CN-121991959-A - RBS element, glucose dehydrogenase expression nucleic acid molecule and NMNH preparation method

Abstract

The invention discloses a RBS element, a glucose dehydrogenase expression nucleic acid molecule and a NMNH preparation method, which belong to the technical field of nucleic acid, wherein the nucleotide sequence of the RBS element is shown as any one of SEQ ID NO.1-10, and the glucose dehydrogenase expression nucleic acid molecule comprises a nucleic acid molecule which sequentially consists of a promoter, the RBS element with the nucleotide sequence shown as any one of SEQ ID NO.3-10 and a glucose dehydrogenase coding nucleic acid molecule. The RBS element can be used for protein expression nucleic acid molecules to effectively improve the protein expression quantity, and the expression quantity of glucose dehydrogenase prepared by the glucose dehydrogenase expression nucleic acid molecules is higher, so that the preparation efficiency of NMNH is higher.

Inventors

• LIU LIHUI
• WANG JINGYI
• YANG PENG
• LI ZHAO
• XIE RUIMIN

Assignees

• 深圳希吉亚生物技术有限公司

Dates

Publication Date

20260508

Application Date

20260409

Claims (10)

1. 1. RBS element, characterized in that the nucleotide sequence is shown in any one of SEQ ID NO. 1-10.
2. 2. An RBS, characterized in that it is transcribed from the RBS element of claim 1.
3. 3. A protein expressing nucleic acid molecule comprising a nucleic acid molecule consisting of, in order, a promoter, the RBS element of claim 1, and a protein encoding nucleic acid molecule.
4. 4. A glucose dehydrogenase expression nucleic acid molecule is characterized by comprising a nucleic acid molecule which sequentially comprises a promoter, an RBS element and a glucose dehydrogenase coding nucleic acid molecule, wherein the nucleotide sequence of the RBS element is shown in any one of SEQ ID NO. 3-10.
5. 5. A glucose dehydrogenase expression plasmid comprising the glucose dehydrogenase expression nucleic acid molecule of claim 4.
6. 6. A glucose dehydrogenase-expressing bacterium comprising the glucose dehydrogenase-expressing nucleic acid molecule of claim 4 and/or the glucose dehydrogenase-expressing plasmid of claim 5.
7. 7. A method for producing glucose dehydrogenase, wherein the glucose dehydrogenase-expressing bacterium according to claim 6 is induced to express glucose dehydrogenase.
8. 8. A preparation method of NMNH, characterized in that glucose dehydrogenase is obtained by inducing expression of the glucose dehydrogenase expression bacteria of claim 6, and then the glucose dehydrogenase is added into a solution containing NMN and glucose for reaction and purification to obtain NMNH.
9. 9. The method for preparing NMNH according to claim 8, wherein the glucose dehydrogenase is directly added to the solution containing NMN and glucose or immobilized on a carrier and then added to the solution containing NMN and glucose; The purification comprises membrane filtration, resin purification, nanofiltration concentration desalination and freeze-drying into powder; The purified solution is further recrystallized, and absolute ethyl alcohol is adopted as a recrystallization solvent; The recrystallization operation comprises the steps of dissolving the purified NMNH in water, dripping the water into absolute ethyl alcohol, stirring for crystal growth, carrying out suction filtration and drying to obtain the recrystallization NMNH.
10. 10. The method of claim 9, wherein the absolute ethanol is at a temperature of 0-10 ℃; the dosage ratio of NMNH to water is 1kg (1-1.1) L; after the purified NMNH is dissolved in water, the pH is adjusted to 9-10; The dosage ratio of NMNH to absolute ethyl alcohol is 1kg (4-4.5) L; the temperature of the stirring and crystal growing is 0-10 ℃ and the time is 5-10h.

Description

RBS element, glucose dehydrogenase expression nucleic acid molecule and NMNH preparation method Technical Field The invention belongs to the technical field of nucleic acid, and in particular relates to a RBS element, a glucose dehydrogenase expression nucleic acid molecule and a NMNH preparation method. Background NMNH (Nicotinamide Mononucleotide in its reduced form, reduced nicotinamide mononucleotide) is a direct precursor of reduced Nicotinamide Adenine Dinucleotide (NADH) and is also a key reduced metabolic intermediate of intracellular important coenzyme nicotinamide adenine dinucleotide (NAD+). NAD+ and its reduced form NADH are widely present in all living cells and are essential as coenzymes involved in thousands of biochemical reactions including energy metabolism, redox reactions, DNA repair and cell signalling, critical to maintaining cell viability and homeostasis. Therefore, NMNH as a novel high-efficiency substrate for improving the intracellular NAD+ level has great application potential in the fields of medicines, health-care products, cosmetics and the like. Currently, mass production of NMNH faces two major core challenges. On the synthetic biology level, the efficiency of the biosynthetic pathway is severely dependent on the expression level and activity of the key catalytic enzyme. Protein expression is regulated by multiple levels of transcription and translation, where RBS located in the translation initiation region directly regulates the translation initiation rate of the gene and protein yield by binding strength to ribosomes. Rational optimization of RBS sequences is a key strategy for enhancing ribosome binding capacity and thereby efficiently increasing the expression level of a target protein. Therefore, the development of an RBS that increases enzyme expression is of urgent need and importance for achieving efficient large-scale production of NMNH. Disclosure of Invention In order to overcome the defects in the prior art, the invention provides a RBS element, a glucose dehydrogenase expression nucleic acid molecule and a NMNH preparation method. The invention carries out rational design and optimization on ribosome binding sites (Ribosome Binding Site, RBS) to obviously improve the translation efficiency and expression level of protein expression nucleic acid molecules, and can effectively improve the protein expression quantity. The technical scheme adopted for solving the technical problems is as follows: the invention provides an RBS element, and the nucleotide sequence is shown in any one of SEQ ID NO. 1-10. The present invention provides an RBS transcribed from the RBS element described above. The invention provides a protein expression nucleic acid molecule, comprising a nucleic acid molecule consisting of a promoter, the RBS element and a protein coding nucleic acid molecule in sequence. The protein expression quantity of the protein expression nucleic acid molecule is obviously improved. Preferably, the protein-encoding nucleic acid molecule is a fluorescent protein-encoding nucleic acid molecule or a glucose dehydrogenase-encoding nucleic acid molecule. The invention provides a fluorescent protein expression nucleic acid molecule, which comprises a nucleic acid molecule consisting of a promoter, the RBS element and a fluorescent protein coding nucleic acid molecule in sequence. The invention provides a fluorescent protein expression plasmid, which comprises the fluorescent protein expression nucleic acid molecule. The invention provides a fluorescent protein expression bacterium, which comprises the fluorescent protein expression nucleic acid molecule and/or the fluorescent protein expression plasmid. The invention provides a glucose dehydrogenase expression nucleic acid molecule, which comprises a nucleic acid molecule consisting of a promoter, RBS elements and a glucose dehydrogenase coding nucleic acid molecule in sequence, wherein the nucleotide sequence of the RBS elements is shown in any one of SEQ ID NO. 3-10. The invention provides a glucose dehydrogenase expression plasmid, comprising the glucose dehydrogenase expression nucleic acid molecule. The present invention provides a glucose dehydrogenase-expressing bacterium comprising the above glucose dehydrogenase-expressing nucleic acid molecule and/or the above glucose dehydrogenase-expressing plasmid. The invention provides a preparation method of glucose dehydrogenase, which is to induce and express the glucose dehydrogenase expression bacteria to obtain the glucose dehydrogenase. The invention provides a preparation method of NMNH, which is to add glucose dehydrogenase obtained by inducing and expressing the glucose dehydrogenase expression bacteria into a solution containing NMN and glucose for reaction and purification to obtain NMNH. Preferably, the glucose dehydrogenase is added directly to the solution containing NMN and glucose, or is immobilized on a carrier and then added to the solution containing