Production method of nucleotide as well as product and application thereof

Abstract

The invention provides a nucleotide production method and a product and application thereof, waste yeast cell residues are used as a raw material, a nucleotide mixture with high purity, high nucleoside triphosphate content and high biological activity is obtained through the steps of cell lysis, washing purification, enzymolysis, filtration and the like, and the mixture can be used in the fields of cell-free synthesis and the like and has good application prospects. The problem of high cost of cell-free biosynthesis raw materials is effectively solved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and specifically relates to a method for producing nucleotides, its products and applications. Background Art

[0002] Cell-free protein synthesis (CFPS) is a type of in vitro biosynthesis. Using exogenous mRNA or DNA as a template, it is a technology to synthesize proteins under in vitro conditions using enzymes, amino acid substrates and energy. Compared with traditional in vivo recombinant expression systems, in vitro cell-free synthesis systems have multiple advantages. For example, they can express special proteins that are toxic to cells or contain unnatural amino acids (such as D-amino acids), can directly use PCR products as templates to simultaneously and parallelly synthesize multiple proteins, and can carry out high-throughput screening research on polypeptides or protein drugs. In addition, in vitro cell-free synthesis systems also have unique advantages in the application of expressing polypeptides or proteins that are toxic to cells. The cell-free protein expression system is an important supplement to the cell-based protein expression system.

[0003] Existing cell-free protein synthesis systems can mainly be divided into two categories: One is to combine and compound the purified ribosomes, enzymes, tRNAs, etc. required for protein synthesis in a certain proportion to endow it with the ability to express proteins in vitro, that is, the purified cell-free protein expression system (PURE). This system has clear component information and can conveniently and customizedly adjust the formula to obtain different protein expression characteristics. However, the disadvantage is that the preparation process is complex and the cost is high. The other category is to obtain the main active substances from the extracts of living cells with protein expression activity, and appropriately add the required enzymes, substrates (such as nucleotides, amino acids), energy substances and other components to endow it with the ability to express proteins in vitro, that is, the cell-free protein expression system based on cell extracts.

[0004] In vitro RNA synthesis is also a means of in vitro biosynthesis. Similar to cell-free protein synthesis, it is also a synthesis reaction that uses DNA as a template to obtain RNA through in vitro transcription. Common mRNA vaccines are mainly prepared through in vitro RNA synthesis reactions. In in vitro synthesis, such as in vitro RNA synthesis, cell-free protein synthesis, etc., a large amount of mRNA is required. The RNA synthesis substrate is four ribonucleoside triphosphates (NTPs), namely ATP, GTP, CTP, and UTP. The pure products of these four ribonucleoside triphosphates are very expensive, which to a certain extent increases the cost of in vitro biosynthesis. Therefore, there is an urgent need to find a method that can replace pure nucleotides and reduce costs. Summary of the Invention

[0005] The object of the present invention is to overcome the problems existing in the above-mentioned prior art, and to provide a method for producing nucleotides. The by-product of yeast fermentation, yeast residue, is used to extract mixed nucleotides. By adjusting the method and parameters, its activity is ensured, it can be used for cell-free protein synthesis, and the cost of nucleotide raw materials is greatly reduced.

[0006] In the first aspect of the present invention, there is provided a method for producing nucleotides for cell-free protein synthesis, characterized in that the method comprises the following steps:

[0007] Step 1), lysing yeast cells: mixing waste yeast cell residue and water in a mass ratio of 1:4 - 1:6 to obtain a yeast cell suspension, heating to lyse the cells, and centrifuging to obtain a crude extract;

[0008] Step 2), precipitation: adding ethanol to the crude extract obtained in Step 1) to wash and precipitate, centrifuging to obtain an RNA extract;

[0009] Step 3), enzymatic hydrolysis: drying the obtained RNA extract into a powder, weighing and adding water to make an 8 - 12% aqueous solution, adding nuclease for enzymatic hydrolysis, and centrifuging to obtain an enzymatic hydrolysate;

[0010] Step 4), filtration and purification: filtering the above enzymatic hydrolysate through an ultrafiltration membrane to remove impurities to obtain a nucleotide mixture.

[0011] Further, in the lysis in Step 1), NaCl or NaOH is also added;

[0012] Specifically for the addition of NaCl, NaCl is added when preparing the yeast cell suspension, and the mass concentration range of NaCl in the suspension is greater than 8% and less than 12%, preferably 10%;

[0013] Specifically for the addition of NaOH, NaOH is added before heating or during heating, and the mass concentration of NaOH in the suspension is 0.1 - 0.5%.

[0014] Further, the method further comprises Step 5), drying: freeze-drying or spray-drying the obtained nucleotide mixture into a mixed nucleotide powder.

[0015] Further, optionally, a purification process such as activated carbon adsorption, column chromatography, etc. is added after enzymatic hydrolysis to further purify the nucleotide mixture.

[0016] Further, the precipitation step in Step 2) specifically comprises: adding ethanol with a concentration of more than 70% and 2 - 4 times the volume of the crude extract to the crude extract, precipitating, centrifuging at 4000 - 8000g for 5 - 10 min, and repeating more than 3 times.

[0017] Furthermore, in the precipitation step described in step 2), LiCl or guanidine hydrochloride is added.

[0018] Furthermore, the precipitation step described in step 2) specifically includes:

[0019] The crude extract obtained in step 1) is centrifuged again at 10,000 g - 20,000 g for 10 min, the supernatant is retained, then 0.25 - 1 volume of LiCl is added and precipitated below -20 °C, centrifuged, 2 - 4 volumes of ethanol with a concentration of more than 70% of the supernatant volume are added to the precipitate, washed more than twice, and then washed with ethanol with a concentration of more than 95%, and the separated precipitate is the RNA extract;

[0020] Or, the crude extract obtained in step 1) is centrifuged again at 10,000 g - 20,000 g for 10 min, the supernatant is retained, 1 - 2 volumes of ethanol with a concentration of more than 70% are added and precipitated below 4 °C, the precipitate is washed with guanidine hydrochloride and 1 - 2 volumes of ethanol with a concentration of more than 70% of the supernatant volume, 2 - 4 volumes of ethanol with a concentration of more than 70% of the supernatant volume are added to the precipitate, washed more than twice, and then washed with ethanol with a concentration of more than 95%, and the separated precipitate is the RNA extract;

[0021] The concentration of the added LiCl is 5 M - 10 M; the concentration of the added guanidine hydrochloride is 1 M - 4 M.

[0022] Furthermore, in step 1), the heating is to heat to 70 °C - 100 °C, and after heating and lysing, it is cooled and the pH is adjusted to 5 - 7;

[0023] And / or, the drying condition in step 3) is to dry at 60 °C - 70 °C for 2 - 6 h;

[0024] And / or, the enzymatic hydrolysis condition in step 3) is to heat and enzymatically hydrolyze at 50 °C - 70 °C for 2 - 16 h; the dosage of the nuclease is ≥ 0.5% based on the dry weight of the RNA extract powder;

[0025] And / or, in step 4), the ultrafiltration is carried out using an ultrafiltration membrane with a pore size less than or equal to 4000 D.

[0026] The second aspect of the present invention provides a nucleotide mixture, which is characterized in that it is prepared according to the method described in the first aspect.

[0027] The second aspect of the present invention provides the application of the nucleotide mixture prepared by the above method in in vitro biosynthesis (such as cell-free protein synthesis, in vitro RNA synthesis, etc.), food, feed, and medicine.

[0028] The third aspect of the present invention provides a biosynthesis system, specifically using the nucleotide mixture prepared by the aforementioned method as the source of nucleotide raw materials.

[0029] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0030] (1) The present invention uses waste yeast cell residue as a raw material for nucleotide extraction. On the one hand, it makes full use of the by-products of yeast fermentation. Compared with the traditional method of directly using the fermentation by-product yeast cells as feed, this method greatly improves the application value of the by-products. On the other hand, in the nucleotide production method of the present invention, the instruments and equipment used are simple, the operation method is easy, the production cycle is about one-third of the conventional single nucleotide production cycle, and the energy consumption is only half of the single nucleotide process, greatly reducing the preparation cost of the nucleotide mixture, and thus reducing the production cost of cell-free protein synthesis.

[0031] (2) In the method of the present invention, the yeast cells are treated with salt or alkali to promote cell lysis, and then mild treatment methods such as ethanol washing are used to obtain a purified RNA extract, and then enzymatic hydrolysis is carried out. Compared with the prior art technical solution of directly adding nuclease for enzymatic hydrolysis after obtaining a crude extract, in the present invention, after washing and purifying with ethanol to obtain a relatively pure RNA extract, the amount of enzyme used can be more precisely controlled, and the amount of enzyme used can be greatly reduced. Moreover, since macromolecular impurities are removed before enzymatic hydrolysis, various purification steps such as decolorization column decolorization and chromatography column separation after traditional extraction and enzymatic hydrolysis can be omitted. The instruments used for the washing and purification treatment of the present invention are simpler, the operation is simple, the cost is lower, and it ensures that the extracted RNA has high biological activity, so that a nucleotide mixture with good biological activity can be obtained after enzymatic hydrolysis. Therefore, the RNA purification method of the present invention has multiple effects.

[0032] (3) When purifying RNA in the present invention, LiCl or guanidine hydrochloride is added, and the corresponding process steps and parameter conditions are adjusted according to their characteristics, thereby obtaining a mixed nucleotide product with high recovery rate and high activity. Its biological activity is very close to the combination of commercially available pure nucleotide products, and it can replace expensive pure nucleotide compositions for in vitro biosynthesis (such as cell-free protein synthesis). Although LiCl or guanidine hydrochloride are known common reagents, both are toxic compounds. In the present invention, the purpose of extracting RNA is to obtain a nucleotide mixture with high activity and high purity through subsequent enzymatic hydrolysis and to be used in an in vitro biosynthesis system. Therefore, the usage method and dosage of LiCl or guanidine hydrochloride need to be adjusted and optimized to ensure that it does not affect subsequent in vitro biosynthesis. After multiple adjustment experiments, the inventors obtained the optimal addition timing and addition amount of LiCl or guanidine hydrochloride, greatly improving the purification effect of RNA. The purity of the obtained RNA is as high as 98%, and the biological activity of the nucleotides obtained through subsequent enzymatic hydrolysis is high.

[0033] (4) In the method of the present invention, the process and parameter conditions adopted during enzymatic hydrolysis are controlled, thereby obtaining a nucleotide mixture with a high content of nucleoside triphosphate, which is beneficial to the progress of in vitro biosynthesis reactions. As is well known, when ribonuclease hydrolyzes RNA, nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate can be obtained simultaneously, and nucleoside triphosphate is required for RNA synthesis. Therefore, only when more nucleoside triphosphate is obtained through enzymatic hydrolysis can it be effectively used in in vitro biosynthesis reactions. The inventors conducted multiple research and adjustments and finally obtained a hydrolysis product with a high content of nucleoside triphosphate, that is, the nucleotide mixture of the present invention.

[0034] (5) The nucleotide mixture obtained by the method of the present invention has high purity and biological activity. It can not only be used for biosynthesis, but also be widely used in various fields such as food and feed, and has broad application prospects.

[0035] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Detailed Embodiments

[0036] Through extensive and in-depth research, and through a large number of screenings and explorations, the present invention first proposes a production method for nucleotides. This method uses waste yeast cells as raw materials to prepare nucleotides. By adjusting the purification method and parameters, the extraction rate and purity of the intermediate product RNA are improved. After reasonable enzymatic hydrolysis processes, a nucleotide mixture with high activity is obtained, which not only makes full use of waste yeast cells, but also can greatly reduce the cost of cell-free protein synthesis.

[0037] The present invention will be further illustrated in conjunction with specific embodiments and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional substitution selections based on the existing technologies on the basis of the technical concept of the present invention, rather than being limited to the specific records of the embodiments of the present invention.

[0038] Term Introduction

[0039] In the present invention, "nucleotide", or "ribonucleotide", is a compound composed of a purine base or a pyrimidine base, ribose or deoxyribose, and phosphoric acid. According to the different bases, there are also adenine nucleotides (adenylic acid, AMP), guanine nucleotides (guanylic acid, GMP), cytosine nucleotides (cytidylic acid, CMP), uracil nucleotides (uridylic acid, UMP), thymine nucleotides (thymidylic acid, TMP), and inosine nucleotides (inosinic acid, IMP), etc. The phosphoric acid in nucleotides also has one, two, and three molecular forms. The substrate for synthesizing RNA is nucleoside triphosphate, so the purpose of the present invention is to obtain a high content of nucleoside triphosphate.

[0040] In the present invention, "in vitro biosynthesis" refers to the reaction of synthesizing various biomolecules using biological raw materials in an environment outside the organism. Common ones include in vitro synthesis of RNA, cell-free protein (including peptides, amino acids) synthesis, etc. Among them, "in vitro RNA synthesis" is a synthesis reaction that obtains RNA by in vitro transcription using DNA as a template. Commonly, it is obtained by using linearized plasmid DNA or PCR amplification products as templates and using RNA polymerase for in vitro transcription synthesis and then modification. Common mRNA vaccines are mainly prepared through in vitro RNA synthesis reactions. Nucleotides are required as synthesis raw materials in this reaction.

[0041] In the present invention, "cell-free protein synthesis", also known as "in vitro protein synthesis", "in vitro cell-free protein synthesis", "in vitro protein synthesis", etc., refers to the reaction of synthesizing proteins in an in vitro cell-free environment synthesis system, which includes at least the translation process. It mainly uses exogenous mRNA or DNA as a template and synthesizes proteins, peptides, etc. under in vitro conditions using enzymes, amino acid substrates, and energy. Nucleotides are also necessary raw materials in the reaction. Cell-free protein synthesis includes but is not limited to IVT reaction (in vitro translation reaction), IVTT reaction (in vitro transcription-translation reaction), and IVDTT reaction (in vitro replication-transcription-translation reaction). In the present invention, the IVTT reaction is preferably used. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to such in vitro protein synthesis systems as D2P systems, D-to-P systems, D_to_P systems, DNA-to-Protein systems; the corresponding in vitro protein synthesis methods are also referred to as D2P methods, D-to-P methods, D_to_P methods, DNA-to-Protein methods.

[0042] In the present invention, "cell-free", or "cell-free system", means that when performing in vitro protein synthesis, it is not through the secretion and expression method of intact cells. It should be noted that in the in vitro cell-free protein synthesis system of the present invention, it is also allowed to add cell components to promote the reaction, but the added cells do not mainly aim at secreting and expressing exogenous target proteins. In addition, in the D2P system without intact cells constructed under the guidance of the present invention, a small amount of intact cells are intentionally added (for example, the protein content provided by them does not exceed 30 wt% compared with the protein content provided by the cell extract). Such an "evasion" method is also included in the protection scope of the present invention.

[0043] In the present invention, one of the specific operation modes of the cell-free protein synthesis system further includes, but is not limited to, for example, the Escherichia coli-based cell-free protein synthesis system described in WO2016005982A1. The in vitro cell-free protein synthesis systems including, but not limited to, those based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus, as described in other cited documents of the present invention and their directly and indirectly cited documents, are also incorporated into the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, the in vitro cell-free protein synthesis systems (In vitro cell-free protein synthesis system) described in the cited documents on pages 27-28 of the section "2.1 Systems and Advantages" in the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" can be used as the in vitro protein synthesis system for implementing the present invention. For example (unless it conflicts with the present invention, the following documents and their cited documents are cited for all purposes and in their entirety), the in vitro cell-free protein synthesis systems, DNA template construction and amplification methods described in documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, etc., and their cited documents can be used as the in vitro protein synthesis system of the present invention and the DNA template construction and amplification methods of the present invention.

[0044] In the present invention, "protein" and "protein" have the same meaning and are both translated as "protein" and can be used interchangeably.

[0045] In the present invention, both "system" and "system" are translated as "system" and can be used interchangeably.

[0046] In the present invention, "activity", "expression activity", "synthesis activity", "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably, and all represent the ability of the cell-free protein synthesis system to synthesize proteins.

[0047] In the present invention, the meanings of "cell extract", "cell extract solution", "cell lysate", etc. are the same and can be used interchangeably. They can be described in English as cell extract, cell lysate, etc.

[0048] In the present invention, "waste yeast", "yeast residue", "yeast cell", "yeast cell residue" or "waste yeast cell", etc. all refer to the by-products mainly composed of yeast cells remaining after extracting active substances when using yeast as a fermentation strain to produce active substances in actual experiments or production. In the examples of the present invention, unless otherwise specified, the cell residue or cell powder used is waste yeast cell, and the cell residue can be a dry product (such as cell powder) or wet (wet residue).

[0049] In the present invention, the "purity" of the RNA refers to the proportion of RNA in the prepared RNA extract. Specifically, the extract is placed on an ultraviolet spectrophotometer to measure the absorbance values at 260 nm and 280 nm, and the nucleic acid concentration and the absorbance ratio of the two are calculated to obtain the purity of the RNA extract.

[0050] In the present invention, "precipitation" or "precipitate" both refer to the solid or semi-solid part obtained after separating and removing the supernatant by means such as centrifugation and filtration.

[0051] In the present invention, the conventional meanings of "lysis", "enzymolysis", "purification", "ultrafiltration", etc. are all those in the prior art and will not be elaborated here one by one. Detailed implementation mode

[0053] The method for producing the nucleotide described in the present invention includes the following steps.

[0054] Step 1), lysing yeast cells: Mix waste yeast cell residue and water at a mass ratio of 1:4 - 1:6 to obtain a yeast cell suspension, heat to lyse the cells, and centrifuge to separate to obtain a crude extract. The mass ratio of yeast cell residue to water can be any one of 1:4, 1:4.5, 1:5, 1:5.5, and 1:6.

[0055] Step 2), precipitation: Add ethanol to the crude extract obtained in Step 1) to wash and precipitate, and centrifuge to separate to obtain an RNA extract.

[0056] Step 3), enzymolysis: Dry the obtained RNA extract into a powder, weigh it and make it into an 8 - 12% aqueous solution by adding water, add nuclease for enzymolysis, and centrifuge to obtain an enzymolysis solution.

[0057] Step 4), filtration and purification: Filter the above enzymolysis solution through an ultrafiltration membrane to remove impurities to obtain a nucleotide mixture.

[0058] In traditional cell-free protein synthesis, in vitro RNA synthesis and other technologies, the nucleotide raw materials used are pure products extracted or synthesized, and their prices are expensive. The applicant found in the research that after extracting the active substances from yeast cells, a large amount of RNA is contained, and nucleotides can be obtained after RNA enzymolysis. Therefore, it is considered that the yeast cell residue can be reused to obtain a mixture of four ribonucleotides, so as to realize the waste utilization of yeast residue and the production of ribonucleotides with high added value. To obtain nucleotides from yeast cells, the yeast cell residue needs to be lysed first to release RNA. There are many methods for yeast lysis in the prior art, such as autolysis, enzymolysis, pulverization, etc. When using autolysis or enzymolysis as the cell wall breaking means, the yeast cell residue needs to be first mixed with water to form a suspension (or suspension), and the cell wall breaking treatment is carried out in an aqueous environment. Therefore, the yeast cell residue is first mixed with water in an appropriate ratio to form a suspension, and then the yeast cell wall is broken by means of heating, adding salt, adding enzyme, adding alkali, etc. After crude extraction by cell wall breaking, the crude extract is first washed and purified with ethanol to obtain a relatively pure RNA powder, and then configured into an aqueous solution, and quantitatively hydrolyzed with nuclease, accurately controlling the dosage of the nuclease and saving the nuclease. In the enzymolysis step, by adjusting appropriate conditions and adding nuclease to hydrolyze RNA, different nucleotide mixtures are obtained, and finally, macromolecular impurities are removed by filtration to obtain a nucleotide mixture solution. Each step of the above method is obtained through in-depth research and adjustment, and each step has an important impact on obtaining a relatively pure nucleotide product.

[0059] In one example, Nacl or NaOH is also added in the lysis in step 1).

[0060] Specifically for the addition of Nacl, Nacl is added when preparing the yeast cell suspension, and the mass concentration of Nacl in the suspension is greater than 8% and less than 12%, preferably 10%.

[0061] Specifically for the addition of NaOH, NaOH is added before heating or during heating, and the mass concentration of NaOH in the suspension is 0.1-0.5%.

[0062] None of the above methods in the prior art can ensure the maximum release of RNA in cells while not damaging the activity of RNA. In the lysis process of the present invention, through repeated experimental adjustments, it is finally determined that autolysis is achieved by using 10% Nacl or NaOH with a concentration of 0.1 - 0.5% in combination with heating, and the best cell wall breaking effect is obtained. The above concentrations are all relative to the total volume of the final yeast cell suspension. In one example, the method further includes step 5), drying: The obtained nucleotide mixture is made into a mixed nucleotide powder by freeze-vacuum drying or spray drying. For convenient storage and subsequent use, the nucleotide mixture can be further dried into powder, and the drying method is preferably a method of drying under low temperature conditions, such as low-temperature drying, freeze-vacuum drying or spray drying, etc. Low temperature conditions are beneficial to maintaining the activity of nucleotides. Optionally, after enzymatic hydrolysis, a further purification process can be added, such as activated carbon adsorption, column chromatography, etc., to further separate macromolecular impurities or pigment impurities to purify the nucleotide mixture. Among them, activated carbon adsorption is a commonly used purification means. Experiments have confirmed that adding the step of activated carbon adsorption can improve the purity of the nucleotide mixture.

[0063] In one example, the precipitation step in step 2) of the method specifically includes: adding ethanol with a concentration 2 - 4 times higher than 70% (V / V, the same below) to the crude extract, precipitating, centrifuging at 4000 - 8000g for 5 - 10 min, and repeating it more than 3 times. The concentration of ethanol determines the precipitation effect. Through multiple experimental adjustments, it is found that only ethanol with a concentration above 70% can precipitate the RNA in the crude extract obtained in step 1) well, and multiple washings can achieve a better purification effect and obtain a more pure RNA extract.

[0064] In one example, in the precipitation step of step 2) of the method, LiCl or guanidine hydrochloride can also be added. Both LiCl and guanidine hydrochloride can effectively precipitate RNA. The main advantage of lithium chloride is that it does not effectively precipitate proteins or DNA, and there are fewer impurities generated in the system. Guanidine hydrochloride can inhibit RNase, selectively precipitate RNA molecules and remove impurities such as DNA to improve the purity of RNA. When combined with ethanol washing, both can further improve the purification effect of RNA.

[0065] In one example, the precipitation method including the step of adding LiCl or guanidine hydrochloride specifically includes:

[0066] Centrifuge the crude extract obtained in step 1) again at 10,000 g - 20,000 g for 10 min, retain the supernatant, then add 0.25 - 1 volume of LiCl and precipitate at below -20°C, centrifuge, add ethanol with a concentration of more than 70% and a volume of 2 - 4 times that of the supernatant to the precipitate, wash it more than twice, and then wash it with ethanol with a concentration of more than 95%. The separated precipitate is the RNA extract;

[0067] Alternatively, centrifuge the crude extract obtained in step 1) again at 10,000 g - 20,000 g for 10 min, retain the supernatant, add ethanol with a concentration of more than 70% and a volume of 1 - 2 times and precipitate at below 4°C. Add guanidine hydrochloride and ethanol with a concentration of more than 70% and a volume of 1 - 2 times that of the supernatant to wash the precipitate. Add ethanol with a concentration of more than 70% and a volume of 2 - 4 times that of the supernatant to the precipitate, wash it more than twice, and then wash it with ethanol with a concentration of more than 95%. The separated precipitate is the RNA extract;

[0068] The concentration of the added LiCl is 5 M - 10 M, preferably 7.5 M; the concentration of the added guanidine hydrochloride is 1 M - 4 M, preferably 4 M. In the above steps, the precipitation mechanisms of LiCl and guanidine hydrochloride are different, so the addition timing and operation methods are different. Through repeated experimental adjustments, the inventor finally found the most suitable operation methods and conditions for the two, so as to ensure the smooth precipitation of RNA. The rotation speed and time of centrifugation are very important parameters for the separation effect. Through repeated experimental adjustments, the inventor obtained the optimal condition combination and finally obtained relatively pure RNA. The applicant screened the concentration of LiCl or guanidine hydrochloride and found that when in the above concentration range, the purity of the RNA extract can reach about 80% or more, and when the subsequent enzymatic hydrolysis product is used for cell-free protein synthesis, its biological activity is relatively high. Especially when the concentration of LiCl is 7.5 M or the concentration of guanidine hydrochloride is 4 M, the RNA purity can reach more than 90%, and the highest can reach 98%, which is very close to pure RNA.

[0069] In one example, in step 1) of the method, the heating is to heat to 70 - 100°C, and after heating and lysing, cool and adjust the pH to 5 - 7; preferably heat to 100°C and the pH value is 5.5. High temperature can promote the rupture of yeast cells and release intracellular RNA, proteins, etc., and adjusting the pH can precipitate and separate macromolecular impurities such as proteins, so as to facilitate the extraction and separation of RNA.

[0070] In one example, the drying conditions in step 3) of the method are drying at a temperature below 60-70°C for 2-6 hours; if the temperature is too high during drying, it is easy for RNA to denature and agglomerate, and if the temperature is too low, the process flow will be extended. Drying the RNA extract under mild conditions can not only maintain its good biological activity but also be suitable for actual production. 60-70°C is a more suitable temperature, and the drying time is adjusted according to the temperature and drying degree. More preferably, it is 65°C and dried for 4 hours. Under these conditions, the drying effect is good and the energy consumption is low.

[0071] In one example, the enzymatic hydrolysis conditions in step 3) of the method are heating and enzymatically hydrolyzing at a temperature of 50-70°C for 2-16 hours; the dosage of the nuclease is ≥0.5% based on the dry weight of the RNA extract powder, and preferably 0.5%-20%. The nuclease needs to act under suitable conditions. Therefore, the temperature needs to be adjusted to 50-70°C, and the enzymatic hydrolysis time is adjusted according to the specific process of enzymatic hydrolysis, which is related to various factors such as the source, activity, and dosage of the enzyme; generally speaking, the larger the enzyme dosage, the more thorough the enzymatic hydrolysis, but the corresponding cost also increases. Especially, the nuclease itself is relatively expensive. Therefore, if the enzyme dosage is reduced, the cost will be greatly reduced. Therefore, the inventor conducted scientific and rigorous experiments to explore the relationship between the temperature, time, and enzyme addition amount of enzymatic hydrolysis, and obtained a better combination. Under the optimal conditions, the enzymatic hydrolysis temperature is 60°C, the enzymatic hydrolysis time is 14 hours, and the enzyme dosage is 0.5%. At this time, the enzyme dosage is the lowest, the temperature is mild, the enzymatic hydrolysis is thorough without being excessive, and the nucleoside triphosphate can be retained to the greatest extent.

[0072] In one example, in step 4) of the method, ultrafiltration is carried out using an ultrafiltration membrane with a pore size less than or equal to 4000D. Most preferably, the pore size of the ultrafiltration membrane is 3000D. Although the nucleotide molecules are small, considering the operability of filtration and the situation of the molecules to be removed, an ultrafiltration membrane with a pore size of 3000D is selected for ultrafiltration. The results prove that it can well retain nucleotides and remove impurities.

[0073] In one example, a nucleotide mixture is also provided, which is prepared according to the foregoing method. The obtained nucleotide product is a mixture, rich in various nucleotides, and the content of nucleoside triphosphate therein is dominant. Therefore, it can be effectively applied in biosynthetic systems such as cell-free protein synthesis systems.

[0074] In one example, the application of the mixed nucleotides prepared by the foregoing method in the fields of biosynthesis, food, feed, medicine, etc. is also provided. That is to say, the mixed nucleotide product obtained by the method of the present invention, due to its high purity and biological activity, does not contain residues of toxic and harmful substances, and has a low production cost. Therefore, its application scope can be wider. In addition to being used for cell-free protein synthesis, in vitro RNA synthesis, and other in vitro biosyntheses, it can also be used in the fields of food, feed, related medicines, etc.

[0075] In one example, an in vitro biosynthesis system is also provided, specifically using the nucleotide mixture prepared by the aforementioned method as the source of nucleotide raw materials. As described above, the main technical problem of the present invention is to prepare nucleotides for in vitro biosynthesis such as cell-free protein synthesis and in vitro RNA synthesis using waste yeast cells. Therefore, the in vitro biosynthesis system containing the nucleotide mixture obtained in the present invention is also part of the present invention. Compared with the system using pure nucleotides as raw materials, the production cost of this system is greatly reduced, but it can maintain good synthesis ability and has higher economic benefits.

[0076] Example 1 An implementation manner of the nucleotide production method of the present invention

[0077] Weigh 400 g of yeast cell residue and place it in a 5 L beaker; add NaCl to the above solution, add 1 L of ultrapure water, stir evenly, and then make up the volume to 2 L with ultrapure water, with the NaCl concentration being 10%; boil the above solution on an induction cooker for 30 min, then immediately cool it, and adjust the pH to 5.5;

[0078] Centrifuge the above solution at 4000G for 10 min and keep the supernatant; wash it twice with 75% ethanol by centrifugation (6000G for 10 min), and for the third wash, centrifuge it with absolute ethanol at 6000G for 10 min and keep the precipitate; place the precipitate in a drying oven and dry it at 65 °C for 4 h until it becomes powdery; after drying, weigh it and measure its purity: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of this RNA sample as 56%. Adjust the crude RNA sample to a 10% aqueous solution, pH 5.5, add 0.5% nuclease based on the dry weight, heat it at 60 °C for 14 h, add 0.1% activated carbon, shake well and then centrifuge at 6000G for 10 min; ultrafilter the sample through a 3000D pore size to obtain a purified mixed nucleotide solution, and obtain mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as raw material, add it to the cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 2457 (the detection methods used in the examples of the present invention are the same, and the results are all expressed by fluorescence values), reaching 42% of the activity of the mixture prepared from commercially available single nucleotides (hereinafter referred to as single nucleotide mixture).

[0079] Example 2 An implementation manner of the nucleotide production method of the present invention

[0080] Weigh 400 g of cell residue and place it in a 5 L beaker; add NaCl to the above solution, add 1 L of ultrapure water, stir evenly, and make up the volume to 2 L with ultrapure water. The NaCl concentration is 10%; boil the above solution on an induction cooker for 30 min, then cool it immediately and adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; wash it twice with 95% ethanol by centrifugation (6000 G for 10 min), and for the third wash, use absolute ethanol and centrifuge at 6000 G for 10 min to keep the precipitate; place the precipitate in a drying oven and dry it at 65 °C for 4 h until it becomes powdery; weigh it after drying. Weight measurement and purity determination after drying: Prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 65%. Adjust the crude RNA sample to a 10% aqueous solution with pH: 5.5, add 20% of nuclease based on the dry weight, heat it at 60 °C for 14 h, shake well and centrifuge at 6000 G for 10 min; ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 3020, reaching 51% of the activity of a single nucleotide mixture.

[0081] Example 3 An implementation mode of the nucleotide production method described in the present invention

[0082] Weigh 400 g of cell residue and place it in a 5 L beaker; add NaCl to the above solution, add 1 L of ultrapure water, stir evenly, and then make up the volume to 2 L with ultrapure water, with the NaCl concentration being 10%; boil the above solution on an induction cooker for 30 min, then cool it immediately and adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; wash it twice with 95% ethanol by centrifugation (6000 G for 10 min), and for the third wash, centrifuge it with absolute ethanol at 6000 G for 10 min and keep the precipitate; place the precipitate in an oven to dry, and dry it at 65 °C for 4 h until it becomes powdery; weigh it after drying, weighing after drying and purity determination: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of this RNA sample as 65%. Adjust the crude RNA sample to a 10% aqueous solution, pH: 5.5, add 20% of nuclease based on the dry weight, heat it at 60 °C for 14 h, add 0.1% of activated carbon, shake well and then centrifuge at 6000 G for 10 min; ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 3363, reaching 57% of the activity of a single nucleotide mixture.

[0083] Example 4 An implementation mode of the nucleotide production method described in the present invention

[0084] Weigh 400 g of cell residue and place it in a 5 L beaker; add 1 L of ultrapure water to the above beaker, stir evenly, and then make up the volume to 2 L with ultrapure water; boil the above solution on an induction cooker for 30 min, then add 0.2% NaOH to the above solution, continue to heat for 30 min, and then quickly cool it to adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; centrifuge the supernatant at 20000 G and then add 0.5 times the volume of 7.5 M LiCl, place it at -80 °C for precipitation for 1 h; centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it with 95% ethanol for the last time; place the precipitate in a drying oven and dry it at 65 °C for 4 h until it becomes powdery; weigh and measure the purity after drying: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 91%. Adjust the crude RNA sample to a 10% aqueous solution with pH: 5.5; add 0.5% nuclease based on the dry weight, heat it at 60 °C for 14 h, ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 4911, reaching 82% of the activity of the single nucleotide mixture.

[0085] Example 5 An implementation mode of the nucleotide production method described in the present invention

[0086] Weigh 400 g of cell residue and place it in a 5 L beaker; add 1 L of ultrapure water to the above beaker, stir evenly, and make up the volume to 2 L with ultrapure water; boil the above solution on an induction cooker for 30 min, then add 0.2% NaOH to the above solution, continue heating for 30 min, then quickly cool it and adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; centrifuge the supernatant at 20000 G and then add 0.5 times the volume of 5.0 M LiCl, place it at -80 °C for precipitation for 1 h; centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it with 95% ethanol for the last time; place the precipitate in a drying oven and dry it at 65 °C for 4 h until it becomes powdery; weigh and determine the purity after drying: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 78%. Adjust the RNA crude sample to a 10% aqueous solution with pH: 5.5; add 0.5% nuclease based on the dry weight, heat it at 60 °C for 14 h, ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 4210, reaching 70% of the activity of a single nucleotide mixture.

[0087] Example 6 An implementation mode of the nucleotide production method described in the present invention

[0088] Weigh 400 g of cell residue and place it in a 5 L beaker; add 1 L of ultrapure water to the above beaker, stir evenly, and make up the volume to 2 L with ultrapure water; boil the above solution on an induction cooker for 30 min, then add 0.2% NaOH to the above solution, continue heating for 30 min, then quickly cool it and adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; centrifuge the supernatant at 20000 G again and add 0.5 times the volume of 10 M LiCl, then place it at -80 °C for precipitation for 1 h; centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it with 95% ethanol for the last time; place the precipitate in a drying oven and dry it at 65 °C for 4 h until it becomes powdery; weigh and measure the purity after drying: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of this RNA sample as 91%. Adjust the RNA crude sample 5 to a 10% aqueous solution with pH: 5.5; add 0.5% nuclease based on the dry weight, heat it at 60 °C for 14 h, ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 4621, reaching 77% of the activity of the single nucleotide mixture.

[0089] Example 7 An implementation mode of the nucleotide production method described in the present invention

[0090] Weigh 400 g of cell residue and place it in a 5 L beaker; add 1 L of ultrapure water to the above beaker, stir evenly, and make up the volume to 2 L with ultrapure water; boil the above solution on an induction cooker for 30 min, then add 0.2% NaOH to the above solution, continue heating for 30 min, and then quickly cool it to adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; centrifuge the supernatant at 20000 G, add 2 volumes of 95% ethanol, place it at 4°C for precipitation for 30 min, add 4 M guanidine hydrochloride, and then add 2 volumes of 95% ethanol for precipitation; centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it once with 95% ethanol for the last time; place the precipitate in a drying oven and dry it at 65°C for 4 h until it becomes powdery; weigh and measure the purity after drying: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 98%. Adjust the crude RNA sample to a 10% aqueous solution with pH 5.5, add 0.5% nuclease based on the dry weight, heat it at 60°C for 14 h, ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 5290, reaching 90% of the activity of a single nucleotide mixture.

[0091] Example 8 An implementation mode of the nucleotide production method described in the present invention

[0092] Weigh 400 g of cell residue and place it in a 5 L beaker; add 1 L of ultrapure water to the above beaker, stir evenly, and make up the volume to 2 L with ultrapure water; boil the above solution on an induction cooker for 30 min, then add 0.2% NaOH to the above solution, continue to heat for 30 min, and then quickly cool it to adjust the pH to 5.5; centrifuge the above solution at 4000 G for 10 min and keep the supernatant; centrifuge the supernatant at 20000 G and then add 2 volumes of 95% ethanol, place it at 4°C for precipitation for 30 min, add 1 M guanidine hydrochloride and then add 2 volumes of 95% ethanol for precipitation; centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it with 95% ethanol for the last time; place the precipitate in an oven to dry, and dry it at 65°C for 4 h until it becomes powdery; weigh and measure the purity after drying: prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 93%; adjust the RNA crude sample to a 10% aqueous solution, pH: 5.5, add 0.5% nuclease based on the dry weight, heat it at 60°C for 14 h, ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 4120, reaching 70% of the activity of a single nucleotide mixture.

[0093] Example 9 An implementation mode of the nucleotide production method described in the present invention

[0094] Weigh 400 g of wet cell residue and place it in a 5-L beaker. Add 1 L of ultrapure water to the above beaker, stir evenly, and make up the volume to 2 L with ultrapure water. Boil the above solution on an induction cooker for 30 min, then add 0.2% NaOH to the above solution, continue heating for 30 min, and then quickly cool it and adjust the pH to 5.5. Centrifuge the above solution at 4000 G for 10 min and retain the supernatant. Centrifuge the supernatant at 20000 G, add 2 volumes of 95% ethanol, place it at 4°C for precipitation for 30 min, add 2 M guanidine hydrochloride, and then add 2 volumes of 95% ethanol for precipitation. Centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it once with 95% ethanol for the last time. Place the precipitate in a drying oven and dry it at 65°C for 4 h until it becomes powdery. After drying, weigh it and measure the purity: Prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 95%. Adjust the crude RNA sample to a 10% aqueous solution with pH 5.5, add 0.5% nuclease based on the dry weight, heat it at 60°C for 14 h, ultrafilter the sample through a 3000D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 4530, reaching 77% of the activity of the single nucleotide mixture.

[0095] Example 10 An implementation mode of the nucleotide production method of the present invention

[0096] Weigh 400 g of wet cell residue and place it in a 5-L beaker. Add 1 L of ultrapure water to the above beaker, stir evenly, and make up the volume to 2 L with ultrapure water. Boil the above solution on an induction cooker for 30 min, then add 10% NaCl to the above solution, continue heating for 30 min, and then quickly cool it and adjust the pH to 5.5. Centrifuge the above solution at 4000 G for 10 min and keep the supernatant. Centrifuge the supernatant at 20000 G, add 2 volumes of 95% ethanol, place it at 4 °C for precipitation for 30 min, add 4 M guanidine hydrochloride, and then add 2 volumes of 95% ethanol for precipitation. Centrifuge the above precipitate at 20000 G for 10 min, wash it twice with 70% ethanol, and wash it once with 95% ethanol for the last time. Place the precipitate in a drying oven and dry it at 65 °C for 4 h until it becomes powdery. After drying, weigh it and measure its purity: Prepare the sample into a solution containing 5 - 50 μg of nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of the RNA sample as 81%. Adjust the crude RNA sample to a 10% aqueous solution with a pH of 5.5, add 0.5% nuclease based on the dry weight, heat it at 60 °C for 14 h, ultrafilter the sample through a 3000 D pore size to obtain a purified mixed nucleotide solution, and obtain a mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthesis activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 4350, reaching 74% of the activity of a single nucleotide mixture.

[0097] Example 11

[0098] Weigh 400 g of cell residue and place it in a 5-L beaker. Add NaCl to the above solution, add 1 L of ultrapure water, stir evenly, and make up the volume to 2 L with ultrapure water, with the NaCl concentration being 8%. Boil the above solution on an induction cooker for 30 min, then immediately cool it and adjust the pH to 5.5.

[0099] Centrifuge the above solution at 4000G for 10 min and keep the supernatant; wash it twice by centrifugation with 75% ethanol (6000G for 10 min), and for the third wash, centrifuge with absolute ethanol at 6000G for 10 min and keep the precipitate; place the precipitate in an oven to dry, and dry it at 65 °C for 4 h until it becomes powdery; after drying, weigh it and determine its purity: prepare the sample into a solution containing 5 - 50 μg nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of this RNA sample as 46%. Adjust the crude RNA sample to a 10% aqueous solution, pH 5.5, add 0.5% nuclease based on the dry weight, heat at 60 °C for 14 h, add 0.1% activated carbon, shake well and then centrifuge at 6000G for 10 min; ultrafilter the sample through a 3000D pore size to obtain a purified mixed nucleotide solution, and obtain mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthetic activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 2057, reaching 35.2% of the activity of a single nucleotide mixture.

[0100] Example 12

[0101] Weigh 400 g of cell residue and place it in a 5 L beaker; add NaCl to the above solution, add 1 L of ultrapure water, stir evenly, and then make up the volume to 2 L with ultrapure water, with the NaCl concentration being 12%; boil the above solution on an induction cooker for 30 min, then immediately cool it and adjust the pH to 5.5;

[0102] Centrifuge the above solution at 4000G for 10 min and keep the supernatant; wash it twice by centrifugation with 75% ethanol (6000G for 10 min), and for the third wash, centrifuge with absolute ethanol at 6000G for 10 min and keep the precipitate; place the precipitate in an oven to dry, and dry it at 65 °C for 4 h until it becomes powdery; after drying, weigh it and determine its purity: prepare the sample into a solution containing 5 - 50 μg nucleic acid / mL, measure the absorbance values at 260 nm and 280 nm on an ultraviolet spectrophotometer, calculate the nucleic acid concentration and the absorption ratio of the two to obtain the purity of this RNA sample as 50%. Adjust the crude RNA sample to a 10% aqueous solution, pH 5.5, add 0.5% nuclease based on the dry weight, heat at 60 °C for 14 h, add 0.1% activated carbon, shake well and then centrifuge at 6000G for 10 min; ultrafilter the sample through a 3000D pore size to obtain a purified mixed nucleotide solution, and obtain mixed nucleotide powder through a vacuum freeze dryer or a spray drying device. Using the obtained mixed nucleotide powder as a raw material, add it to a cell-free protein synthesis system, detect its synthetic activity, use green fluorescent protein as the target protein, and detect its fluorescence value as 2257, reaching 38.6% of the activity of a single nucleotide mixture.

[0103] From the experimental results of Example 1 and Examples 11 and 12, it can be seen that the concentration of NaCl added during cell lysis directly affects the purity of the extracted RNA. At a concentration of 10%, the purity of the obtained RNA can reach over 50%, and the activity of the nucleotides after final enzymatic hydrolysis can reach over 40%. When the concentration is lower or higher than this value, the extraction effect is poor, and the obtained nucleotide products are not suitable for in vitro biosynthesis due to low activity and many impurities.

[0104] Comparing the experimental results of RNA extraction and purification between Example 1 and Example 2, it can be known that appropriately increasing the alcohol concentration for washing and purification can improve the purity of RNA. When the amount of nuclease is increased, to a certain extent, the yield of nucleotides can be improved.

[0105] The method parameters and results of Example 2 and Example 3 in the RNA extraction part are the same. Since an additional impurity removal step of activated carbon adsorption is added after enzymatic hydrolysis in Example 3, the purity of the obtained nucleotide mixture is improved, and the final expression activity is also improved (here referring to the cell-free protein synthesis amount corresponding to the same mass of nucleotide products).

[0106] NaOH-assisted lysis was used in Examples 4 - 6, and LiCl was further added during RNA purification. Compared with the methods of Examples 1 - 3, the purity of RNA and the biological activity of the mixed nucleotide products were significantly improved. It can be seen that LiCl has outstanding effects in washing and purifying RNA. However, from the comparison between Examples 4 - 6, an overly high concentration of LiCl will not only not further improve the extraction rate but also affect the activity of nucleotides, resulting in a decrease in the biological activity of the mixed nucleotides. Therefore, the concentration of LiCl is preferably between 5 - 10 M, and the effect is optimal at 7.5 M.

[0107] NaOH-assisted lysis was used in Examples 7 - 9, and guanidine hydrochloride was further added during RNA purification. Comparing with the results of Examples 4 - 6, it can be seen that the method of guanidine hydrochloride has better overall effects, and a purer RNA extract and a nucleotide mixture with higher biological activity can be obtained. Moreover, as the concentration of guanidine hydrochloride increases, the purification effect is further improved. When the concentration of guanidine hydrochloride is 4 M, the purity of the obtained RNA reaches 98%. However, considering production costs and residue problems, it is not recommended to further increase the concentration of guanidine hydrochloride.

[0108] The main difference between Example 10 and Example 7 is that NaCl was used instead during cell lysis, and its extraction effect decreased significantly compared with Example 7. It can be seen that adding NaOH during cell lysis has better effects than adding NaCl. At the same time, obviously, NaCl is safer, easier to operate, and has lower costs. Therefore, both have their own advantages, and can be selected according to requirements during actual production.

[0109] In summary, the technical solution protected by the present invention can obtain a nucleotide mixture with good biological activity, is applicable to reactions such as cell-free protein synthesis, and has broad application prospects.

[0110] The above are only partial embodiments of the present invention, and the present invention is not limited to the content of the above embodiments.

[0111] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for producing nucleotides for cell-free protein synthesis, characterized in that, the method comprises the following steps: Step 1), lysing yeast cells: Mixing waste yeast cell residue with water at a mass ratio of 1:4 - 1:6 to obtain a yeast cell suspension, heating to lyse the cells, and centrifuging to obtain a crude extract; Step 2), precipitation: Adding ethanol to the crude extract obtained in Step 1) to wash the precipitate, centrifuging to obtain an RNA extract; Step 3), enzymatic hydrolysis: Drying the obtained RNA extract into a powder, weighing and adding water to make an 8 - 12% aqueous solution, adding nuclease for enzymatic hydrolysis, and centrifuging to obtain an enzymatic hydrolysate; Step 4), filtration and purification: Filtering the above enzymatic hydrolysate through an ultrafiltration membrane to remove impurities to obtain a nucleotide mixture.

2. The method for producing nucleotides according to claim 1, characterized in that, NaCl or NaOH is further added during the lysis in Step 1); Specifically for adding NaCl, NaCl is added when preparing the yeast cell suspension, and the mass concentration range of NaCl in the suspension is greater than 8% and less than 12%, preferably 10%; Specifically for adding NaOH, NaOH is added before heating or during heating, and the mass concentration of NaOH in the suspension is 0.1 - 0.5%.

3. The method for producing nucleotides according to claim 1, characterized in that, It further includes Step 5), drying: Freeze-drying or spray-drying the obtained nucleotide mixture to make a mixed nucleotide powder.

4. The method for producing nucleotides according to claim 1 or 2, characterized in that, The precipitation step in Step 2) specifically includes: Adding ethanol with a concentration of more than 70% in an amount of 2 - 4 times the volume of the crude extract to the crude extract, precipitating, centrifuging at 4000 - 8000g for 5 - 10 min, and repeating this process more than 3 times.

5. The method for producing nucleotides according to claim 1 or 2, characterized in that, LiCl or guanidine hydrochloride is added in the precipitation step of Step 2).

6. The method for producing nucleotides according to claim 5, characterized in that, the precipitation step in Step 2) specifically includes: Centrifuging the crude extract obtained in Step 1) again at 10000g - 20000g for 10 min, leaving the supernatant, then adding 0.25 - 1 times the volume of LiCl to precipitate at a temperature below -20°C, centrifuging, adding ethanol with a concentration of more than 70% in an amount of 2 - 4 times the volume of the supernatant to the precipitate for washing more than twice, and then washing with ethanol with a concentration of more than 95%. The separated precipitate is the RNA extract; Alternatively, centrifuge the crude extract obtained in step 1) again at 10,000 g - 20,000 g for 10 min, retain the supernatant, add ethanol with a concentration of more than 70% and a volume 1 - 2 times that of the supernatant, precipitate at a temperature below 4°C, wash the precipitate with guanidine hydrochloride and ethanol with a concentration of more than 70% and a volume 1 - 2 times that of the supernatant, add ethanol with a concentration of more than 70% and a volume 2 - 4 times that of the supernatant to the precipitate, wash it more than twice, and then wash it with ethanol with a concentration of more than 95%. The separated precipitate is the RNA extract; The concentration of the added LiCl is 5 M - 10 M; the concentration of the added guanidine hydrochloride is 1 M - 4 M.

7. The production method of the nucleotide according to any one of claims 1 - 6, characterized in that, in step 1), the heating is: heating to 70°C - 100°C, and after heating and cracking, cooling and adjusting the pH to 5 - 7; and / or, the drying condition in step 3) is drying at 60°C - 70°C for 2 - 6 h; and / or, the enzymatic hydrolysis condition in step 3) is enzymatic hydrolysis by heating at a temperature of 50°C - 70°C for 2 - 16 h; the dosage of the nuclease is ≥0.5% based on the dry weight of the RNA extract powder; and / or, in step 4), the ultrafiltration is carried out using an ultrafiltration membrane with a pore size less than or equal to 4000 D.

8. A nucleotide mixture, characterized in that, it is prepared by the method according to any one of claims 1 - 7.

9. The application of the nucleotide mixture according to claim 8 in biosynthesis, food, feed, and pharmaceuticals.

10. An in vitro biosynthesis system, characterized in that, using the nucleotide mixture according to claim 8 as the nucleotide raw material.