阅读说明：本技术 苏氨酸醛缩酶、其编码基因和在屈昔多巴生物合成中的应用 (Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis ) 是由 赵文艳 杨碧玲 潘银平 倪德春 王伯初 戚娜 祝连彩 于 2019-10-12 设计创作，主要内容包括：本发明涉及L-苏氨酸醛缩酶及其编码基因与应用,属于生物技术领域。所述L-苏氨酸醛缩酶,为SEQ ID NO.1所示的蛋白,或将SEQ ID NO.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加所获得的功能相同的蛋白。所述L-苏氨酸醛缩酶能够以3-/4-位羟基取代的苯甲醛为底物,以5-磷酸吡哆醛为辅酶,以甘氨酸为辅底物,在适当的条件和介质内进行酶催化反应,生物合成屈昔多巴(L-threo-DOPS),该方法的非对映选择性能达到30％。本发明具有较高选择性的L-苏氨酸醛缩酶对于屈昔多巴生物催化具有重要的意义。(The invention relates to an L-threonine aldolase, a coding gene and application thereof, belonging to the technical field of biology. The L-threonine aldolase is a protein shown in SEQ ID NO.1, or a protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in SEQ ID NO. 1. The L-threonine aldolase can perform enzyme catalytic reaction in a proper condition and medium by using 3-/4-hydroxyl-substituted benzaldehyde as a substrate, pyridoxal 5-phosphate as a coenzyme and glycine as an auxiliary substrate to biosynthesize droxidopa (L-threo-DOPS), and the diastereoselectivity of the method reaches 30%. The L-threonine aldolase with higher selectivity has important significance for droxidopa biocatalysis.)

1. An L-threonine aldolase represented by a1 or a 2:

a protein shown as SEQ ID NO. 1;

a2. the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

2. The gene encoding L-threonine aldolase as set forth in claim 1.

3. The encoding gene of claim 2, wherein: the coding gene is any one of the following b1-b 3:

b1. a DNA molecule represented by SEQ ID NO. 2;

b2. a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in b1 and which encodes the L-threonine aldolase of claim 1;

b3. a DNA molecule having more than 90% identity to the DNA molecule defined in b1 or b2 and encoding the L-threonine aldolase of claim 1.

4. A recombinant vector, expression cassette or recombinant cell comprising the coding gene of claim 2 or 3.

5. Use of the L-threonine aldolase of claim 1 for catalyzing a reaction.

6. A catalyst, characterized by: comprising the L-threonine aldolase of claim 1.

7. A method for carrying out a selective aldehyde condensation reaction of a substrate, characterized in that: a substrate is subjected to a catalytic reaction using the L-threonine aldolase of claim 1 or the catalyst of claim 6.

8. The method of claim 7, wherein: the catalytic reaction takes 3, 4-dihydroxy benzaldehyde as a substrate, takes pyridoxal 5-phosphate as a coenzyme and glycine as an auxiliary substrate to synthesize droxidopa.

9. The method of claim 8, wherein: the dosage of the L-threonine aldolase is 100-2000mg/mL, the concentration of the 3, 4-dihydroxybenzaldehyde is 1-100g/L, the concentration of the glycine is 5-100g/L, and the concentration of the 5-pyridoxal phosphate is 0.01-0.2 g/L.

10. The method according to any one of claims 7-9, wherein: the temperature of the catalytic reaction is controlled to be 5-42 ℃, and the stirring speed is 70-300 rpm.

Technical Field

The invention relates to the field of biotechnology, in particular to L-threonine aldolase, a coding gene thereof and application thereof in droxidopa biosynthesis.

Background

Droxidopa (L-threo-DOPS) is a novel anti-Parkinson disease drug. For improving gait stiffness and orthostatic dizziness caused by Parkinson's disease; ameliorating orthostatic hypotension, orthostatic dizziness and fainting caused by Shy-Drager syndrome or familial amyloid neuropathy; improve dizziness and hypodynamia of hemodialysis patients caused by orthostatic hypotension.

At present, the synthetic method of droxidopa mainly comprises chemical synthesis and enzymatic catalysis. The chemical synthesis method mainly obtains chiral droxidopa through addition reaction, esterification reaction and chemical resolution, although the chemical method is simple to operate, a large amount of water, heavy metals and virulent hydrogen sulfide are used in the reaction process, and a large amount of optical isomers are used as byproducts, so that the method does not meet the atomic economy and environmental protection policies advocated and promoted by the current and future countries. The enzyme catalysis method has the advantages of mild conditions, small water consumption (only one tenth of that of the chemical method), no heavy metal pollution, no use of highly toxic chemicals, high chiral selection and the like, and has good application prospect.

Threonine Aldolase (TA) has very wide application potential, and can catalyze and synthesize chiral pharmaceutical key intermediate beta-hydroxy-alpha-amino acid. Threonine aldolase can be classified into two types, L-type and D-type, according to its stereospecificity at the α carbon of the threonine substrate. However, the stability of threonine aldolase is poor, and the chiral selectivity, especially the chiral selectivity of beta-hydroxy is insufficient, so that the application of the threonine aldolase in chiral synthesis is limited. It is therefore highly necessary to develop threonine aldolases with high selectivity for the synthesis of droxidopa.

Disclosure of Invention

The invention utilizes metagenome sequencing technology to separate a new L-threonine aldolase gene from excrement samples of healthy black bears in Sichuan black bear protection and incubation bases, the gene is named as L-TA Sz-1-2 gene, the nucleotide sequence of the gene is shown as a sequence 2 in a sequence table, an open reading frame is arranged from the 1 st position to the 1032 th position of the 5' end, the length is 1032bp, and the coded amino acid of the L-TA Sz-1-2 protein is shown as a sequence 1 and consists of 343 amino acid sequences.

The L-threonine aldolase belongs to a biocatalyst, can use benzaldehyde substituted by 3-/4-hydroxyl as a substrate, pyridoxal 5-phosphate as a coenzyme and glycine as an auxiliary substrate to carry out enzyme catalytic reaction in a proper condition and medium, and is used for biosynthesizing droxidopa (L-threo-DOPS), and the diastereoselectivity of the method can reach 30%. Because droxidopa is mainly synthesized by chemical synthesis under the harsh conditions and the enzyme catalysis does not realize industrial production due to a low de value, the L-threonine aldolase has important significance for the biological catalysis of droxidopa.

In one aspect, the present invention provides an L-threonine aldolase represented by a1 or a 2:

a protein shown as SEQ ID NO. 1;

a2. the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

The invention also provides a coding gene of the L-threonine aldolase. Preferably, the coding gene is any one of the following b1-b 3:

b1. a DNA molecule represented by SEQ ID NO. 2;

b2. a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in b1 and which encodes said L-threonine aldolase;

b3. a DNA molecule having 90% or more identity to the DNA molecule defined in b1 or b2 and encoding said L-threonine aldolase.

Recombinant vectors, expression cassettes or recombinant cells containing said coding genes also belong to the scope of protection of the present invention. The vector can be a cloning vector and comprises an L-TA Sz-1-2 gene and other elements required for plasmid replication; it may also be an expression vector comprising the L-TA Sz-1-2 gene and other elements that enable successful expression of the protein. Recombinant cells refer to transgenic cell lines or recombinant bacteria, and may be recombinant cells comprising a cloning vector, e.g., e.coli DH5 α; or a recombinant cell comprising an expression vector, such as e.coli BL 21. The expression of L-TA Sz-1-2 can be induced by culturing the recombinant cells under appropriate conditions.

The application of the L-threonine aldolase in catalytic reaction also belongs to the protection scope of the invention.

The invention also provides a catalyst, which comprises the L-threonine aldolase. The catalyst may be used simultaneously with other suitable catalysts to increase the efficiency of the enzyme catalysis or to carry out two catalytic reactions one after the other in the same reaction system.

In another aspect, the present invention provides a method for effecting selective aldehyde condensation of a substrate by using the L-threonine aldolase or the catalyst to catalyze the reaction of the substrate.

Preferably, the catalytic reaction takes 3, 4-dihydroxybenzaldehyde as a substrate, takes pyridoxal-5-phosphate as a coenzyme and glycine as an auxiliary substrate to synthesize droxidopa.

Preferably, the amount of L-threonine aldolase is 100-2000mg/mL, the concentration of 3, 4-dihydroxybenzaldehyde is 1-100g/L, the concentration of glycine is 5-100g/L, and the concentration of pyridoxal-5-phosphate is 0.01-0.2 g/L.

Preferably, the temperature of the catalytic reaction is controlled to be 5-42 ℃, and the stirring speed is 70-300 rpm.

Drawings

FIG. 1 is a schematic diagram of the selective synthesis of droxidopa by L-threonine aldolase.

FIG. 2 is an agarose gel electrophoresis image of the isolated L-TA Sz-1-2 gene.

FIG. 3 is an SDS-PAGE electrophoresis of L-TA Sz-1-2 protein, wherein lane 1 is a Marker of 14kDa to 120kDa, and lane 2 is L-TA Sz-1-2 protein.

FIG. 4 shows the alignment of L-TA Sz-1-2 with the existing E.coli L-threonine aldolase.

FIG. 5 is an HPLC chromatogram of synthetic droxidopa with peak 1 being L-erythro-DOPS and peak 2 being L-threo-DOPS (droxidopa).

FIG. 6 shows the effect of pH on de-value of the L-TA Sz-1-2 enzymatic reaction.

FIG. 7 shows the effect of temperature on de value of the L-TA Sz-1-2 enzymatic reaction.

Detailed Description

The invention is further described below in connection with specific examples, which are to be construed as merely illustrative and explanatory and not limiting the scope of the invention in any way.

Biological material：

Coli DH5 α competent cells, purchased from tiangen biochemical technology (beijing) ltd, cat #: CB 101;

coli BL21 competent cells, purchased from tiangen biochemical technology (beijing) ltd, cat #: CB 105.

Experimental reagent：

Fecal DNA genome extraction kit, purchased from Qiagen, germany, cat #: 51604, respectively;

prime STAR HS DNA Polymerase, purchased from TaKaRa, cat # stock: DR 010A;

agarose gel recovery kit, purchased from Omega, cat No.: d2500-01;

vector pGEX-6p-2, purchased from Youbao: cargo number VT 1259;

plasmid extraction Kit OMEGA Plasmid Mini Kit I, purchased from OMEGA, cat #: d6943;

restriction enzyme BamHI, purchased from gangbao biotechnology limited, cat #: 1010S;

restriction enzyme XhoI, purchased from gangbao biotechnology limited, cat #: 1094S;

t4DNA Ligase, purchased from british biotechnology limited, cat #: D2011A; 10 XT 4Ligase buffer, purchased from Dalianbao Biotechnology Ltd;

lysis buffer: prepared, 10mM PBS with pH 7.3 contains PMSF 0.1mM and Leupeptin 0.5 mg/mL;

glutaminone Sepharose 4B, purchased from GE Healthcare, cat No.: 10223836, respectively;

PreScission Protease, purchased from GenScript, Cat. No.: z02799-100;

BCA kit, purchased from Beyotime, cat # s: p0006;

L-threo-DOPS, available from Profenor technologies, Cathaka: d4235;

glycine, purchased from bio-engineering (shanghai) gmbh, cat #: a502065;

3, 4-dihydroxybenzaldehyde, purchased from bio-engineering (shanghai) gmbh, cat #: a601406;

PLP, purchased from bio-engineering (shanghai) gmbh, cat #: a610455;

EB, purchased from carbofuran technologies, cat #: 242101, respectively;

the black bear feces used for DNA extraction are frozen and stored in the laboratory.

The biochemical reagents not specifically described in the following examples are all conventional reagents in the art, can be prepared according to conventional methods in the art or are commercially available, and are all chemically pure and biologically pure.