaJ gene was deleted for nucleotides 112-1246 (GenBank: CP001509.3 (16-FEB-2017) as position 2019078 of the WcaJ gene) compared to E.coli BL21 (DE 3) ΔLacZ Δwcaj, thereby causing a frameshift to knock out the WcaJ gene.

1.3 construction of the Strain BL 21. Delta. LacZ. Delta. WcaJ. Delta. FucIK

The method of reference 1.1 uses a CRISPR-Cas9 gene knockout system to knock out Δfucik gene in recombinant escherichia coli bl21Δlacz Δwcaj to obtain recombinant escherichia coli bl21Δlacz ΔwcajΔfucik deleted of FucIK gene. The primer sequences involved are shown in Table 1. Wherein, the FucI gene (nucleotide sequence (CDS) is GenBank: CP001509.3 (16-FEB-2017) nucleotide 2773864-2775639, and the coding amino acid sequence is GenBank: ACT44469.1 protein (fucose isomerase/fucokinase)); fucK gene (nucleotide sequence (CDS) is nucleotide 2775748-2777166 of GenBank: CP001509.3 (16-FEB-2017), and encoded amino acid is protein (fucose isomerase/fucokinase) of GenBank: ACT 44470.2).

1.3.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 'GAATTTTTCTTCGCAAGCAG-3'), resulting in a pTargetF plasmid with sgRNA targeting the FucIK gene, designated pTarg etF-FucIK. LacZ-sg-F and LacZ-sg-R were replaced with FucIK-sg-F and FucIK-sg-R (Table 1), and the rest was 1.1.1.

1.3.2 preparation of donor DNA fragments. The upstream homology arm of the FucIK gene was amplified by PCR using the E.coli BL21 star (DE 3) genome as a template, using FucIK-up-F (5 '-TGTGCTTCTTTATTGGTCGTTT-3') and FucIK-up-R (5 '-AATAATTCAGAGGCCTCGCCATATTCATTGTTTGTTC-3'), using FucIK-down-F (5 '-CAATGAATATGGCGAGGCCTCTGAATTATTGTTAGTCG-3') and FucIK-down-R (5 '-TCAGGACACGGGGCTTGTAGTG-3'), and the downstream homology arm of the FucIK gene was amplified by PCR, and the above fragment was recovered by gel. And then, using an upstream homology arm of the FucIK gene and a downstream homology arm of the FucIK gene as templates, obtaining a complete donor DNA fragment for carrying out homologous recombination with the FucIK gene by overlapping PCR by using FucIK-up-F and FucIK-down-R, and recovering the DNA fragment by gel.

1.3.3 is the same as 1.1.3.

1.3.4 100ng of the pTargetF-FucIK plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), incubated at 30℃for 24h, and PCR (primers FucIK-F and FucIK-R) was performed to verify the FucI gene knockout effect. The result shows that the positive clone bacteria obtain 1000bp PCR product, the wild type escherichia coli BL21 star (DE 3) obtains 3337bp PCR product, and the strain BL21 delta LacZ delta WcaJ obtains 3337bp PCR product.

1.3.5 pTargetF-FucIK plasmid and pCas plasmid in positive clone were removed according to the method of 1.1.5 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK (recombinant E.coli ΔLacZ ΔFucIK or strain BL21 ΔLacZ ΔWcaJ ΔFucIK for short). The 34 th-1776 th nucleotide of the FucI gene is deleted (the 1 st position of the FucI gene is marked by GenBank: CP001509.3 rd position 2773864) compared with E.coli BL21 (DE 3) DeltaLacZDeltaWcaJ, and the 38 th-416 th amino acid of the GenBank: ACT43804.1 is deleted to knock out the FucI gene; the FucK gene is deleted from nucleotides 1 to 480 (GenBank: CP001509.3, position 2775748 is designated as position 1 of the FucK gene), and the encoded protein is deleted from amino acids 1 to 160 of GenB ank ACT44470.2, so that the FucK gene is knocked out.

1.4 construction of the Strain BL 21. Delta. LacZ. DELTA.WcaJ. DELTA. FucIK. DELTA.AraA

The method of reference 1.1 knocks out the AraA gene (nucleotide sequence (CDS) is nucleotide 69640-71142 of GenBank: CP001509.3 (16-FEB-2017), and the encoded amino acid sequence is protein (arabinose isomerase) of GenBank: ACT 41965.1) in recombinant E.coli BL 21. DELTA.LacZ. DELTA.WcaJ. FucI. DELTA.FucK using a CRISPR-Cas9 gene knockout system to obtain the AraA gene-deleted recombinant E.coli BL 21. DELTA.LacZ. DELTA.WcaJ. DELTA.FucIK. DELTA.AraA. The primer sequences involved are shown in Table 1.

1.4.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 'GAATTTTTCTTCGCAAGCAG-3'), resulting in a pTargetF plasmid with sgRNA targeting the ΔAraA gene (GenBank: nucleotides 69640-71142 of CP001509.3 (16-FEB-2017), the encoded amino acid sequence being the protein of GenBank: ACT41965.1 (arabinose isomerase)), designated pTargetF-AraA. LacZ-sg-F and LacZ-sg-R were replaced with AraA-sg-F and AraA-sg-R (Table 1), and the rest was 1.1.1.

1.4.2 preparation of donor DNA fragments. The upstream homology arm of the AraA gene was PCR amplified using the Escherichia coli BL21 star (DE 3) genome as a template, using AraA-up-F (5 '-GATCTTAACCTCGCTACCGACG-3') and AraA-up-R (5 '-CGTTACACACCTGATGCGACTACTATCGTGTCCTTATAGAGTCGCAAC-3'), and the downstream homology arm of the AraA gene was PCR amplified using AraA-down-F (5 '-GTAGTCGCATCAGGTGTGTAACG-3') and AraA-down-R (5 '-TATCGTCAGCGGTCATGATGC-3'), and the fragments were recovered by gel. And then, by taking an upstream homology arm of the AraA gene and a downstream homology arm of the AraA gene as templates and adopting AraA-up-F and AraA-down-R, obtaining a complete donor DNA fragment for carrying out homologous recombination with the AraA gene through overlap PCR, and recovering the DNA fragment by glue.

1.4.3 is the same as 1.1.3.

1.4.4 100ng of the pTargetF-AraA plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24 hours, and PCR (primers AraA-F and AraA-R) was performed to verify the FucI knockout effect. The result shows that the positive cloning strain obtains a PCR product of 1000bp, the wild type escherichia coli BL21 star (DE 3) obtains a PCR product of 1384bp, and the strain BL21 delta LacZ delta WcaJ delta FucIK obtains a PCR product of 1384 bp.

1.5 pTargetF-AraA plasmid and pCas plasmid in positive clone were removed according to the method of 1.1.5 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA (recombinant E.coli ΔLacZ ΔFucI or strain BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA for short). The AraA gene was deleted for nucleotides 117 to 500 (GenB ank: CP001509.3, position 69640 was designated as position 1 of the AraA gene) compared to E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA, and was subjected to a frame shift to knock out the AraA gene.

1.5 construction of the Strain BL 21. Delta. LacZ. DELTA.WcaJ. DELTA. FucIK. DELTA. AraA. DELTA.RhaA

The method of reference 1.1 knocks out the RhaA gene (nucleotide sequence (CDS) is nucleotide 4002645-4003904 of GenBank: CP001509.3 (16-FEB-2017), the encoded amino acid sequence is protein (rhamnose isomerase) of ACT 45582.2) in recombinant E.coli BL 21. DELTA.LacZ. DELTA.WcaJ. FucI. DELTA.FucK. DELTA.AraA using a CRISPR-Cas9 gene knockout system to obtain recombinant E.coli BL 21. DELTA.LacZ. DELTA.WcaJ. DELTA.FucIK. DELTA.AraA deleted RhaA. The primer sequences involved are shown in Table 1.

1.5.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-CCAACGTCACGATACCCCAG-3'), resulting in a pTargetF plasmid with sgRNA targeting the RhaA gene (GenBank: nucleotide 4002645-4003904 of CP001509.3 (16-FEB-2017), the encoded amino acid sequence being the protein of GenBank: ACT45582.2 (rhamnose isomerase)), named pTargetF-RhaA). LacZ-sg-F and LacZ-sg-R were replaced with RhaA-sg-F and RhaA-sg-R (Table 1), and the rest was 1.1.1.

1.5.2 preparation of donor DNA fragments. The upstream homology arm of the RhaA gene was PCR amplified using the E.coli BL21 star (DE 3) genome as a template, rhaA-up-F (5 '-TATTAACCCTGACGAGATGTGCAG-3') and RhaA-up-R (5 '-GCCGCATCCGGCAGTGTGCGCAAAGCTCCTTTGTCTG-3'), the downstream homology arm of the RhaA gene was PCR amplified using RhaA-down-F (5 '-ACACTGCCGGATGCGGC-3') and RhaA-down-R (5 '-GACGCCTAAGTTAGCTGCAGGA-3'), and the fragments were recovered by PCR. And then, taking an upstream homology arm of the AraA gene and a downstream homology arm of the AraA gene as templates, adopting RhaA-up-F and RhaA-down-R to obtain a complete donor DNA fragment for carrying out homologous recombination with the RhaA gene through overlap PCR, and recovering the DNA fragment by glue.

1.5.3 is the same as 1.1.3.

1.5.4 100ng of the pTargetF-RhaA plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24 hours, and PCR (primers RhaA-F and RhaA-R) was performed to verify the FucI knockout effect. The result shows that the positive cloning bacteria obtain 1000bp PCR product, the wild type escherichia coli BL21 star (DE 3) obtains 1502bp PCR product, and the strain BL21 delta LacZ delta WcaJ delta FucIK delta AraA obtains 1502bp PCR product.

1.5.5 removal of pTargetF-AraA plasmid and pCas plasmid from positive clones was performed according to the method of 1.1.5 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA (recombinant E.coli ΔLacZ ΔFucI or strain BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA for short). The RhaA gene was deleted for nucleotides 147 to 648 (GenBank: CP001509.3, position 4002645 was designated as position 1 of the RhaA gene) compared to E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA, and was subjected to a frameshift to knock out the RhaA gene. Strain FBL06-1 (bl21 Δlacz ΔwcajΔfucikΔaraaΔrhaa) was obtained. The above gene knockout results are shown in FIG. 1.

2. Construction of recombinant E.coli FBL00, FBL01, FBL02, FBL03, FBL04, FBL05 and FBL06 (engineering bacteria of E.coli producing 2' -fucosyllactose)

2.1 recombinant expression vector pCOLADuet-1-FutC-Fkp for expressing artificially synthesized Fkp gene and FutC gene

Construction of the recombinant expression vector pCOLADuet-1-FutC-Fkp: fkp gene (L-fucose kinase/GDP-L-fucose pyrophosphorylase gene) and FutC gene (α -1, 2-fucosyltransferase gene) were synthesized by Shanghai JieR bioengineering Co., ltd.) after codon optimization. The recombinant vector pCOLADuet-1-FutC-Fkp was obtained by replacing the fragment (small fragment) between the recognition sites of restriction endonucleases NcoI and BamHI of pCOLADuet-1 (available from Wohan vast biological sciences Co., ltd.) with the FutC gene whose nucleotide sequence is SEQ ID NO.6 (the coding amino acid sequence is alpha-1, 2-fucosyltransferase of SEQ ID NO. 2), and replacing the fragment (small fragment) between the recognition sites of restriction endonucleases NdeI and PacI of pCOLADuet-1 (Wohan vast biological sciences Co., ltd.) with the Fkp gene whose nucleotide sequence is SEQ ID NO.10 (the coding amino acid sequence is L-fucoskinase/GDP-L-fucose pyrophosphorylase of SEQ ID NO. 1), and the expression vector containing the FutC gene and the Fkp gene was obtained while keeping the other nucleotide sequence of OLADuet-1.

2.2 construction of engineering bacteria of E.coli producing 2' -fucosyllactose

pCOLADuet-1-FutC-Fkp was transferred into the recombinant E.coli BL21 star (DE 3), BL21 DeltaLacZ DeltaWcaJ DeltaFucI, BL21 DeltaLacZ DeltaWcaJ DeltaFucIK DeltaAraA, BL21 DeltaLacZ DeltaWcaJ DeltaFucIK DeltaArgA, FBL06-1 of step 1, respectively, to give E.coli engineering bacteria FBL00, FBL01, FBL02, FBL03, FBL04, FBL05 and FBL06 capable of synthesizing 2' -fucosyllactose (Table 4). Fkp is an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, and FutC is an alpha-1, 2-fucosyltransferase gene derived from helicobacter pylori.

3. Construction of recombinant expression vectors pCOLADuet-1-WbgL-Fkp, pCOLADuet-1-WcfB-Fkp and pCOLADuet-1-Te2FT-Fkp for expression of L-fucose kinase/GDP-L-fucose pyrophosphorylase gene and alpha-1, 2-fucosyltransferase gene

3.1 construction of recombinant plasmid pCOLADuet-1 containing alpha-1, 2-fucosyltransferases of different origins

Three genes, wbgL, wcfB and Te2FT, were synthesized by Shanghai JieRui Biotechnology Co., ltd.

Construction of the recombinant expression vector pCOLADuet-1-WbgL-Fkp: the recombinant vector pCOLADuet-1-WbgL-Fkp is an expression vector containing the WbgL gene and Fkp gene, which is obtained by replacing the fragment (small fragment) between the recognition sites of the restriction endonuclease NcoI and BamHI of pCOLADuet-1-FutC-Fkp with the WbgL gene (the coding amino acid sequence of which is the alpha-1, 2-fucosyltransferase of SEQ ID NO. 3. Coli) having the nucleotide sequence of SEQ ID NO.7, and keeping the other nucleotide sequence of pCOLADuet-1-FutC-Fkp unchanged.

Construction of the recombinant expression vector pCOLADuet-1-WcfB-Fkp: the recombinant vector pCOLADuet-1-WcfB-Fkp is an expression vector containing the WcfB gene and Fkp gene, which is obtained by replacing the fragment (small fragment) between the recognition sites of the restriction endonuclease NcoI and BamHI of pCOLADuet-1-FutC-Fkp with the WcfB gene (the coding amino acid sequence of which is the alpha-1, 2-fucosyltransferase of the sequence SEQ ID NO.4, derived from Bacteroides fragilis) having the nucleotide sequence of SEQ ID NO.8, keeping the other nucleotide sequence of pCOLADuet-1-FutC-Fkp unchanged.

Construction of recombinant expression vector pCOLADuet-1-Te2 FT-Fkp: the recombinant vector pCOLADuet-1-WcfB-Fkp is an expression vector containing Te2FT gene and Fkp gene, which is obtained by replacing the fragment (small fragment) between the recognition sites of restriction endonuclease NcoI and BamHI of pCOLADuet-1-FutC-Fkp with Te2FT gene (the coding amino acid sequence is alpha-1, 2-fucosyltransferase of sequence SEQ ID NO.5, derived from Haematococcus elongatus) whose nucleotide sequence is sequence SEQ ID NO.9, keeping the other nucleotide sequence of pCOLADuet-1-FutC-Fkp unchanged.

The WbgL gene is derived from escherichia coli, the WcfB gene is derived from bacteroides fragilis, and the Te2FT gene is derived from Synechococcus elongatus.

3.2. Construction of recombinant strains

The three plasmids were transferred into FBL06-1 strain by shock transformation (1 mm electric shock cup, 1.8 KV) to obtain FBL07 (pCOLADuet-1-WbgL-Fkp), FBL08 (pCOLADuet-1-WcfB-Fkp) and FBL09 (pCOLADuet-1-Te 2 FT-Fkp), respectively.

FBL07 contains the alpha-1, 2-fucosyltransferase and the L-fucose kinase/GDP-L-fucose pyrophosphorylase genes derived from E.coli.

FBL08 contains the alpha-1, 2-fucosyltransferase and the L-fucose kinase/GDP-L-fucose pyrophosphorylase genes derived from Bacteroides fragilis.

FBL09 contains the α -1, 2-fucosyltransferase and the L-fucoskinase/GDP-L-fucose pyrophosphorylase genes from Haematococcus elongatus.

4. Production of 2' -fucosyllactose using E.coli engineering bacteria

4.1 preparation of seed liquid

Coli FBL00, FBL01, FBL02, FBL03, FBL04, FBL05, FBL06, FBL07, FBL08 and FBL09 were inoculated into LB liquid medium (containing the corresponding antibiotics) and cultured overnight at 37 ℃ at 200rpm, respectively, to obtain seed liquid.

4.2 fermentation production of 2' -fucosyllactose Using E.coli engineering bacteria

1mL of the seed solution was added to a 250mL Erlenmeyer flask containing 50mL of fermentation medium, and cultured at 37℃and 200rpm to OD ₆₀₀ =0.6-0.8, IPTG solution, lactose solution and fucose solution were added to the culture system at a final concentration of 0.1mM, lactose at a final concentration of 5g/L and fucose at a final concentration of 5g/L. Incubated at 25℃for 72h, samples were taken at regular time and the yield of 2' -FL was quantitatively determined by HPLC.

Experimental results show that 2'-FL can be synthesized in a small amount after the 2' -FL salvage synthesis pathway is integrated into escherichia coli BL21 star (DE 3), and the yield is 0.32g/L after shaking flask fermentation for 72 hours. After LacZ is knocked out, the content of lactose used for synthesizing 2'-FL in cells is increased, and the yield of 2' -FL is increased to 1.56g/L after the escherichia coli engineering strain is fermented for 72 hours. Along with knocking out genes of metabolic fucose and GDP-L-fucose, the 2' -FL shake flask fermentation yield of engineering bacteria is gradually increased. When the WcaJ, fucIK gene cluster, araA and RhaA are knocked out, the 2' -FL shake flask fermentation yield of the engineering strain is improved to 1.85g/L. When FutC is replaced by alpha-1, 2-fucosyltransferase genes from other sources, the shake flask fermentation yield of engineering bacteria 2' -FL is changed, and the shake flask fermentation yields of engineering bacteria 2' -FL replaced by WbgL, wcfB and Te2FT are respectively 2.88g/L, 1.94g/L and 1.31g/L (the yields of different engineering strains 2' -FL are shown in FIG. 2 and Table 4).

TABLE 1 primers for gene knockout

Example 2: influence of expression intensity of genes WbgL and Fkp on synthesis of 2' -fucosyllactose by engineering bacteria

The expression intensity of the genes WbgL and Fkp is important for the synthesis of 2' -FL by engineering bacteria. The present example uses two ways of regulating gene expression to optimize the expression intensity of WbgL and Fkp. In this example, plasmids were constructed by means of restriction ligation and seamless cloning, and the primer sequences involved are shown in Table 2.

1. The first is to organize Fkp and WbgL for expression as operons, pseudooperons and monocistronic forms.

Construction of plasmid pCOLADuet-1-WbgL-Fkp-operon vector: the plasmid pCOLADuet-1-WbgL-Fkp is used as a template, and the primer WbgL-F/R is used for amplification to obtain a gene fragment RBS-WbgL. The plasmid pCOLADuet-1-WbgL-Fkp is used as a template and amplified by a primer Fkp-F/R to obtain a gene fragment RBS-Fkp. The plasmid pCOLADuet-1 is used as a template, and the primer pCOLADuet-F/R is used for amplification to obtain the fragment pCOLADuet. Fragments WbgL, fkp and pCOLADuet were assembled into plasmid pCOLADuet-1-WbgL-Fkp-operon using the C113 kit (Nanjinouzan Biotech Co., ltd.).

Construction of plasmid pCOLADuet-1-WbgL-Fkp-pseudoooperon: the plasmid pCOLADuet-1-WbgL-Fkp is used as a template, and the primer WbgL-PO-F/R is used for amplification to obtain a gene fragment T7-RBS-WbgL. The plasmid pCOLADuet-1-WbgL-Fkp is used as a template, and the primer Fkp-PO-F/R is used for amplification to obtain a gene fragment T7-RBS-Fkp. The plasmid pCOLADuet-1 is used as a template, and the primer pCOLADuet-PO-F/R is used for amplification to obtain the fragment pCOLADuet-PO. Fragments T7-RBS-WbgL, T7-RBS-Fkp and pCOLADuet-PO were assembled into plasmids using the C113 kit (Nanjinouzan Biotechnology Co., ltd.), pCOLADuet-1-T7-RBS-WbgL-T7-RBS-Fkp-operon being abbreviated as pCOLADuet-1-WbgL-Fkp-operon.

Construction of plasmid pCOLADuet-1-WbgL-Fkp-monocyclonic: the plasmid pCOLADuet-1 was used as a template and amplified with the primer T7ter-F/R to give fragment T7ter. The plasmid pCOLADuet-1-WbgL-Fkp is used as a template, and the primer Gi-WbgL-F/R is used for amplification to obtain a fragment T7-RBS-WbgL. The plasmid pCOLADuet-1-WbgL-Fkp was used as a template and amplified with the primer Gi-Fkp-F/R to give the fragment T7-RBS-Fkp. Overlapping extension PCR was performed using the fragments T7ter and T7-RBS-WbgL as templates and the primers Gi-WbgL-F and T7ter-R to obtain the fragment T7-RBS-WbgL-T7ter. The plasmid pCOLADuet-1 is used as a template, and the primer pCOLADuet-M-F/R is used for amplification to obtain the fragment pCOLADuet-M. Fragments T7-RBS-WbgL-T7ter, T7-RBS-Fkp and pCOLADuet-M were assembled into plasmids pETDuet-1-T7-RBS-WbgL-T7ter-T7-RBS-Fkp, pETDuet-1-WbgL-Fkp-monoccistronic using the C113 kit (Nanjinovirzan Biotechnology Co., ltd.).

2. Construction of recombinant vectors with different copy strengths of genes WbgL and fkp

As described above, the second was to select high copy pRSFDuet-1, medium copy number pETDuet-1 and low copy pCDFDuet-1 plasmids to optimize the expression intensities of the genes WbgL and Fkp.

Construction of recombinant vectors with different copy numbers of WbgL and Fkp genes

Construction of pRSFDuet-1-WbgL vector: the pRSFDuet-1-WbgL vector is a WbgL gene expression vector obtained by replacing a fragment (small fragment) between the recognition sites of restriction endonuclease NcoI and BamHI of pRSFDuet-1 (WHANGSHU vast Biotechnology Co., ltd.) with the WbgL gene whose nucleotide sequence is SEQ ID NO.7 (the encoded amino acid sequence is alpha-1, 2-fucosyltransferase of SEQ ID NO. 3) to maintain the other nucleotide sequence of pRSFDuet-1.

Construction of pRSFDuet-1-Fkp vector: the pRSFDuet-1-Fkp vector is a Fkp gene expression vector in which the restriction endonuclease NcoI and BamHI recognition site fragment (small fragment) of pRSFDuet-1 (Wuhan vast Biotechnology Co., ltd.) are replaced with Fkp gene whose nucleotide sequence is SEQ ID NO.10 (the encoded amino acid sequence is L-fucoskinase/GDP-L-fucose pyrophosphorylase of SEQ ID NO. 1) and the other nucleotide sequence of pRSFDuet-1 is kept unchanged.

Construction of pRSFDuet-1-WbgL-fkp vector: the pRSFDuet-1-WbgL-fkp vector is an expression vector containing the WbgL gene and the Fkp gene, which is obtained by replacing a fragment (small fragment) between the restriction endonuclease NdeI and PacI recognition sites of pRSFDuet-1-WbgL with the Fkp gene having the nucleotide sequence of SEQ ID NO.10 (the encoded amino acid sequence is L-fucoskinase/GDP-L-fucose pyrophosphorylase having the sequence of SEQ ID NO. 1).

Construction of pETDuet-1-WbgL vector: the pETDuet-1-WbgL vector is a WbgL gene expression vector obtained by replacing a fragment (small fragment) between the recognition sites of restriction endonuclease NcoI and BamHI of pETDuet-1 (WHan vast, biotechnology Co., ltd.) with the WbgL gene whose nucleotide sequence is SEQ ID NO.7 (the encoded amino acid sequence is alpha-1, 2-fucosyltransferase of SEQ ID NO. 3), and keeping the other nucleotide sequence of pETDuet-1 unchanged.

Construction of pETDuet-1-Fkp vector: the pETDuet-1-Fkp vector is a Fkp gene expression vector in which the Fkp gene (the encoded amino acid sequence is the L-fucoskinase/GDP-L-fucose pyrophosphorylase enzyme of SEQ ID NO. 1) having the nucleotide sequence of SEQ ID NO.10 is substituted for the fragment (small fragment) between the recognition sites of restriction endonuclease NcoI and BamHI of pETDuet-1 (Wohan vast, biotechnology Co., ltd.) and the other nucleotide sequence of pETDuet-1 is kept unchanged.

Construction of pETDuet-1-WbgL-Fkp vector: the pETDuet-1-WbgL-fkp vector is an expression vector containing the WbgL gene and the Fkp gene, wherein the nucleotide sequence of the expression vector is Fkp gene with the sequence SEQ ID NO.10 (the coding amino acid sequence is L-fucose kinase/GDP-L-fucose pyrophosphorylase with the sequence SEQ ID NO. 1), and the fragment (small fragment) between the restriction endonuclease NdeI and PacI recognition sites of pETDuet-1-WbgL is replaced, and the other nucleotide sequence of pETDuet-1-WbgL is kept unchanged.

Construction of pCDFDuet-1-WbgL vector: the pCDFDuet-1-WbgL vector is a WbgL gene expression vector obtained by replacing the fragment (small fragment) between the restriction endonuclease NdeI and PacI recognition site of pCDFDuet-1 (WHan dynasty vast, biotechnology Co., ltd.) with the WbgL gene whose nucleotide sequence is SEQ ID NO.7 (the encoded amino acid sequence is alpha-1, 2-fucosyltransferase of SEQ ID NO. 3) and keeping the other nucleotide sequence of pCDFDuet-1 unchanged.

Construction of pCDFDuet-1-Fkp vector: the pCDFDuet-1-Fkp vector is a Fkp gene expression vector in which the restriction endonuclease NdeI and PacI recognition site fragment (small fragment) of pCDFDuet-1 (Wohan vast Biotechnology Co., ltd.) was replaced with Fkp gene (the coding amino acid sequence of which is the L-fucose kinase/GDP-L-fucose pyrophosphorylase of SEQ ID NO. 1) having the nucleotide sequence of SEQ ID NO.10, and the other nucleotide sequence of pCDFDuet-1 was kept unchanged.

Construction of pCDFDuet-1-WbgL-Fkp vector: the pCDFDuet-1-WbgL-fkp vector is an expression vector containing the WbgL gene and the Fkp gene, which is obtained by replacing the fragment (small fragment) between the restriction endonuclease NdeI and PacI recognition site of pCDFDuet-1-WbgL with Fkp gene whose nucleotide sequence is SEQ ID NO.10 (the coding amino acid sequence is L-fucoskinase/GDP-L-fucose pyrophosphorylase of SEQ ID NO. 1).

The constructed recombinant plasmids are pRSFDuet-1-WbgL, pRSFDuet-1-Fkp, pRSFDuet-1-WbgL-Fkp, pETDuet-1-WbgL, pETDuet-1-Fkp, pETDuet-1-WbgL-Fkp, pCDFDuet-1-WbgL, pCDFDuet-1-Fkp and pETDuet-1-WbgL-Fkp respectively.

The constructed vectors were pooled and combined as follows:

Combining: pCDFDuet-1-WbgL-Fkp;

and (2) combining two: pCDFDuet-1-WbgL, pETDuet-1-Fkp;

and (3) combining three: pCDFDuet-1-WbgL, pRSFDuet-1-Fkp;

combination four: pETDuet-1-WbgL-Fkp;

and (5) combining: pETDuet-1-WbgL, pCDFDuet-1-Fkp;

and (3) combining six: pETDuet-1-WbgL, pRSFDuet-1-Fkp;

combination seven: pRSFDuet-1-WbgL-Fkp;

combination eight: pRSFDuet-1-WbgL, pCDFDuet-1-Fkp;

combination nine: pRSFDuet-1-WbgL, pETDuet-1-Fkp.

3. Construction of recombinant strains

The above pCOLADuet-1-WbgL-Fkp-operon, pCOLADuet-1-WbgL-Fkp-pseudoopero, pCOLADuet-1-WbgL-Fkp-monocyclonic and the above recombinant vectors of one to nine combinations were transferred into the strain FBL06-1 of example 1 in order by electric shock transformation (1 mm electric shock cup, 1.8 KV) to obtain 12 recombinant strains, which were named as FBL10 to FBL21, respectively (see Table 4, in particular). The WbgL gene is derived from an alpha-1, 2-fucosyltransferase gene derived from Escherichia coli. The Fkp gene is an L-fucose kinase/GDP-L-fucose pyrophosphorylase gene.

4. Production of 2' -fucosyllactose using E.coli engineering bacteria

4.1 preparation of seed liquid

Recombinant E.coli FBL10 to FBL21 were inoculated into LB liquid medium (containing the corresponding antibiotics) and cultured overnight at 37℃at 200rpm, respectively, to obtain seed solutions.

1mL of the seed solution was added to a 250mL Erlenmeyer flask containing 50mL of fermentation medium, and cultured at 37℃and 200rpm to OD ₆₀₀ =0.6-0.8, IPTG solution, lactose solution and fucose solution were added to the culture system at a final concentration of 0.1mM, lactose concentration of 5g/L and fucose concentration of 5g/L. Incubated at 25℃for 72h, samples were taken at regular time and the yield of 2' -FL was quantitatively determined by HPLC.

The experimental results show that: the expression intensity of the target gene is regulated to balance the expression quantity of different enzymes in a multienzyme system, so that the metabolic burden of thalli is reduced. The expression intensity of the target gene is regulated and controlled in two different modes, and the obtained recombinant strain: FBL10-FBL21. Recombinant bacteria FBL10, FBL11 and FBL12 are obtained by organizing WbgL and Fkp into operon, pseudooperon and monocistronic forms for expression, wherein the 2' -FL shake flask fermentation yield of FBL10 is 3.42g/L at maximum, FBL11 times and FBL12 is lowest.

Optimizing the copy number of the plasmid to obtain the maximum 4.16 g/L2' -FL shake flask fermentation yield of FBL20 in 9 strains; the 2' -FL of FBL18 had the lowest shake flask fermentation yield of only 0.66g/L. The 2' -FL shake flask fermentation yield of the other strains is between 0.80g/L and 2.25 g/L. pRSFDuet-1 high copy number plasmid is selected from FBL19 bacterial strain, wbgL and Fkp are expressed simultaneously, and the 2' -FL shake flask fermentation yield is only 0.80g/L; when Fkp is expressed by using a copy number plasmid in pETDuet-1, and a plasmid for expressing WbgL is unchanged, the shake flask fermentation yield of the strain FBL21 is improved to 0.98g/L, which shows that the reduction of the expression intensity of Fkp can improve the yield of 2' -FL; when pCDFDuet-1 was used to express Fkp, the 2' -FL shake flask fermentation yield of the strain FBL20 was increased to 4.16g/L, which was 5.2 times that of the strain FBL 19. The above results indicate that the expression levels of WbgL and Fkp were regulated, and the yield of strain 2'-FL was further increased (see FIG. 3 and Table 4 for the yields of different engineering strains 2' -FL).

TABLE 2 primers used in plasmid construction

Example 3: increasing the effect of lactose and fucose intake on engineering bacteria to synthesize 2' -fucosyllactose

The availability of engineering bacteria intracellular fucose and lactose has an important influence on the synthesis of 2' -fucosyl lactose.

1. Construction of LacY Gene over-expressing lactose-transporting and FucP Gene recombinant vector transporting fucose

Construction of pRSFDuet-1-WbgL-LacY vector: the pRSFDuet-1-WbgL-LacY vector is a WbgL gene and LacY gene expression vector in which the restriction endonuclease NdeI of pRSFDuet-1-WbgL and the PacI recognition site fragment (small fragment) are replaced with LacY gene whose nucleotide sequence is SEQ ID NO.11 (lactose permease encoding amino acid sequence GenBank: NP-414877.1 (Mar8, 2022)) and the other nucleotide sequence of pRSFDuet-1-WbgL is kept unchanged.

Construction of pRSFDuet-1-WbgL-FucP vector: the pRSFDuet-1-WbgL-FucP vector is a WbgL gene and FucP gene expression vector in which the fragment (small fragment) between the restriction endonuclease NdeI and PacI recognition sites of pRSFDuet-1-WbgL is replaced with a FucP gene whose nucleotide sequence is SEQ ID NO.12 (the encoding amino acid sequence is GenBank: NP-417281.1 (Mar8, 2022)), and the other nucleotide sequences of pRSFDuet-1-WbgL are kept unchanged.

Construction of pRSFDuet-1-WbgL-FucP-LacY vector: pRSFDuet-1-WbgL-FucP-LacY vector is a WbgL gene, fucP gene and LacY gene expression vector obtained by inserting a LacY gene (lactose permease encoding amino acid sequence GenBank: NP-414877.1 (Mar8, 2022)) having a nucleotide sequence of SEQ ID NO.11 into the restriction endonuclease PacI recognition site of pRSFDuet-1-WbgL-FucP and keeping the other nucleotide sequences of pRSFDuet-1-WbgL-FucP unchanged.

The recombinant plasmids constructed were pRSFDuet-1-WbgL-FucP, pRSFDuet-1-WbgL-LacY, pRSFDuet-1-WbgL-FucP-LacY, respectively.

The constructed vectors were pooled and combined as follows:

combining: pRSFDuet-1-WbgL-FucP, pCDFDuet-1-Fkp;

and (2) combining two: pRSFDuet-1-WbgL-LacY, pCDFDuet-1-Fkp;

and (3) combining three: pRSFDuet-1-WbgL-FucP-LacY, pCDFDuet-1-Fkp;

2. construction of recombinant strains

The recombinant vectors of the above combinations one to three were transferred into the strain FBL06-1 in example 1 in sequence using electric shock transformation (1 mm electric shock cup, 1.8 KV) to obtain 3 recombinant strains, which were named FBL22 to FBL24 (see Table 4, in particular).

3. Production of 2' -fucosyllactose using E.coli engineering bacteria

3.1 preparation of seed liquid

Recombinant E.coli FBL22 to FBL24 were inoculated into LB liquid medium (containing the corresponding antibiotics) and cultured overnight at 37℃at 200rpm, respectively, to obtain seed solutions.

3.2 fermentation production of 2' -fucosyllactose Using E.coli engineering bacteria

The experimental results show that: when the FucP gene is over-expressed, the 2' -FL shake flask fermentation yield of the engineering bacterium FBL22 is 5.24g/L; the 2' -FL shake flask fermentation yield of FBL23 and FBL24 was lower than that of FBL22. In addition, the dry cell weight after 72h of shake flask fermentation of FBL23 and FBL24 was significantly lower than that of FBL22, which may be the cause of low 2' -FL production. Studies have shown that when the lactose permease LacY gene is overexpressed in E.coli, considerable toxic effects on E.coli cell growth are produced, and thus the strain growth is significantly inhibited. The above results indicate that the 2'-FL yield of the strain was further increased by overexpressing the FucP gene (see FIG. 4 and Table 4 for the 2' -FL yields of the different engineering strains).

Example 4: effect of outer membrane lipid Synthesis and modification of related Gene knockout on engineering bacteria Synthesis of 2' -fucosyllactose

1. Construction of recombinant E.coli FBL25

Based on recombinant escherichia coli FBL06-1, lpxL, lpxM, lpxP and LpxT genes are knocked out by using a CRISPR-Cas9 gene knockout system, so that recombinant escherichia coli FBL06-1 delta LpxL delta LpxM delta LpxP delta LpxT (namely E.coli BL21 star (DE 3) delta LacZ delta WcaJ delta FucIK delta AraA delta RhaA delta LpxL delta LpxM delta LpxP delta LpxT) is obtained, and the related primer sequences are shown in Table 3.LpxL, lpxM, lpxP and LpxT are genes involved in outer membrane lipid synthesis and modification.

1.1 construction of Strain FBL06-1 DeltaLpxL

The method of reference example 1 knocks out the LpxL gene (nucleotide sequence (CDS) is nucleotide 1118229-1119149 of GenBank: CP001509.3 (16-FEB-2017)), the encoded amino acid sequence is protein of GenBank: ACT42945.1 in recombinant E.coli FBL 06-1. DELTA.LpxL, the primer sequences involved in which the LpxL gene was deleted, were obtained by using CRISPR-Cas9 gene knockout system are shown in Table 3.

1.1.1 construction of the pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-CCAGAGTGTTCTCCGCCACT-3'), resulting in a pTargetF plasmid with sgRNA targeting the LpxL gene (nucleotide sequence (CDS) is GenBank: CP001509.3 (16-FEB-2017) nucleotide 1118229-1119149, the coding amino acid sequence is GenBank: ACT42945.1 protein (lauroyl carrier protein (ACP) -dependent acyltransferase)), designated pTargetF-LpxL. LacZ-sg-F and LacZ-sg-R were replaced with LpxL-sg-F and LpxL-sg-R (Table 3), and the rest was the same as in example 1 at 1.1.1.

1.1.2 preparation of donor DNA fragments. The upstream homology arm of the LpxL gene was PCR amplified using the E.coli BL21 star (DE 3) genome as a template, lpxL-up-F (5 '-TTTCGGCCTTTACGGTCGG-3') and LpxL-up-R (5 '-GGAAGTGATCGGCATGGAACATGATGGCACCAGAGCAGTATATG-3'), and the downstream homology arm of the LpxL gene was PCR amplified using LpxL-down-F (5 '-GTTCCATGCCGATCACTTCC-3') and LpxL-down-R (5 '-ACATCCACACATTTTACGCTACATT-3'), and the fragments were recovered by gel. And then, taking an upstream homology arm of the LpxL gene and a downstream homology arm of the LpxL gene as templates, obtaining a complete donor DNA fragment for carrying out homologous recombination with the LpxL gene by overlapping PCR by adopting LpxL-up-F and LpxL-down-R, and recycling the DNA fragment.

1.1.3 the procedure is as in example 1, 1.1.3.

1.1.4 100ng of the pTargetF-LpxL plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24 hours, and PCR (primers LpxL-F and LpxL-R) was performed to verify the effect of LpxL gene knockout. The result shows that the positive cloning bacteria obtain 1000bp PCR product and the colibacillus FBL06-1 obtains 1500bp PCR product.

1.1.5 pTargetF-LpxL plasmid and pCas plasmid in positive clone were removed as in 1.1.5 of example 1 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL (recombinant E.coli BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL or strain FBL06-1 ΔLpxL). The LpxL gene was deleted from nucleotide 344 to nucleotide 843 (GenBank: CP001509.3, position 1118229 was designated as position 1 of the LpxL gene) compared to E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔRhaA ΔLpxL, thereby causing a frameshift knockout to knock out the LpxL gene.

1.2 construction of strain FBL06-1 DeltaLpxLDeltaLpxM

The method of reference 1.1 knocks out the LpxM gene (nucleotide sequence (CDS) is nucleotide 1884946-1885917 of GenBank: CP001509.3 (16-FEB-2017)) in recombinant E.coli FBL 06-1. DELTA.LpxL by using a CRISPR-Cas9 gene knock-out system, and the encoded amino acid sequence is protein (myristoyl carrier protein (ACP) -dependent acylase) of GenBank: ACT 43680.1), thereby obtaining recombinant E.coli FBL 06-1. DELTA.LpxL.LpxM deleted in the LpxM gene. The primer sequences involved are shown in Table 3.

1.2.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-ACAGATTGATTAACGCACGA-3'), resulting in a pTargetF plasmid with sgRNA targeting the LpxM gene (nucleotide sequence (CDS) is GenBank: CP001509.3 (16-FEB-2017) nucleotide 1884946-1885917, the encoded amino acid sequence is GenBank: ACT43680.1 protein), named pTargetF-LpxM. LacZ-sg-F and LacZ-sg-R were replaced with LpxM-sg-F and LpxM-sg-R (Table 3), and the rest was the same as in example 1 at 1.1.1.

1.2.2 preparation of donor DNA fragments. The upstream homology arm of the LpxM gene was PCR amplified using the E.coli BL21 star (DE 3) genome as a template, lpxM-up-F (5 '-GGTGACGGTGAAGTGGTGGTT-3') and LpxM-up-R (5 '-CATCCATCGGTGGGCGCATTTATCAAACTCAGGAATGTATTCGCTATTATTTTTTTTC-3'), and the downstream homology arm of the LpxM gene was PCR amplified using LpxM-down-F (5 '-TGCGCCCACCGATGGATG-3') and LpxM-down-R (5 '-GTGGCGTTACGCCGGTG-3'), and the fragments were recovered by gel. And then, taking an upstream homology arm of the LpxM gene and a downstream homology arm of the LpxM gene as templates, obtaining a complete donor DNA fragment for carrying out homologous recombination with the LpxM gene by overlapping PCR by adopting LpxM-up-F and LpxM-down-R, and recycling the DNA fragment.

1.2.3 the procedure is as in example 1, 1.1.3.

1.2.4 100ng of the pTargetF-LpxM plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24 hours, and PCR (primers LpxM-F and LpxM-R) was performed to verify the effect of LpxM gene knockout. The result shows that the positive cloning bacteria obtain 1000bp PCR product, the Escherichia coli FBL06-1 obtains 1735bp PCR product, and the Escherichia coli FBL06-1 delta LpxL obtains 1735bp PCR product.

1.2.5 pTargetF-LpxM plasmid and pCas plasmid in positive clone were removed as in 1.1.5 of example 1 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM (recombinant E.coli BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM or strain FBL06-1 ΔLpxL ΔLpxM). The LpxM gene is deleted from nucleotide 49-784 (1 st position of LpxM gene in GenBank: CP 001509.3) compared with E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL, so that the encoded protein is deleted from amino acids 17-262 in GenBank: ACT 43680.1.

1.3 construction of the strain FBL 06-1. DELTA.LpxLDELTA.LpxM DELTA.LpxP

The method of reference 1.1 knocks out the LpxP gene (nucleotide sequence (CDS) is nucleotide 2373530-2374450 of GenBank: CP001509.3 (16-FEB-2017)) in recombinant E.coli FBL 06-1. DELTA. LpxL. DELTA. LpxM by using CRISPR-Cas9 gene knock-out system, and the encoded amino acid sequence is protein (palmitoyl Acyl Carrier Protein (ACP) -dependent acyl transferase) of GenBank: ACT 44109.1), to obtain recombinant E.coli FBL 06-1. DELTA. LpxL. DELTA. LpxM.DELTA.LpxP deleted in LpxP gene. The primer sequences involved are shown in Table 3.

1.3.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-ATCGGCAGAAATAATCTGCG-3'), to obtain pTargetF plasmid with sgRNA targeting LpxP gene (GenBank: CP001509.3 (16-FEB-2017) nucleotide 2373530-2374450, encoded amino acid sequence is GenBank: ACT44109.1 protein), named pTargetF-LpxP. LacZ-sg-F and LacZ-sg-R were replaced with LpxP-sg-F and LpxP-sg-R (Table 3), and the rest was the same as in example 1 at 1.1.1.

1.3.2 preparation of donor DNA fragments. The upstream homology arm of the LpxP gene was amplified by PCR using the E.coli BL21 star (DE 3) genome as a template, lpxP-up-F (5 '-TCAACCTCCTGCCGCGCTAAA-3') and LpxP-up-R (5 '-GCGGCTTGATTTTCATCTGTCGGGTAATGGTCGCGCCATTGCG-3'), and the downstream homology arm of the LpxP gene was amplified by PCR using LpxP-down-F (5 '-TACCCGACAGATGAAAATCAAGCCGC-3') and LpxP-down-R (5 '-TCACTTTACATCTCTCCGCGTAATTACT-3'), and the fragments were recovered by gel. And then, using an upstream homology arm of the LpxP gene and a downstream homology arm of the LpxP gene as templates, obtaining a complete donor DNA fragment for carrying out homologous recombination with the LpxP gene by overlapping PCR with LpxP-up-F and LpxP-down-R, and recovering the DNA fragment by gel.

1.3.3 the procedure is as in example 1, 1.3.

1.3.4 100ng of pTargetF-LpxP plasmid of step 1.1.1 and 400ng of donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), cultured at 30℃for 24 hours, and PCR (primers LpxP-F and LpxP-R) was performed to verify the effect of LpxP gene knockout. The result shows that the positive cloning bacteria obtain a PCR product of 1000bp, the escherichia coli FBL06-1 obtains a PCR product of 1629bp, and the escherichia coli FBL06-1 delta LpxL delta LpxM obtains a PCR product of 1629 bp.

1.3.5 pTargetF-LpxP plasmid and pCas plasmid in positive clone were removed as in 1.1.5 of example 1 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxP (recombinant E.coli BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxP or strain FBL06-1 ΔLpxL ΔLpxM ΔLpxP). The LpxP gene is deleted from nucleotide 152 to 780 (GenBank: CP001509.3, position 2373530 is designated as LpxP gene 1) compared with E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM, so that the encoded protein is deleted from amino acids 51 to 260 of ACT44109.1, thereby causing a frame shift to knock out the LpxP gene.

1.4 construction of the strain FBL 06-1. DELTA.LpxLDELTA.LpxM DELTA.LpxP DELTA.LpxT

The method of reference 1.1 knocks out the LpxT gene (nucleotide sequence (CDS) is nucleotide 2163703-2164416 of GenBank: CP001509.3 (16-FEB-2017)) in recombinant E.coli FBL 06-1. DELTA.LpxL. DELTA.LpxM. DELTA.LpxP by using a CRISPR-Cas9 gene knock-out system, and the encoded amino acid sequence is the protein of GenBank: ACT43927.1 (lipid A1-diphosphate synthase/undecylenic pyrophosphate: lipid A1-phosphate phosphotransferase)), to obtain the recombinant E.coli L06-1. DELTA.LpxL. DELTA.LpxM.LpxP. DELTA.LpxT FBT deleted. The primer sequences involved are shown in Table 3.

1.4.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-ACCAGGAAAGAAACAGCGCC-3'), resulting in a pTargetF plasmid with sgRNA targeting LpxT gene (GenBank: CP001509.3 (16-FEB-2017) nucleotide 2163703-2164416, encoded amino acid sequence is GenBank: ACT43927.1 protein), named pTargetF-LpxT. LacZ-sg-F and LacZ-sg-R were replaced with LpxT-sg-F and LpxT-sg-R (Table 3), and the rest was the same as in example 1 at 1.1.1.

1.4.2 preparation of donor DNA fragments. The upstream homology arm of the LpxT gene was amplified by PCR using the E.coli BL21 star (DE 3) genome as a template, lpxT-up-F (5 '-GGATTTGCCGCGTCGCAA-3') and LpxT-up-R (5 '-GGAAATGTTTGTTTTTTCCCGGTAGTGATTTAGGCCGACAATATTCAACAACACTATTTG-3'), and the downstream homology arm of the LpxT gene was amplified by PCR using LpxT-down-F (5 '-AAATCACTACCGGGAAAAAACAAACATTTCC-3') and LpxT-down-R (5 '-GGAATCCCGCGCAAGATATATCTCAAAA-3'), and the fragments were recovered by gel. And then, taking an upstream homology arm of the LpxT gene and a downstream homology arm of the LpxT gene as templates, obtaining a complete donor DNA fragment for carrying out homologous recombination with the LpxT gene by overlapping PCR by adopting LpxT-up-F and LpxT-down-R, and recycling the DNA fragment.

1.4.3 the procedure is as in example 1, 1.1.3.

1.4.4 100ng of the pTargetF-LpxT plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), incubated at 30℃for 24 hours, and PCR (primers LpxT-F and LpxT-R) was used to verify the LpxP gene knockout effect. The result shows that the positive cloning bacteria obtain a PCR product of 1000bp, the escherichia coli FBL06-1 obtains a PCR product of 1625bp, and the escherichia coli FBL06-1 delta LpxL delta LpxM delta LpxP obtains a PCR product of 1625 bp.

1.4.5 pTargetF-LpxT plasmid and pCas plasmid in positive clone were removed as in 1.1.5 of example 1 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxP ΔLpxT (recombinant E.coli BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxT or strain FBL06-1 ΔLpxL ΔLpxM ΔLpxP ΔLpxT). The LpxT gene was deleted at nucleotide 48-672 (1 st position of LpxT gene designated as GenBank: CP 001509.3) by comparison with E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxT and by deletion of amino acids 16-224 of GenBank: ACT43927.1, the protein encoded by the gene was knocked out. The strain FBL 06-1. DELTA.LpxLΔLpxMΔLpxP. DELTA.LpxT was obtained. The above gene knockout results are shown in FIG. 5.

2. Construction of recombinant strains

The plasmids pRSFDuet-1-WbgL-FucP and pCDFDuet-1-Fkp were co-transformed into recombinant E.coli FBL 06-1. DELTA.LpxL. DELTA.LpxM. DELTA.LpxP. DELTA.LpxT by electric shock transformation to give strain FBL25.

3. Production of 2' -fucosyllactose using E.coli engineering bacteria

3.1 preparation of seed liquid

Coli FBL25 was inoculated into LB liquid medium (containing the corresponding antibiotics) and cultured overnight at 37 ℃ at 200rpm to obtain a seed solution.

The results show that the fermentation yield of the strain FBL25 from which the outer membrane lipid synthesis and modification related genes (LpxL, lpxM, lpxP and LpxT) are knocked out is 5.83g/L, and the yield is obviously improved (the 2' -FL yields of different engineering strains are shown in FIG. 5 and Table 4).

Example 5: effect of outer membrane lipopolysaccharide Synthesis, modification and transport related Gene knockout on engineering Strain Synthesis of 2' -fucosyllactose

The reasonable modification of the cell membrane of the escherichia coli can not only reduce the consumption of nutrient substances, but also improve the growth speed of the escherichia coli and increase the amount of auxiliary factors and the yield of some chemicals.

1. Construction of recombinant E.coli FBL26, FBL27

Based on recombinant E.coli FBL06-1 ΔLpxL ΔLpxM ΔLpxT, waaF, waaL, waaV, waaW, waaY, waaT, waaO, waaP, waaG and WaaQ genes were knocked out using CRISPR-Cas9 gene knockout system, i.e., recombinant E.coli FBL06-1 ΔLpxL ΔLpxM ΔLpxT ΔWaaF and FBL06-1 ΔLpxM ΔLpxT ΔWaaF ΔWaaL ΔWaaW ΔWaaY ΔWaaT ΔWaaP ΔWaaG ΔWaaQ (i.e., E.coli BL21 r (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaL ΔLpxM ΔLpxT ΔWaaF and E.i BL21 r (3) ΔLacaZ ΔWaaW ΔWaaL) were related to the WapWapWapWapΔWaaL ΔWaaL WaaL by WapWaaL ΔWaaL. WaaF, waaL, waaV, waaW, waaY, waaT, waaO, waaP, waaG and WaaQ are genes involved in the synthesis and modification of outer membrane lipopolysaccharide.

1.1 construction of strain FBL06-1 DeltaLpxLDeltaLpxM DeltaLpxP DeltaLpxT DeltaWaaF

The method of reference example 1 knocks out the WaaF gene (nucleotide sequence (CDS) is nucleotide 3662908-3663954 of GenBank: CP001509.3 (16-FEB-2017), and the encoded amino acid sequence is protein (ADP-heptose: LPS-heptyl transferase II) of recombinant E.coli FBL 06-1. Delta. LpxL. Delta. LpxM. Delta. LpxP. Delta. LpxT. Delta. WaaF) using a CRISPR-Cas9 gene knockout system to obtain recombinant E.coli FBL 06-1. Delta. LpxL. Delta. LpxM. Delta. LpxT. WaaF deleted. The primer sequences involved are shown in Table 3.

1.1.1 construction of the pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-CTGCTGCTCGGTATTCAAAG-3'), resulting in a pTargetF plasmid with sgRNA targeting LpxT gene (GenBank: CP001509.3 (16-FEB-2017) nucleotide 3662908-3663954, encoding the protein whose amino acid sequence is GenBank: ACT 45276.1), named pTargetF-WaaF. LacZ-sg-F and LacZ-sg-R were replaced with WaaF-sg-F and WaaF-sg-R (Table 3), and the procedure was the same as in example 1 at 1.1.1.

1.1.2 preparation of donor DNA fragments. The upstream homology arm of the WaaF gene was PCR amplified using the E.coli BL21 star (DE 3) genome as a template, waaF-up-F (5 '-ACTCGCAGATTGTTGGCTTCC-3') and WaaF-up-R (5 '-GGTGATAACCCTCCGCAGCGTCAATCTTGTGCGGTACGCATCAC-3'), the downstream homology arm of the WaaF gene was amplified using WaaF-down-F (5 '-TGACGCTGCGGAGGGTTATCACC-3') and WaaF-down-R (5 '-ACAATAGCGCGTTGAGTTCTTCC-3'), and the fragments were recovered by PCR. And then, by taking an upstream homology arm of the WaaF gene and a downstream homology arm of the WaaF gene as templates and adopting WaaF-up-F and WaaF-down-R, obtaining a complete donor DNA fragment for carrying out homologous recombination with the WaaF gene through overlap PCR, and recycling the DNA fragment by gel.

1.1.3 the procedure is as in example 1, 1.1.3.

1.1.4 100ng of the pTargetF-LpxT plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), incubated at 30℃for 24 hours, and PCR (primers WaaF-F and WaaF-R) was performed to verify the LpxP gene knockout effect. The result shows that the positive cloning bacteria obtain 1000bp PCR product, the Escherichia coli FBL06-1 obtains 1500bp PCR product, and the Escherichia coli FBL06-1 delta LpxL delta LpxM delta LpxP delta LpxT obtains 1500bp PCR product.

1.1.5 pTargetF-WaaF plasmid and pCas plasmid in positive clone were removed according to the method of 1.1.5 in example 1 to obtain E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpLpxL ΔLpxM ΔLpxPΔLpxT ΔWaaF (recombinant E.coli BL21 ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxT ΔWaaF or strain FBL06-1 ΔLpxL ΔLpxPΔLpxT ΔWaaF). Coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxT ΔWaaF the WaaF gene was deleted for nucleotides 450-950 (indicated as position 1 of the WaaF gene in GenBank: CP001509.3 3662908) compared to E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxT, thereby causing a frameshift deletion to knock out the WaaF gene.

1.2 construction of strain FBL06-1 ΔLpxL ΔLpxM ΔLpxP ΔLpxT ΔWaaF ΔWaaL ΔWaaV ΔWaaW ΔWaaY ΔWaaT ΔWaaO ΔWaaP ΔWaaG ΔWaaQ

The method of reference 1.1 knocks out the recombinant escherichia coli FBL06-1 Δlpxl ΔlpxmΔlpxΔlpxtΔlpxtΔwaaf waawwaaywaawawawaawaawaagwaaq gene cluster (abbreviated as WaaLQ) using the CRISPR-Cas9 gene knock-out system to obtain a recombinant escherichia coli FBL06-1 ΔlpxlΔlpxmΔlpxΔlpΔwaaq gene cluster deleted of the wanaawwaaywaaywaatwaawaawaagwaaq gene cluster. The primer sequences involved are shown in Table 3. Wherein the WaaL gene sequence is GenBank:CP001509.3 (16-FEB-2017) nucleotide 3665008-3666261, and the coded amino acid sequence is GenBank:ACT45278.1 protein (lipid A core-surface polymer ligase)); the WaaV gene sequence is GenBank: CP001509.3 (16-FEB-2017) nucleotide 3666307-3667290, and the coded amino acid sequence is GenBank: ACT45279.1 protein (beta-1, 3-glucosyltransferase)); the WaaW gene sequence is GenBank: CP001509.3 (16-FEB-2017) nucleotide 3667375-3668400, and the coded amino acid sequence is GenBank: ACT45280.1 protein (UDP-galactose (galactosyl) LPS alpha-1, 2-galactosyltransferase)); the WaaY gene sequence is GenBank: CP001509.3 (16-FEB-2017) nucleotide 3668426-3668974, and the coded amino acid sequence is GenBank: ACT45281.1 protein (participating in the phosphorylation activity of core oligosaccharide heptose)); the WaaT gene sequence IS GenBank: CP001509.3 (16-FEB-2017) nucleotide 3669128-3670800, and the coding amino acid sequences are GenBank: ACT45282.1 and GenBank: ACT45283.1 proteins (IS 1 protein InsA/InsB activity)); the WaaO gene sequence is GenBank: CP001509.3 (16-FEB-2017) nucleotide 3670916-3671932, and the coded amino acid sequence is GenBank: ACT45284.1 protein (UDP-glucose (glucosyl) LPSalpha-1, 3-glucosyltransferase)); the WaaP gene sequence is GenBank: CP001509.3 (16-FEB-2017) nucleotide 3671948-3672745, and the coded amino acid sequence is GenBank: ACT45285.2 protein (kinase for phosphorylating lipopolysaccharide core heptose)); the WaaG gene sequence is GenBank:CP001509.3 (16-FEB-2017) nucleotide 3672738-3673862, and the coded amino acid sequence is GenBank:ACT45286.1 protein (UDP-glucose (heptyloxy) lipopolysaccharide alpha-1, 3-glucosyltransferase/lipopolysaccharide core biosynthesis protein/lipopolysaccharide glucosyltransferase I)); the WaaQ gene sequence is GenBank: CP001509.3 (16-FEB-2017) nucleotide 3673859-3674917, and the coded amino acid sequence is GenBank: ACT45287.1 protein (lipopolysaccharide core biosynthesis protein activity).

1.2.1 construction of pTargetF plasmid containing sgRNA. The N20 sequence in 1.1.1 was replaced with (5 '-ACCAGGAAAGAAACAGCGC-3'), resulting in a pTargetF plasmid with sgRNA targeting the WaaLQ gene, designated pTargetF-WaaLQ. LacZ-sg-F and LacZ-sg-R were replaced with WaaLQ-sg-F and WaaLQ-sg-R (Table 3), and the rest was the same as in example 1 at 1.1.1.

1.2.2 preparation of donor DNA fragments. The upstream homology arm of the WaaLQ gene was PCR amplified using E.coli BL21 star (DE 3) genome as a template, waaLQ-up-F (5 '-GGGATTCTATGTATTTAGCTGTGGC-3') and WaaLQ-up-R (5 '-CGTTTTTCCAGTGATTGTCCTATTTCGATTTTTGCGTCAGGGTAAT-3'), the downstream homology arm of the WaaL-Q gene was amplified by PCR using WaaLQ-down-F (5 '-AATAGGACAATCACTGGAAAAACG-3') and WaaLQ-down-R (5 '-TCAATTGACAGCAAATGCTGTTAAG-3'), and the above fragments were recovered by gel. And then, by taking an upstream homology arm of the WaaLQ gene and a downstream homology arm of the WaaLQ gene as templates and adopting WaaLQ-up-F and WaaLQ-down-R, obtaining complete donor DNA fragments for carrying out homologous recombination with the WaaLQ gene through overlap PCR, and recycling the DNA fragments.

1.2.3 the procedure is as in example 1, 1.1.3.

1.2.4 100ng of the pTargetF-WaaLQ plasmid of step 1.1.1 and 400ng of the donor DNA fragment of 1.2.2 were electrotransferred to recombinant E.coli BL21 star (DE 3)/pCas competent cells prepared in step 1.1.3, plated on LB plates (50. Mu.g/mL kanamycin and 50. Mu.g/mL spectinomycin), incubated at 30℃for 24h, and PCR (primers WaaLQ-F and WaaLQ-R) was performed to verify the WaaLQ gene knockout effect. The result shows that the positive cloning bacteria obtain 1000bp PCR product, the Escherichia coli FBL06-1 obtains 10430bp PCR product, and the Escherichia coli FBL06-1 delta LpxL delta LpxM delta LpxP delta LpxT delta WaaF obtains 10430bp PCR product.

1.2.5 removal of the pTargetF-WaaLQ plasmid and the pCas plasmid from the positive clones as described in example 1 at 1.1.5 gave E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxP ΔLpxT ΔWaaF ΔWaaL ΔWaaV ΔWaaW ΔWaaY ΔWaaT ΔWaaO ΔWaaP ΔWaaG ΔWaaQ short recombination coli BL21 ΔLacZ ΔWcaJ ΔFucaxL ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxP ΔLpxT ΔWaaF ΔWaaL ΔWaaV ΔWaaW ΔWaaY ΔWaaO ΔWaaP ΔWaaG ΔWaaQ or strain FBL06-1 ΔLpxL ΔLpxM ΔLpxT ΔWaaF ΔWaaL ΔWaaV ΔWaaY ΔWaaT ΔWaaO ΔWaaP ΔWaaQ. Coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxM ΔLpxT ΔWaaF ΔWaaL ΔWaaV ΔWaaY ΔWaaT ΔWaaO ΔWaaP ΔWaaG ΔWaaQ compared with E.coli BL21 (DE 3) ΔLacZ ΔWcaJ ΔFucIK ΔAraA ΔRhaA ΔLpxL ΔLpxT ΔWaaF, waaL deleted nucleotide 1-890 (GenBank: CP001509.3 position 3665008 was designated as position 1 of the WaaL gene), resulting in a frameshift deletion to knock out the WaaL gene; waaV lacks 1 st-984 th nucleotide (GenBank: CP001509.3 3666307 st 1 st nucleotide of WaaV gene) and the encoded protein lacks 1 st-327 th amino acid of GenBank: ACT45279.1, so that the WaaV gene is knocked out; the WaaW is deleted from 1 st to 1026 th nucleotides (GenBank: CP001509.3, 3667375 st is marked as 1 st of the WaaW gene) so as to cause the deletion of the amino acid and the WaaW gene to be knocked out, so that the encoded protein is deleted from 1 st to 341 st amino acid of GenBank: ACT45280.1 so as to knock out the WaaW gene; waaY lacks 1 st-549 th nucleotide (GenBank: CP001509.3, 3668426 st 1 st of WaaY gene), so that the encoded protein lacks 1 st-182 nd amino acids of GenBank: ACT45281.1, thereby knocking out the WaaY gene; waaT is deleted from nucleotides 1 to 1673 (GenBank: CP001509.3, 3669128 is taken as 1 st of the WaaT gene), thereby causing a frameshift deletion to knock out the WaaT gene; waaO lacks 1 st-1017 th nucleotide (GenBank: CP001509.3 3670916 st 1 st of WaaO gene), so that the encoded protein lacks 1 st-228 st amino acid of GenBank: ACT45284.1, thereby knocking out the WaaO gene; waaP lacks 1 st-798 th nucleotide (GenBank: CP001509.3 3671948 st bit of WaaP gene 1 st bit), so that the encoded protein lacks all amino acids of GenBank: ACT45285.2, thereby knocking out the WaaP gene; waaG lacks 1 st-1125 th nucleotide (GenBank: CP001509.3 3672738 st bit of WaaG gene 1 st bit), so that the encoded protein lacks all amino acids of GenBank: ACT45286.1, thereby knocking out the WaaG gene; waaQ has been deleted from nucleotide 117 to nucleotide 1059 (GenBank: CP001509.3, position 3673859 is designated as position 1 of the WaaQ gene), thereby causing a frameshift deletion to knock out the WaaQ gene. The strain FBL 06-1. DELTA.LpxL. DELTA.LpxM. DELTA.LpxT. DELTA.WaaFDELTA.WaaL. DELTA.WaaV. DELTA.WaaW. DELTA.WaaY. DELTA.WaaT. WaaO. DELTA.WaaP. WaaG. DELTA.WaaQ was obtained. The above gene knockout results are shown in FIG. 6

2. Construction of recombinant strains

The plasmids pRSFDuet-1-WbgL-FucP and pCDFDuet-1-Fkp were co-transformed into recombinant E.coli FBL 06-1. DELTA.LpxL. DELTA.LpxM. DELTA.LpxT. DELTA.WaaF and FBL 06-1. DELTA.LpxL. DELTA.LpxM. DELTA.LpxP. DELTA.LpxT. DELTA.WaaF. DELTA.WaaL. DELTA.WaaV. DELTA.WaaW. DELTA.WaaY. DELTA.WaaT. DELTA.WaaO. DELTA.WaaP. DELTA.WaaG. WAaQ to give the strains FBL26 and FBL27.

3. Production of 2' -fucosyllactose using E.coli engineering bacteria

3.1 preparation of seed liquid

Coli FBL26 and FBL27 were inoculated in LB liquid medium (containing the corresponding antibiotics) and cultured overnight at 37 ℃ at 200rpm to obtain seed solution.

In order to understand the influence of cell membrane engineering on the strain, the growth rate of the engineering bacteria after transformation and the utilization rate of fucose and lactose are measured, and HPLC is adopted for quantitative analysis of fucose and lactose.

Determination of strain growth rate: the activated strain was picked up and the OD at different time points was measured on 96-well plates containing LB ₆₀₀ Values.

The results show that the 2' -fucosyllactose shake flask fermentation yield of the strains FBL26 and FBL27 obtained by knocking out the genes related to outer membrane lipopolysaccharide synthesis, modification and transportation is 6.84g/L and 7.28g/L, and the yield is obviously improved. The effect of knocking out the growth of the outer membrane lipopolysaccharide synthesis, modification and transport related gene strains was less pronounced (see FIG. 6 and Table 4 for the 2' -FL yields of the different engineering strains).

TABLE 3 primers for cell membrane defect engineering experiments

Example 6: fed-batch fermentation synthesis of 2' -fucosyllactose in 5L fermentation tank

1. Batch fed-batch fermentation experiments with strain FBL27 in 5L fermentors

1.1 preparation of seed liquid

The stored strain of FBL27 was removed from the-80℃refrigerator, streaked onto LB plates (containing the corresponding antibiotics), single colonies were picked up in 5mLLB (containing the corresponding antibiotics), cultured overnight at 37℃at 220rpm, and 3mL of primary seed solution in 150mL of feed medium. Culturing at 37℃and 220rpm for 16 hours, and obtaining a seed solution.

1.2 fermentation production of 2' -fucosyllactose Using E.coli FBL27

The seed solution was added to a 5L fermenter containing 1.35L of feed medium. To improve the growth of the strain, the availability of NADPH in the tricarboxylic acid cycle was increased, and sodium thiosulfate was fed to the feed medium to a final concentration of 3.2g/L. Culturing at 37deg.C and 800rpm to OD ₆₀₀ 20, cooling to 25deg.C, adding IPTG to a final concentration of 0.1mM, adding lactose to a final concentration of 10g/L, addingFucose was introduced to give a final concentration of 10g/L. Dissolved oxygen was set at 30%, cultured by a strategy of dissolved oxygen feed linkage, maintained at pH 6.80, sampled every 5 hours, and assayed for 2' -fucosyllactose, fucose, lactose and glycerol content by HPLC method, and cell dry weight by absorbance conversion method (cell dry weight=0.36X OD ₆₀₀ )。

And (5) displaying fermentation results. After 48h fermentation the 2' -fucosyllactose yield reached 55g/L and its dry cell weight reached 43g/L (FIG. 7). When the FBL27 strain ferments to produce 2' -FL, the conversion rate of fucose is 0.78mol 2' -FL/mol fucose, the conversion rate of lactose is 0.85mol 2' -FL/mol lactose, and the conversion rate of the two substrates is obviously improved compared with that of the FBL24 strain, so that the method has great application potential.

TABLE 4 details of engineering bacteria and 2' -FL yields (please supplement corresponding data for E.coli BL21 star (DE 3))

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. Recombinant E.coli, characterized in that it is A5), A4), A3), A2) or A1) described below:

2. The recombinant escherichia coli of claim 1, wherein:

and/or, the fucose transporter is derived from escherichia coli;

and/or, the lactose permease is derived from escherichia coli.

3. The recombinant escherichia coli of claim 1 or 2, wherein the outer membrane lipid synthesis and modification-related gene is at least one of an LpxL gene, an LpxM gene, an LpxP gene, and an LpxT gene;

4. A recombinant escherichia coli as claimed in any one of claims 1 to 3 wherein:

the beta-galactosidase is any one of the following proteins:

h1 Amino acid sequence is a protein shown in ACT42197.1 of GenBank;

j1 Amino acid sequence is a protein shown in ACT43804.1 of GenBank;

and/or, the fucose isomerase is any one of the following proteins:

k1 Amino acid sequence is a protein shown in ACT44469.1 of GenBank;

and/or, the fucoidan is any one of the following proteins:

L1) amino acid sequence is a protein shown in GenBank ACT 44470.2;

and/or, the arabinose isomerase is any one of the following proteins:

m1) the amino acid sequence is a protein shown in GenBank ACT 41965.1;

and/or the rhamnose isomerase is any one of the following proteins:

n1) the amino acid sequence is a protein shown in GenBank ACT 45582.2;

o1) the amino acid sequence is a protein shown in the sequence SEQ ID NO. 1;

p1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 2;

q1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 3;

r1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 4;

S1) the amino acid sequence is a protein shown as a sequence SEQ ID NO. 5;

and/or lactose permease is any one of the following proteins:

t1) the amino acid sequence is a protein shown in GenBank NP-414877.1;

and/or the fucose transporter is any one of the following proteins:

u1) the amino acid sequence is a protein with a sequence shown as GenBank NP-417281.1;

and/or, the protein encoded by the LpxL gene is any one of the following:

v1) the amino acid sequence is a protein shown in GenBank ACT 42945.1;

and/or, the protein encoded by the LpxM gene is any one of the following:

w1) is a protein with GenBank as ACT 43680.1;

and/or the protein encoded by the LpxP gene is any one of the following:

X1) the amino acid sequence is a protein shown in GenBank ACT 44109.1;

and/or, the protein encoded by the LpxT gene is any one of the following:

y1) the amino acid sequence is a protein shown in GenBank ACT 43927.1;

and/or, the WaaF gene encodes a protein as follows:

z1) the amino acid sequence is a protein shown in GenBank ACT 45276.1;

and/or, the WaaL gene encodes a protein as follows:

z1) the amino acid sequence is a protein shown in GenBank ACT 45278.1;

and/or, the WaaV gene encodes a protein as follows:

t1) the amino acid sequence is a protein shown in GenBank ACT 45279.1;

And/or, the WaaW gene encodes any one of the following proteins:

n1) the amino acid sequence is a protein shown in GenBank ACT 45280.1;

and/or the protein encoded by the WaaY gene is any one of the following:

o1) the amino acid sequence is a protein shown in GenBank ACT 45281.1;

and/or, the WaaT gene encodes a protein as follows:

l1) the amino acid sequence is a protein shown in GenBank ACT45282.1 and GenBank ACT45283.1 (16-FEB-2017);

and/or, the WaaO gene encodes a protein as follows:

r1) amino acid sequence is protein shown in GenBank ACT 45284.1;

and/or, the WaaP gene encodes a protein as follows:

f1 Amino acid sequence is a protein shown in ACT45285.2 of GenBank;

and/or, the WaaG gene encodes a protein as follows:

g1 Amino acid sequence is a protein shown in ACT45286.1 of GenBank;

and/or, the WaaQ gene encodes a protein as follows:

h1 Amino acid sequence is a protein shown in ACT45287.1 of GenBank;

5. The recombinant escherichia coli of any one of claims 1-4, wherein the recombinant escherichia coli comprises a combination of plasmids of different copy numbers for regulating expression of the α -1, 2-fucosyltransferase gene and the L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, the plasmids being pcdfdur-1, petdiet-1, or prsfpdur-1;

6. Recombinant E.coli according to claims 1 to 5, characterized in that it is constructed according to the method of claim 7 or 8.

7. A method of constructing recombinant escherichia coli, said method comprising performing any one of the following procedures on a recipient escherichia coli:

c1 Introducing the α -1, 2-fucosyltransferase gene and the L-fucokinase/GDP-L-fucoidanase gene of any one of claims 1 to 5 into a recipient escherichia coli to obtain the recombinant escherichia coli of A1) of claims 1 to 5;

8. The method of claim 7, wherein the α -1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene are introduced into the recipient escherichia coli by a recombinant plasmid expressing the α -1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, the recombinant plasmid regulating expression of the α -1, 2-fucosyltransferase gene and L-fucose kinase/GDP-L-fucose pyrophosphorylase gene, the recombinant plasmid being pcdfdur-1, petdout-1 or prsduet-1;

And/or the number of the groups of groups,

9. Use of any of the following in the preparation of 2 '-fucosyllactose or in the construction of a recombinant E.coli producing 2' -fucosyllactose;

d1 A recombinant E.coli according to any one of claims 1 to 6 or a method according to claim 7 or 8;

d2 A nucleic acid molecule that is any one of:

d21 A G1 gene according to any one of claims 1 to 5;

d22 A G1 gene according to any one of claims 1 to 5 and a G2 gene according to any one of claims 1 to 5;

d3 D2) an expression cassette comprising said nucleic acid molecule;

e11 A K1 gene according to any one of claims 1 to 5;

e12 A K1 gene according to any one of claims 1 to 5 and said outer membrane lipid synthesis and modification related gene;

e13 A K1 gene, said outer membrane lipid synthesis and modification related gene, and knock-out said outer membrane lipopolysaccharide synthesis, modification and transport related gene as defined in any one of claims 1 to 5;

e2 An expression cassette comprising E1) the nucleic acid composition;

10. A method for producing 2 '-fucosyllactose, characterized in that the method comprises culturing the recombinant escherichia coli according to any one of claims 1-6 to obtain a fermentation product, and obtaining 2' -fucosyllactose from the fermentation product.