CN112225785B - GntR family transcription inhibitor mutant, mutant gene and application thereof in preparation of vitamin B2In (1) - Google Patents

GntR family transcription inhibitor mutant, mutant gene and application thereof in preparation of vitamin B2In (1) Download PDF

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CN112225785B
CN112225785B CN202011146278.1A CN202011146278A CN112225785B CN 112225785 B CN112225785 B CN 112225785B CN 202011146278 A CN202011146278 A CN 202011146278A CN 112225785 B CN112225785 B CN 112225785B
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张大伟
孙宜文
刘川
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Tianjin National Synthetic Biotechnology Innovation Center Co ltd
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Abstract

The invention discloses a GntR family transcription repression factor mutant, a mutant gene and application thereof in preparing vitamin B2. Wherein the amino acid sequence of the mutant has the following mutations relative to the sequence shown in SEQ ID No. 3: substitution of amino acid 89 with M. The gene engineering bacterium obtained by carrying out site-directed mutagenesis on the 89 th amino acid coding nucleotide in the GntR family transcription repression factor gene on the chromosome of the bacillus subtilis to code the amino acid M has greatly improved capability of producing the vitamin B2, is favorable for the growth of the bacterium and has larger application and popularization values.

Description

GntR family transcription inhibitor mutant, mutant gene and application thereof in preparation of vitamin B2In (1)
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a GntR family transcription repressing factor mutant, a genetic engineering bacterium thereof, and application thereof in preparing vitamin B2.
Background art:
riboflavin (Riboflavin) also called vitamin B2Molecular formula is C17H20O6N4The vitamin B is a water-soluble vitamin in a vitamin B group, exists in two forms of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) in organisms, participates in redox reaction as a coenzyme of some important oxidoreductases in organisms, and plays a role in delivering hydrogen. The lack of riboflavin causes the metabolic disorder of organisms, but neither humans nor animals can synthesize riboflavin by themselves and only ingest riboflavin from food, so that the production of riboflavin has a very wide market in the food, feed and pharmaceutical industries.
Currently, the industrial production methods of riboflavin include plant extraction methods, chemical synthesis methods, semi-synthesis methods, and microbial synthesis methods. Among them, the microbial synthesis method has advantages of low cost, environmental friendliness, renewable energy and the like, and gradually takes a leading position, and becomes a main method for most industrial production. Among a plurality of microorganisms capable of producing riboflavin, bacillus subtilis is taken as a non-pathogenic microorganism, has clear physiological metabolism and genetic background, is convenient to determine a metabolic target point and modify genetic engineering, has reliable safety, has long-term application of fermentation products in food and feed industries, and is very important for environmental, medical and industrial fermentation production. Secondly, the bacillus subtilis genetic engineering strain can excessively synthesize folic acid, inosine or guanosine and has the potential of providing enough precursors for excessive synthesis of riboflavin, so the bacillus subtilis genetic engineering strain gradually shows strong vitality in the microbial fermentation production of riboflavin and becomes a main production strain.
Riboflavin synthesis in bacillus subtilis (b.subtilis) requires two precursor substances, ribulose-5-phosphate (Ru5P) and guanine-5' -triphosphate (GTP), where Ru5P is derived from the pentose phosphate pathway and GTP is derived from the de novo purine synthesis pathway. Both of them finally synthesize riboflavin from the riboflavin synthesis pathway through 7-step reactions under the action of a number of riboflavin operons.
The GntR family is a Gluconate operator transcription repression (GntR) which is first found in Bacillus subtilis and is one of The widely occurring Transcription Factor (TFs) families in prokaryotes (Fujita Y, Fujita T, Miwa Y, et al, organization and transcription of The Gluconate operator, gnt, of Bacillus subtilis [ J ]. The Journal of biological chemistry,1986,261(29): 13744) -13753). Members of The GntR family regulate a variety of biological processes such as metabolic pathways, morphogenesis, sporulation, cell envelope stress or The production of secondary metabolites such as antibiotics (Rigali S, Derouaux A, Giannotta F, et al. Subdivision of The helix-turn-helix GntR in The FadR, HutC, MocR, and YtrA subfamilies [ J ]. The Journal of biological chemistry,2002,277(15): 12507-. YtrA is a sub-family of GntR prototyped by Bacillus subtilis YtrA TF, the entire operon including YtrA, ytrB, ytrC, ytrD, ytrE and ytrF, the first gene (YtrA) in this family encoding the negative regulatory factor of the ytr operon, the following five genes encoding ABC transporter proteins (Yoshida K I, Fujita Y, Ehrlich S d. an operator for a reactive ATP-binding cassette transport system protein inactivation of Bacillus subtilis J. Journal of bacteriology 2000,182(19): 5454-5461). YtrA is an additional regulator of cell membrane stress, and in addition to regulating the stress of the cell wall to antibiotics, regulates acetoin utilization (Salzberg L I, Luo Y, Hachmann Anna-Barbara, et al. the Bacillus subtilis GntR family pressor YtrA responses to cell walls antibodies [ J ]. Journal of bacteriology,2011,193(20): 5793-. The production of acetoin consumes the intermediate metabolites of the EMP pathway, enhances the competition with the pentose phosphate pathway and reduces the synthesis of vitamin B2 precursor substances. In addition, the regulation of the transcriptional expression level of the gene ytrA may contribute to the growth of the strain to a certain extent, thereby increasing the yield of the desired target. At present, no report has been made on the use of the GntR family transcription repressing factor mutant gene ytrA for vitamin B2 synthesis.
The invention content is as follows:
the inventor selects a high-yield vitamin B strain in the earlier stage2The Bacillus subtilis CGMCC NO.16132 strain (see Chinese patent application 201910604170.3) can be fermented to produce vitamin B2. The present inventors have further studied to find that vitamin B is produced2Genes with an effect on competence. Through research, the mutation of the GntR family transcription repressing factor coding gene is discovered, and the capacity of the strain for producing vitamin B2 can be improved by verifying that the GntR family transcription repressing factor coding gene is subjected to point mutation in bacillus subtilis to form the mutant gene.
First, the present invention provides a mutant of a GntR family transcription repressing factor, characterized in that its polypeptide amino acid sequence has the following mutations based on the original sequence shown in SEQ ID No. 3: amino acid 26 was replaced with H.
Preferably, the amino acid sequence is shown as SEQ ID No. 4.
Secondly, the invention provides a coding gene of the GntR family transcription repressing factor mutant.
Preferably, the nucleotide sequence is shown as SEQ ID No. 2.
Accordingly, the third aspect of the present invention also provides an expression cassette, a recombinant vector containing a gene encoding the GntR family transcription repressing factor mutant. The recombinant vector is not particularly limited to the starting vector, and may be any vector known in the art as long as it can replicate in a host. For example, the vector includes, but is not limited to, a plasmid, a phage. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, integrate into the genome itself.
More preferably a recombinant expression vector, more preferably a prokaryotic re-expression vector. Most preferred are expression vectors suitable for expression in Bacillus subtilis.
In a fourth aspect, the invention provides a recombinant host cell containing a gene encoding a mutant of said GntR family transcriptional repressor. Wherein said "host cell" is a cell having a meaning generally understood in the art, which is capable of introducing a gene encoding a mutant of the invention, the introduction being hereinafter referred to as recombinant host cell. The host cell of the invention may be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, more preferably Bacillus subtilis.
In a fifth aspect, the invention provides the use of a gene encoding a GntR family transcription repressing factor mutant for the manufacture of vitamin B2.
In a sixth aspect, the invention provides a method for enhancing vitamin B2 production of Bacillus subtilis, which is to perform site-directed mutagenesis on a GntR family transcription inhibitor encoding gene on a chromosome to obtain the Bacillus subtilis with enhanced vitamin B2 production capacity, wherein the site-directed mutagenesis is to replace an 89 th amino acid encoding nucleotide of the encoding gene with an encoding nucleotide of an encoding amino acid M, and more specifically to replace T with C at a 266 th nucleotide of the encoding gene. The site-directed mutagenesis can be carried out by various methods known in the art.
Preferably, the original strain of Bacillus subtilis is Bacillus subtilis BS168 ribCmThe strain of (1).
In a seventh aspect of the present invention, there is provided a method for producing vitamin B2 using the bacillus subtilis obtained by the method of the above sixth aspect, which comprises the steps of culturing the bacillus subtilis, and collecting vitamin B2, and more preferably further comprises the step of purifying vitamin B2.
Wherein the cultivation of Bacillus subtilis can be carried out according to conventional methods in the art, such as shake flask cultivation, batch cultivation, continuous cultivation, fed-batch cultivation, etc., and suitable cultivation conditions such as temperature, time, pH of the medium, etc., can be selected according to the actual situation. In addition, the vitamin B2 can be recovered or purified from the cells or culture medium by methods conventional in the art, such as filtration, anion exchange chromatography, crystallization, and HPLC.
The invention has the beneficial effects that: the research proves that the genetic engineering bacteria containing the GntR family transcription inhibiting factor mutant gene are biologically safe, and the experiment shows that the genetic mutation on the chromosome not only does not influence the growth of the bacteria, but also is beneficial to the growth of the bacteria, so that the capacity of producing the vitamin B2 by the bacillus subtilis can be effectively improved. Experimental data show that the BS168 ribC is combinedmThe change of the gene ytrA coded by the middle GntR family transcription repressing factor into the mutant gene ytrA (T89M) can improve the capacity of producing vitamin B2 by 11.6 percent, thereby having great application value in preparing vitamin B2.
Drawings
FIG. 1: VB of different bacillus subtilis strains after fermentation for 41 hours2And (4) yield.
FIG. 2: biomass after 41h fermentation of different bacillus subtilis strains.
Detailed Description
The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these schemes and figures should not be understood as limiting the invention and any changes made without departing from the principle and spirit of the invention are within the scope of protection of the invention.
The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.
The formula of the culture medium is as follows:
LB medium (g/L): 10 parts of sodium chloride, 10 parts of tryptone, 5 parts of yeast extract and 18 parts of agar powder added into a solid culture medium.
Fermentation medium (g/L): 10 parts of corn steep liquor dry powder, 30 parts of cane sugar, 2 parts of magnesium sulfate, 7 parts of ammonium sulfate, 3 parts of dipotassium phosphate and 1 part of monopotassium phosphate, and adjusting the pH value to 7.2-7.4 by NaOH.
Vitamin B2Is detected by
Mixing the fermentation liquor uniformly, diluting the fermentation liquor to a proper multiple by using 0.01mol/L NaOH, mixing uniformly, carrying out light-shielding alkali dissolution for 20min, centrifuging at 12000rpm for 2min, taking supernate, taking 0.01mol/L NaOH as a blank, measuring absorbance (the display value is controlled between 0.2 and 0.8) at 444nm, and calculating the content of riboflavin according to the following formula: FB (mg/L) ═ (dilution times absorbance)/0.0321.
Example 1: bacterial strain CGMCC NO.16132 and BS168 ribCmPerforming comparative genomic analysis
With the strain BS168 ribCm(see Chinese patent application 201910604170.3 for a specific construction process) as a reference genome, and sending the strain CGMCC NO.16132 to Jinzhi Biotech limited to perform whole genome re-sequencing and differential analysis to find genes which have an effect on the ability of producing vitamin B2. Through sequence alignment analysis, a great amount of mutations are found in the strain CGMCC NO.16132, and the strain comprises 338 mutation sites: through mutation data statistics, 72 genes are involved in mutation. The mutations in the coding region involved 59 genes in total, of which 8 were synonymous mutations, 41 were nonsynonymous mutations, 2 were nonsense mutations, 5 were frameshift mutations, and 3 were non-frameshift mutations; mutations within the non-coding region involved a total of 13 genes.
Removing synonymous mutation and gene mutation with unknown gene function, searching metabolic pathways involved in biosynthesis from glucose to vitamin B2, focusing on researching gene mutation involved in enzyme catalysis, excretion and metabolic regulation in relevant metabolic pathways by means of pathway enrichment analysis, and primarily screening genes possibly influencing the capacity of producing vitamin B2, wherein the genes comprise mutant genes directly related to vitamin B2 synthetic pathway, purine efflux pump mutant genes, purine synthetic pathway regulation mutant genes, purine degradation pathway mutant genes and the like. Among them, it was analyzed that the gene ytrA encoding the GntR family transcription repressing factor was point-mutated and the 266 th nucleotide thereof was substituted with C for T. To verify the effect of the mutation site on vitamin B2 production, the original strain BS168 ribC was inoculatedmIn (b) the ytrA gene is point mutated。
Example 2: construction of a Strain containing the ytrA mutant
Bacillus subtilis 168 chromosome is taken as a template, and a primer UPytrA is usedm-F、UPytrAmUPytrA with point mutation and with linker for R (containing DR) amplificationm(containing DR) fragment, amplifying the adapter-carrying araR (containing DR) fragment by using a primer araR-F, araR-R (containing DR); using pC194 plasmid as a template, and amplifying a cat fragment with a joint by using a primer cat-F, cat-R; using chromosome of strain CGMCC NO.16132 as template, and using primer DNytrA-F, DNytrA-R to amplify downstream homologous arm fragment DNytrA, wherein the nucleotide sequence of the mutant ytrA is shown as SEQ ID No.2, and the coded amino acid sequence is shown as SEQ ID No. 4.
With UPytrAmThe fragment, cat fragment, araR fragment and DNytrA fragment are used as templates, and a primer UPytrA is usedm-F, DNytrA-R fusion PCR, to obtain the assembly fragment UCR-ytrAmThe nucleic acid electrophoresis detection is correct, and the gel is recovered to obtain purified UCR-ytrAmAnd (3) fragment.
Subjecting UCR-ytrAmTransformation of BS168 ribC by fragment SpizizenmSpreading the mixture on LB solid plate containing 8mg/L chloramphenicol, culturing for 24h, performing colony PCR verification, carrying out gold-fed intelligent sequencing with correct nucleic acid electrophoresis, and obtaining intermediate strain BS168 UCR-ytrA with point mutation of ytrA gene after correct sequencingmWherein the nucleotide sequence of said mutant ytrA is shown in SEQ ID No.2 and the encoded amino acid sequence is shown in SEQ ID No. 4.
Picking an intermediate strain BS168 UCR-ytrAmThe single colony is put in a test tube containing 5mL LB, after shaking culture is carried out for 8h at 37 ℃,200 uL bacterial liquid is taken and coated on an LB solid plate containing 40mg/L neomycin, colony PCR verification is carried out after 24h of culture, gold-feeding intelligent sequencing is carried out when nucleic acid electrophoresis is correct, and after the sequencing is correct, the strain BS168 ytrA which removes the screening marker cat-araR and has point mutation of the ytrA gene through homologous recombination of DR in the chromosome is obtainedmWherein the nucleotide sequence of said mutant ytrA is shown in SEQ ID No.2 and the encoded amino acid sequence is shown in SEQ ID No. 4.
The primers used in this section were as follows:
Figure BDA0002739797210000051
the strains and plasmids used in this section were as follows:
Figure BDA0002739797210000052
Figure BDA0002739797210000061
example 3: evaluation of vitamin B in different strains2Capacity of production
1. The strain culture conditions are as follows:
the original strain BS168 ribCmAnd the engineered strain BS168 ytrAmLB solid plates containing 25mg/L erythromycin were streaked with an inoculating needle under aseptic conditions, and inverted in an incubator at 37 ℃ for 24 hours to obtain freshly activated single colonies. A single colony was picked with an inoculating needle, streaked on an LB solid slant containing 25mg/L erythromycin, and cultured in an incubator at 37 ℃ for 48 hours. Scraping 1/3 thallus Porphyrae on the inclined plane, inoculating into 500mL baffle triangular flask containing 70mL fermentation medium (3 parallel strains), culturing at 37 deg.C under shaking at 200rpm for 41 hr, and measuring OD600 and vitamin B in the fermentation broth2And (4) yield.
2. Different strains OD600 and vitamin B2Comparison of yields
With the starting strain BS168 ribCmIn contrast, the engineered strain BS168 ytrA containing the GntR family transcription repressing factor mutant gene ytrA (T89M)mVitamin B of (2)2The yield increased by 11.6% (see table 3 below and figure 1).
Figure BDA0002739797210000062
The experimental result shows that the gene ytrA coded by the GntR family transcription inhibitor is changed into the mutant gene ytrA(T89M) such genetic modification increases the production of vitamin B by the strain2The ability of the cell to perform. In addition, the engineered strain BS168 ytrA containing the GntR family transcription repressing factor coding gene ytrA (T89M)mThe biological quantity of the strain is higher than that of the original strain BS168 ribCmIt was shown that point mutations in the ytrA gene are beneficial for bacterial growth (see Table 3 and FIG. 2).
Sequence listing
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<120> GntR family transcription repressing factor mutant, mutant gene and application thereof in preparing vitamin B2
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Claims (9)

1. A mutant of a GntR family transcription repressing factor, characterized in that the polypeptide amino acid sequence thereof has only the following mutations with respect to the sequence shown in SEQ ID No. 3: substitution of amino acid 89 with M.
2. A gene encoding a mutant of a GntR family transcription repressing factor of claim 1.
3. The encoding gene of claim 2, wherein the nucleotide sequence is as shown in SEQ ID No. 2.
4. A recombinant vector comprising a gene encoding a mutant of a GntR family transcription repressing factor according to claim 2.
5. A recombinant host cell comprising a gene encoding a mutant of a GntR family transcription repressing factor according to claim 2.
6. Use of a mutant of a GntR family transcription repressing factor as defined in claim 1, or a gene encoding it, for the manufacture of vitamin B2.
7. A method for enhancing vitamin B2 production of Bacillus subtilis, characterized in that a gene encoding GntR family transcription repressing factor on chromosome is subjected to site-directed mutagenesis, the polypeptide amino acid sequence of the GntR family transcription repressing factor is shown as SEQ ID No.3, thus obtaining the Bacillus subtilis with enhanced vitamin B2 production capability, wherein the site-directed mutagenesis is to site-directed mutagenesis of the coding nucleotide of the 89 th amino acid T of the coding gene to code for the amino acid M.
8. The method of claim 7, wherein the Bacillus subtilis is Bacillus subtilis BS168 ribCmStrain as strainAnd (4) fermenting the strain.
9. A method for producing vitamin B2 using Bacillus subtilis obtained by the method of claim 7 or 8, comprising the steps of culturing the Bacillus subtilis, and collecting vitamin B2.
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