CN117946984A - Pantothenate synthetase mutant and preparation method thereof, construction method thereof, pantothenate production strain and application thereof, and pantothenate preparation method - Google Patents

Pantothenate synthetase mutant and preparation method thereof, construction method thereof, pantothenate production strain and application thereof, and pantothenate preparation method Download PDF

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CN117946984A
CN117946984A CN202410349134.8A CN202410349134A CN117946984A CN 117946984 A CN117946984 A CN 117946984A CN 202410349134 A CN202410349134 A CN 202410349134A CN 117946984 A CN117946984 A CN 117946984A
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pantothenate
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CN117946984B (en
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钟锦潮
马立清
陈必钦
张璐
赵珍
朱志春
祝俊
李丹
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Jindawei Biotechnology Jiangsu Co ltd
INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd
Xiamen Kingdomway Group Co
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Abstract

The invention belongs to the technical field of D-pantothenic acid synthesis, and discloses a pantothenic acid synthetase mutant, a preparation method, a construction body and a construction method thereof, a pantothenic acid production strain and application and a pantothenic acid preparation method. The pantothenate synthetase mutant is an original pantothenate synthetase comprising a T29K mutation and/or an L135P mutation, the original pantothenate synthetase comprising an amino acid fragment having a sequence as set forth in SEQ ID NO. 1 and/or a variant sequence having at least 80% homology with the sequence set forth in SEQ ID NO. 1. Compared with the original pantothenic acid synthetase, the pantothenic acid synthetase mutant provided by the invention has obviously enhanced enzyme activity, can enhance the capability of the strain for metabolizing and synthesizing pantothenic acid when being applied to the construction of pantothenic acid fermentation strains, and has good application prospect.

Description

Pantothenate synthetase mutant and preparation method thereof, construction method thereof, pantothenate production strain and application thereof, and pantothenate preparation method
Technical Field
The invention belongs to the technical field of D-pantothenic acid synthesis, and particularly relates to a pantothenic acid synthetase mutant, a preparation method, a construct and a construction method thereof, a pantothenic acid production strain and application and a pantothenic acid preparation method.
Background
Pantothenic acid (pantothenic acid), also known as vitamin B5, a polyacid, is a constituent of CoA and Acyl Carrier Protein (ACP); it mainly has the functions of participating in vivo energy production, helping cell formation, fat and sugar conversion, antibody synthesis, maintaining adrenal gland normal function and the like, and is one of the necessary nutrient substances for the normal growth of organisms.
Wherein, the natural pantothenic acid (D-pantothenic acid) has right optical activity, is an important food additive and feed additive, is also an important vitamin medicament, and is clinically used for treating diseases such as vitamin B deficiency, peripheral neuritis, postoperative ileus, streptomycin poisoning, rheumatoid disease and the like. The D-pantothenic acid biosynthesis pathway includes pantoic acid synthesis, beta-alanine synthesis, and condensation of pantoic acid and beta-alanine under pantothenic acid synthase (panC), and the biosynthesis pathway mainly includes pantoic acid biosynthesis pathway and beta-alanine biosynthesis pathway. Therefore, enhancing the expression of key genes in the favorable product synthesis pathway is one of the important ideas for strain engineering.
At present, along with the completion of whole genome sequencing and genetic engineering annotation, metabolic engineering transformation is carried out on microorganisms by genetic engineering means, and a method for constructing D-pantothenate producing bacteria is becoming more and more important. The high-efficiency escherichia coli protein expression system is adopted in CN106676051A, a plurality of pantothenic acid synthetases (panC) with different sources are expressed in a heterologous way, so that a pantothenic acid synthetase strain with high activity is obtained, and the engineering bacteria are fermented for 38 hours to produce 101.2g/L pantothenic acid. CN109868254A repairs ilvG gene weakening feedback regulation and strengthening pantoic acid synthesis path by strengthening expression of key enzymes PanB, panC, panE and IlvC in D-pantothenic acid biological generation path, knockout avtA and knockout ilvE weaken competition branch to obtain a plasmid-free high-yield strain, D-pantothenic acid titer is increased from 0.48g/L to 1.54g/L. However, the resulting D-pantothenate producing bacteria have low pantothenate synthase activity, which is detrimental to efficient pantothenate synthesis and still have significant limitations.
Disclosure of Invention
The invention aims to overcome the defect of low pantothenic acid synthetase activity in the existing D-pantothenic acid producing bacteria and realize efficient synthesis of pantothenic acid, and provides a pantothenic acid synthetase mutant and a preparation method, a construction body and a construction method thereof, a pantothenic acid producing strain and application and a pantothenic acid preparation method.
In a first aspect, the invention provides a mutant pantothenate synthetase comprising a T29K mutation and/or an L135P mutation, wherein the original pantothenate synthetase comprises an amino acid fragment having a sequence as set forth in SEQ ID NO. 1.
In some embodiments, the pantothenate synthetase mutants include a T29K mutation and the pantothenate synthetase mutants include an amino acid fragment having a sequence as set forth in SEQ ID NO. 2.
In some embodiments, the pantothenate synthetase mutants include a T135K mutation and the pantothenate synthetase mutants include an amino acid fragment having a sequence as set forth in SEQ ID NO. 3.
In some embodiments, the pantothenate synthetase mutants include a T29K mutation and an L135P mutation, and the pantothenate synthetase mutants include an amino acid fragment having a sequence as set forth in SEQ ID NO. 4.
In a second aspect, the present invention provides a construct comprising a nucleotide fragment encoding the above pantothenate synthetase mutant.
In some embodiments, the construct comprises one or more of the nucleotide fragments set forth in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7.
In a third aspect, the present invention provides a pantothenate producing strain capable of expressing the above pantothenate synthetase mutants.
In a fourth aspect, the present invention provides a method for constructing the construct according to the second aspect of the present invention, which comprises recombining a gene encoding an original pantothenate synthetase of a marine propionic acid bacterium (Propionigenium maris) into a plasmid vector to obtain an original plasmid, and performing site-directed mutagenesis on the obtained original plasmid using a mutation primer to obtain the construct.
In some specific embodiments, the mutation primer comprises a T29K mutation primer and/or an L135P mutation primer; the T29K mutant primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 8 and/or a reverse primer with a nucleotide sequence shown as SEQ ID NO. 9; the L135P mutation primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 10 and/or a reverse primer with a nucleotide sequence shown as SEQ ID NO. 11.
In a fifth aspect, the present invention provides a process for the preparation of a pantothenate synthetase mutant, comprising transforming a host cell with the above construct to obtain a pantothenate producing strain; pantothenate synthase mutants are expressed using pantothenate producing strains.
In some specific embodiments, the host cell is selected from one or more of E.coli FV5069, E.coli FV5069/pFV31 or E.coli MG 1655.
In some specific embodiments, the construct is contained on a pTrc-cycA plasmid vector.
In a sixth aspect, the present invention provides the use of the above pantothenate synthetase mutants and/or pantothenate producing strains in fermentation culture for the synthesis of D-pantothenate.
In a seventh aspect, the present invention provides a process for producing pantothenic acid, comprising: beta-alanine is fermented in the presence of the above-described pantothenate synthetase mutants and/or pantothenate producing strains to give pantothenate.
The beneficial effects are that:
Compared with the original pantothenic acid synthetase, the obtained pantothenic acid synthetase mutant has obviously enhanced enzyme catalytic activity by introducing T29K and/or L135P mutation into the original pantothenic acid synthetase, and the pantothenic acid synthetase mutant can be applied to construction of pantothenic acid fermentation strains, so that the capacity of synthesizing and expressing D-pantothenic acid of the strains can be enhanced, and the pantothenic acid synthetase mutant has good application prospect.
Detailed Description
In the invention, based on the deep understanding of the three-dimensional structure and the catalytic activity of pantothenic acid synthetase, the inventor takes the original pantothenic acid synthetase with specific amino acid fragments as a mutation object, and determines the mutation amino acid residue position and mutation type through intensive and extensive research, and finally constructs and obtains the pantothenic acid synthetase mutant with high enzyme activity.
In the present invention, the raw pantothenate synthetase can be derived from marine propionic acid bacterium (Propionigenium maris). The original pantothenic acid synthetase derived from the marine propionic acid bacterium (Propionigenium maris) refers to pantothenic acid synthetase synthetically expressed in the marine propionic acid bacterium cells, and the original pantothenic acid synthetase has at least an amino acid fragment with a sequence shown as SEQ ID NO. 1 and/or a variant sequence with at least 80% homology with the sequence shown as SEQ ID NO. 1. Wherein the variant sequence is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1, and the substitution, deletion or addition does not affect the biological activity of pantothenic acid synthetase.
In the present invention, numbering of amino acid residue positions is carried out with reference to the amino acid sequence of SEQ ID NO. 1, said pantothenate synthetase mutant comprising a T29K mutation and/or an L135P mutation as compared to the original pantothenate synthetase.
In the present invention, the T29K mutation is defined as a mutation of the amino acid residue at position 29 of SEQ ID NO. 1 from threonine to lysine. The L135P mutation is defined as a leucine to proline mutation of the amino acid residue at position 135 of SEQ ID NO. 1.
In some embodiments, the pantothenase mutant has a T29K mutation, and the pantothenase mutant has at least an amino acid fragment having a sequence as set forth in SEQ ID NO.2 and/or a variant sequence having at least 90% homology with the sequence set forth in SEQ ID NO. 2. Wherein the variant sequence is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO.2, and the substitution, deletion or addition does not affect the biological activity of pantothenic acid synthetase, namely the amino acid fragment shown in the sequence shown in SEQ ID NO.2 has the same biological activity as the variant sequence. In some preferred embodiments, the pantothenate synthetase mutants have at least the amino acid fragment sequence shown in SEQ ID NO. 2.
In some embodiments, the pantothenase mutant has an L135P mutation, and the pantothenase mutant has at least an amino acid fragment having a sequence as set forth in SEQ ID NO. 3 and/or a variant sequence having at least 90% homology with the sequence set forth in SEQ ID NO. 3. Wherein the variant sequence is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 3, and the substitution, deletion or addition does not affect the biological activity of pantothenic acid synthetase, namely the amino acid fragment shown in the sequence shown in SEQ ID NO. 3 has the same biological activity as the variant sequence. In some preferred embodiments, the pantothenate synthetase mutants have at least the amino acid fragment sequence set forth in SEQ ID NO. 3.
In some embodiments, the pantothenase mutant has a T29K mutation and an L135P mutation, and the pantothenase mutant has at least an amino acid fragment having a sequence as set forth in SEQ ID NO. 4 and/or a variant sequence having at least 90% homology with the sequence set forth in SEQ ID NO. 4. Wherein the variant sequence is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 4, and the substitution, deletion or addition does not affect the biological activity of pantothenic acid synthetase, namely the amino acid fragment shown in the sequence shown in SEQ ID NO. 4 has the biological activity equivalent to that of the variant sequence. In some preferred embodiments, the pantothenate synthetase mutants have at least the amino acid fragment sequence shown in SEQ ID NO. 4.
In the invention, the T26K and L135P mutations generated by the original pantothenate synthetase can jointly influence the space structure formation of the active site and the conformational change in catalyzing the D-pantothenate synthesis, and compared with single T26K mutation and L135P mutation, the affinity of the enzyme active site to ATP can be better increased, the catalysis efficiency can be improved, and the activity of the pantothenate enzyme mutant with higher enzyme activity can be obtained.
In the present invention, for the purpose of obtaining the above pantothenate synthetase mutants, there is provided a construct comprising at least a nucleotide fragment encoding the above pantothenate synthetase mutants.
In the present invention, the construct is a type of biochemical substance carrying genetic information, and may specifically be, but not limited to, one or more of DNA, RNA or cDNA.
In some embodiments, the construct encodes a mutant pantothenate synthetase having a T29K mutation, which construct has at least a nucleotide fragment having a sequence set forth in SEQ ID NO. 5 and/or a variant sequence having at least 70% homology with the sequence set forth in SEQ ID NO. 5. Wherein the variant sequence is obtained by substituting one or more nucleotides for the sequence shown in SEQ ID NO. 5; the substitutions that occur do not alter the encoded amino acids and do not affect the progress of transcription and translation.
In some embodiments, the construct encodes a mutant pantothenate synthetase having an L135P mutation, which construct has at least a nucleotide fragment having a sequence as set forth in SEQ ID NO. 6 and/or a variant sequence having at least 70% homology to the sequence set forth in SEQ ID NO. 6. Wherein the variant sequence is obtained by substituting one or more nucleotides for the sequence shown in SEQ ID NO. 6, and the substitution does not change the encoded amino acid and does not affect the transcription and translation.
In some embodiments, the construct encodes a mutant pantothenate synthetase having T29K and L135P mutations, and has at least a nucleotide fragment having a sequence as set forth in SEQ ID NO. 7 and/or a variant sequence having at least 70% homology with the sequence set forth in SEQ ID NO. 7. Wherein the variant sequence is obtained by substituting one or more nucleotides for the sequence shown in SEQ ID NO. 7, the substitution does not change the encoded amino acid and does not affect the transcription and translation.
In the present invention, the constructs also include nucleotide sequences that perform different functions. In some embodiments, the nucleotide sequences that perform different functions may specifically be, but are not limited to, one or more of regulatory sequences such as promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, homologous recombination sites, poly (A) signals, and the like, for both coding and non-coding regions. In some embodiments, the nucleotide sequences that perform the different functions may specifically be, but are not limited to, one or more of the NdeI cleavage site, xhoI cleavage site, bamHI cleavage site, hindII cleavage site, and the like.
In the invention, based on the purpose of obtaining the pantothenate synthetase mutant, a construction method of a construction body is provided, wherein the construction method comprises the steps of recombining a gene fragment encoding the original pantothenate synthetase of marine propionic acid bacteria (Propionigenium maris) into a plasmid vector to obtain an original plasmid, and carrying out site-directed mutagenesis on the obtained original plasmid by using a mutation primer to obtain the construction body.
In the present invention, the gene fragment encoding the original pantothenic acid synthetase of Marine propionic acid bacterium (Propionigenium maris) may be obtained by directly extracting from marine propionic acid bacterium (Propionigenium maris), or may be obtained by performing codon optimization according to the protein sequence of the original pantothenic acid synthetase and then performing gene synthesis technology.
In some embodiments, the gene fragment encoding the original pantothenate synthetase of marine propionic acid bacterium (Propionigenium maris) is preferably obtained by gene synthesis techniques after codon optimization based on the protein sequence of the original pantothenate synthetase, and comprises at least the nucleotide sequence set forth in SEQ ID NO. 12.
In the present invention, the plasmid vector is preferably a pET series plasmid, and specifically can be, but is not limited to, one or more of pET-3a, pET-3b, pET-3c, pET-24a, pET-24b or pET-24 c.
In the invention, the mutation primer is an oligonucleotide containing a predetermined mutation site, and specifically comprises a T29K mutation primer and/or an L135P mutation primer.
In the present invention, the T29K mutation primer is defined as an oligonucleotide capable of directed mutation of the codon of the 29 th amino acid residue in the fragment of the gene encoding the original pantothenate synthetase of marine propionic acid bacterium to a codon encoding lysine. The L135P mutation primer is defined as an oligonucleotide capable of performing a directed mutation of the codon encoding the 135 th amino acid residue in the fragment of the original pantothenate synthetase gene of marine propionic acid bacterium to a codon encoding proline.
In some specific embodiments, the T29K mutant primer comprises a forward primer having a nucleotide sequence shown in SEQ ID NO. 8 and a reverse primer having a nucleotide sequence shown in SEQ ID NO. 9.
In some specific embodiments, the L135P mutant primer comprises a forward primer having a nucleotide sequence shown as SEQ ID NO. 10 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 11.
In the present invention, site-directed mutagenesis is performed on the original plasmid using a T29K mutant primer to obtain a construct encoding a mutant pantothenate synthase having a T29K mutation.
In the present invention, site-directed mutagenesis is performed on the original plasmid using an L135P mutation primer to obtain a construct encoding a mutant pantothenate synthase having an L135P mutation.
In the present invention, site-directed mutagenesis is performed on the original plasmid using T29K mutation primers and L135P mutation primers to obtain constructs encoding pantothenate synthase mutants having T29K and L135P mutations.
In the present invention, the site-directed mutagenesis method is a PCR induction method, and more specifically may be, but is not limited to, one or more of an overlap extension PCR method, a large primer PCR method, a recombinant PCR method, or a single primer PCR method.
In the present invention, based on the object of obtaining the above pantothenate synthetase mutants, there is provided a pantothenate producing strain capable of expressing a pantothenate synthetase mutant.
In the present invention, based on the object of obtaining the above pantothenate synthetase mutants, there is provided a process for producing pantothenate synthetase mutants, which comprises: transforming a host cell with the above construct to obtain a pantothenate producing strain; pantothenate synthase mutants are expressed using pantothenate producing strains.
In the present invention, the host cell is a eukaryotic cell and/or a prokaryotic cell capable of expressing and synthesizing D-pantothenic acid, and specifically can be, but is not limited to, one or more of Escherichia coli, yeast, lactic acid bacteria, or Streptomyces.
In some embodiments, the host cell is preferably E.coli, which may specifically be, but is not limited to, one or more of E.coli BL21 (DE 3), E.coli MG1655, or E.coli DH 5. Alpha.
In some embodiments, the host cell is preferably an existing pantothenate producing strain with high uptake capacity for beta-alanine, and may specifically be, but is not limited to, one or more of E.coli FV5069, E.coli FV5069/pFV31, or E.coli MG 1655.
In some specific embodiments, the construct is preferably contained on a pTrc-cysA plasmid vector. That is, specifically, the transformation of the construct into a host cell comprises: recombinant plasmid pTrc-cycA-panCx was obtained by recombining the construct into pTrc-cycA plasmid vector, and the above-described host cells were transformed with recombinant plasmid pTrc-cycA-panCx. More specifically, the recombinant plasmid pTrc-cycA-panCx comprises, according to the variant pantothenate synthetases with different mutations that can be encoded: recombinant plasmid pTrc-cycA-panC1, recombinant plasmid pTrc-cycA-panC2 and recombinant plasmid pTrc-cycA-panC3. Wherein the recombinant plasmid pTrc-cycA-panC1 comprises a construct encoding a mutant pantothenate synthetase having a T29K mutation. The recombinant plasmid pTrc-cycA-panC2 comprises a construct which codes for a pantothenate synthetase mutant having an L135P mutation. The recombinant plasmid pTrc-cycA-panC3 comprises a construct which codes for a pantothenate synthetase mutant with a T29K/L135P mutation.
In the present invention, when a pantothenic acid-producing strain is obtained by introducing a recombinant plasmid pTrc-cycA-panC3 into "E.coli Fv5069/pFV31", the pantothenic acid-producing strain is expressed which not only has a high catalytic activity for the reaction of pantoic acid with beta-alanine, but also can regulate the intracellular pantothenic acid biosynthesis pathway, and the resulting pantothenic acid-producing strain has an excellent pantothenic acid-producing ability.
In the present invention, the method for transforming a host cell with the construct may be, in particular but not limited to, chemical transformation and/or electroporation. The chemical conversion method specifically comprises the following steps: the host cells are treated by adopting calcium chloride, and the permeability of cell membranes of the host cells is changed so as to improve the absorption capacity of the cells to exogenous DNA and obtain competent cells. The electroporation method comprises the following steps: the high voltage electric field is used to form small holes in the cell membrane of host cell, and the exogenous DNA enters the cell through the small holes to raise the absorption capacity of the cell to exogenous DNA.
In the invention, the pantothenate synthetase mutant has high catalytic activity, and the pantothenate production strain has high pantothenate production capacity, can be well applied to the fermentation culture synthesis of D-pantothenic acid, and has good application prospect.
In the present invention, based on the object of efficiently synthesizing pantothenic acid, there is provided a process for producing pantothenic acid, which comprises: beta-alanine is fermented in the presence of the above-described pantothenate synthetase mutants and/or pantothenate producing strains to give pantothenate.
The amino acid sequences and nucleotide sequences involved in the present invention are shown in Table 1:
TABLE 1 amino acid and nucleotide sequence listing
The following detailed description of embodiments of the invention is intended to be illustrative of the invention and is not to be taken as limiting the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The following examples obtained the preparation of recombinant plasmids pTrc-cyc, E.coli FV5069 competent cells, E.coli FV5069/pFV31 competent cells and E.coli MG1655 competent cells used in the comparative examples ：Schneider F, Krämer R, Burkovski A. Identification and characterization of the main beta-alanine uptake system in Escherichia coli. Appl Microbiol Biotechnol. 2004 Oct;65(5):576-82.
Example 1
This example illustrates the construction of recombinant strains containing pantothenate synthase mutant constructs, comprising the steps of:
S1, construction and amplification of original plasmid
(1) Codon optimization is carried out on a gene fragment of encoding original pantothenic acid synthetase (amino acid is shown as SEQ ID NO: 1) in a genome of marine propionic acid bacteria (Propionigenium maris), and the optimized gene fragment (nucleotide is shown as SEQ ID NO: 12) is synthesized.
(2) The optimized gene fragment was synthesized by adding BamHI and HindII cleavage sites at both ends, and recombined onto pET24a vector after cleavage with BamHI and HindII to obtain the original plasmid.
(3) E.coli DH 5. Alpha. Competent cells were transformed with the original plasmid and plated on solid LB plates containing 50. Mu.g/mL kana resistance, cultured overnight at 37℃and part of the cells in each single colony were taken for PCR identification and sequencing to obtain recombinant strain PS-0 with the original plasmid.
(4) Single colonies of the recombinant strain PS-0 were picked up to LB liquid medium containing 50. Mu.g/mL kana resistance, cultured in a shaker at 37℃and 200rpm until the bacterial concentration OD 600 was greater than 0.8, and then the bacterial solution was extracted using a plasmid extraction kit (Aishi/AXYGEN, cat. No. AP-MN-P-250) to obtain a large amount of original plasmids.
S2 construction of the construct
(1) Respectively utilizing a forward/reverse T29K mutation primer (the nucleotide sequences are respectively shown as SEQ ID NO:8 and SEQ ID NO: 9), a forward/reverse L135P mutation primer (the nucleotide sequences are respectively shown as SEQ ID NO:10 and SEQ ID NO: 11) and a mixture of the forward and reverse T29K mutation primer and the forward/reverse L135P mutation primer, and carrying out PCR mutation amplification on the original plasmid by using TAKARA PRIMESTAR max high-fidelity polymerase to obtain a mutation amplification product;
The PCR procedure was: pre-denaturation at 98℃for 3min,30 cycles of amplification (98℃10s,55℃5s,72℃70 s), 72℃10min.
(2) The mutant amplification product was digested with DpnI at 37℃for 1 hour, and the template was removed to obtain construct 1 (containing the nucleotide fragment of SEQ ID NO: 5), construct 2 (containing the nucleotide fragment of SEQ ID NO: 6) and construct 3 (containing the nucleotide fragment of SEQ ID NO: 7).
S3 construction of recombinant strains
Constructs 1, 2 and 3 were transformed with E.coli BL21 (DE 3) competent cells, respectively, plated onto LB plates containing 50. Mu.g/mL kana resistance, cultured overnight at 37℃and part of the cells from each single colony were subjected to PCR identification and sequencing to obtain recombinant strain PS-1 (containing construct 1), recombinant strain PS-2 (containing construct 2) and recombinant strain PS-3 (containing construct 3).
Example 2
This example illustrates the preparation of a crude enzyme comprising pantothenate synthase mutants, comprising the steps of:
(1) Single colonies of the recombinant strains PS-1, PS-2 and PS-3 obtained in example 1 were picked, inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL kana resistance, and cultured in a shaker at 37℃and 200rpm for 7 hours, respectively, to obtain seed solutions;
(2) Transferring the seed solution into 50mL of TB liquid culture medium containing 50 mug/mL kana resistance according to the inoculum size of 2%, culturing for 2h in a shaking table at 37 ℃ and 200rpm, adding 0.2mmol/L IPTG after culturing until the bacterial concentration OD 600 is more than 0.6, cooling to 25 ℃, continuously culturing for 16h, centrifuging for 15min at 4 ℃ and 10000rpm, and collecting bacterial cells;
(3) To the cells, 15mL of a sodium phosphate buffer solution having a concentration of 50mmol/L was added and resuspended, and the cells were broken by using an ultrasonic breaker, centrifuged at 4℃and 10000rpm for 15 minutes, and the supernatant was collected to obtain crude enzymes T29K, L P and T29K/L135P.
Example 3
This example is intended to illustrate the enzymatic activity assay of pantothenate synthase mutants, using the original pantothenate synthase as a control, and specifically:
10 mu L of crude enzyme T29K, L P and T29K/L135P prepared in example 2 and the original pantothenic acid synthetase are respectively added into an enzyme activity measuring system, reacted in a water bath at 37 ℃ for 15min, 100 mu L of NaOH solution with the concentration of 1M is added after the reaction is finished to stop the reaction, supernatant is centrifugally taken, the content of pantothenic acid is detected by adopting high performance liquid chromatography after the supernatant is filtered, the pantothenic acid content generated by the reaction is calculated, and the enzyme activity (defined as the enzyme amount required for catalyzing the reaction to generate 1 mu mol of pantothenic acid per minute at 37 ℃) is calculated according to the obtained pantothenic acid content, and the result is shown in a table 2.
Wherein, the enzyme activity measurement system specifically comprises: 40mM beta-alanine, 40mM sodium pantoate, 15mM magnesium chloride, 15mM potassium chloride, 10mM ATP and 50mM sodium carbonate-sodium bicarbonate buffer (pH 8.5).
Conditions for high performance liquid chromatography include: the chromatographic column is Eclipse XD8-C18 (5 μm,4.6mm multiplied by 250 mm), the column temperature is 30 ℃, the detector is an ultraviolet detector, the detection wavelength is 210nm, the volume ratio of mobile phase A (phosphoric acid): B (acetonitrile): C (water) =1:50:950, the flow rate is 0.9mL/min, and the sample injection amount is 10 μl.
TABLE 2 enzyme Activity
As can be seen from the results of the enzyme activity assay, the pantothenate synthetase mutants having the T29K mutation, the L135P mutation and the T29K/L135P mutation obtained in example 2 of the present invention had higher enzyme activities than the original pantothenate synthetase derived from Propionigenium maris. It is worth noting that the pantothenate synthetase mutant with T29K/L135P mutation had an increase in enzyme activity of about 47% compared to the original pantothenate synthetase, i.e., the activity was significantly increased.
Example 4
This example illustrates the construction of pantothenate-producing strains, comprising the steps of:
(1) Amplification of the construct: single colonies of the recombinant strains PS-1, PS-2 and PS-3 obtained in example 1 were picked up, respectively inoculated into LB liquid medium containing 50. Mu.g/mL kana resistance, cultured in a shaker at 37℃and 200rpm until the bacterial concentration OD 600 was greater than 2.0, and then the bacterial solution was extracted using a plasmid extraction kit to obtain a large number of recombinant plasmids with constructs 1,2 and 3.
(2) Construction of pantothenate-producing strains: the recombinant plasmids carrying constructs 1, 2 and 3 were treated with BamHI and HindII enzymes, electrophoresed to obtain gel bands carrying constructs 1, 2 and 3, and the gel bands were recovered using a DNA gel recovery kit (AXYGEN, accession number AP-GX-250) to obtain a large number of constructs 1, 2 and 3; constructs 1, 2 and 3 were recombined onto plasmids pTrc-cyc to obtain recombinant plasmids pTrc-cycA-panC1, pTrc-cycA-panC2 and pTrc-cycA-panC3.
(3) The recombinant plasmids pTrc-cycA-panC1, pTrc-cycA-panC2 and pTrc-cycA-panC3 are respectively transformed into competent cells of E.coli FV5069 to obtain pantothenate production strains PA-M.11, PA-M.12 and PA-M.13.
Example 5
In this example, pantothenate producing strains were constructed according to the method provided in example 4, except that in step (3), recombinant plasmids pTrc-cycA-panC1, pTrc-cycA-panC2 and pTrc-cycA-panC3 were used to transform competent cells of E.coli Fv5069/pFV31, respectively, to give pantothenate producing strains PA-M.21, PA-M.22 and PA-M.23, under the same conditions.
Example 6
In this example, pantothenate-producing strains were constructed according to the method provided in example 4, except that in step (3), competent cells of E.coli MG1655 were transformed with the recombinant plasmids pTrc-cycA-panC1, pTrc-cycA-panC2 and pTrc-cycA-panC3, respectively, to give pantothenate-producing strains PA-M.31, PA-M.32 and PA-M.33, under the same conditions.
Comparative example 1
This comparative example was constructed as provided in example 4, except that in step (2), the original plasmid obtained in example 1 was treated with BamHI and HindII enzymes, and after electrophoresis and recovery, a large amount of gene fragments encoding the original pantothenic acid synthase were obtained, and the gene fragments were recombined onto the plasmid pTrc-cyc to obtain the recombinant plasmid pTrc-cycA-panC0; in the step (3), the recombinant plasmid pTrc-cycA-panC0 is transformed into competent cells of E.coli FV5069 to obtain pantothenic acid producing strain D-1 under the same conditions.
Comparative example 2
This comparative example was constructed as provided in example 4, except that in step (2), the original plasmid obtained in example 1 was treated with BamHI and HindII enzymes, and after electrophoresis and recovery, a large amount of gene fragments encoding the original pantothenic acid synthase were obtained, and the gene fragments were recombined onto the plasmid pTrc-cyc to obtain the recombinant plasmid pTrc-cycA-panC0; in step (3), the recombinant plasmid pTrc-cycA-panC0 was transformed into competent cells of E.coli Fv5069/pFV31 to give pantothenate-producing strain D-2, under otherwise identical conditions.
Comparative example 3
This comparative example was constructed as provided in example 4, except that in step (2), the original plasmid obtained in example 1 was treated with BamHI and HindII enzymes, and after electrophoresis and recovery, a large amount of gene fragments encoding the original pantothenic acid synthase were obtained, and the gene fragments were recombined onto the plasmid pTrc-cyc to obtain the recombinant plasmid pTrc-cycA-panC0; in the step (3), the recombinant plasmid pTrc-cycA-panC0 was transformed into competent cells of E.coli MG1655 to give pantothenate-producing strain D-3, under the same conditions.
Example 7
This example is for illustrating the use of the pantothenate production strains and their expressed pantothenate synthetase mutants provided in examples 4-6 in the fermentative synthesis of D-pantothenic acid, and specifically comprises the following steps:
(1) Strain activation: a loop of the strain liquid provided in examples 4-6 is dipped from an glycerol pipe by an inoculating loop and inoculated in a flat plate culture medium by streaking, and the culture is carried out for 24 hours at 37 ℃ to obtain a mature single colony.
The plate medium comprises: 10g/L sodium chloride, 8g/L tryptone, 4g/L yeast extract, 50. Mu.g/mL kana and 20g/L agar powder; and the plate medium was sterilized 25 min at 121 ℃ before use.
(2) Shake flask culture: and (3) respectively inoculating the mature single colonies into 100mL of shake flask culture medium, and culturing in a shaking table at 32 ℃ and 180rpm until the bacterial concentration OD 600 is more than or equal to 5.0 to obtain shake flask bacterial liquid.
The shake flask medium comprises: 20g/L glucose, 10g/L corn steep liquor, 0.6g/L dipotassium hydrogen phosphate, 0.3g/L sodium dihydrogen phosphate, 5g/L calcium carbonate, 50. Mu.g/mL kana and 10g/L ammonium chloride; and sterilizing the shake flask culture medium at 121deg.C for 25min before use, and adjusting pH of the shake flask culture medium to about 7.0 before sterilization.
(3) Seed pot culture: transferring the shake flask bacterial liquid into a seed tank culture medium according to the inoculation amount of 2%, and performing expansion culture until the bacterial concentration OD 600 is more than or equal to 5.5 under the conditions that the temperature is 28 ℃, the tank pressure is 0.04MPa, the aeration ratio is 0.5VVM and the rotating speed is 300rpm, thereby obtaining the seed bacterial liquid.
The seed tank medium comprises: 30g/L glucose, 20g/L corn steep liquor, 1.0g/L dipotassium hydrogen phosphate, 0.8g/L sodium dihydrogen phosphate, 6g/L calcium carbonate, 50. Mu.g/mL kana and 15g/L ammonium chloride; and sterilizing the seed tank culture medium at 121deg.C for 25min before use, and adjusting pH of the seed tank culture medium to about 7.0 before sterilization.
(4) Fermentation culture: inoculating the seed bacterial liquid into a fermentation culture medium according to the inoculation amount of 12wt%, and carrying out fermentation culture, wherein the liquid loading amount after inoculation is 23L; after the thalli grow to the mid-logarithmic growth phase, adding isopropyl thiogalactoside (IPTG) with the final concentration of 0.1mM, continuously culturing for 6 hours, starting to add beta-alanine into the fermentation broth in a divided mode, wherein the total adding amount of the beta-alanine is 10wt%, and stopping fermentation when the pantothenic acid synthesis rate is lower than 2.5g/L per hour, so as to obtain D-pantothenic acid. Wherein the pantothenic acid synthesis rate is obtained by measuring the pantothenic acid content in the fermentation broth and calculating.
Conditions of fermentation culture: the pH of the fermentation liquid is about 7.0, the dissolved oxygen is 30% -50%, and the concentration of residual sugar is 1-7 g/L.
The fermentation medium comprises: 35g/L glucose, 22g/L corn steep liquor, 18g/L ammonium chloride, 1.2g/L sodium dihydrogen phosphate, 2.0g/L magnesium sulfate, 1.2g/L sodium chloride and 0.05mg/L zinc sulfate; the fermentation medium is sterilized at 121deg.C for 30min before use, and the pH of the fermentation medium is adjusted to about 7.0 before sterilization.
After the fermentation, D-pantothenate production and beta-alanine conversion rate of each pantothenate-producing strain were counted, and the same fermentation conditions were applied to the pantothenate-producing strains provided in comparative examples 1 to 3 and to E.coli Fv5069/pTrc-cycA, fv5069/pFV31/pTrc-cycA and MG1655/pTrc-cycA as controls, and the results are shown in Table 3.
TABLE 3 pantothenate production ability of pantothenate producing strains
As shown by the test results, the pantothenic acid production strains provided in examples 4 to 6 of the present invention have significantly improved ability to take in and utilize beta-alanine and synthesize D-pantothenic acid, and have higher D-pantothenic acid yield and beta-alanine conversion rate than comparative examples 1 to 3 and E.coli Fv5069/pTrc-cycA, E.coli Fv5069/pFV31/pTrc-cycA and E.coli MG 1655/pTrc-cycA.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims (13)

1. A pantothenate synthetase mutant, characterized in that the pantothenate synthetase mutant is an original pantothenate synthetase comprising a T29K mutation and/or an L135P mutation, and the original pantothenate synthetase comprises an amino acid fragment having a sequence as shown in SEQ ID NO. 1.
2. The mutant pantothenate synthetase according to claim 1, wherein said mutant pantothenate synthetase comprises a T29K mutation and said mutant pantothenate synthetase comprises an amino acid fragment having the sequence set forth in SEQ ID NO. 2.
3. The mutant pantothenate synthetase according to claim 1, wherein said mutant pantothenate synthetase comprises a T135K mutation and said mutant pantothenate synthetase comprises an amino acid fragment having the sequence set forth in SEQ ID NO. 3.
4. The pantothenate synthetase mutant according to claim 1, wherein said pantothenate synthetase mutant comprises a T29K mutation and an L135P mutation, and said pantothenate synthetase mutant comprises an amino acid fragment having a sequence as set forth in SEQ ID NO. 4.
5. A construct comprising a nucleotide fragment encoding the pantothenate synthetase mutant of any one of claims 1-4.
6. The construct of claim 5, wherein the construct comprises one or more of the nucleotide fragments set forth in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7.
7. A pantothenate producing strain, which is capable of expressing the pantothenate synthase mutant according to any one of claims 1 to 4.
8. The method for constructing a construct according to claim 5 or 6, wherein the method comprises recombining a gene fragment encoding an original pantothenate synthetase of a marine propionic acid bacterium (Propionigenium maris) into a plasmid vector to obtain an original plasmid, and performing site-directed mutagenesis on the obtained original plasmid by using a mutation primer to obtain the construct.
9. The method of construction of a construct according to claim 8, wherein the mutation primer comprises a T29K mutation primer and/or an L135P mutation primer; the T29K mutant primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 8 and/or a reverse primer with a nucleotide sequence shown as SEQ ID NO. 9; the L135P mutation primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 10 and/or a reverse primer with a nucleotide sequence shown as SEQ ID NO. 11.
10. A process for producing a mutant pantothenate synthetase, which comprises transforming a host cell with a construct according to claim 5 or 6 to obtain a pantothenate-producing strain; pantothenate synthase mutants are expressed using pantothenate producing strains.
11. The process for the preparation of a mutant pantothenate synthetase according to claim 10, wherein said host cell is selected from one or more of E.coli FV5069, E.coli FV5069/pFV31 or E.coli MG 1655.
12. The pantothenate synthetase mutants according to claim 1 to 4 and/or the pantothenate production strain according to claim 7, for use in the fermentative synthesis of D-pantothenic acid.
13. A process for the preparation of pantothenic acid, comprising: fermenting beta-alanine in the presence of the pantothenate synthetase mutants according to any one of claims 1 to 4 and/or the pantothenate production strain according to claim 7 to obtain pantothenate.
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