CN112852691B - Recombinant escherichia coli producing MK-7 and construction method and application thereof - Google Patents

Recombinant escherichia coli producing MK-7 and construction method and application thereof Download PDF

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CN112852691B
CN112852691B CN201911210070.9A CN201911210070A CN112852691B CN 112852691 B CN112852691 B CN 112852691B CN 201911210070 A CN201911210070 A CN 201911210070A CN 112852691 B CN112852691 B CN 112852691B
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林白雪
高全秀
陶勇
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Institute of Microbiology of CAS
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Abstract

The invention discloses recombinant escherichia coli for producing MK-7 and a construction method and application thereof. The invention firstly discloses a construction method of recombinant escherichia coli, which comprises the steps of introducing genes related to MK-7 synthesis and/or 1, 2-diacylglycerol 3-glucosyltransferase genes into receptor escherichia coli to obtain recombinant escherichia coli; the acceptor escherichia coli is an escherichia coli mutant or wild type escherichia coli. Further discloses the recombinant escherichia coli constructed by the method and application of the recombinant escherichia coli in preparation of MK-7. The invention realizes MK-7 production by escherichia coli for the first time, and the used raw materials are relatively cheap and are easy for industrial large-scale production; the recombinant escherichia coli for producing MK-7 constructed by the invention can reduce the time required by fermentation and improve the production efficiency; meanwhile, the strain transformation is carried out by utilizing the metabolic engineering principle, so that the yield of MK-7 is greatly improved, and the method has important application value for MK-7 production.

Description

Recombinant escherichia coli producing MK-7 and construction method and application thereof
Technical Field
The invention relates to the field of genetic engineering, in particular to recombinant escherichia coli for producing MK-7 and a construction method and application thereof.
Background
MK-7(menaquinone-7) is vitamin K2 with most biological activity in animal body, and the structure of the vitamin K2 is composed of a menaquinone mother ring and a side chain containing 7 isoprene units positioned at the C-3 position of the mother ring, has wide biological functions, and has specific health care and treatment effects of improving low prothrombin syndrome, preventing and treating Parkinson's disease and osteoporosis, reducing the risk of liver cancer of female patients with viral cirrhosis, and the like. Has wide application prospect and higher market value in the aspects of health care and medicine. Is a novel international third-generation health care product for preventing and treating osteoporosis, has safety which is approved by authorities of the American FDA, the Chinese SFDA, the American drug research institute, the European parliament and the European Union council, and has no side effect even if being eaten excessively.
At present, MK-7 is currently expensive (about $1200 per gram MK-7) and, due to the extremely low levels of Mk-7 in natural foods, only food-derived amounts are not in therapeutic demand. Scientists have therefore attempted to synthesize MK-7 chemically, but chemical synthesis presents two serious problems: firstly, chemically synthesized MK-7 contains cis-trans isomers, and only trans MK-7 has bioactivity; secondly, the chemical synthesis precursor menadione has serious toxicity to human tissues, so that MK-7 for human consumption does not favor chemical synthesis at present. The synthesis of MK-7 therefore depends mainly on biosynthesis and extraction from natural products. Although the natural product is safe to extract, the MK-7 is expensive and the supply is short due to the low content of the natural product MK-7. The Pichia pastoris is adopted to convert and synthesize Mk-3 by taking menadione as a substrate, but the biotransformation method is only limited to synthesizing MK-3, and the half life of the MK-3 in a human body is not as long as that of MK-7. Both gram-positive (B.subtilis) and gram-negative (E.coli) bacteria can synthesize vitamin K2. Most studied is the fermentation with bacillus, and the sources of the high-producing strains are mainly Japanese natto and Chinese fermented soybeans. Furthermore, the MK-7 synthesis pathway of Bacillus is well studied. Bacillus subtilis natto is the most commonly used strain for synthesizing MK-7, and the conditions for producing MK-7 by fermentation are referred by high-yield MK-7 bacillus which is developed later. At present, the MK-7 synthetic pathway of bacillus subtilis is subjected to metabolic engineering transformation by a Songhong task group at Tianjin university to obtain a high-yield genetic engineering strain, and the strain is fermented for 144 hours, wherein MK-7 reaches about 69.5 mg/L. However, the chassis needs 6 days for starting bacteria and fermenting, the period is long, and the production intensity is low.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a high-yield MK-7 strain so as to realize high-efficiency synthesis of MK-7.
In order to solve the technical problems, the invention firstly provides a construction method of recombinant escherichia coli.
The construction method of the recombinant escherichia coli comprises the steps of introducing genes related to MK-7 synthesis into receptor escherichia coli to obtain recombinant escherichia coli; the acceptor escherichia coli is an escherichia coli mutant or wild type escherichia coli;
the gene involved in MK-7 synthesis may be heptameric isopentenyl pyrophosphate synthase gene (HepPPS) and/or 1, 4-dihydroxy dinaphthoic acid octameric prenyl transferase gene (MenA) and/or S-adenosyl-L-methionine/demethylnaphthoquinone methyltransferase gene (Ubie) and/or 1, 2-diacylglycerol 3-glucosyltransferase gene (Almgs);
in the above construction method, the escherichia coli mutant is obtained by modifying the genome of the wild type escherichia coli from all, any seven, any six, any five, any four, any three, any two or any one of the following m1) -m 8):
m1) overexpressing isopentenyl pyrophosphate isomerase gene (idi);
m2) knock-out of the myristoyl carrier protein dependent acyltransferase gene (msbB);
m3) introduction of a heterologous mevalonate pathway (MVA pathway);
m4) overexpressing the methionine adenosyltransferase gene (metK);
m5) overexpressing the S-adenosyl-L-methionine/desmethyl naphthoquinone methyltransferase gene (Ubie);
m6) overexpressing the isochorismate synthase gene (menF);
m7) overexpressing a DNA junction transcriptional dual regulatory protein gene (fadR);
m8) knocking out lipoprotein gene (nlpI).
In a specific embodiment of the invention, the m1) is used for over-expressing the isopentenyl pyrophosphate isomerase gene (idi) by integrating the CPA1 promoter into the upstream promoter region of the gene idi (i.e. the 3032865-3033064 site of the whole genome sequence GenBank number NC-000913.3 of Escherichia coli BW 25113) by the crispr method;
m2) knocking out myristoyl carrier protein dependent acyltransferase gene (msbB) and m3) and introducing a heterologous mevalonate pathway (MVA pathway) by integrating an MVA pathway tandem gene fragment E2SKPM (namely MvaE-MvaS-MVK-PMK-MVD) with a pBAD promoter into a knocked-out gene msbB region (namely 1939222-0191943 of the whole genome sequence GenBank number NC-000913.3 of Escherichia coli BW 25113) by a crispr method; specifically, the MVA pathway tandem gene segment E2SKPM with the pBAD promoter is obtained by taking plasmids (namely pYESKs) of an MVA pathway constructed by von Van and the like as templates (von Van, xu Yang, Dougong, and the like; increasing the synthesis of isoprene by Escherichia coli through the MVA pathway. Biotechnology report, 2015, 31 (7): 1073-; wherein the MVA pathway tandem gene segment E2SKPM with the pBAD promoter comprises a pBAD promoter (shown in SEQ ID No.12), MvaE (1338888-1341299 of GeneBank No. NC-004668.1 (28-AUG-2016) position), MvaS (1337551-1338702 of GeneBank No. NC-004668.1 (28-AUG-2016) position), MVK (2101873-2102778 of GeneBank No. NC-003901.1 (29-MAR-2017) position), PMK (DQ 331671.1 of GeneBank No. DQ331671.1 (26-APR-2007) position) and MVD (NC 001146.8 of GeneBank No. NC-7070705-3085 position) which are connected in sequence.
The m4) overexpression methionine adenosyltransferase gene (metK) and m5) overexpression of S-adenosyl-L-methionine/demethylnaphthoquinone methyltransferase gene (UbiE) is achieved by integrating CPA1-UbiE into the upstream promoter region of metK gene (i.e., 3086506-3086706 site of the whole genome sequence GenBank of Escherichia coli BW25113, NC-000913.3) by the crispr method;
m6) the method for overexpressing the isochorismate synthetase gene (menF) is to integrate the pBAD promoter into the upstream promoter region of the menF gene (i.e., the whole genome sequence GenBank of Escherichia coli BW25113 is No. 2379148 and No. 2379348 of NC-000913.3) by the crispr method;
m7) the method for over-expressing DNA joint transcription dual regulatory protein gene (fadR) is to integrate a promoter CPA1 into an upstream promoter region of the fadR gene (namely, the whole genome sequence GenBank of Escherichia coli BW25113 is 1234738-1234938 of NC-000913.3) by a crispr method;
m8) is the method for knocking out the lipoprotein gene (nlpI) by the criprpr method (i.e. 3308040-3308924 of the whole genome sequence GenBank of Escherichia coli BW25113 with NC-000913.3).
Wherein the sequence of the promoter CPA1 is shown as SEQ ID No. 11; the sequence of the pBAD promoter is shown in SEQ ID No. 12.
In a specific embodiment of the present invention, the escherichia coli mutant is any one of:
m1) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with M1) -M8) (M06);
m2) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with M1) -M7) (M05);
m3) the Escherichia coli mutant is an Escherichia coli mutant (M04) obtained by modifying the wild type Escherichia coli with the M1) -M6) and M8);
m4) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with M1) -M6) (M03);
m5) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with M1) -M5) (M02);
m6) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with M1) -M3) (M01);
m7) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with the M1) (M00).
In the above construction method, the wild type escherichia coli is escherichia coli K12, specifically escherichia coli K12 strain BW 25113.
In the construction method, the HepPPS gene and the Ubie gene are derived from bacillus subtilis, namely BsHepPPS and BsUbie; the MenA gene is derived from Escherichia coli, namely EcMenA. Specifically, the HepPPS gene and the UbiE gene are derived from Bacillus subtilis 168; the MenA gene is derived from escherichia coli BW 25113.
In the above construction method, the HepPPS gene, the Ubie gene and the MenA gene are constructed in one expression cassette, and the positional relationship of the three genes in the expression cassette is that the HepPPS gene is located at the upstream of the MenA gene, and the Ubie gene is located at the downstream of the MenA gene.
In a specific embodiment of the present invention, the HepPPS gene, UbiE gene and MenA gene are introduced into the recipient escherichia coli through a recombinant vector a; the recombinant vector A is a recombinant expression vector pSB1a-BsHepPPS obtained by replacing a fragment between NcoI and EcoRI sites of the pSB1a vector with a HepPPS gene and keeping other sequences of the pSB1a vector unchanged, and then the pSB1a-BsHepPPS is treated by EcoRI and CIP to be connected with RBS-EcMenA-RBS-EcUbiE to obtain the recombinant expression vector pSB1 a-BsHepPPS-EcMenA-EcUbiE.
The Almgs gene is introduced into the acceptor escherichia coli through a recombinant vector B; the recombinant vector B is a recombinant expression vector pLB1s-Almgs obtained by replacing a fragment between XhoI and SacI sites of the pLB1s vector with Almgs gene and keeping other sequences of the pLB1s vector unchanged.
In the above construction method, the heptameric isoprene pyrophosphate synthase gene (HepPPS) comprises a HepS gene and a HepT gene, and the protein encoded by the HepS gene is A1) or A2):
A1) a protein (SEQ ID No.1) encoded by the DNA molecule shown in SEQ ID No. 5;
A2) a protein having 90% or more identity and function identity to the protein represented by A1) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A1);
the protein which can be coded by the HepT gene is shown as A3) or A4):
A3) the protein (SEQ ID No.2) coded by the DNA molecule shown in SEQ ID No. 6;
A4) a protein having 90% or more identity and function identity to the protein represented by A3) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A3);
the protein which can be coded by the 1, 4-dihydroxy dinaphthyl octa-isoprenyl transferase gene (MenA) is shown as A5) or A6):
A5) a protein (SEQ ID No.3) encoded by the DNA molecule shown in SEQ ID No. 7;
A6) a protein having 90% or more identity and function identity to the protein represented by A5) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A5);
the protein which can be coded by the S-adenosine-L-methionine/demethyl naphthoquinone methyltransferase gene (Ubie) is shown as A7) or A8):
A7) a protein (SEQ ID No.4) encoded by the DNA molecule shown in SEQ ID No. 8;
A8) a protein having 90% or more identity and function identity to the protein represented by A7) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A7);
the 1, 2-diacylglycerol 3-glucosyltransferase gene (Almgs) can code proteins shown as A9) or A10):
A9) a protein (SEQ ID No.9) encoded by the DNA molecule shown in SEQ ID No. 10;
A10) a protein having 90% or more identity and function identity to the protein represented by A9) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A9);
the isopentenyl pyrophosphate isomerase gene (idi) can encode proteins shown as A11) or A12):
A11) a protein coded by a DNA molecule shown in the position 3033065-3033613 of the GenBank number NC-000913.3 (the update date is 11-OCT-2018) (the Genbank protein ID is CTS32993.1 (the update date is 22-AUG-2015));
A12) a protein having 90% or more identity and function identity to the protein represented by A11) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A11);
the myristoyl carrier protein dependent acyltransferase gene (msbB) may encode a protein as shown in A13) or A14):
A13) a protein encoded by a DNA molecule shown in the position 1939222-1940193 of GenBank No. NC-000913.3 (the update date is 11-OCT-2018) (the GenBank protein ID is AAA24181.1 (the update date is 26-APR-1993));
A14) a protein having 90% or more identity and function identity to the protein represented by A13) which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A13);
the protein encoded by the methionine adenosyltransferase gene (metK) is shown in A15) or A16):
A15) a protein coded by a DNA molecule shown in the position No. NC-000913.3 (the update date is 11-OCT-2018) No. 3086706-3087860 of GenBank (the Genbank protein ID is AAC75979.1 (the update date is 24-SEP-2018));
A16) a protein having 90% or more identity and function identity to the protein represented by A15) which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A15);
the protein which can be coded by the isochorismate synthetase gene (menF) is shown as A17) or A18);
A17) a protein coded by a DNA molecule shown in the position 2379348-;
A18) a protein having 90% or more identity and function identity to the protein represented by A17) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A17);
the protein which can be coded by the DNA junction transcription dual regulatory protein gene (fadR) is shown as A19) or A20);
A19) a protein encoded by a DNA molecule shown in positions 1235657 of 1234938 and 1235657 of GenBank number NC-000913.3 (the update date is 11-OCT-2018) (the Genbank protein ID is AY605712 (the update date is 26-JUL-2016));
A20) a protein having 90% or more identity and function identity to the protein represented by A19) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein of A19);
the lipoprotein gene (nlpI) can encode proteins shown as A21) or A22):
A21) a protein coded by a DNA molecule shown in the position 3308040-3308924 of the GenBank number NC-000913.3 (the update date is 11-OCT-2018) (the Genbank protein ID is AYG17882.1 (the update date is 08-OCT-2018));
A22) a protein having 90% or more identity and function identity to the protein represented by A21) obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein represented by A21).
In the above construction method, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.
In the above construction method, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
In the above construction method, the heptameric isoprene pyrophosphate synthase gene (HepPPS) comprises a HepS gene and a HepT gene, and the HepS gene can be B1) or B2):
B1) the coding sequence is DNA molecule shown in SEQ ID No. 5;
B12) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.5 and has the same function as SEQ ID No. 5;
the HepT gene can be B3) or B4):
B3) the coding sequence is DNA molecule shown in SEQ ID No. 6;
B4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.6 and has the same function as SEQ ID No. 6;
the 1, 4-dihydroxy dinaphthoic acid octa-polyisoprene transferase gene (MenA) can be B5) or B6):
B5) the coding sequence is DNA molecule shown in SEQ ID No. 7;
B6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.7 and has the same function as SEQ ID No. 7;
the S-adenosine-L-methionine/demethyl naphthoquinone methyltransferase gene (Ubie) can be B7) or B8):
B7) the coding sequence is DNA molecule shown in SEQ ID No. 8;
B8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID No.8 and has the same function as SEQ ID No. 8;
the 1, 2-diacylglycerol 3-glucosyltransferase gene (Almgs) is B9) or B10):
B9) the coding sequence is DNA molecule shown in SEQ ID No. 10;
B10) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.10 and has the same function as SEQ ID No. 10;
the isopentenyl pyrophosphate isomerase gene (idi) can be B11) or B12):
B11) a DNA molecule with the coding sequence shown as the No. 3033065-3033613 site of NC-000913.3 (the update date is 11-OCT-2018);
B12) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the 3033613 th 3033065 th site of the GenBank number NC-000913.3 (the update date is 11-OCT-2018) and has the same function with the 3033065 th 3033613 th 3033065 th site of the GenBank number NC-000913.3 (the update date is 11-OCT-2018);
the myristoyl carrier protein dependent acyltransferase gene (msbB) can be B13) or B14):
B13) the coding sequence is DNA molecule shown in the 1939222-1940193 site with the GenBank number of NC-000913.3 (the update date of 11-OCT-2018);
B14) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides at 1939222-1940193 with the GenBank number of NC-000913.3 (the update date of 11-OCT-2018) and has the same function as the 1939222-1940193 with the GenBank number of NC-000913.3 (the update date of 11-OCT-2018);
the methionine adenosyltransferase gene (metK) can be B15) or B16):
B15) the coding sequence is DNA molecule shown in the No. 3086706-3087860 of GenBank number NC-000913.3 (the update date is 11-OCT-2018);
B16) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides at the No. 3086706-3087860 (the No. NC-000913.3 (the No. 11-OCT-2018) position) of the GenBank, and has the same function with the No. 3086706-3087860 (the No. NC-000913.3 (the No. 11-OCT-2018) position of the GenBank;
the isochorismate synthetase gene (menF) can be B17) or B18):
B17) a DNA molecule with the coding sequence shown in the position 2379348-2380643 of GenBank number NC-000913.3 (update date is 11-OCT-2018);
B18) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides at positions 2379348 and 2380643 of GenBank No. NC-000913.3 (the update date is 11-OCT-2018) and have the same functions as the positions 2379348 and 2380643 of GenBank No. NC-000913.3 (the update date is 11-OCT-2018);
the DNA jointing transcription dual-regulatory protein gene (fadR) can be B19) or B20):
B19) the coding sequence is a DNA molecule shown as the No. 1234938-1235657 of GenBank No. NC-000913.3 (the update date is 11-OCT-2018);
B20) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the 1234938-1235657 position of NC-000913.3 (the update date is 11-OCT-2018) of GenBank and has the same function with the 1234938-1235657 position of NC-000913.3 (the update date is 11-OCT-2018);
the lipoprotein gene (nlpI) can be B21) or B22):
B21) the coding sequence is a DNA molecule shown in the 3308040-3308924 position of the GenBank number NC-000913.3 (the update date is 11-OCT-2018);
B22) a DNA molecule which has the same functions with the 3308040-.
In the invention, the SEQ ID No.5 consists of 756 nucleotides, the coding sequence of the SEQ ID No.5 is shown in 1-756 bits, and the coding sequence of the SEQ ID No.5 encodes a protein with the Genbank protein ID of CAB14192.1 (the update date of 08-FEB-2018);
the SEQ ID No.6 consists of 1047 nucleotides, the coding sequence of the SEQ ID No.6 is shown as 1-1047 bits, and the coding sequence of the SEQ ID No.6 codes protein of which the Genbank protein ID is CAB14190.1 (the update date is 08-FEB-2018);
the SEQ ID No.7 consists of 927 nucleotides, the coding sequence is shown in positions 1-927, and the coding sequence encodes protein with the Genbank protein ID of BAE77380.1 (the update date is 29-SEP-2018);
the SEQ ID No.8 consists of 702 nucleotides, the coding sequence of the SEQ ID No.8 is shown as 1-702 bits, and the coding sequence of the SEQ ID No.8 codes a Genbank protein ID: protein of AQR82285.1(update date 28-FEB-2017);
the SEQ ID No.10 consists of 1197 nucleotides, the coding sequence of the SEQ ID No.10 is shown in positions 1-1197, and the coding sequence of the SEQ ID No.10 codes for a protein with GenBank protein ID of AAK38877.2(update date of 20-JUN-2001);
the coding sequence of the gene idi is shown in the No. 3033065-3033613 of a DNA molecule shown in the GenBank number NC-000913.3 (the update date is 11-OCT-2018), and consists of 549 nucleotides, and the Genbank protein ID of the coded protein is CTS32993.1 (the update date is 22-AUG-2015);
the coding sequence of the gene msbB is shown in 1939222-1940193 of a DNA molecule shown by GenBank number NC-000913.3 (update date is 11-OCT-2018), consists of 972 nucleotides, and the Genbank protein ID of the coded protein is AAA24181.1(update date is 26-APR-1993);
the coding sequence of the gene metK is represented by the No. 3086706-3087860 of the DNA molecule represented by the GenBank number NC-000913.3 (the update date is 11-OCT-2018), and consists of 1155 nucleotides, and the Genbank protein ID of the coded protein is AAC75979.1 (the update date is 24-SEP-2018);
the coding sequence of the gene menF is represented by the position 2379348-;
the coding sequence of the gene fade R is shown in 1234938-1235657 of a DNA molecule shown by GenBank number NC-000913.3 (the update date is 11-OCT-2018), and consists of 720 nucleotides, and the Genbank protein ID of the coded protein is AY605712 (the update date is 26-JUL-2016);
the coding sequence of the nlpI gene is shown in 3308040-3308924 of a DNA molecule shown by GenBank number NC-000913.3 (update date is 11-OCT-2018), and consists of 885 nucleotides, and the Genbank protein ID of the coded protein is AYG17882.1(update date is 08-OCT-2018).
The SEQ ID No.11 consists of 178 nucleotides and is a promoter sequence;
the SEQ ID No.12 consists of 1308 nucleotides and is a promoter sequence.
The recombinant Escherichia coli obtained by the construction method and the application of the recombinant Escherichia coli in preparing MK-7 are also within the protection scope of the invention.
The invention further discloses a method for preparing MK-7.
The method for preparing MK-7 comprises the following steps: the recombinant escherichia coli is used for catalyzing glucose reaction to obtain MK-7.
Specifically, the recombinant escherichia coli is subjected to arabinose induction culture to obtain induced recombinant escherichia coli, and the induced recombinant escherichia coli is used for catalyzing glucose reaction to obtain MK-7.
In the method, the arabinose induction culture is carried out in a culture medium containing arabinose, and the temperature of the induction culture is 30 ℃ and the time is 20 h.
In the above method, the arabinose is L-arabinose.
The invention utilizes the metabolic engineering principle to carry out strain transformation, firstly adopts an MVA way to supply main precursor isoprene, utilizes escherichia coli to strengthen methyl circulation to promote methyl supply, improves the yield of MK-7 by knocking out lipoprotein NlpI (NlpI) and overexpressing DNA splicing transcription dual regulatory protein FadR, saves one prenyl group in the obtained product MK-7 compared with MK-8 generated by the escherichia coli, utilizes a broader spectrum in the product MK-7 compared with MK-8, adopts whole cell catalysis to produce short cycle, has simple operation and 9h reaction, and ensures that the yield of MK-7 can reach 224 +/-5.8 mu m/L and 22.6 +/-0.5 mg/g DCW.
The invention realizes MK-7 production by escherichia coli for the first time, and the used raw materials are relatively cheap and are easy for industrial large-scale production; the recombinant escherichia coli for producing MK-7 provided by the invention can reduce the time required by fermentation and improve the production efficiency; meanwhile, the strain is transformed by utilizing the metabolic engineering principle, the MK-7 synthesis path and the methylation cycle of DMK-7 are enhanced, so that the synthesis of competitive products q-8 and MK-8 is reduced, the yield of MK-7 is greatly improved, and the method has important application value for MK-7 production.
Drawings
FIG. 1 is a schematic diagram of the recombinant vector pSB1 a-HAE.
FIG. 2 is an HPLC profile of MK-7 standard.
FIG. 3 is an HPLC chromatogram of the conversion product.
FIG. 4 shows MK-7 yields of genetically engineered bacteria MK00, MKC1, MK01 and MK 02.
FIG. 5 shows the MK-7 yields of genetically engineered bacteria MK02, MK03, MK04, MK05, MK06 and MK 07.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Coli K12 strain BW25113 in the following examples is described in the literature "Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H: construction of Escherichia coli K-12in-frame, single-gene knock out variants: the Keio collection. mol Syst Biol 2006, 2: 2006.0008 ", it is a non-pathogenic bacterium, with clear genetic background, short generation time, easy culture and cheap culture medium raw material. GenBank number of the whole genome sequence of Escherichia coli K12 is NC-000913.3 (11-OCT-2018 for update date). The biological material is only used for repeating the relevant experiments of the present invention and is not used for other purposes.
Plasmids such as pTarget used for CRISPR-Cas9 knockout technology in the following examples are described in the literature "Jiang, y., b.chen, c.duan, b.sun, j.yang and s.yang (2015)" Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system, "apple Environ Microbiol 81 (7): 2506-2514 ", the biological material is only used for repeating the related experiments of the present invention and is not used for other purposes.
Example 1 construction of recombinant bacterium producing MK-7
Firstly, constructing recombinant plasmid for simultaneously expressing HepPPS, MenA and Ubie
1. Construction of recombinant plasmid pSB1a-BsHepPPS
Bacillus subtilis 168(,(s) (s))
Figure BDA0002293267870000081
23857 TM ) Strain number) as a template, two pairs of primers (H1F and H3R, H3F and H2R) were designed and amplified separately to obtain fragments of genes respectively comprising the two subunits HepS and HepT of heptameric isoprene pyrophosphate synthetase (HepPPS), and sequencing analysis showed that the nucleotide sequence of the HepS gene is shown in SEQ ID No.5, consisting of 756 nucleotides whose coding sequence is shown in positions 1-756, and the coding sequence of Genbank protein ID is CAB14192.1(update date: 08-FEB-2018), namely the protein shown in SEQ ID No. 1; the nucleotide sequence of the hepT gene is shown as SEQ ID No.6 and consists of 1047 nucleotides, the coding sequence of the hepT gene is shown as positions 1-1047, and the protein with the Genbank protein ID of CAB14190.1 (the update date of 08-FEB-2018), namely the protein shown as SEQ ID No.2, is coded. Then the two fragments are recovered by glue, the two fragments are taken as templates,the overlap PCR was performed using H1F and H2R as primers to obtain fragment NcoI-hepS-RBS-hepT-EcoRI, while plasmid pSB1a (Liu, B., S.M.Xiang, G.ZHao, B.J.Wang, Y.H.Ma, W.F.Liu and Y.Tao (2019) "instant product of 3-hydroxyproprionate from fatty acids fed stock in Escherichia coli 51: 121. and fragment NcoI-hep-hepT-EcoRI were digested simultaneously with NcoI and EcoRI, and the digested products were separately recovered and ligated with T4 ligase T4, the ligation product was sequenced T1, cultured for 12 hours, and the sequence was inverted to confirm correct cloning. The recombinant vector with the correct sequence was named pSB1 a-BsHepPPS.
Wherein the primer sequences are as follows:
H1F:5’-cttgccatggggatgcaagacatctacggaactttag-3’
H2R:5’-ccggaattcttaaaattttcttttaccgatatattttg-3’
H3F:5’-gaagaagggtaagagctcaggaggaattaacatgttaaatatcattcgtttac-3’
H3R:5’-gttaattcctcctgagctcttacccttcttccactttctg-3’
2. construction of recombinant plasmid pSB1a-HAE
Taking the genome DNA of escherichia coli BW25113 as a template, designing a primer (the specific primer sequence is EcmenA-F: 5'-ggtaaaagaaaattttaagaattcaggaggaattaacatgactgaacaacaaattagccg-3'; EcmenA-R: 5'-TTCTTTTGAGTCCTGCATGTTAATTCCTCCTGAGCTCTTATTTATGCTGCCCACTGGCTTAGG-3'), and amplifying to obtain a fragment containing EcmenA; bacillus subtilis 168 genomic DNA is used as a template, and primers (the specific primer sequences are Bscubi E-F: 5'-atgcaggactcaaaagaacagcgcg-3'; Bscubi E-R: 5'-CGGCACCAGCTGCAGACCGAGCTCACCGAATTCTCATTTCCATCCGATATGCGTGGCAGC-3') are designed for amplification to obtain a Bsubi E-containing fragment. Sequencing analysis shows that the nucleotide sequence of EcMenA is shown as SEQ ID No.7, the coding sequence is shown as 1-927, the EcMenA consists of 927 nucleotides, and the coding protein of which the Genbank protein ID is BAE77380.1(update date is 29-SEP-2018), namely the protein shown as SEQ ID No. 3; BsubiE has a nucleotide sequence shown in SEQ ID No.8, a coding sequence shown in positions 1-702, consists of 702 nucleotides and codes a protein with Genbank protein ID of AQR82285.1(update date of 28-FEB-2017), namely a protein shown in SEO ID No. 4. Then, the two fragments are recovered by glue, overlap PCR is carried out by taking the two fragments as templates and using EcmenA-F and BsmuE-R as primers, RBS-EcmenA-RBS-Bsubie is obtained by amplification, pSB1 a-BseIPPPS is treated by EcoRI and CIP, and then a Gibson ligation method is used, and the specific steps are as follows: the fragment RBS-EcMenA-RBS-BsUbie and the plasmid pSB1a-BsHepPPS of which the phosphate is cut off by enzyme are taken and mixed into 4ul, 6ul of Gibson connecting solution (NEB, E5510) is added to form a10 ul connecting system, the 10ul connecting system is reacted for 50min at 50 ℃, then the connecting system is transformed into T1 chemical competence according to the method of the step 1, positive clones are screened and sequenced by a bacteria liquid PCR mode, and the plasmid with correct sequencing is named as pSB1a-BsHepPPS-EcMenA-BsUbie, which is called pSB1a-HAE for short, and the structural schematic diagram is shown in figure 1.
3. Construction of recombinant plasmid pSB1a-AHE
Primers (specific primer sequences are EcubiE-F and EcubiE-R) are designed by taking genome DNA of Escherichia coli BW25113 as a template, and a fragment 1 containing UbiE (EcUbiE) is amplified. Sequencing analysis shows that the coding sequence of EcUbiE is represented by 4018855-4019610 of GenBank number NC-000913.3 (update date is 11-OCT-2018), consists of 756 nucleotides and encodes protein with Genbank protein ID of AUP44431.1(update date is 22-JAN-2018). Simultaneously, primers EcmenA-2F and EcmenA-2R are used for amplifying EcmenA from the constructed plasmid pSB1a-HAE to obtain a fragment 2 containing EcmenA; BshepST was amplified from the above-described constructed plasmid pSB1a-HAE using primers H3F and H3R to give fragment 3 containing BshepST. Plasmid pSB1a was double digested with XhoI and SacI to give fragment 4. Fragments 1,2, 3 and 4 were each gel recovered. As with the above GIbson construction method, fragments 1,2, 3 and 4 were combined into 4ul system, and 6ul Gibson ligation solution was added to react to obtain recombinant plasmid pSB1a-EcMenA-BsHepPPS-EcUbiE, abbreviated as pSB1 a-AHE.
Wherein the primer sequences are as follows:
EcubiE-F:5’-cggtaaaagaaaattttaaaggaggaattaacatggtggataagtcacaa-3’
EcubiE-R:5’-CTGCCGCGCGGCACCAGCTGCAGACCGAGCTCTCAGAACTTATAACCACG-3’
EcmenA-2F:5’-cagcggcctggtgccgcgcggcagcctcgagatgactgaacaacaaatta-3’
EcmenA-2R:5’-AGATGTCTTGCATGTTAATTCCTCCTGAGCTCTTATGCTGCCCACTGGCTTAGGAATATC-3’
H3F:5’-atgcaagacatctacggaacttt-3’
H3R:5’-TTAAAATTTT CTTTTACCGATAT-3’
secondly, constructing recombinant plasmid for expressing Almgs
Constructing a recombinant plasmid pLB1 s-Almgs:
the method comprises the steps of carrying out gene synthesis on the Almgs from Acholeplasia laidwii, synthesizing the Almgs gene with the sequence shown as SEQ ID No.10 (consisting of 1197 nucleotides, the coding sequence of which is shown in positions 1-1197 and the protein shown as SEQ ID No.9, wherein the protein is encoded with GenBank protein ID of AAK38877.2 (20-JUN-2001), using a plasmid containing the synthesized Almgs gene sequence as a template, designing primers (the specific primer sequence is Almgs-F: 5'-gcctggtgccgcgcggcagcctcgagatgcgtattggtatcttcag-3'; Almgs-R: 5'-GGCACCAGCTGCAGACCGAGCTCTTATTTCTTGTTCAGTTTCTTG-3'), amplifying to obtain the Almgs, connecting the fragments Almgs with a plasmid pLB1s which is subjected to double digestion by XhoI and SacI by using the above bGison reaction solution, connecting the Almgs to pLB1s (Liu, B., S.M.X.J.ha., Wang.J.H.Wang.W.W.W.2019. origin and molecular protein of molecular protein, namely Tauchi-34. reaction solution of protein, and protein of Tauchi: 31, the recombinant plasmid pLB1s-Almgs was obtained.
Thirdly, constructing escherichia coli mutant
1. Construction of E.coli mutants M00 and M01
1.1 Escherichia coli BW25113 is used as a starting strain to over-express the gene idi. The CRISPR method is used for integrating a promoter CPA1 into an upstream promoter region of a gene idi so as to over-express the gene idi (the coding sequence of the gene idi is 3065-3033613 th position of NC-000913.3 (11-OCT-2018) in GenBank, the coding sequence of the gene idi consists of 549 nucleotides, the coding sequence of the gene bank protein ID is CTS32993.1 (22-AUG-352015 in update date) and the promoter sequence of the gene bank is 3032865-3034 th position of NC-000913.3 (11-OCT-2018 in update date), and the specific steps are as follows:
taking genome DNA of Escherichia coli BW25113 as a template, designing primers (CPA1-F and CPA1-R) for amplification to obtain a fragment containing a promoter CPA1 sequence (the sequence is SEQ ID No.11) of 178bp, and simultaneously respectively designing primers (idiup-F, idiup-R, ididw-F and ididw-R) for amplification to obtain a fragment U (idi) of an upstream homology arm of about 500bp and a fragment D (idi) of a downstream homology arm of about 500bp of a gene idi promoter; taking the three fragments as templates, performing overlap PCR by using primers idiup-F and ididw-R to obtain a homologous fragment of the idi promoter, wherein CPA1 is sandwiched between upstream and downstream homologous arms, and the homologous fragment is named as U (idi) -CPA1-D (idi). pCAS9 (purchased from Clontech) was transformed into E.coli BW25113 by calcium chloride transformation to give E.coli BW25113 harboring plasmid pCAS9, designated pCas9/K12 electroporation competent.
Primers pidi-F and pidi-R are designed, linear pTarget-upidi is obtained by amplification with pTarget as a template, then the linear pTarget-upidi is transferred into Escherichia coli T1 chemical competent cells, T1/pTarget-upidi positive strains are obtained, bacteria are shaken, plasmids are extracted from the strains, and the obtained plasmids are named as pTarget-upidi. Electrically transferring U (idii) -CPA1-D (idii) and pTarget-upidii plasmid with a positioned cutting position into pCas9/K12 electrically transferring competence (capable of being positioned on a promoter of the gene idii and cut under the action of pTarget-upidii, then obtaining a recombinant fragment CPA 1-idii under the action of homologous fragments U (idii) -CPA1-D (idii) and Cas9, culturing to obtain a bacterial liquid, then respectively adding 200bp on the basis of the upper and lower homologous arms which are designed before, designing identification primers (JDidii-F and JDi-R), using genomic DNA of the bacterial liquid as a template, amplifying to obtain a strip with about 1.6Kb for sequencing, comparing to determine that the upstream promoter region of the gene idii is replaced by CPA1 (corresponding to the whole genome sequence BW of Escherichia coli 25113 is NC-000913.3 No. 2865 No. 3034,3034,3060.30625) and then adding IPT-upidii plasmid with 30 ℃ for culturing, coli BW25113 in which the promoter of gene idi was CPA1 was obtained, and cultured at 40 ℃ to eliminate pCas9 to obtain a strain overexpressing idi.
Wherein the primer sequences are as follows:
CPA1-F:5’-ttatcaaaaagagtattgac-3’
CPA1-R:5’-TATATCTCCTTCTTAAAAGATC-3’
idiup-F:5’-ctagtgatgttcggcatggt-3’
idiup-R:5’-GTCAATACTCTTTTTGATAAGATGCCGACAGGTAAAAACC-3’
ididw-F:5’-cttttaagaaggagatataatgcaaacggaacacgtcattt-3’
ididw-R:5’-TCGTTTTCTGGCTTCGCGAT-3’
pidi-F:5’-gttttagagctagaaatagc-3’
pidi-R:5’-AGTGAGCGTTTATCGACAACactagtattatacctaggac-3’
JDidi-F:5’-atgcttcatggtttgcgattg-3’
JDidi-R:5’-CAAATGTCGGGGTTTTTTTATTTA-3’
1.2 Using E.coli BW25113 and the strain overexpressing idi as starting strains, msbB was knocked out and MVA pathway was introduced at the msbB site. The method comprises the following specific steps:
a plasmid of the MVA pathway constructed by Von Van et al (pYESKs) as a template (Von Van, Xylon, Dougong, et al., enhanced Escherichia coli for isoprene synthesis by the MVA pathway BioEngineering, 2015, 31 (7): 1073-1081)), and amplified with primers MVA-F and MVA-R to obtain an MVA pathway tandem gene fragment E2SKPM (MvaE-MvaS-MVK-PMK-MVD) with a pBAD promoter (Yang, Y.L., S.Zhang, K.Ma, Y.Xu, Q.Tao, Y.Chen, J.Chen, S.Guo, J.ren, W.Wang, Y.Tao, W.B.Yin and H.Liu (2017), "Discovery and Characterization of a New Faterpen cycles Bacteria and organism, Inc. 56 Ed 17): 4749-4752.). And amplifying by using primers pMVA-F and pMVA-R and pTarget as a template to obtain a plasmid pTarget-MVA. Amplifying by mBup-F and mBup-R with Escherichia coli BW25113 genome as template to obtain msbB upstream 1000bp (named as UmsbB), amplifying by mBdw-F and mBdw-R with Escherichia coli BW25113 genome as template to obtain msbB downstream 1000bp (named as DmsbB), amplifying by primers mBup-F and mBdw-R with E2SKPM, UmsbB and DmsbB three fragments as templates to obtain segment UmsbB-E2SKPM-DmsbB, and transferring pTarget-MVA and segment UmsbB-E2SKPM-DmsbB into strains respectively overexpressing idii in transfer-competent Escherichia coli BW25113 and 1.1 by electrotransformation, replacing E2SKPM into msbB site (19322, NC 000913.3, NC-019923 site corresponding to whole genome sequence GenBank of Escherichia coli 25113) (for improving organism synthesis of Escherichia coli BW, Van. No. 2015 1073, Van. 3, Van. Wa-MRB), an escherichia coli strain (named as an escherichia coli mutant M00) which knocks out msbB and introduces MVA pathway at the msbB site and an escherichia coli strain (named as an escherichia coli mutant M01) which overexpresses idi, knocks out msbB and introduces MVA pathway at the msbB site are obtained respectively.
Wherein the primer sequences are as follows:
MVA-F:5’-ttatccgaaactggaaaagc aatgtgcctgtcaaatggacgaa-3’
MVA-R:5’-TCTCCTCGCGAGAGGCTTTTCGGCACCACCGAAATTCAGTAAGC-3’
pMVA-F:5’-GCACGGCTGGGACGTTTTGCgttttagagctagaaatagc-3’
pMVA-R:5’-GCAAAACGTCCCAGCCGTGCactagtattatacctaggac-3’
mBup-F:5’-aactgcagcgcctcacctggga-3’
mBup-R:5’-GCTTTTCCAGTTTCGGATAA-3’
mBdw-F:5’-aaaagcctctcgcgaggaga-3’
mBdw-R:5’-CTCTCCAACAATAAAGGTAT-3’
2. construction of E.coli mutant M02
Escherichia coli mutant M01 is used as a starting strain to overexpress methyl cycle gene MetK (the coding sequence of the gene MetK is represented by position 3086706-3087860 of NC-000913.3 (update date is 11-OCT-2018), the gene MetK consists of 1155 nucleotides, the coding sequence of the gene MetK is protein of AAC75979.1(update date is 24-SEP-2018), the promoter sequence of the gene MetK is represented by position 3086506-3086705 of NC-000913.3 (update date is 11-OCT-2018) and BsubiE gene (the nucleotide sequence of BsubiE is represented by SEQ ID No.8, the promoter sequence of the gene BsubiE is represented by position 702, the coding sequence of the gene BsubiE is represented by position 1-702, and the coding sequence of the gene MetK is protein of AQR82285.1(update date is 28-FEB-2017). CPA1-BsUbiE was integrated into the upstream promoter region of gene metK by the crispr method to overexpress genes BsUbiE and MetK as follows:
a fragment containing 178bp of promoter CPA1 sequence (SEQ ID No.11) was obtained according to the method 1.1 in step three, and a fragment containing Bsubie was obtained by the method 2in step one of example 1; performing overlap PCR amplification by using CPA1-F and Bsubie-R as primers to obtain CPA1-Bsubie, performing amplification by using primers pMetK-F and pMetK-R and pTarget as a template to obtain plasmid pTarget-MetK, performing amplification by using Mup-F and Mup-R and using Escherichia coli BW25113 genome as a template to obtain upstream 1000bp (named UM) of the metK promoter region, performing amplification by using Mdw-F and Mdw-R and using Escherichia coli BW25113 genome as a template to obtain downstream 1000bp (named DM) of the metK promoter region, performing amplification by using primers Mup-F and Mdw-R and using CPA 1-UBsbbie, UM and DM as templates to obtain a fragment UM-CPA1-Bsubie-DM, and performing state transition on the plasmid and the fragment UM-Bsubie 1-Bsubie-DM by using primers 01, CPA 1-BsiBioE was integrated into the upstream promoter region of the gene metK of E.coli mutant M01 genome (corresponding to position 3086506-3086706 of the whole genome sequence GenBank No. NC-000913.3 (update date is 11-OCT-2018)) i.e.the upstream promoter region of the gene metK (position 3086506-3086705 of the whole genome sequence GenBank No. NC-000913.3 (update date is 11-OCT-2018)) was replaced with CPA 1-BsiBioE, and E.coli mutant over expressing BsiBioE and metK was obtained and named E.coli mutant M02.
Wherein the primer sequences are as follows:
CPA1-F:5’-ttatcaaaaagagtattgac-3’
BsUbiE-R:5’-TCATTTCCATCCGATATGCGTGGCAGC-3’
pMetK-F:5’-GTGTGGATTTTCTGTGGTAGgttttagagctagaaatagc-3’
pMetK-R:5’-CTACCACAGAAAATCCACACactagtattatacctaggac-3’
Mup-F:5’-gagctcattgcaacctcctg-3’
Mup-R:5’-GTCAATACTCTTTTTGATAAATTTTCTGCCATTGTGGGGTAT-3’
Mdw-F:5’-gctgccacgcatatcggatggaaatga atggcaaaacacctttttacgtc-3’
Mdw-R:5’-CGGGCAGAATTGGCTTGATG-3’
3. construction of E.coli mutant M03
Escherichia coli mutant M02 is used as a starting strain to overexpress a menF gene cluster (the coding sequence of the menF gene is represented by position 2379348 2380643 of NC _000913.3(update date is 11-OCT-2018), the coding sequence consists of 1296 nucleotides, the coding protein ID of the GenBank protein is AAC75325.2(update date is 24-SEP-2018), and the promoter sequence is represented by position 2379148 2379347 of NC _000913.3(update date is 11-OCT-2018)).
Taking plasmid pSB1a as a template, obtaining pBAD promoter (SEQ ID No.12) by PCR, using primers pMenF-F and pMenF-R and pTarget as a template to amplify to obtain plasmid pTarget-MenF, then using MFup-F and MFup-R and using Escherichia coli BW25113 genome as a template to amplify to obtain 500bp (named as UMF) at the upstream of menF promoter region, using MFdw-F and MFdw-R and using BW25113 genome as a template to amplify to obtain 500bp (named as DMF) at the downstream of menF promoter region, then using primers MFup-F and MFdw-R and using three fragments of pBAD, UMF and DMF as templates to amplify to obtain fragment UMF-pBAD-DMF, then using primers MFup-F and MFdw-R to convert into electric state M02 by electricity to integrate pBAD promoter into upstream of menF gene (the whole genome sequence of Escherichia coli BW equivalent NC 237913 as NC 2352), namely, the upstream promoter region of the menF gene (position 2379148-.
Wherein the primer sequences are as follows:
pBAD-F:5’-aatgtgcctgtcaaatggacgaag-3’
pBAD-R:5’-GGTTAATTCCTCCTGTTAGCCCAA-3’
pMenF-F:5’-GCAAGGAATTGGTGTGGGCGgttttagagctagaaatagc-3’
pMenF-R:5’-CGCCCACACCAATTCCTTGCactagtattatacctaggac-3’
MFup-F:5’-aacaaccaaagtccgctctg-3’
MFup-R:5’-CGTCCATTTGACAGGCACATTTTAACGGCGTGCCAGCAACA-3’
MFdw-F:5’-gctaacaggaggaattaaccatgcaatcacttactacggcgc-3’
MFdw-R:5’-CGTTGGCTGCAACTGATGTA-3’
4. construction of E.coli mutant M04
Taking an Escherichia coli mutant M03 as an original strain, knocking out nlpI (the coding sequence of the nlpI of the gene is shown as the 3308924 th 3308040 th of NC _000913.3(update date is 11-OCT-2018), consists of 885 nucleotides, and codes a protein with Genbank protein ID of AYG17882.1(update date is 08-OCT-2018)), and specifically comprising the following steps:
the plasmid pTarget-nlpI is obtained by using primers pnlpI-F and pnlpI-R and using pTarget as a template for amplification, then the gene nlpI upstream 500bp (named UN) is obtained by using Nup-F and Nup-R and using Escherichia coli BW25113 genome as a template for amplification, the gene nlpI downstream 500bp (named DN) is obtained by using Ndw-F and Ndw-R and using Escherichia coli BW25113 genome as a template for amplification, then a fragment UN-DN is obtained by using primers Nup-F and Ndw-R and using UN and DN as templates, then the plasmid pTarget-nlpI and the fragment UN-DN are electrically transferred into an electrotransformation competent M03, and the nlpI (3304 th 33040 th 3304 th of NC (NC-000913.3) corresponding to the whole genome sequence of the Escherichia coli BW25113 is knocked out to obtain an E.coli knockout E.coli M04 mutant.
Wherein the primer sequences are as follows:
pnlpI-F:5’-GGGATCGCATTATATTACGGgttttagagctagaaatagc-3’
pnlpI-R:5’-CCGTAATATAATGCGATCCCactagtattatacctaggac-3’
Nup-F:5’-caaagagatcatgcaggttg-3’
Nup-R:5’-GGGCTGATGTGTACGTCAGTTCCCACTCCCGAAGACCAC-3’
Ndw-F:5’-gtggtcttcgggagtgggaa ctgacgtacacatcagccc-3’
Ndw-R:5’-TCAGCTTCATCCAGAACCAG-3’
5. construction of E.coli mutant M05
The Escherichia coli mutant M03 is used as a starting strain, the overexpression fadR (the coding sequence of the gene fadR is represented by the 1234938-1235657 th position of the GenBank number NC-000913.3 (update date is 11-OCT-2018), the overexpression fadR consists of 720 nucleotides, the coding protein of the Genbank protein ID is AY605712(update date is 26-JUL-2016), the promoter sequence of the overexpression fadR is represented by the 1234738-1234937 th position of the GenBank number NC-000913.3 (update date is 11-OCT-2018)), and the specific steps are as follows:
amplifying by using primers pR-F and pR-R and pTarget as a template to obtain plasmid pTarget-fadR, then amplifying by using Rup-F and Rup-R and Escherichia coli BW25113 as a template to obtain 500bp (named as UR) at the upstream of the fadR promoter region, amplifying by using Rdw-F and Rdw-R and Escherichia coli BW25113 as a template to obtain 500bp (named as DR) at the downstream of the fadR promoter region, then amplifying by using primers Rup-F and Rdw-R and CPA1, UR and DR as templates to obtain a fragment UR-CPA1-DR, then electrically transferring the plasmid pTarget-fadR and the fragment UR-CPA1-DR to a transduction competent M03, and integrating the promoter 1 into the upstream promoter region of the fadR gene (namely, the whole genome sequence GenBank number of Escherichia coli 25113 is NC-000913.3 (the number of date is 11-201478 of OCT 11-1238), namely, the upstream promoter region of the fadR gene (1234738-1234938 of GenBank NC-000913.3 (update date 11-OCT-2018)) is replaced by the promoter CPA1, so as to obtain the E.coli mutant for over-expressing fadR, and the E.coli mutant is named as E.coli mutant M05.
Wherein the primer sequences are as follows:
pR-F:5’-ATAACACAGCAAAACAAAGTgttttagagctagaaatagc-3’
pR-R:5’-ACTTTGTTTTGCTGTGTTATactagtattatacctaggac-3’
Rup-F:5’-agaggctacgcggtgataaatac-3’
Rup-R:5’-GTCAATACTCTTTTTGATAAGAAAAAGGGATCAAGGCTTC-3’
Rdw-F:5’-tcttttaagaaggagatatatatggtcattaaggcgcaaag-3’
Rdw-R:5’-CGACCAATACGCGTATACAG-3’
6. construction of E.coli mutant M06
Taking an Escherichia coli mutant M05 as an original strain, knocking out nlpI (the coding sequence of the nlpI of the gene is shown as 3308924 of 3308040 of the gene with the GenBank number of NC-000913.3 (the update date of 11-OCT-2018), the nlpI consists of 885 nucleotides, and the coding protein ID of the Genbank protein is AYG17882.1 (the update date of 08-OCT-2018)), and specifically comprising the following steps:
amplifying by using primers pnlpI-F and pnlpI-R and pTarget as a template to obtain a plasmid pTarget-nlpI, then using Nup-F and Nup-R to amplify by taking an Escherichia coli BW25113 genome as a template to obtain the nlpI upstream 500bp (named as UN), using Ndw-F and Ndw-R to amplify by taking an Escherichia coli BW25113 genome as a template to obtain the nlpI downstream 500bp (named as DN), then, primers Nup-F and Ndw-R are utilized to amplify two fragments UN and DN to obtain a fragment UN-DN, then the plasmid pTarget-nlpI and the fragment UN-DN are transferred into an electrotransformation competence M05 by electrotransformation to knock out nlpI (corresponding to 3308040-3308924 of the whole genome sequence GenBank number NC-000913.3 (update date 11-OCT-2018) of the Escherichia coli BW 25113), so as to obtain the nlpI-knocked-out Escherichia coli mutant which is named as an Escherichia coli mutant M06.
Wherein the primer sequences are as follows:
pnlpI-F:5’-GGGATCGCATTATATTACGGgttttagagctagaaatagc-3’
pnlpI-R:5’-CCGTAATATAATGCGATCCCactagtattatacctaggac-3’
Nup-F:5’-caaagagatcatgcaggttg-3’
Nup-R:5’-GGGCTGATGTGTACGTCAGTTCCCACTCCCGAAGACCAC-3’
Ndw-F:5’-gtggtcttcgggagtgggaactgacgtacacatcagccc-3’
Ndw-R:5’-TCAGCTTCATCCAGAACCAG-3’
fourth, constructing recombinant bacteria producing MK-7
Transforming the recombinant plasmid pSBIa-AHE into Escherichia coli M00 by a calcium chloride method to obtain MK 00; transforming the recombinant plasmid pSB1a-AHE into Escherichia coli M01 by a calcium chloride method to obtain MKC 1; transforming the recombinant plasmid pSB1a-HAE into Escherichia coli M01 by a calcium chloride method to obtain MK 01; transforming the recombinant plasmid pSB1a-HAE into Escherichia coli M02 by a calcium chloride method to obtain MK 02; transforming the recombinant plasmid pSB1a-HAE into Escherichia coli M03 by a calcium chloride method to obtain MK 03; transforming the recombinant plasmid pSB1a-HAE into Escherichia coli M04 by a calcium chloride method to obtain MK 04; transforming the recombinant plasmid pSB1a-HAE into Escherichia coli M05 by a calcium chloride method to obtain MK 05; transforming the recombinant plasmids pSB1a-HAE and pLB1s-Almgs into Escherichia coli M03 by a calcium chloride method to obtain MK 06; the recombinant plasmid pSB1a-HAE was transformed into E.coli M06 by the calcium chloride method to obtain MK 07.
Example 2 MK-7 production Using recombinant MK-7 producing bacteria
Induction of recombinant bacteria producing MK-7
Self-induction medium: recombinant bacteria MK00, MKC1, MK01, MK02, MK03, MK04, MK05 and MK07 producing MK-7 are streaked on agar containing 1.5% by mass and ampicillin LB containing 50ug/mL respectively, and recombinant bacteria MK06 producing MK-7 are streaked on agar containing 1.5% by mass and ampicillin and 50ug/mL respectively, and then cultured at 37 ℃ for 12 h. Respectively picking MK00, MKC1, MK01, MK02, MK03, MK04, MK05 and MK07 monoclonals from the plate to be respectively inoculated into a liquid LB culture medium containing 50ug/mL ampicillin, picking MK06 monoclonals from the plate to be inoculated into a liquid LB culture medium containing 50ug/mL ampicillin and 50ug/mL streptomycin, and carrying out overnight shaking culture at 37 ℃ and 250rpm rotation to obtain an overnight culture; inoculating the overnight culture into a self-induction culture medium ZYM in an inoculation amount of 1% by volume, and performing shake culture at 30 ℃, wherein the rotation speed is 250rpm, and the culture time is 20h, so as to obtain the induced recombinant bacteria.
The formula of the self-induction culture medium ZYM-5052 is as follows: 100mL A +2mL B +2mL C + 200. mu. L D + 100. mu. L E (the following are concentrations in mass percent, i.e.% represent g/100 mL);
a, ZY: 1% tryptone, 0.5% yeast powder;
B.50×M:1.25M Na 2 HPO 4 ,1.25M KH 2 PO 4 ,2.5M NH 4 cl and 0.25M Na 2 SO 4
C.50 × 5052: 25% glycerol, 2.5% glucose, 10% L-arabinose;
D.500×MgSO 4 :1M MgSO 4
e.1000 × microelements: 50mM FeCl 3 ,20mM CaCl 2 ,10mM MnCl 2 ,10mM ZnSO 4 ,CoCl 2 、NiCl 2 、Na 2 MoO 4 、Na 2 SeO 3 And H 3 BO 3 2mM each.
Second, preparation of MK-7 by biotransformation
Centrifuging the recombinant bacteria induced in the step one for 5-15 min at 4 ℃ at 4000 rpm, washing the bacteria for 2 times by using a sodium chloride aqueous solution with the mass percentage concentration of 0.85%, and collecting the bacteria under the same centrifugation condition. Resuspending in appropriate volume of transformation substrate solution (1 × M9, 100mM glucose), the concentration of transformation bacterial solution of MK00, MKC1, MK01 and MK02 was OD600 ═ 10, the transformation time was 14h, 200rpm, pH7, and the transformation time was 14h, to obtain transformation solution; the transformed cell suspension was transformed at OD600 ═ 20, 37 ℃, 200rpm, pH7 for 9h with MK02, MK03, MK04, MK05, MK06, and MK07 to obtain transformed cells.
The resulting transformation solution was centrifuged at 12000rpm at 4 ℃ for 5min to remove the supernatant, and then the mixture was mixed with 1: 2 isopropanol: and (3) resuspending the thallus by using n-hexane, simultaneously carrying out votex, then centrifuging at 8000rpm for 10min, taking the upper n-hexane phase in a new 1.5mL centrifuge tube, then rapidly volatilizing the n-hexane by using a vacuum rotary evaporator, adding 1mL isopropanol into the 1.5mL centrifuge tube in which the n-hexane is volatilized, and dissolving the product. After filtration through a 0.22 μm filter, the MK-7 production was measured by HPLC. HPLC used an Agilent 1200 high performance liquid chromatograph (equipped with a quaternary pump, DAD detector and workstation). Chromatographic conditions are as follows: ascentis Express C18(15 cm. times.4.6 mm); mobile phase: 100% methanol, flow rate: 1mL/min, and the column temperature is 50 ℃; the sample amount is 5ul, and the detection wavelength is 254 nm. MK-7 standards were purchased from MCE, USA. The experiment was repeated three times and the average was taken. At the same time, three 4.5mL portions of MK06 transformation solution with OD600 ═ 20 were weighed, the three bacteria were collected in parallel and placed in an oven at 50 ℃ to be dried to constant weight, the three constant weights were weighed to be 29mg, 30mg and 28mg respectively, and the conversion relationship between the concentration OD600 of the bacteria and the dry weight obtained by averaging the three parallel measurements was 1mL of the dry weight of the bacteria with OD600 ═ 20, which was about 6.44 mg.
As a result: the HPLC profile of the MK-7 standard is shown in FIG. 2, the retention time of MK-7 is 18.4, the HPLC profile of the MK06 conversion product is shown in FIG. 3, and the retention time is about 18.4, which is the peak of MK-7;
the MK-7 yields are as follows:
as shown in FIG. 4, under the condition that the concentration of the transformed bacterial liquid is OD600 ═ 10 whole-cell catalysis for 14h, the recombinant bacteria MK00 is 2.25 mu M, the intensity of idi is enhanced by MK00 to MKC1, the recombinant bacteria MKC1 is 16.6 mu M, and the yield of MK-7 is improved by 6.3 times. MKC 1-MK 01 changed species sources of MK-7 synthetic genes MenA and Ubie and the combination sequence of BsHepPPS, EcMenA and BsUbie, used E.coli-derived MenA and Bacillus subtilis-derived Ubie and HepPPS, and changed the sequence of genes on plasmid from EcMenA-BsHepPPS-EcUbie to BsHepPPS-EcMenA-BsUbie, and the gene engineering bacterium MK01 was 70.3 μ M, so that the yield of MK-7 was increased by more than 3 times. The genome of MK01 to MK02 strengthens Ubie and MetK, the recombinant bacterium MK02 is 86.8 mu M, and the yield of MK-7 is increased by 16 mu M.
As shown in fig. 5, under the condition that the concentration of the transformed bacterial liquid is OD600 ═ 20 whole cell catalysis for 9h, 53.8 μ M for recombinant bacterial MK02, 144 μ M for recombinant bacterial MK03 and 1.6-fold for MK-7 yield, was enhanced by the menF gene cluster through MK02 to MK 03; nlpI is knocked out from MK03 to MK04, the recombinant strain MK04 is 205 mu M, and the yield of MK-7 is improved by 60 mu M; the MK 03-MK 05 overexpress fadR, the recombinant strain MK05 is 198 mu M, and the yield of MK-7 is improved by more than 40 mu M; the MK03 to MK06 over-expresses Almgs, the recombinant bacterium MK06 is 185 mu M, and the yield of MK-7 is improved by more than 40 mu M; MK03 to MK07 over-express fadR and knock out nlpI, the recombinant bacterium MK07 is 224 muM, the yield of MK-7 is improved by 80 muM, the yield is the highest, and when the yield is 224 muM, the yield is equivalent to 22.6mg/g DCW according to the conversion relation of dry weight bacterium concentration.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
SEQUENCE LISTING
<110> institute of microbiology of Chinese academy of sciences
<120> MK-7 producing recombinant escherichia coli, and construction method and application thereof
<130> GNCFY192234
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 251
<212> PRT
<213> Bacillus subtilis 168)
<400> 1
Met Gln Asp Ile Tyr Gly Thr Leu Ala Asn Leu Asn Thr Lys Leu Lys
1 5 10 15
Gln Lys Leu Ser His Pro Tyr Leu Ala Lys His Ile Ser Ala Pro Lys
20 25 30
Ile Asp Glu Asp Lys Leu Leu Leu Phe His Ala Leu Phe Glu Glu Ala
35 40 45
Asp Ile Lys Asn Asn Asp Arg Glu Asn Tyr Ile Val Thr Ala Met Leu
50 55 60
Val Gln Ser Ala Leu Asp Thr His Asp Glu Val Thr Thr Ala Arg Val
65 70 75 80
Ile Lys Arg Asp Glu Asn Lys Asn Arg Gln Leu Thr Val Leu Ala Gly
85 90 95
Asp Tyr Phe Ser Gly Leu Tyr Tyr Ser Leu Leu Ser Glu Met Lys Asp
100 105 110
Ile Tyr Met Ile Arg Thr Leu Ala Thr Ala Ile Lys Glu Ile Asn Glu
115 120 125
His Lys Ile Arg Leu Tyr Asp Arg Ser Phe Lys Asp Glu Asn Asp Phe
130 135 140
Phe Glu Ser Val Gly Ile Val Glu Ser Ala Leu Phe His Arg Val Ala
145 150 155 160
Glu His Phe Asn Leu Pro Arg Trp Lys Lys Leu Ser Ser Asp Phe Phe
165 170 175
Val Phe Lys Arg Leu Met Asn Gly Asn Asp Ala Phe Leu Asp Val Ile
180 185 190
Gly Ser Phe Ile Gln Leu Gly Lys Thr Lys Glu Glu Ile Leu Glu Asp
195 200 205
Cys Phe Lys Lys Ala Lys Asn Ser Ile Glu Ser Leu Leu Pro Leu Asn
210 215 220
Ser Pro Ile Gln Asn Ile Leu Ile Asn Arg Leu Lys Thr Ile Ser Gln
225 230 235 240
Asp Gln Thr Tyr His Gln Lys Val Glu Glu Gly
245 250
<210> 2
<211> 348
<212> PRT
<213> Bacillus subtilis 168)
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Met Leu Asn Ile Ile Arg Leu Leu Ala Glu Ser Leu Pro Arg Ile Ser
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Asp Gly Asn Glu Asn Thr Asp Val Trp Val Asn Asp Met Lys Phe Lys
20 25 30
Met Ala Tyr Ser Phe Leu Asn Asp Asp Ile Asp Val Ile Glu Arg Glu
35 40 45
Leu Glu Gln Thr Val Arg Ser Asp Tyr Pro Leu Leu Ser Glu Ala Gly
50 55 60
Leu His Leu Leu Gln Ala Gly Gly Lys Arg Ile Arg Pro Val Phe Val
65 70 75 80
Leu Leu Ser Gly Met Phe Gly Asp Tyr Asp Ile Asn Lys Ile Lys Tyr
85 90 95
Val Ala Val Thr Leu Glu Met Ile His Met Ala Ser Leu Val His Asp
100 105 110
Asp Val Ile Asp Asp Ala Glu Leu Arg Arg Gly Lys Pro Thr Ile Lys
115 120 125
Ala Lys Trp Asp Asn Arg Ile Ala Met Tyr Thr Gly Asp Tyr Met Leu
130 135 140
Ala Gly Ser Leu Glu Met Met Thr Arg Ile Asn Glu Pro Lys Ala His
145 150 155 160
Arg Ile Leu Ser Gln Thr Ile Val Glu Val Cys Leu Gly Glu Ile Glu
165 170 175
Gln Ile Lys Asp Lys Tyr Asn Met Glu Gln Asn Leu Arg Thr Tyr Leu
180 185 190
Arg Arg Ile Lys Arg Lys Thr Ala Leu Leu Ile Ala Val Ser Cys Gln
195 200 205
Leu Gly Ala Ile Ala Ser Gly Ala Asp Glu Lys Ile His Lys Ala Leu
210 215 220
Tyr Trp Phe Gly Tyr Tyr Val Gly Met Ser Tyr Gln Ile Ile Asp Asp
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Ile Leu Asp Phe Thr Ser Thr Glu Glu Glu Leu Gly Lys Pro Val Gly
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Gly Asp Leu Leu Gln Gly Asn Val Thr Leu Pro Val Leu Tyr Ala Leu
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Lys Asn Pro Ala Leu Lys Asn Gln Leu Lys Leu Ile Asn Ser Glu Thr
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Thr Gln Glu Gln Leu Glu Pro Ile Ile Glu Glu Ile Lys Lys Thr Asp
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Ala Ile Glu Ala Ser Met Ala Val Ser Glu Met Tyr Leu Gln Lys Ala
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<210> 3
<211> 308
<212> PRT
<213> Escherichia coli K12(E.coli K12)
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Arg Pro Lys Thr Leu Pro Leu Ala Phe Ala Ala Ile Ile Val Gly Thr
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Ala Leu Ala Trp Trp Gln Gly His Phe Asp Pro Leu Val Ala Leu Leu
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Ala Leu Ile Thr Ala Gly Leu Leu Gln Ile Leu Ser Asn Leu Ala Asn
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Asp Tyr Gly Asp Ala Val Lys Gly Ser Asp Lys Pro Asp Arg Ile Gly
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Pro Leu Arg Gly Met Gln Lys Gly Val Ile Thr Gln Gln Glu Met Lys
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Arg Ala Leu Ile Ile Thr Val Val Leu Ile Cys Leu Ser Gly Leu Ala
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Leu Val Ala Val Ala Cys His Thr Leu Ala Asp Phe Val Gly Phe Leu
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Asn Arg Pro Tyr Gly Tyr Ile Gly Leu Gly Asp Ile Ser Val Leu Val
145 150 155 160
Phe Phe Gly Trp Leu Ser Val Met Gly Ser Trp Tyr Leu Gln Ala His
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Thr Leu Ile Pro Ala Leu Ile Leu Pro Ala Thr Ala Cys Gly Leu Leu
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Ala Arg Arg Tyr His Ala Cys Leu Leu Met Gly Ser Leu Val Cys Leu
225 230 235 240
Ala Leu Phe Asn Leu Phe Ser Leu His Ser Leu Trp Gly Trp Leu Phe
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Leu Leu Ala Ala Pro Leu Leu Val Lys Gln Ala Arg Tyr Val Met Arg
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Glu Met Asp Pro Val Ala Met Arg Pro Met Leu Glu Arg Thr Val Lys
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Gly Ala Leu Leu Thr Asn Leu Leu Phe Val Leu Gly Ile Phe Leu Ser
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Gln Trp Ala Ala
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<210> 4
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<212> PRT
<213> Bacillus subtilis 168)
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35 40 45
Ala Lys Ala Leu Asp Val Cys Cys Gly Thr Ala Asp Trp Thr Ile Ala
50 55 60
Leu Ala Lys Ala Ala Gly Lys Ser Gly Glu Ile Lys Gly Leu Asp Phe
65 70 75 80
Ser Glu Asn Met Leu Ser Val Gly Glu Gln Lys Val Lys Asp Gly Gly
85 90 95
Phe Ser Gln Ile Glu Leu Leu His Gly Asn Ala Met Glu Leu Pro Phe
100 105 110
Asp Asp Asp Thr Phe Asp Tyr Val Thr Ile Gly Phe Gly Leu Arg Asn
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Val Pro Asp Tyr Leu Thr Val Leu Lys Glu Met Arg Arg Val Val Lys
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Pro Gly Gly Gln Val Val Cys Leu Glu Thr Ser Gln Pro Glu Met Phe
145 150 155 160
Gly Phe Arg Gln Ala Tyr Phe Met Tyr Phe Lys Tyr Ile Met Pro Phe
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Phe Gly Lys Leu Phe Ala Lys Ser Tyr Lys Glu Tyr Ser Trp Leu Gln
180 185 190
Glu Ser Ala Arg Asp Phe Pro Gly Met Lys Glu Leu Ala Gly Leu Phe
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<210> 5
<211> 756
<212> DNA
<213> Bacillus subtilis 168)
<400> 5
atgcaagaca tctacggaac tttagccaat ctgaacacga aattaaaaca aaagctgtct 60
catccttatt tagcgaagca tatttctgcg ccgaaaattg atgaggataa gcttcttctt 120
tttcatgctt tatttgaaga agccgacata aaaaacaacg acagagaaaa ttatattgta 180
acagcgatgc ttgtacaaag cgcccttgat acccatgatg aagtgacgac agctagagtc 240
ataaaacgag acgaaaacaa aaaccgccaa ttgactgttc tcgcgggcga ttatttcagc 300
gggctgtact actctttact atctgaaatg aaggatatct acatgattcg gacgcttgct 360
acagccatta aagaaatcaa cgaacataaa attcgtctgt atgaccgttc tttcaaggac 420
gaaaacgatt ttttcgaaag tgtcggcatc gttgaatcag ctttattcca tcgtgtggcg 480
gaacacttca acctcccgcg ctggaaaaag ctgtcgagtg atttttttgt atttaagcgg 540
cttatgaacg gaaatgatgc atttctggat gtgatcggca gttttataca gctgggaaaa 600
acaaaagaag agatattaga agattgtttt aaaaaagcga aaaacagcat tgagtcactt 660
ctgcctctaa attcacctat tcagaacatt ttaataaacc gtctgaagac aatcagccaa 720
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<210> 6
<211> 1047
<212> DNA
<213> Bacillus subtilis 168)
<400> 6
atgttaaata tcattcgttt actggcggag tcgctgccac gcatatcgga tggaaatgaa 60
aacacagatg tttgggtgaa tgatatgaaa tttaaaatgg cctactcttt tttaaatgac 120
gatattgatg taatcgaaag agaacttgaa caaaccgtac gttccgatta cccgctttta 180
agcgaggcag gtcttcacct gctgcaggcc ggagggaaac gtattcggcc tgttttcgtg 240
ctgctttctg gcatgtttgg cgattatgat attaataaga ttaaatatgt cgccgtcact 300
ctggaaatga ttcacatggc atctttggtt catgatgatg tcattgatga tgcagagctt 360
cgccgaggaa aaccgacaat caaagcaaag tgggacaatc gtattgcgat gtacacaggc 420
gattatatgc ttgcgggatc tcttgaaatg atgacgagaa ttaacgaacc gaaagcccat 480
aggattttgt cacagacgat cgttgaagtt tgtctagggg aaattgagca gatcaaagac 540
aaatacaaca tggaacaaaa tctcagaacg tatctccgcc gtatcaaaag aaaaacagct 600
ctcttgatcg cggtcagctg ccagcttggt gccattgcgt ctggagctga tgagaagatt 660
cataaggcat tgtactggtt tgggtattac gtcggcatgt cttatcagat tattgatgat 720
attcttgatt ttacttcaac tgaggaagag ctgggtaaac ccgtaggagg agatttgctt 780
caaggaaacg tcacattgcc agtgctgtat gccctgaaaa atcctgcatt aaaaaaccag 840
cttaaattga ttaacagtga gacaacgcag gaacagcttg aaccaatcat tgaagaaatc 900
aaaaaaacag atgcaattga agcatctatg gcagtaagcg aaatgtatct gcagaaagct 960
tttcagaaat taaacacgct tcctcgaggg cgcgcacgct cgtctcttgc agccatcgca 1020
aaatatatcg gtaaaagaaa attttaa 1047
<210> 7
<211> 927
<212> DNA
<213> Escherichia coli K12(E.coli K12)
<400> 7
atgactgaac aacaaattag ccgaactcag gcgtggctgg aaagtttacg acctaaaacc 60
ctccccctcg cctttgctgc aattatcgtc gggacagcgc tggcatggtg gcaaggtcac 120
ttcgatccgc tggtcgccct gctggcacta attaccgccg ggctattaca gatcctttct 180
aacctcgcca atgattacgg cgatgcggta aaaggcagcg ataaacctga ccgcattggg 240
ccgctacgcg gcatgcaaaa aggggtcatt acccagcaag agatgaaacg ggcgctcatt 300
attaccgtcg tgctcatctg tctctccggg ctggcactgg ttgcagtggc atgccatacg 360
ctggccgatt ttgtcggttt cctgattctt ggcgggttgt cgatcattgc cgctatcacc 420
tacaccgtgg gcaatcgtcc ttatggttat atcggtctgg gtgatatttc cgtactggtt 480
ttctttggct ggttgagtgt catggggagc tggtatttac aggctcatac attgattccg 540
gcactgatcc ttccggcgac cgcatgcggc ctgctggcaa cggcagtact gaatattaat 600
aacctgcgtg atatcaatag cgaccgcgaa aatggcaaaa acacgctggt ggtgcgctta 660
ggtgaagtga acgcgcgtcg ttatcatgcc tgcctgctga tgggctcgct ggtgtgtctg 720
gcgctgttta atctcttttc gctgcatagc ctgtggggct ggctgttcct gctggcggca 780
ccattactgg tgaagcaagc ccgttatgtg atgcgggaaa tggacccggt ggcgatgcga 840
ccaatgctgg aacgtactgt caagggagcg ttactgacta acctgctgtt tgttttaggg 900
atattcctaa gccagtgggc agcataa 927
<210> 8
<211> 702
<212> DNA
<213> Bacillus subtilis 168)
<400> 8
atgcaggact caaaagaaca gcgcgtacac ggagtatttg aaaaaatata taaaaattat 60
gaccaaatga actctgtcat cagttttcag cagcataaaa aatggcgcga caaaacgatg 120
cgcatcatga atgtaaagga aggcgcaaaa gcacttgatg tctgctgcgg aacggctgac 180
tggacgatcg ctcttgcaaa agcggcaggc aaaagcggcg agatcaaggg cttggatttc 240
agtgaaaata tgctgagtgt cggcgagcag aaagtaaaag acggcggatt cagccaaatt 300
gaactgctgc acggaaatgc gatggagctt ccttttgatg atgatacatt tgattatgtc 360
accattggct tcggactccg caatgtccct gattacttga ctgtactgaa agagatgaga 420
cgtgtagtga agccgggcgg gcaggtggta tgtctggaaa cgtcccagcc ggaaatgttc 480
ggattcagac aggcttactt tatgtacttt aagtatatta tgccgttttt cgggaaactg 540
tttgcgaaga gctataaaga atattcttgg cttcaagaat cagccagaga tttccctgga 600
atgaaggaac tggcaggcct gtttgaagag gcgggcctga aaaatgttaa atatcattcg 660
tttactggcg gagtcgctgc cacgcatatc ggatggaaat ga 702
<210> 9
<211> 398
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Arg Ile Gly Ile Phe Ser Glu Ala Tyr Leu Pro Leu Ile Ser Gly
1 5 10 15
Val Val Thr Ser Val Val Asn Leu Lys Glu Gly Leu Glu Ala Leu Gly
20 25 30
His Glu Val Tyr Val Ile Thr Pro Ile Pro Ser Lys Asp Lys Phe Glu
35 40 45
Asn Asp Pro Ser Val Ile Arg Ile Pro Gly Trp Val Ile Pro Arg Lys
50 55 60
Ser Leu Lys Gly Phe Arg Leu Val Leu Phe Val Lys Arg Tyr Val Arg
65 70 75 80
Lys Met Arg Lys Leu Lys Leu Asp Val Val His Ile His Thr Glu Phe
85 90 95
Ser Met Gly Lys Leu Gly Leu Ala Val Ala Lys Lys Glu Arg Ile Pro
100 105 110
Ser Val Tyr Thr Leu His Thr Ser Tyr Gln Asp Tyr Thr His Tyr Val
115 120 125
Ser Lys Leu Leu Thr Arg Phe Ala Pro Asn Ala Ala Lys Lys Leu Ala
130 135 140
Gly Lys Ile Asn Asn Gln Tyr Thr Lys Asn Cys His Met Thr Ile Val
145 150 155 160
Pro Thr Lys Lys Ile Tyr Asp Lys Met Ile Arg Leu Lys His Asp Gly
165 170 175
Glu Phe Thr Ile Ile Pro Ser Gly Ile Asn Leu Lys Pro Phe Tyr Lys
180 185 190
Ser Ser Tyr Thr Ser Glu Gln Val Gln Ala Leu Lys Asp Lys Leu Gly
195 200 205
Ile Arg Asn Asp Glu Phe Val Ala Ile Leu Val Ala Arg Ile Ala Lys
210 215 220
Glu Lys Ser Ile Gly Asp Leu Val Glu Ala Phe Val Glu Phe Tyr Lys
225 230 235 240
Ser Tyr Pro Asn Ser Arg Phe Ile Ile Ile Gly Asp Gly Pro Asp Lys
245 250 255
Pro Val Leu Asp Lys Leu Ile Asp Ser Lys Lys Ala Ser Lys Tyr Ile
260 265 270
Asn Thr Leu Gly Phe Val Lys Asn Ala Glu Val Gly Leu Tyr Tyr Gln
275 280 285
Ile Ala Asp Val Phe Leu Asn Ala Ser Thr Thr Glu Thr Gln Gly Leu
290 295 300
Thr Tyr Val Glu Ala Leu Ala Ala Ser Leu Pro Ile Ile Val Arg Tyr
305 310 315 320
Asp Asp Val Phe Asp Ala Phe Val Glu Asp Gly Lys Asn Gly Ile Phe
325 330 335
Phe Asn Lys Asn Glu Glu Leu Val Lys His Leu Ile His Ile Arg Gln
340 345 350
Asn Pro Glu Ile Leu Gly Thr Leu Ser Lys Asn Ala Glu Ile Ser Thr
355 360 365
Lys Pro Tyr Ala Lys Glu Val Tyr Ala Lys Ser Cys Glu Thr Leu Tyr
370 375 380
Leu Asp Leu Ile Asp Lys Asn Asn Lys Lys Leu Asn Lys Lys
385 390 395
<210> 10
<211> 1197
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgcgtattg gtatcttcag cgaggcgtat ctgccgctga ttagcggcgt ggttaccagc 60
gtggttaacc tgaaagaggg tctggaagcg ctgggccacg aggtgtacgt tattaccccg 120
atcccgagca aggacaaatt tgaaaacgat ccgagcgtta ttcgtatccc gggttgggtg 180
atcccgcgta agagcctgaa aggcttccgt ctggtgctgt ttgttaagcg ttatgttcgt 240
aaaatgcgta agctgaaact ggacgtggtt cacattcaca ccgagttcag catgggtaag 300
ctgggcctgg cggttgcgaa gaaagaacgt atcccgagcg tgtacaccct gcacaccagc 360
taccaggatt atacccacta cgttagcaaa ctgctgaccc gttttgcgcc gaacgcggcg 420
aagaaactgg cgggcaagat caacaaccaa tacaccaaga actgccacat gaccatcgtg 480
ccgaccaaga aaatttacga caagatgatc cgtctgaaac acgatggcga gttcaccatc 540
attccgagcg gcatcaacct gaagccgttt tataaaagca gctacaccag cgaacaggtt 600
caagcgctga aggacaaact gggtattcgt aacgatgagt tcgtggcgat tctggttgcg 660
cgtatcgcga aggagaagag catcggcgac ctggtggagg cgttcgttga attttataaa 720
agctacccga acagccgttt tatcattatc ggtgacggcc cggataagcc ggtgctggac 780
aaactgattg atagcaagaa agcgagcaag tatatcaaca ccctgggttt cgtgaaaaac 840
gcggaagtgg gcctgtacta tcagattgcg gatgtttttc tgaacgcgag caccaccgag 900
acccaaggtc tgacctatgt ggaagcgctg gcggcgagcc tgccgattat cgtgcgttac 960
gacgatgttt tcgacgcgtt tgtggaagat ggcaagaacg gcatcttctt taacaagaac 1020
gaggaactgg ttaaacacct gattcacatc cgtcagaacc cggagattct gggcaccctg 1080
agcaaaaacg cggaaatcag caccaagccg tatgcgaaag aggtgtacgc gaagagctgc 1140
gaaaccctgt acctggacct gatcgataaa aacaacaaga aactgaacaa gaaataa 1197
<210> 11
<211> 178
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ttatcaaaaa gagtattgac ataaagtcta acctatagat aattacagcc atcgagaggg 60
acacggcgat ttgctgtcac cggatgtgct ttccggtctg atgagtccgt gaggacgaaa 120
cagcctctac aaataatttt gtttaagaat tcaaaagatc ttttaagaag gagatata 178
<210> 12
<211> 1308
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc 60
cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt 120
cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 180
agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 240
tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 300
taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 360
gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 420
cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 480
gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540
gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600
ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660
cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720
agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780
gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840
ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900
ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960
ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020
attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080
ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140
tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200
ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260
ctactgtttc tccatacccg ttttttgggc taacaggagg aattaacc 1308

Claims (7)

1. The construction method of the recombinant escherichia coli is characterized in that: the construction method comprises the steps of introducing genes related to MK-7 synthesis into a receptor bacterium to obtain recombinant escherichia coli; the recipient bacterium is any one of Escherichia coli mutants M1-M6;
wherein the genes involved in MK-7 synthesis are heptapolyisoprene pyrophosphate synthase gene, 1, 4-dihydroxydinaphthoic acid octapolyisoprene transferase gene and S-adenosyl-L-methionine/demethylnaphthoquinone methyltransferase gene;
the Escherichia coli mutant M1 is obtained by modifying wild Escherichia coli M1) -M8);
the Escherichia coli mutant M2 is obtained by modifying wild Escherichia coli M1) -M7);
the Escherichia coli mutant M3 is obtained by modifying wild Escherichia coli with the following M1) -M6) and M8);
the Escherichia coli mutant M4 is obtained by modifying wild Escherichia coli M1) -M6);
the Escherichia coli mutant M5 is obtained by modifying wild Escherichia coli M1) -M5);
the Escherichia coli mutant M6 is obtained by modifying wild Escherichia coli M1) -M3);
the modifications of m1) -m8) are respectively as follows:
m1) overexpressing the isopentenyl pyrophosphate isomerase gene;
m2) knockout of msbB gene
m3) introducing genes MvaE, MvaS, MVK, PMK and MVD related to the heterologous mevalonate pathway;
m4) overexpressing the methionine adenosyltransferase gene;
m5) overexpressing the S-adenosyl-L-methionine/desmethyl naphthoquinone methyltransferase gene;
m6) overexpressing an isochorismate synthase gene;
m7) overexpressing a DNA-junction transcriptional dual regulatory protein gene;
m8) knocking out nlpI gene;
the hepta-isoprene pyrophosphate synthetase gene comprises a HepS gene and a HepT gene, and the protein coded by the HepS gene is the protein coded by the DNA molecule shown in SEQ ID No. 5;
the protein coded by the HepT gene is the protein coded by the DNA molecule shown in SEQ ID No. 6;
the protein coded by the 1, 4-dihydroxy-dinaphthoic acid octa-polyprenyl transferase gene is the protein coded by a DNA molecule shown in SEQ ID No. 7;
the protein coded by the S-adenosine-L-methionine/demethyl naphthoquinone methyltransferase gene is the protein coded by the DNA molecule shown in SEQ ID No. 8;
the protein coded by the isopentenyl pyrophosphate isomerase gene is a protein coded by a DNA molecule shown in the GenBank No. NC-000913.3 No. 3033065-3033613;
the MvaE is a DNA molecule shown in the 1338888-1341299 position of GeneBank No. NC-004668.1;
the MvaS is a DNA molecule shown in the position 1337551-1338702 of GeneBank number NC-004668.1;
the MVK is a DNA molecule shown in the position 2101873-2102778 of the GeneBank number NC-003901.1;
the PMK is a DNA molecule shown in GeneBank with the number of DQ 331671.1;
the MVD is a DNA molecule shown in the 701895-703085 site of GeneBank No. NC-001146.8;
the protein coded by the methionine adenosyltransferase gene is the protein coded by the DNA molecule shown in the position with the GenBank number of NC-000913.3 No. 3086706-3087860;
the protein coded by the isochorismate synthetase gene is the protein coded by the DNA molecule shown in the GenBank number NC-000913.3 2379348-2380643;
the protein coded by the DNA jointing transcription dual regulatory protein gene is the protein coded by the DNA molecule shown in the GenBank number NC-000913.3 No. 1234938-1235657;
the wild type escherichia coli is escherichia coli K12BW 25113.
2. The construction method of the recombinant escherichia coli is characterized in that: the construction method comprises the steps of introducing genes related to MK-7 synthesis into a receptor bacterium to obtain recombinant escherichia coli; the recipient bacterium is an escherichia coli mutant;
wherein the genes involved in MK-7 synthesis are heptapolyisoprene pyrophosphate synthase gene, 1, 4-dihydroxydinaphthoic acid octapolyisoprene transferase gene, S-adenosyl-L-methionine/demethylnaphthoquinone methyltransferase gene and 1, 2-diacylglycerol 3-glucosyltransferase gene;
the Escherichia coli mutant is obtained by transforming wild Escherichia coli m1) -m 6);
the modifications of m1) -m6) are respectively as follows:
m1) overexpressing the isopentenyl pyrophosphate isomerase gene;
m2) knockout of msbB gene
m3) introducing genes MvaE, MvaS, MVK, PMK and MVD related to the heterologous mevalonate pathway;
m4) overexpressing the methionine adenosyltransferase gene;
m5) overexpressing the S-adenosyl-L-methionine/desmethyl naphthoquinone methyltransferase gene;
m6) overexpressing an isochorismate synthase gene;
the hepta-isoprene pyrophosphate synthetase gene comprises a HepS gene and a HepT gene, wherein the protein coded by the HepS gene is the protein coded by the DNA molecule shown in SEQ ID No. 5;
the protein coded by the HepT gene is the protein coded by the DNA molecule shown in SEQ ID No. 6;
the protein coded by the 1, 4-dihydroxy dinaphthoic acid octa-polyisoprene transferase gene is the protein coded by the DNA molecule shown in SEQ ID No. 7;
the protein coded by the S-adenosine-L-methionine/demethyl naphthoquinone methyltransferase gene is the protein coded by the DNA molecule shown in SEQ ID No. 8;
the protein coded by the 1, 2-diacylglycerol 3-glucosyltransferase gene is the protein coded by the DNA molecule shown in SEQ ID No. 10;
the protein coded by the isopentenyl pyrophosphate isomerase gene is a protein coded by a DNA molecule shown in the GenBank No. NC-000913.3 No. 3033065-3033613;
the MvaE is a DNA molecule shown in the 1338888-1341299 position of GeneBank number NC-004668.1;
the MvaS is a DNA molecule shown in the position 1337551-1338702 of GeneBank number NC-004668.1;
the MVK is a DNA molecule shown in the position 2101873-2102778 of the GeneBank number NC-003901.1;
the PMK is a DNA molecule shown in GeneBank with the number of DQ 331671.1;
the MVD is a DNA molecule shown in the 701895-703085 site of the GeneBank number NC-001146.8;
the protein coded by the methionine adenosyltransferase gene is the protein coded by the DNA molecule shown in the position with the GenBank number of NC-000913.3 No. 3086706-3087860;
the protein coded by the gene of the isochorismate synthetase is the protein coded by the DNA molecule shown in the position 2379348 and 2380643 of the GenBank number NC-000913.3;
the wild type escherichia coli is escherichia coli K12BW 25113.
3. The construction method according to claim 1 or 2, characterized in that: the heptameric isoprene pyrophosphate synthetase gene, the S-adenosine-L-methionine/demethyl naphthoquinone methyltransferase gene and the 1, 4-dihydroxy dinaphthoic acid octa-polyisoprene transferase gene are constructed in an expression cassette, the positional relationship of the three genes in the expression cassette is that the heptameric isoprene pyrophosphate synthetase gene is positioned at the upstream of the 1, 4-dihydroxy dinaphthoic acid octa-polyisoprene transferase gene, and the S-adenosine-L-methionine/demethyl naphthoquinone methyltransferase gene is positioned at the downstream of the 1, 4-dihydroxy dinaphthoic acid octa-polyisoprene transferase gene.
4. The construction method according to claim 1 or 2, characterized in that:
the protein coded by the msbB gene is a protein coded by a DNA molecule shown in the sequence 1939222-1940193 with the GenBank number of NC-000913.3;
the protein coded by the nlpI gene is a protein coded by a DNA molecule shown in the position 3308040-3308924 of GenBank No. NC-000913.3.
5. A recombinant Escherichia coli constructed by the method according to any one of claims 1 to 4.
6. Use of the recombinant E.coli of claim 5 for the preparation of MK-7.
7. A method of making MK-7, characterized by: comprising catalyzing a glucose reaction with the recombinant Escherichia coli of claim 5 to obtain MK-7.
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