阅读说明：本技术 尿卟啉原ⅲ合成酶突变体、突变基因及其在制备维生素b12中的应用 (Uroporphyrinogen III synthetase mutant, mutant gene and application of mutant gene in preparation of vitamin B12 ) 是由 张大伟 董会娜 马延和 于 2020-01-07 设计创作，主要内容包括：本发明公开一种尿卟啉原Ⅲ合成酶突变体、突变基因及在制备维生素B<Sub>12</Sub>中的应用。在苜蓿中华根瘤菌中过表达的尿卟啉原Ⅲ合成酶基因<I>hemD</I>和突变基因的基因工程菌,其生产维生素B<Sub>12</Sub>的能力得到了大幅提高,具有较大的应用推广价值。(The invention discloses uroporphyrinogen III synthetase mutant, mutant gene and application thereof in preparing vitamin B 12 The use of (1). Uroporphyrinogen III synthase gene overexpressed in Sinorhizobium meliloti hemD And mutant gene, producing vitamin B 12 Has greatly improved capability ofGreater application and popularization value.)

1. A mutant uroporphyrinogen iii synthase having an amino acid sequence that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 4 has the following mutations: the amino acid substitution at position 53 is V, and the amino acid substitution at position 173 is S.

2. A gene encoding the uroporphyrinogen iii synthase mutant according to claim 1.

3. The gene encoding an uroporphyrinogen iii synthase mutant according to claim 2, having the nucleotide sequence of SEQ ID NO: 3, respectively.

4. Process for preparing vitamin B from uroporphyrinogen III synthetase coding gene₁₂Wherein the uroporphyrinogen iii synthase-encoding gene encodes a protein having the amino acid sequence of SEQ ID NO: 6.

5. The use of claim 4, wherein overexpression is achieved by introducing the coding gene into Sinorhizobium meliloti via an expression vector comprising the coding gene.

6. The use of claim 5, wherein the Sinorhizobium meliloti strain has a accession number of CGMCC No. 9638.

7. The use of claim 4, wherein said mutant uroporphyrinogen III synthase encoding gene has the amino acid sequence of SEQ ID NO: 3.

8. The use of any one of claims 4 to 7, wherein the introduced coding gene is located on a plasmid or chromosome.

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to uroporphyrinogen III synthetase mutant, a coding gene thereof and application thereof in preparing vitamin B₁₂The use of (1).

Background

Vitamin B₁₂(VB₁₂) The vitamin B derivative has wide application in the pharmaceutical and food industries, is called cobalamin, belongs to corrin compounds, is the only vitamin compound containing metal elements, and is a macromolecular organic compound with the latest discovery of B vitamins. Vitamin B, depending on the type of ligand (R group) above the corrin ring₁₂The method can be divided into the following steps: hydroxycobalamin, deoxyadenosylcobalamin and methylcobalamin. Vitamin B₁₂Participate in a large number of biochemical processes including DNA synthesis and regulation, fatty acid synthesis, amino acid metabolism and ability generation.

Due to vitamin B₁₂The molecular structure is complex, the artificial synthesis by a chemical method needs to consume a large amount of manpower and material resources, and the synthesis period is long. The requirements on operators during the synthesis process are too high, so that the large-scale production cannot be realized. Microbial fermentation currently produces vitamin B₁₂The method (2) can be mass-produced and popularized for use.

At present, vitamin B is targeted at home and abroad₁₂Biosynthesis of vitamin B by producing bacteria₁₂The research mainly focuses on the optimization of the fermentation process, and mainly relates to the optimization of a culture medium comprising a carbon nitrogen source and metal ions, the addition of betaine and the addition of rotenone, and the control of process conditions comprising pH and oxygen supply and the like. There are reports in the literature of increased vitamin B production by cells expressing a single copy of the Vitreoscilla vgb gene on the Pseudomonas denitrificans genome₁₂(vii) ability (Chenoporyl et al, expression of uroporphyrinogen III transmethylase from different sources in Pseudomonas denitrificans and its effect on vitamin B12 production. Industrial microorganism, 2017, Vol.47, No. 3).

The inventor selects a high-yield vitamin B strain in the earlier stage₁₂The Sinorhizobium meliloti CGMCC NO.9638 strain (CN 104342390A) can be fermented to produce vitamin B₁₂. Whereas in Sinorhizobium meliloti ALA (5-aminolevulinic acid) and uroporphyrinogen III are vitamin B₁₂Key precursors of the synthetic pathway. ALA in ALA dehydratase (HemB, gene)hemBCode) is catalyzed to generate porphobilinogen, and then the porphobilinogen is synthesized in hydroxymethyl cholestrinEnzyme (HemC, gene ofhemCEncoded) by the enzyme hydroxymethylcholine, and by uroporphyrinogen III synthase (HemD, gene-encoded)hemDEncoding) catalytic production of uroporphyrinogen III containing tetrapyrrole rings (Huang Fang, Jie Kang, Dawei Zhang. Microbial production of vitamin B)₁₂: A review and future perspectives.Microbial Cell Factories2017 Jan 30, 16(1):15. doi: 10.1186/s 12934-017-0631-y.). At present, uroporphyrinogen III synthetase gene is not availablehemDFor vitamin B₁₂And (5) reporting the synthesis.

Disclosure of Invention

The inventor screens high-yield vitamin B in the early stage₁₂The Sinorhizobium meliloti strain CGMCC NO.9638 (CN 104342390A) is subjected to mutagenesis to obtain a strain capable of producing vitamin B₁₂A mutagenized strain with improved capacity. The present inventors have further studied to find that vitamin B is produced₁₂Genes with an effect on competence. Research shows that uroporphyrinogen III synthetase genehemDAnd the mutant gene is introduced into Sinorhizobium meliloti to be over-expressed, so that the vitamin B production of the Sinorhizobium meliloti can be improved₁₂The ability of the cell to perform.

First, the present invention provides a mutant of uroporphyrinogen iii synthase gene, characterized in that the amino acid sequence thereof is represented by SEQ ID NO: 4 has the following mutations: the amino acid substitution at position 53 is V, and the amino acid substitution at position 173 is S. More preferably, the amino acid sequence is as shown in SEQ ID NO: and 6.

The present invention also provides a gene encoding a mutant uroporphyrinogen III synthase as described above. More preferably, the nucleotide sequence is as set forth in SEQ ID NO: 3, respectively.

In a third aspect, the present invention provides a uroporphyrinogen III synthetase encoding gene or a mutant encoding gene thereof for use in the preparation of vitamin B₁₂The use of (1).

In a specific embodiment, the coding gene is introduced into Sinorhizobium meliloti through an expression vector containing the coding gene for overexpression. More preferably, the Sinorhizobium meliloti has a preservation number of CGMCC NO. 9638.

Preferably, the uroporphyrinogen iii synthase encoding gene encodes a gene having the sequence of SEQ ID NO: 4; the mutant coding gene of uroporphyrinogen III synthetase has the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, preferably the uroporphyrinogen iii synthase encoding gene has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. Wherein the introduced coding gene is located in a plasmid or chromosome.

Proved by research, the invention overexpresses uroporphyrinogen III synthetase genehemDThe engineering bacteria with mutant genes are biologically safe, and can effectively improve the vitamin B production of sinorhizobium meliloti₁₂The ability of the cell to perform. Through experiments, data show that the over-expression of the original gene in sinorhizobium meliloti can improve the vitamin B production₁₂The capacity of the mutant gene reaches 12.5 percent, and the over-expression of the mutant gene in Sinorhizobium meliloti can further improve the vitamin B production₁₂The capacity of the strain is improved by more than 20 percent.

Drawings

FIG. 1: plasmid vector pBBR-P21-hemDThe structure of (1).

FIG. 2: VB of different sinorhizobium meliloti strains fermented for 144h₁₂And (4) yield.

FIG. 3: biomass of different sinorhizobium meliloti strains after fermentation for 144 h.

FIG. 4: vitamin B₁₂Standard graph of (2).

Detailed Description

The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope 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 15 parts of agar powder added into a solid culture medium.

Seed medium (g/L): sucrose 40, corn steep liquor 20, betaine 5, (NH)₄)₂SO₄1，(NH₄)₂HPO₄2，MnSO₄·H₂O 0.8，CoCl₂·6H₂O 0.02，MgO 0.3，DMBI 0.01，ZnSO₄·7H₂O 0.01，CaCO₃1.5, and controlling the pH value to be 7.0-7.4 by NaOH.

Fermentation medium (g/L): sucrose 80, corn steep liquor 30, betaine 15, (NH)₄)₂SO₄2，MgSO₄1.5, K₂HPO₄0.75，CoCl₂·6H₂O 0.14, DMBI 0.075，ZnSO₄·7H₂O 0.08，CaCO₃1, controlling the pH value to be 7.0-7.4 by NaOH.

Vitamin B₁₂Is detected by

(1) Sample pretreatment

Taking 1mL of fermentation liquor, adding 8% sodium nitrite solution and glacial acetic acid, shaking up 0.25mL each, and placing in a water bath at 95-100 ℃ for 30-40 min; after cooling to room temperature, centrifugation was carried out at 10000 rpm for 1 minute, and the supernatant was filtered through a 0.22 μm membrane (⌀ = 0.22 μm) filter into an upper flask, followed by addition of 20 μ l of 2% NaCN (w/v) to 1mL of the supernatant. The addition amount of the sodium nitrite solution and the glacial acetic acid can be correspondingly adjusted along with the amount of the fermentation liquor.

(2) Preparation of standards

Configuring gradient vitamin B₁₂Standard substance (20 mg/L, 50mg/L, 100mg/L, 150 mg/L).

(3) HPLC detection conditions

C18-250A column (Agilent, 4.6 mmid 9X 250 mm, 5 μm). The mobile phase is 70% organic phase (acetonitrile) and 30% inorganic phase (sodium acetate aqueous solution), the absorption wavelength is 361 nm, the column temperature is 35 ℃, the flow rate is 0.8 mL/min, and the sample amount is 20 mu L.

(4) Vitamin B₁₂Drawing of standard curve

Performing HPLC detection on the standard substances with different concentrations according to the above conditions, and drawing peak area A-VB₁₂Concentration standard curve. Using the measured peak area A as the ordinate, vitamin B₁₂The mass concentration C (mg/L) is recorded as the abscissa and vitamin B is plotted₁₂A standard curve. See fig. 4, resulting in regression equation y =19.846x-80.857, R²= 0.999, the absorbance is in a good linear relationship with the mass concentration. After the liquid phase is finished, according to vitamin B₁₂The standard curve calculates the sample yield.