CN117004543A - Genetically engineered bacterium for producing methyl selenocysteine and preparation and application thereof - Google Patents

Genetically engineered bacterium for producing methyl selenocysteine and preparation and application thereof Download PDF

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CN117004543A
CN117004543A CN202311131720.7A CN202311131720A CN117004543A CN 117004543 A CN117004543 A CN 117004543A CN 202311131720 A CN202311131720 A CN 202311131720A CN 117004543 A CN117004543 A CN 117004543A
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gene
gene encoding
genetically engineered
methyltransferase
methionine
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殷娴
王凤寰
廖永红
赵美怡
周瑜
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Beijing Technology and Business University
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Abstract

The invention relates to a gene engineering bacterium for producing methyl selenocysteine, which is a recombinant bacillus subtilis containing one or more of a gene for encoding serine O-acetyltransferase SATp, a gene for encoding selenocysteine methyltransferase SMT, a gene for encoding S-adenosylmethionine synthetase SAM2 and a gene for encoding methionine S-methyltransferase Mmt in genome, and one or more of a gene for encoding cysteine desulphurase IscSB, a gene for encoding aminotransferase YhdR and a gene cluster for encoding serine dehydratase SdaA are omitted. The bacteria can be used for producing methyl selenocysteine, can greatly reduce the production cost, and is not easily influenced by factors such as environmental temperature, pH and the like; no toxic product is produced in the conversion process, and no other byproducts are produced; the bacteria is expected to realize the industrialized SeMCys production with low energy consumption, high efficiency, high purity and no pollution.

Description

Genetically engineered bacterium for producing methyl selenocysteine and preparation and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a genetically engineered bacterium for producing methyl selenocysteine, and preparation and application thereof.
Background
Selenium (Se) is an important trace element in many organisms and can be inserted into proteins and nucleic acids by selenocysteine (sels) and 2-selenoprotein. Selenium-methylselenocysteine (SeMCys) as a direct precursor of methylselenol can be used as a dietary selenium supplement and in combination chemotherapy by reducing tumor growth and metastatic capacity.
At present, the preparation method of SeMCys is mainly a chemical synthesis method, but the chemical synthesis methods have the problems of complex steps, lengthy process, harsh reaction conditions, unfriendly environment and the like.
Biosynthesis of SeMCys occurs naturally in broccoli and astragalus, which are super-accumulating plants of selenium. Selenocysteine Methyltransferase (SMT) is a key enzyme responsible for the methylation of SeCys to synthesize SeMCys. Expression of plant-derived SMT in Saccharomyces cerevisiae overproducing S-adenosylmethionine (SAM) as a methyl donor provided a SeMCys yield of 1.140. Mu.g/g stem cell weight (DCW) for this strain. Overexpression and expression of SMT-based S-adenosylmethionine synthetase (SAM 2) derived from Saccharomyces cerevisiae increased SAM yield, by and in addition to increasing intracellular SeCys levels, the applicant has optimized production of SeMCys in Bacillus subtilis (Yin X, zhou Y, yang H, liao Y, ma T, wang F.enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis. Appl Microbiol Biotechnol.2023;107 (9): 2843-54.https:// doi. Org/10.1007/S00253-023-12482-8), and produced SeMCys in fed-batch culture at a rate of 18. Mu.g/L with the heterologous gene-integrating strain GBACB, however, the yield of the strain was still low.
Therefore, there is a need to construct a genetically engineered bacterium that increases the yield of methylselenocysteine.
Disclosure of Invention
In order to solve the technical problems, the invention provides the genetically engineered bacterium for producing the methylselenocysteine, which can greatly reduce the production cost, is not easily influenced by factors such as environmental temperature, pH and the like in the preparation process, and is convenient to use; no toxic product is produced in the conversion process, and no other byproducts are produced; the genetic engineering bacteria is expected to realize the industrialized SeMCys production with low energy consumption, high efficiency, high purity and no pollution.
The first aspect of the invention provides a genetically engineered bacterium for producing methylselenocysteine, which is synthesized by the following steps:
(1) Serine (Ser) synthesizes O-acetylserine by action of serine O-acetyltransferase (SATp) from arabidopsis thaliana;
(2) O-acetylserine releases the expression inhibition of O-acetylserine thiol lyase (CysK), and synthesizes selenocysteine (SeCys) under the action of CysK;
(3) Synthesizing S-adenosylmethionine (SAM) by methionine under the action of S-adenosylmethionine synthetase (SAM 2) derived from Saccharomyces cerevisiae;
(4) Selenocysteine (SeCys) is methylated by S-adenosylmethionine (SAM) under the action of Selenocysteine Methyltransferase (SMT) derived from Astragalus membranaceus to form methylselenocysteine;
(5) S-adenosylmethionine (SAM) and methionine form methyl methionine (MMet) under the action of methionine S-methyltransferase (Mmt);
(6) Selenocysteine (sels) is methylated by methyl methionine (MMet) under the action of Selenocysteine Methyltransferase (SMT) to form methylselenocysteine.
According to some embodiments of the invention, the genetically engineered bacterium is a recombinant host bacterium whose genome comprises a gene encoding serine O-acetyltransferase, SATp, a gene encoding selenocysteine methyltransferase, SMT, a gene encoding S-adenosylmethionine synthetase SAM2, the host bacterium being bacillus subtilis 168; wherein the genome of the genetically engineered bacterium further comprises a gene encoding methionine S-methyltransferase Mmt.
In some embodiments of the invention, the gene encoding methionine S-methyltransferase Mmt includes a gene encoding methionine S-methyltransferase CpMmt (abbreviated as CpMmt protein) derived from B.isodomain candidates (Candidatus Peregrinibacteria), a gene encoding methionine S-methyltransferase RiMmt (abbreviated as RiMmt protein) derived from B.roseum (Roseovarius indicus), and a gene encoding methionine S-methyltransferase NmMmt (abbreviated as NmMmt protein) derived from MBES 04.
Preferably, the nucleotide sequence of the gene encoding methionine S-methyltransferase CpMmt is shown in SEQ ID NO. 1; and/or the nucleotide sequence of the gene encoding methionine S-methyltransferase RiMmt is shown as SEQ ID NO. 2; and/or the nucleotide sequence of the gene encoding methionine S-methyl transferase NmMmt is shown in SEQ ID NO. 3.
According to other embodiments of the invention, the genetically engineered bacterium is a genetically engineered bacterium which is subjected to chassis modification and produces methylselenocysteine; the chassis modification comprises the knockout of one or more of a gene encoding cysteine desulphurase IscSB (abbreviated as IscSB protein), a gene encoding aminotransferase YhdR (YhdR protein) and a gene cluster encoding serine dehydratase SdaA; wherein the gene cluster for coding the serine dehydratase SdaA comprises a gene for coding the serine dehydratase SdaAA (SdaAA protein for short) and a gene for coding the serine dehydratase SdaAB (SdaAA protein for short).
In some embodiments of the invention, the nucleotide sequence of the gene encoding cysteine desulphurase IscSB is shown in SEQ ID No. 7; and/or the nucleotide sequence of the gene encoding aminotransferase YhdR is shown in SEQ ID NO. 9; and/or the nucleotide sequence of the gene encoding serine dehydratase SdaAA is shown in SEQ ID NO. 11; and/or the nucleotide sequence of the gene encoding serine dehydratase SdaAB is shown in SEQ ID NO. 12.
A second aspect of the invention provides a method as described in the first aspect of the inventionThe construction method of the genetically engineered bacterium comprises the steps of taking bacillus subtilis 168 as a host bacterium, and placing genes CpMmt, riMmt and NmMmt for encoding methionine S-methyltransferase at P xylA Formation of P under promoter xylA -CpMmt、P xylA RiMmt and P xylA Inserting the expression frames into KinB sites of a bacillus subtilis 168 genome respectively, and knocking out one or more of a gene encoding cysteine desulphurase IscSB, a gene encoding aminotransferase YhdR and a gene cluster encoding serine dehydratase SdaA in a strain modified by the genome to obtain a genetically engineered bacterium producing methyl selenocysteine; wherein the gene cluster encoding serine dehydratase SdaA comprises a gene encoding serine dehydratase SdaAA and a gene encoding serine dehydratase SdaAB.
The third aspect of the invention provides an application of the genetically engineered bacterium according to the first aspect of the invention or the genetically engineered bacterium constructed by the construction method according to the second aspect of the invention in preparation of methylselenocysteine; the application comprises the steps of fermenting and culturing the genetically engineered bacteria to prepare the methylselenocysteine.
In some embodiments of the invention, the genetically engineered bacterium is inoculated into the fermentation medium in the form of a seed solution during fermentation culture; the fermentation culture conditions are as follows: the temperature is 30-38 ℃, and the rotating speed of the shaking table is 150-300 rpm.
Preferably, the OD of the seed solution of the genetically engineered bacterium 600 Not less than 1.0.
The beneficial effects of the invention are as follows:
(1) The invention greatly improves the synthesis of methyl selenocysteine in recombinant bacillus subtilis. The method for preparing the methylselenocysteine can greatly reduce the production cost, is not easily influenced by factors such as ambient temperature, pH and the like, and is convenient to use; no toxic product is produced in the conversion process, and no other byproducts are produced; is expected to realize the industrialized SeMCys production with low energy consumption, high efficiency, high purity and no pollution.
(2) The invention can lead the extracellular yield of the methylselenocysteine produced by bacillus subtilis fermentation to reach the mg/L level, which is far higher than the extracellular yield of the methylselenocysteine reported in the prior art, and is only in the mu g/L level.
(3) Since bacillus subtilis (Bacillus subtilis) belongs to a food-safe strain, methylselenocysteine produced by using recombinant bacillus subtilis obtained by taking bacillus subtilis (Bacillus subtilis) as a host can reach a food grade.
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The invention is described in further detail below with reference to the accompanying drawings:
FIG. 1 shows the way of synthesizing methylselenocysteine by the genetically engineered bacterium provided by the invention.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail below with reference to the accompanying drawings. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Embodiment I
As described above, the yield of methylselenocysteine in the existing strain is still low, and for this reason, the present inventors have made extensive studies on methylselenocysteine-producing strains.
The inventors have observed through research that, although SAM is the primary methyl donor for many transmethylations, it has been demonstrated that (Neuhierl B, thanbichler M, lottspeich F, bock A.A family of S-methyl-dependent thiol/selenol methyl transfer ferases. Role in selenium tolerance and evolutionary concern. J Biol chem.1999;274 (9): 5407-14.Https:// doi. Org/10.1074/jbc.274.9.5407), SMT from Astragalus bifidus has a 4-fold higher specific activity than SAM when S-methyl methionine (MMet) is used as the methyl donor substrate. MMet can be detected in angiosperms and some marine heterotrophic bacteria, with the corresponding catalytic enzyme being Mmt. The applicant therefore hoped to increase the synthesis of SeMCys by expressing three Mmt of marine heterotrophic bacterial origin.
To increase the production of SeMCys, it is important to continuously supply high levels of SeMCys precursor. The formation of SeCys depends on the sulfur metabolic pathway, and the corresponding metabolite is cysteine (Cys), which is a key point in the carbon metabolic pathway, amino acid synthesis pathway, and cell growth. Not only is the synthesis of SeCys tightly controlled, but the degradation pathway is strong, resulting in the fact that SeCys cannot accumulate in large amounts within the cell. Thus, by knocking out the degradation pathway genes IscSB, yhdR of the related SeCys, the synthesis of SeMCys can be improved.
The inventors have found that serine (Ser) is a precursor for the synthesis of Cys and SeCys, and facilitates Cys production by preventing Ser degradation reactions catalyzed by the serine dehydratases encoded by SdaAA and SdaAB. Thus, by knocking out the SdaA gene cluster, it is expected that SeMCys production will be further enhanced by increasing SeCys accumulation.
In addition, the invention considers that the food-grade genetically engineered bacteria are utilized to strengthen the synthesis of the methylselenocysteine, which is beneficial to improving the industrialized application potential of the genetically engineered bacteria.
Therefore, the invention provides a new way for synthesizing the methylselenocysteine, which realizes the efficient synthesis of the methylselenocysteine through a genetically engineered bacterium for high yield of the methylselenocysteine.
Specifically, the way of synthesizing the methylselenocysteine by the genetically engineered bacteria for producing the methylselenocysteine is shown in fig. 1, and the method comprises the following steps:
(1) Serine (Ser) synthesizes O-acetylserine by action of serine O-acetyltransferase (SATp) from arabidopsis thaliana;
(2) O-acetylserine releases the expression inhibition of O-acetylserine thiol lyase (CysK), and synthesizes selenocysteine (SeCys) under the action of CysK;
(3) Synthesizing S-adenosylmethionine (SAM) by methionine under the action of S-adenosylmethionine synthetase (SAM 2) derived from Saccharomyces cerevisiae;
(4) Selenocysteine (SeCys) is methylated by S-adenosylmethionine (SAM) under the action of Selenocysteine Methyltransferase (SMT) derived from Astragalus membranaceus to form methylselenocysteine;
(5) S-adenosylmethionine (SAM) and methionine form methyl methionine (MMet) under the action of methionine S-methyltransferase (Mmt);
(6) Selenocysteine (SeCys) is methylated by methyl methionine (MMet) under the action of Selenocysteine Methyltransferase (SMT) to generate methyl selenocysteine;
in order to realize the technical scheme, the invention provides a host strain capable of producing methylselenocysteine, which expresses genes in a methylselenocysteine synthesis path in original or modified bacteria and fungi cells to form genetically engineered bacteria capable of synthesizing the methylselenocysteine.
The reaction mechanism of biosynthesis of methylselenocysteine in the present invention is shown in FIG. 1, and it can be understood from FIG. 1 that the genes in the methylselenocysteine synthesis pathway include recombinant host bacteria encoding serine O-acetyltransferase SATp, selenocysteine methyltransferase SMT, S-adenosylmethionine synthetase SAM2, and methionine S-methyltransferase Mmt.
Based on the above, it is easily understood that the genetically engineered bacterium for producing methylselenocysteine according to the present invention is a recombinant host bacterium comprising a gene encoding serine O-acetyltransferase SATp, a gene encoding selenocysteine methyltransferase SMT, a gene encoding S-adenosylmethionine synthetase SAM2, and a gene encoding methionine S-methyltransferase Mmt.
The inventors examined and studied the effect of knocking out the gene encoding cysteine desulphurase IscSB, the gene encoding aminotransferase YhdR, the gene cluster encoding serine dehydratase SdaA on the yield of methylselenocysteine of genetically engineered bacteria.
Specifically: (1) Knocking out a cysteine desulphurase (IscSB) gene, which catalyzes the degradation of SeCys to alanine; (2) Knocking out an aminotransferase (YhdR) gene, wherein the enzyme catalyzes the degradation of SeCys into mercaptopyruvic acid; (3) The gene cluster of serine dehydratase (SdaA) is knocked out, and the enzyme catalyzes the degradation of Ser into 2-amino acrylic ester.
The inventors have further studied and found that the yield of methylselenocysteine can be further increased by knocking out one or more of the gene encoding cysteine desulphurase IscSB, the gene encoding aminotransferase YhdR and the gene cluster encoding serine dehydratase SdaA.
In the present invention, the host bacterium is Bacillus subtilis 168, which has a deposit number of ATCC 23857 (American type culture Collection).
In some embodiments of the present invention, the serine O-acetyltransferase SATp gene from Arabidopsis thaliana (gene expression is initiated by Pgrad) and selenocysteine methyltransferase SMT gene from Astragalus bifidus (gene expression is initiated by P43), i.e. Pgrad-SATp-P43-SMT expression cassette, are integrated at the genomic amyE locus with Bacillus subtilis 168 as host bacteria; meanwhile, S-adenosylmethionine synthetase SAM2 gene (the gene is started to express by Pgrad) derived from Saccharomyces cerevisiae and another copy of SMT gene, namely Pgrad-SAM 2-SMT expression frame are integrated at XylA locus to obtain first recombinant bacillus subtilis, which is called bacillus subtilis GBACB in the invention. The strain has SeMCys synthesis capacity and yield of 18.4 mug/L.
In some further specific embodiments of the present invention, the recombinant bacillus subtilis uses bacillus subtilis GBACB as a host, integrates a PxylA-Mmt expression frame into a bacillus subtilis genome KinB site, and knocks out one or more of a gene encoding cysteine desulphurase IscSB, a gene encoding aminotransferase YhdR and a gene cluster encoding serine dehydratase SdaA to obtain the genetically engineered bacterium for producing the methylselenocysteine, which is also called as the recombinant bacillus subtilis for producing the methylselenocysteine.
In one embodiment of the invention, the methionine S-methyltransferases are CpMmt from Candidatus Peregrinibacteria, riMmt from Roseovarius indicus and NmMmt from Novosphingobium sp.MBES04.
In one embodiment of the invention, the amino acid sequence of the CpMmt protein is shown as SEQ ID NO. 4; the nucleotide sequence of the encoding CpMmt protein is shown as SEQ ID NO. 1.
In one embodiment of the invention, the amino acid sequence of the RiMmt protein is shown as SEQ ID NO. 5; the nucleotide sequence of the coding RiMmt protein is shown as SEQ ID NO. 2.
In one embodiment of the invention, the amino acid sequence of the nmmt protein is shown in SEQ ID No. 6; the nucleotide sequence of the NmMmt protein is shown as SEQ ID NO. 3.
In one embodiment of the invention, the amino acid sequence of the cysteine desulphurase IscSB protein is shown in SEQ ID No. 8; the nucleotide sequence of the IscSB protein is shown in SEQ ID NO. 7.
In one embodiment of the invention, the amino acid sequence of the aminotransferase YhdR protein is shown in SEQ ID No. 10; the nucleotide sequence for encoding the YhdR protein is shown in SEQ ID NO. 9.
In the present invention, the gene cluster encoding serine dehydratase SdaA includes a gene encoding serine dehydratase SdaAA and a gene encoding serine dehydratase SdaAB. In one embodiment of the invention, the amino acid sequences of the serine dehydratase SdaAA and SdaAB proteins are shown in SEQ ID NO.13 and SEQ ID NO.14 respectively; the nucleotide sequence for coding the SdaAA protein is shown as SEQ ID NO.11, and the nucleotide sequence for coding the SdaAB protein is shown as SEQ ID NO. 12.
The invention also relates to a construction method of the gene engineering bacteria for producing the methylselenocysteine, which comprises the steps of taking bacillus subtilis 168 as a host bacteria, placing genes CpMmt, riMmt and NmMmt for encoding methionine S-methyltransferase under a PxylA promoter to form PxylA-CpMmt, pxylA-RiMmt and PxylA-NmMmt expression frames, respectively inserting the expression frames into KinB sites of a bacillus subtilis 168 genome, and knocking out one or more of genes for encoding cysteine desulphurase IscSB, genes for encoding aminotransferase YhdR and gene clusters for encoding serine dehydratase SdaA in a strain modified by the genome to obtain the gene engineering bacteria for producing the methylselenocysteine; wherein the gene cluster encoding serine dehydratase SdaA comprises a gene encoding serine dehydratase SdaAA and a gene encoding serine dehydratase SdaAB.
In some embodiments of the present invention, constructing the above genetically engineered bacteria producing methylselenocysteine includes:
constructing bacillus subtilis GBACB by taking bacillus subtilis 168 as host bacteria
(1) Integrating serine O-acetyltransferase SATp gene (the gene is expressed by Pgrad) from Arabidopsis thaliana and selenocysteine methyltransferase SMT gene (the gene is expressed by P43) from astragalus bicolor at a genome amyE locus by taking bacillus subtilis 168 as a host bacterium, namely a Pgrad-SATp-P43-SMT expression frame; meanwhile, integrating S-adenosylmethionine synthetase SAM2 gene (the gene is started to express by Pgrad) from Saccharomyces cerevisiae and another copy SMT gene, namely Pgrad-SAM 2-SMT expression frame at XylA site to obtain bacillus subtilis GBACB;
(II) constructing genetically engineered bacteria for high-yield methylselenocysteine by taking bacillus subtilis 168 as host bacteria
(1) When 3 coded Mmt proteins are synthesized by the genes, enzyme cutting sites SpeI and SmaI are respectively added at two ends of the genes, and then the genes are connected with a plasmid pSTOP1622 subjected to enzyme cutting by the SpeI and the SmaI to obtain vectors pSTOP-CpMmt, pSTOP-RiMmt and pSTOP-NmMmt, wherein the genes are positioned at the downstream of a PxylA promoter; cloning an upstream fragment LB and a downstream fragment RB of KinB respectively, connecting the LB fragments to a pMD19 cloning vector to obtain pMD19-LB, carrying out digestion on the vector by Kpn I and EcoR I, recovering the vector, and connecting the vector with RB which is also digested by Kpn I and EcoR I by using T4 ligase to obtain a pMD-LB-RB vector; pxylA-CpMmt expression cassette fragments are amplified by primers Mmt-kineB-F and CpMmt-kineB-R, pxylA-RiMmt expression cassette fragments are amplified by primers Mmt-kineB-F and RiMmt-kineB-R, pxylA-NmMmt expression cassette fragments are amplified by primers Mmt-kineB-F and NmMmt-R, pMD-LB-RB vector is cut into linear fragments by Kpn I, and vectors pMD-LB-CpMmt-RB, pMD-LB-RiMmt-RB and pMD-LB-NmMmt-RB are obtained by Gibson assembly. LB-CpMmt-RB, LB-RiMmt-RB and LB-NmMmt-RB fragments were amplified as donor DNA, respectively. A23 bp KinB fragment was selected and ligated into the pcrF11 vector to construct a targeting RNA plasmid. Firstly, converting plasmid pHT-XCR6 containing recombinase into bacillus subtilis GBACB, respectively co-converting guide RNA plasmid and three LB-Mmt-RB fragments in the transformants, and screening genome-integrated transformants GBACBC, GBACBR and GBACBM;
(2) Cloning and fusing PCR (polymerase chain reaction) respectively an upstream segment LBi and a downstream segment RBi of the IscSB gene to obtain a segment LBi-RBi; the upstream fragment LBy and the downstream fragment RBy of the YhdR gene were cloned and fused into one fragment, LBy-RBy, respectively. The 23bp IscSB and YhdR fragments were selected separately, each ligated into the pcrF11 vector, and three guide RNA plasmids were constructed. Firstly, converting plasmid pHT-XCR6 containing recombinase into bacillus subtilis GBACBM, respectively co-converting guide RNA plasmid and corresponding LB-RB fragment in the transformants, and screening gene knocked-out transformants GMC1 and GMC3;
(3) The upstream fragment LBs of the SdaAB gene and the downstream fragment RBs of the SdaAA gene were cloned and fused into one fragment LBs-RBs, respectively. A23 bp SdaAB fragment was selected and ligated into the pcrF11 vector to construct a guide RNA plasmid. Firstly, converting plasmid pHT-XCR6 containing recombinase into bacillus subtilis GBACBM, respectively co-converting guide RNA plasmid and LBs-RBs fragment in the transformant, and screening the transformant GMS with knocked-out gene; in addition, plasmid pHT-XCR6 containing recombinase is firstly transformed into bacillus subtilis GMC1, guide RNA plasmid and LBs-RBs fragment are respectively transformed in the transformant, and the transformant GMSC with knocked-out gene is screened to obtain the genetically engineered bacterium with high yield of methylselenocysteine.
Research results show that the gene cluster of the coding cysteine desulphurase IscSB and the coding serine dehydratase SdaA are knocked out from the recombinant genetically engineered bacteria, and the obtained genetically engineered bacteria have the highest yield of methylselenocysteine.
The construction method of the genetically engineered bacterium for producing the methylselenocysteine can be understood as a method for improving the synthesis of the methylselenocysteine by bacillus subtilis. According to the invention, by constructing the recombinant bacillus subtilis taking bacillus subtilis as a host, expressing a gene for encoding methionine S-methyltransgerase Mmt protein, knocking out a cysteine desulphurase IscSB gene, knocking out an amino transferase YhdR gene and knocking out a serine dehydratase SdaA gene cluster, preferably knocking out IscSB and SdaA gene clusters, the intracellular of methylselenocysteine in the recombinant bacillus subtilis is enhanced. The method for preparing the methylselenocysteine can greatly reduce the production cost, is not easily influenced by factors such as ambient temperature, pH and the like, and is convenient to use; no toxic product is produced in the conversion process, and no other byproducts are produced; is expected to realize the industrialized SeMCys production with low energy consumption, high efficiency, high purity and no pollution.
The application of the genetically engineered bacterium or the genetically engineered bacterium constructed by the construction method in preparing the methylselenocysteine can be understood as a method for preparing the methylselenocysteine by using the genetically engineered bacterium or the genetically engineered bacterium constructed by the construction method, which comprises the steps of fermenting and culturing the genetically engineered bacterium to prepare the methylselenocysteine.
In some embodiments of the present invention, the seed solution of the recombinant bacillus subtilis is inoculated into a fermentation medium, and cultured at a temperature of 30-38 ℃ and a rotation speed of 150-300 rpm, so as to finally obtain the expressed methylselenocysteine thallus. Preferably, the seed solution bacterial concentration of the recombinant bacillus subtilis is not lower than OD 1.0.
The invention also provides application of the recombinant bacillus subtilis or the recombinant bacillus subtilis obtained by the construction method or the method for preparing the methylselenocysteine in preparing the methylselenocysteine or a product containing the methylselenocysteine.
II, examples
The present invention will be specifically described below by way of specific examples. The experimental methods described below, unless otherwise specified, are all laboratory routine methods. The experimental materials described below, unless otherwise specified, are commercially available.
The pSTOP1622 plasmid referred to in the examples below was purchased from pravastatin biotechnology (beijing) inc.
The following examples relate to the following media:
LB medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.
LB medium was used for both seed medium and fermentation medium, and at OD 600 When the solution reaches 0.4, an inducer IPTG is added and the solution is at OD 600 When the concentration reaches 0.7, 6mg/L sodium selenite is added.
The detection method involved in the following examples is as follows:
and (3) detecting the content of methyl selenocysteine: the detection conditions of the UPLC-MS method are as follows:
instrument: waters XenvoTQS-micro ultra-high performance liquid chromatography-triple quadrupole mass spectrometer; chromatographic conditions: the chromatographic Column was ACQUITY UPLC HSS T C18 Column, 2.1X100 mm 1.8 μm, column temperature 40 ℃, sample chamber temperature 10 ℃, sample volume 5. Mu.L, flow rate 0.2mL/min, run time 7min, mobile phase A0.1% formic acid aqueous solution, mobile phase B acetonitrile, mobile phase concentration gradient as shown in Table 1.
TABLE 1 Mobile phase gradient Table
Example 1: construction of recombinant Bacillus subtilis GBACBC, GBACBR and GBACBM
Chemically synthesizing a gene (the nucleotide sequence is shown as SEQ ID NO. 1) for encoding the CpMmt protein, and chemically synthesizing a gene (the nucleotide sequence is shown as SEQ ID NO. 2) for encoding the RiMmt protein; chemically synthesizing a gene encoding NmMmt protein (the nucleotide sequence is shown as SEQ ID NO. 3); adding SpeI restriction enzyme sites (ACTAGT) to the 5 'ends of genes for encoding three Mmt proteins, and adding SmaI restriction enzyme sites (CCCGGG) to the 3' ends of genes for encoding three Mmt proteins to obtain three Mmt fragments respectively; pSTOP1622 was digested and recovered by SpeI and SmaI to give pSTOP1622 fragment; the Mmt fragment was ligated with pSTOP1622 fragment using T4 ligase to give vectors pSTOP-CpMmt, pSTOP-RiMmt and pSTOP-NmMmt. At this time, all three Mmt genes were located downstream of the PxylA promoter.
The primer KinBL-F (the nucleotide sequence is shown as SEQ ID NO. 15) and the primer KinBL-R (the nucleotide sequence is shown as SEQ ID NO. 16) are utilized, bacillus subtilis thalli are used as templates, and an upstream fragment LB (the nucleotide sequence is shown as SEQ ID NO. 17) of bacillus subtilis KinB is cloned by PCR and constructed on a pMD19 cloning vector to obtain a vector pMD19-LB; PCR cloning a downstream fragment RB of bacillus subtilis KinB (nucleotide sequence shown as SEQ ID NO. 20) by using a primer KinBR-F (nucleotide sequence shown as SEQ ID NO. 18) and a primer KinBR-R (nucleotide sequence shown as SEQ ID NO. 19), and carrying out enzyme digestion by using Kpn I and EcoR I, and connecting the RB fragment with pMD19-LB digested by the same restriction enzyme with T4 ligase to obtain a pMD-LB-RB vector, wherein the vector contains the fragment LB-RB (nucleotide sequence shown as SEQ ID NO. 21).
Amplifying the PxylA-CpMmt sequence (the nucleotide sequence is shown as SEQ ID NO. 24) of the expression frame by using primers Mmt-kinB-F (the nucleotide sequence is shown as SEQ ID NO. 22) and CpMmt-kinB-R (the nucleotide sequence is shown as SEQ ID NO. 23); primers Mmt-kineB-F (nucleotide sequence shown as SEQ ID NO. 22) and RiMmt-kineB-R (nucleotide sequence shown as SEQ ID NO. 25) amplify the expression cassette PxylA-RiMmt sequence (nucleotide sequence shown as SEQ ID NO. 26); the primers Mmt-kineB-F (nucleotide sequence shown as SEQ ID NO. 22) and NmMmt-kineB-R (nucleotide sequence shown as SEQ ID NO. 27) were used to amplify the expression cassette PxylA-NmMmt sequence (nucleotide sequence shown as SEQ ID NO. 28). The pMD-LB-RB was digested with restriction enzymes KpnI and XbaI to obtain a linear vector fragment. And respectively assembling the three expression frame sequences with the linear vector fragment through Gibson to obtain a pMD-LB-CpMmt-RB vector, a pMD-LB-RiMmt-RB vector and a pMD-LB-NmMmt-RB vector.
PCR amplifying LB-CpMmt-RB fragment as donor DNA (called CpMmt donor for short) by using primers KinBL-F and KinBR-R and pMD-LB-CpMmt-RB vector as template, wherein the nucleotide sequence is shown as SEQ ID NO. 29; PCR amplifying LB-RiMmt-RB fragment as donor DNA (called RiMmt donor for short) by using primers KinBL-F and KinBR-R and pMD-LB-RiMmt-RB vector as template, wherein the nucleotide sequence is shown as SEQ ID NO. 30; PCR amplification of LB-NmMmt-RB fragment as donor DNA (abbreviated as NmMmt, nucleotide sequence shown in SEQ ID NO. 31) using primers KinBL-F and KinBR-R and pMD-LB-NmMmt-RB vector as template
Construction of guide RNA vector: the primers KinBg-F (nucleotide sequence shown as SEQ ID NO. 32) and KinBg-R (nucleotide sequence shown as SEQ ID NO. 33) are synthesized, and the annealed product is connected with the pcrF11 plasmid digested by Eco31I by using T4 DNA ligase to obtain the guide RNA plasmid of KinB site.
Plasmid pHT-XCR6 containing recombinase is firstly transformed into bacillus subtilis GBACB, guide RNA plasmids of KinB site are respectively co-transformed with LB-CpMmt-RB fragment, LB-RiMmt-RB fragment and LB-NmMmt-RB fragment in the transformants, and genome-integrated transformants GBACBC, GBACBR and GBACBM are selected. GBACBC, GBACBR and GBACBM were cultured in LB containing 0.005% SDS, and the non-resistant plates were plated, and the bacteria on the non-resistant plates were picked up to a chloramphenicol resistant plate, and colonies that did not grow on chloramphenicol resistance were selected as GBACBC, GBACBR and GBACBM without plasmids.
Example 2: verification of recombinant Bacillus subtilis GBACBC, GBACBR and GBACBM
Bacillus subtilis GBACB is taken as a host, and bacillus subtilis GBACBC, GBACBR and GBACBM are respectively obtained according to the method of example 1; respectively inoculating GBACB, GBACBC, GBACBR and GBACBM into LB culture medium as seed culture medium, and culturing overnight at 37 ℃ to obtain seed solution;
inoculating the seed solution into LB fermentation medium at 5% inoculum size, and culturing OD at 33deg.C at 200r/min 600 IPTG and xylose were added to induce gene expression at 0.4 and OD 600 To 0.7, 6mg/L sodium selenite was added. Fermenting for 7h to obtain fermentation liquor.
The obtained fermentation broths were centrifuged, and the supernatants were filtered with microporous filters, and then subjected to ultrafiltration, and the filtrate was subjected to detection of intracellular methylselenocysteine content by UPLC-MS method, and the results are shown in table 1.
The results show that the yield of methylselenocysteine of the initial strain GBACB is only 18.4 mug/L, and the intracellular yields of methylselenocysteine of the recombinant bacillus subtilis strain GBACBC, GBACBR and GBACBM are improved, which indicates that the synthesis of methyl methionine greatly promotes the synthesis of methylselenocysteine, and the invention successfully constructs recombinant bacillus subtilis for synthesizing a large amount of methylselenocysteine in cells. Wherein the yield of the bacillus subtilis GBACBM strain is highest, and the yield of the bacillus subtilis GBACBC strain is inferior.
TABLE 2 production of methylselenocysteine by recombinant GBACBC, GBACBR and GBACBM
Example 3: construction of recombinant Bacillus subtilis GMC1, GMC3 and GMS
PCR cloning of an upstream fragment LBi of bacillus subtilis IscSB (nucleotide sequence shown as SEQ ID NO. 36) by using a primer IscSBL-F (nucleotide sequence shown as SEQ ID NO. 34) and a primer IscSBL-R (nucleotide sequence shown as SEQ ID NO. 35) and using bacillus subtilis thalli as templates; PCR cloning a downstream fragment RBi (nucleotide sequence shown as SEQ ID NO. 39) of the bacillus subtilis IscSB by using a primer IscSBR-F (the nucleotide sequence is shown as SEQ ID NO. 37) and a primer IscSBR-R (the nucleotide sequence is shown as SEQ ID NO. 38) and using bacillus subtilis thalli as templates; the LBi fragment and RBi fragment were mixed and ligated into a fragment LBi-RBi (nucleotide sequence shown as SEQ ID NO. 40) by fusion PCR using primers IscSBL-F and IscSBR-R.
PCR cloning of an upstream fragment LBy of the bacillus subtilis YhdRL by using a primer YhdRL-F (the nucleotide sequence of which is shown as SEQ ID NO. 41) and a primer YhdRL-R (the nucleotide sequence of which is shown as SEQ ID NO. 42) and using bacillus subtilis thalli as templates; PCR cloning of a downstream fragment RBy of the bacillus subtilis YhdRR (the nucleotide sequence of which is shown as SEQ ID NO. 46) by using a primer YhdRR-F (the nucleotide sequence of which is shown as SEQ ID NO. 44) and a primer YhdRR-R (the nucleotide sequence of which is shown as SEQ ID NO. 45) and using bacillus subtilis thalli as templates; the LBy fragment and RBy fragment were mixed and ligated into a fragment LBy-RBy (nucleotide sequence shown in SEQ ID NO. 47) by fusion PCR using the primers YhdRL-F and YhdRR-R.
PCR cloning of an upstream fragment LBs (nucleotide sequence shown as SEQ ID NO. 50) of bacillus subtilis SdaA by using a primer SdaAL-F (nucleotide sequence shown as SEQ ID NO. 48) and a primer SdaAL-R (nucleotide sequence shown as SEQ ID NO. 49) and using bacillus subtilis thalli as templates; PCR cloning a downstream fragment RBs (nucleotide sequence shown as SEQ ID NO. 53) of bacillus subtilis SdaA by using a primer SdaAR-F (the nucleotide sequence is shown as SEQ ID NO. 51) and a primer SdaAR-R (the nucleotide sequence is shown as SEQ ID NO. 52) and using bacillus subtilis thalli as templates; the LBs fragment and RBs fragment were mixed and ligated into a fragment LBs-RBs (nucleotide sequence shown as SEQ ID NO. 54) by fusion PCR using primers SdaAL-F and SdaAR-R.
Construction of guide RNA vector: synthesizing primers IscSBg-F (the nucleotide sequence is shown as SEQ ID NO. 55) and IscSBg-R (the nucleotide sequence is shown as SEQ ID NO. 56), and connecting annealed products with the pcrF11 plasmid digested by the Eco31I by using a T4 DNA ligase line to obtain a guide RNA plasmid of an IscSB site; synthesizing primers YhdRg-F (the nucleotide sequence is shown as SEQ ID NO. 57) and YhdRg-R (the nucleotide sequence is shown as SEQ ID NO. 58), and connecting annealed products with the pcrF11 plasmid digested by Eco31I by using a T4 DNA ligase to obtain a guide RNA plasmid of a YhdR site; the primers SdaAg-F (nucleotide sequence shown as SEQ ID NO. 59) and SdaAg-R (nucleotide sequence shown as SEQ ID NO. 60) are synthesized, and the annealed product is connected with the ecrF 11 plasmid digested by the Eco31I enzyme by using a T4 DNA ligase line to obtain the guide RNA plasmid of the SdaA locus.
The plasmid pHT-XCR6 containing the recombinase is firstly transformed into bacillus subtilis GBACBM, matched guide RNA plasmids and LB-RB fragments are subjected to co-transformation in the transformants, and transformants GMC1 of which IscSB genes are knocked out, transformants GMC3 of which YhdR genes are knocked out and transformants GMS of which SdaA gene clusters are knocked out are screened. GMC1, GMC3 and GMS were cultured in LB containing 0.005% SDS, and the non-resistant plates were coated, and the bacteria on the non-resistant plates were picked up to a chloramphenicol resistant plate, and colonies that did not grow on chloramphenicol resistance were selected as GMC1, GMC3 and GMS without plasmids.
Example 4: verification of recombinant Bacillus subtilis GMC1, GMC3 and GMS
The fermentation was carried out under the same conditions as in example 2, and the results are shown in Table 2.
The result shows that the yield of the methylselenocysteine of the recombinant bacillus subtilis strain GMC1 is improved by 1 time compared with that of GBACBM, and the fact that the IscSB gene is knocked out effectively reduces the degradation of SeCys so as to improve the yield of the methylselenocysteine; GMC3 knocks out the gene YhdR of the aminotransferase, the yield of the strain is improved, and as the aminotransferase catalyzes amino acid to convert amino acid into corresponding alpha-keto acid, the result of the invention shows that the aminotransferase has obvious amino conversion effect on SeCys, so that the gene is knocked out to obviously improve the yield of methylselenocysteine; after the Ser precursor degradation gene SdaA gene cluster is knocked out, the yield of the obtained methylselenocysteine of the strain GMS is improved most obviously, and the yield is improved by more than 3.5 times.
TABLE 3 production of methylselenocysteine by recombinant bacteria GMC1, GMC3 and GMS
Example 5: construction of recombinant Bacillus subtilis GMSC
The plasmid pHT-XCR6 containing the recombinase is firstly transformed into bacillus subtilis GMC1, the guide RNA plasmid positioned on the SdaAB and the LBs-RBs fragment are subjected to co-transformation in the transformant, and the transformant GMSC with the SdaA gene cluster knocked out is selected. GMSC was cultured in LB containing 0.005% SDS, and the non-resistant plate was plated, and the bacteria on the non-resistant plate were picked up and single colonies were transferred to a chloramphenicol resistant plate, and colonies that did not grow on chloramphenicol resistance were selected as GMSC without plasmid.
EXAMPLE 6 verification of recombinant Bacillus subtilis GMSC
The fermentation was carried out under the same conditions as in example 2, and the results are shown in Table 3. Meanwhile, the recombinant strain of IscSB and SdaA gene clusters is knocked out, the yield of methylselenocysteine is further improved, and 4120.3 mug/L is finally achieved. The final strain obtained by the invention has 225 times higher yield than the GBACB of the original strain.
TABLE 4 production of methylselenocysteine by recombinant GMSC
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, not to be construed as limiting the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims (10)

1. A genetic engineering bacterium for producing methyl selenocysteine has the following synthetic route:
(1) Serine (Ser) synthesizes O-acetylserine by action of serine O-acetyltransferase (SATp) from arabidopsis thaliana;
(2) O-acetylserine releases the expression inhibition of O-acetylserine thiol lyase (CysK), and synthesizes selenocysteine (SeCys) under the action of CysK;
(3) Synthesizing S-adenosylmethionine (SAM) by methionine under the action of S-adenosylmethionine synthetase (SAM 2) derived from Saccharomyces cerevisiae;
(4) Selenocysteine (SeCys) is methylated by S-adenosylmethionine (SAM) under the action of Selenocysteine Methyltransferase (SMT) derived from Astragalus membranaceus to form methylselenocysteine;
(5) S-adenosylmethionine (SAM) and methionine form methyl methionine (MMet) under the action of methionine S-methyltransferase (Mmt);
(6) Selenocysteine (sels) is methylated by methyl methionine (MMet) under the action of Selenocysteine Methyltransferase (SMT) to form methylselenocysteine.
2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is a recombinant host bacterium whose genome comprises a gene encoding serine O-acetyltransferase, SATp, a gene encoding selenocysteine methyltransferase, SAM2, and the host bacterium is bacillus subtilis 168; wherein the genome of the genetically engineered bacterium further comprises a gene encoding methionine S-methyltransferase Mmt.
3. The genetically engineered bacterium of claim 2, wherein the genes encoding methionine S-methyltransferase Mmt include a candidate heteromycota (Candidatus Peregrinibacteria) -derived gene encoding methionine S-methyltransferase CpMmt, a rose color-changing bacterium (Roseovarius indicus) -derived gene encoding methionine S-methyltransferase RiMmt, and a new sphingosine bacterium (novospingobium) MBES 04-derived gene encoding methionine S-methyltransferase nmmt.
4. The genetically engineered bacterium of claim 3, wherein,
the nucleotide sequence of the gene encoding methionine S-methyltransferase CpMmt is shown as SEQ ID NO. 1;
and/or the nucleotide sequence of the gene encoding methionine S-methyltransferase RiMmt is shown as SEQ ID NO. 2;
and/or the nucleotide sequence of the gene encoding methionine S-methyl transferase NmMmt is shown in SEQ ID NO. 3.
5. The genetically engineered bacterium of any one of claims 2-4, wherein the genetically engineered bacterium is a chassis-engineered bacterium that produces methylselenocysteine; the chassis modification comprises the knocking out of one or more of a gene encoding cysteine desulphurase IscSB, a gene encoding aminotransferase YhdR and a gene cluster encoding serine dehydratase SdaA; wherein the gene cluster encoding serine dehydratase SdaA comprises a gene encoding serine dehydratase SdaAA and a gene encoding serine dehydratase SdaAB.
6. The genetically engineered bacterium of claim 5, wherein,
the nucleotide sequence of the gene encoding cysteine desulphurase IscSB is shown in SEQ ID NO. 7;
and/or the nucleotide sequence of the gene encoding aminotransferase YhdR is shown in SEQ ID NO. 9;
and/or the nucleotide sequence of the gene encoding serine dehydratase SdaAA is shown in SEQ ID NO. 11;
and/or the nucleotide sequence of the gene encoding serine dehydratase SdaAB is shown in SEQ ID NO. 12.
7. The construction method of a genetically engineered bacterium according to any one of claims 1 to 6, comprising the steps of taking bacillus subtilis 168 as a host bacterium, placing genes CpMmt, riMmt and NmMmt encoding methionine S-methyltransferase under a PxylA promoter to form PxylA-CpMmt, pxylA-RiMmt and PxylA-NmMmt expression frames, inserting the expression frames into KinB sites of a genome of bacillus subtilis 168 respectively, and knocking out one or more of a gene encoding cysteine desulphurase IscSB, a gene encoding aminotransferase YhdR and a gene cluster encoding serine dehydratase SdaA in a genetically modified strain to obtain a genetically engineered bacterium for producing methylselenocysteine; wherein the gene cluster encoding serine dehydratase SdaA comprises a gene encoding serine dehydratase SdaAA and a gene encoding serine dehydratase SdaAB.
8. The use of the genetically engineered bacterium according to any one of claims 1 to 6 or the genetically engineered bacterium constructed by the construction method according to claim 7 in the preparation of methylselenocysteine; the application comprises the steps of fermenting and culturing the genetically engineered bacteria to prepare the methylselenocysteine.
9. The use according to claim 8, wherein during the fermentation culture the genetically engineered bacterium is inoculated into the fermentation medium in the form of a seed solution; the fermentation culture conditions are as follows: the temperature is 30-38 ℃, and the rotating speed of the shaking table is 150-300 rpm.
10. The use according to claim 8, wherein the OD of the seed solution of the genetically engineered bacterium 600 Not less than 1.0.
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