CN114134093B - Recombinant microorganism producing cytosine and method for producing cytosine - Google Patents

Abstract

The present application discloses a recombinant microorganism for producing cytosine and a method for producing cytosine, the recombinant microorganism having at least one of the following features: 1) Recombinant microorganisms do not degrade or utilize cytosine; 2) Accumulating cytosine in vitro by the recombinant microorganism; 3) The recombinant microorganism overexpresses a gene encoding ribose kinase; 4) The recombinant microorganism can simultaneously utilize glucose and ribose; 5) Genes encoding regulatory proteins of ribose and related compounds of recombinant microorganisms are deleted or have no function. The recombinant microorganism strain can be used for fermenting and independently producing cytosine or producing a mixture of cytosine and cytidine.

Description

Recombinant microorganism producing cytosine and method for producing cytosine

The application relates to a split application of China patent application No. 201910602231.2, which is a recombinant microorganism for producing cytosine and has the name of "a method for producing cytosine" and has the application date of 2019, 07 and 05.

Technical Field

The application belongs to the technical field of biology, and particularly relates to a method for producing cytosine by a biological fermentation method and an enzymatic method. The method mainly comprises the steps of constructing recombinant microorganisms for producing cytosine and establishing a fermentation production method, and secondly, producing cytidine hydrolase liquid by utilizing genetically engineered microorganisms, and then, utilizing the enzyme liquid to hydrolyze cytidine to prepare cytosine.

Background

Cytosine is one of pyrimidine bases in nucleic acid, and has the chemical name of 4-amino-2-carbonyl pyrimidine, CAS RN of 71-30-7 and molecular formula of C ₄ H ₅ N ₃ O. White or white-like crystalline powder, which has low solubility in water at normal temperature, is slightly soluble in ethanol and insoluble in diethyl ether.

Cytosine is an important intermediate of fine chemical industry, pesticides and medicines, and is mainly used for synthesizing anti-AIDS medicines and anti-hepatitis B medicines lamivudine, anticancer medicines gemcitabine, enotabine, 5-fluorocytosine and the like in the medicine field. At present, cytosine is synthesized by a chemical method at home and abroad, and main production is concentrated at home. Cytosine is a heterocyclic compound containing carbon and nitrogen, and strong alkali and organic solvent are required to be used in the chemical synthesis process, so that the wastewater treatment difficulty is high. With the improvement of the national environmental protection requirements for chemical synthesis, domestic enterprises start to stop the chemical synthesis method to produce cytosine, or transfer the production to areas with low environmental protection requirements. There is therefore a need to develop a cytosine production process that can replace chemical synthesis and reduce pollution.

Humans have been fermenting nucleoside products such as beta-thymidine, guanosine, inosine, and adenosine with microorganisms, but cytidine, cytosine, etc. have not been industrially produced by the microbiological method. In the earlier study of the company, the technology of genetic engineering is utilized, and the biological method is utilized to produce cytidine (patent application number: 201710003833.7), and the constructed recombinant strain can produce 35g/L cytidine in a 5L fermentation tank. The application further improves cytidine producing bacteria on the basis of earlier work, and is expected to realize biological production of cytosine.

Disclosure of Invention

The application aims to: (1) Constructing genetically engineered strains capable of producing cytosine; (2) An enzyme for converting cytidine to produce cytosine, a method for constructing a genetically engineered strain to produce relevant enzyme solution and a method for converting cytidine to produce cytosine are provided.

The technical scheme is as follows:

the applicant provides recombinant microorganisms capable of producing cytidine by fermentation in domestic patent application (application number 201710003833.7), and takes escherichia coli genetic engineering bacteria as an example, the application is modified into new recombinant escherichia coli to produce cytosine on the basis of the bacteria, meets the requirement of industrial fermentation to produce cytosine, provides an optimal cytosine biosynthesis path and achieves the lowest cost of industrial fermentation production of cytosine. Since the genetically engineered E.coli bacteria of the above-mentioned patent application (application No. 201710003833.7) can ferment to produce cytidine, the simplest method is to ferment with the above-mentioned recombinant microorganism, and hydrolyze cytidine to cytosine and ribose by nucleoside hydrolase at the end of fermentation, or after obtaining purified cytidine. However, the market for produced ribose is small, ribose is not sold, and byproduct ribose also causes increased cost and time for processing. Thus, the present application contemplates the direct production of cytosine upon fermentation of recombinant E.coli, while allowing the byproducts to be further utilized without accumulation of ribose as a byproduct.

As shown in FIG. 1, the present application also contemplates that the gene encoding nucleoside hydrolase is recovered in cytidine-producing E.coli, or that the gene encoding nucleoside hydrolase is overexpressed in cells to directly transform cytidine into cytosine and ribose, whereas ribose is hardly utilized by E.coli when glucose is used as a carbon source (ribose is accumulated to a certain extent when glycerol is used as a carbon source), which results in low yield of cytosine, and the accumulated ribose in fermentation broth increases the treatment time and cost of fermentation waste liquid, so that the metabolic regulation of cells needs to be changed to allow the cells to return ribose produced in the process of producing cytosine to the synthesis of pyrimidine nucleosides when glucose or glycerol is utilized. The second way of producing cytosine is by nucleoside phosphorylase in the presence of inorganic phosphate to produce cytosine and 1-ribose phosphate, which can be returned to the pyrimidine nucleoside via isomerase to 5-ribose phosphate fermentation pathway, however this reaction is reversible, whether it is capable of converting cytidine to cytosine completely in the cell or not.

In the above domestic patent application (application No. 201710003833.7) disclosed that the intracellular synthesis of Cytidine Monophosphate (CMP) from Cytidine Triphosphate (CTP) is increased, the present application can directly use Cytidine Monophosphate (CMP) as a substrate to produce cytosine and 5' -phosphoribosyl (R5P) by nucleotide 5' -phosphoribosyl enzymes, which are returned to the synthesis pathway of nucleotides after synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP) under the catalysis of 5' -phosphoribosyl diphosphate kinase (Prs), as shown in fig. 2.

Coli not only has the ability to synthesize pyrimidine nucleosides de novo, but also has a salvage pathway to generate pyrimidine nucleotides from uracil by uracil phosphoribosyl transferase (Upp). Cytosine in a cell is deaminated first to form uracil and then participates in a salvage pathway for pyrimidine nucleotide synthesis. Thus, neither cytidine nor CMP is a direct precursor of cytosine, and the resulting cytosine cannot be degraded to uracil, which requires disruption of the gene encoding cytosine deaminase in E.coli.

1. Metabolic pathway of cytosine in E.coli

In the applicant's domestic patent application (application No. 201710003833.7), in the de novo synthesis pathway of pyrimidine nucleotides, cytidine Triphosphate (CTP) is hydrolyzed by an overexpressed CTP pyrophosphorohydrolase (NudG of escherichia coli itself) to produce Cytidine Monophosphate (CMP). Meanwhile, the CMP generated by RNA enzymolysis in cells and the CMP synthesized by NudG are degraded by CMP hydrolase such as PHM8 (Kuznetsova 2015) and the self-nucleotidase of escherichia coli (UshA, mmpG, mmpH (www.ecocyc.org) and the like) to generate cytidine. Although E.coli is the most deeply studied microorganism, it is not known to date which nucleotide contributes most to the hydrolysis of CMP, and this may be why Saccharomyces cerevisiae-derived PHM8 was borrowed from domestic patent application (application No. 201710003833.7). Cytidine is produced in escherichia coli and then is involved in pyrimidine nucleotide salvage synthesis by various ways, firstly cytidine can be deaminated to generate uridine, cytidine and uridine generate corresponding CMP and MMP under the action of uridine kinase (Udk), and CDP is produced by phosphorylation of CMP kinase (Cmk), and CDP and MMP are intermediates in pyrimidine nucleotide synthesis. Secondly, cytidine can be degraded into cytosine by various cytidine hydrolases in the cell (see description in 2A), which produce uracil by deaminase (CodA). Uracil can produce uridine and MMP, respectively, under the action of uridine phosphorylase (Udp) or uracil phosphoribosyl transferase (Upp). In domestic patent application (application number 201710003833.7), most of the genes encoding cytidine hydrolase, uridine phosphorylase (Udp) and uridine kinase (Udk) have been knocked out by genetic engineering.

Coli can express different transport proteins, some of which are responsible for transporting out the extra material produced in the cell (outer transport proteins) and some of which are responsible for transporting extracellular nutrients from the cell to the cell (inner transport proteins). Among the known extracellular transport proteins are NupG, nupC, tsx and CodB, which are intracellular transport proteins, etc., and the extracellular transport proteins are not known. If an extracellular transporter can be found, overexpression of the extracellular transporter will reduce accumulation of cytosine in genetically engineered strains (with the codB gene knocked out), which may not only increase the yield of cytosine extraction and purification, but may also further increase the yield of cytosine.

Synthesis of cytosine by extracellular hydrolysis of cytidine

Two pathways are available for modification of the strain to produce cytosine based on cytidine-producing strains (as shown in FIG. 1), one is to produce cytosine by hydrolysis of cytidine, and the other is to produce cytosine by hydrolysis from CMP.

Most wild-type organisms are themselves capable of producing many nucleoside hydrolases and nucleoside phosphorylases for different types of nucleoside substrates. For example, E.coli can synthesize several nucleoside hydrolases, such as RihA, rihB, rihC (Petersen 2001) and nucleoside phosphorylases, such as PpnP (Sevin 2017). Wherein RhiA, rihB and RihC can hydrolyze various nucleosides to generate corresponding purine or pyrimidine and ribose, while PpnP requires the participation of phosphate ions to generate 1-ribose phosphate in addition to corresponding purine or pyrimidine. Many nucleoside hydrolases capable of hydrolyzing cytidine in other organisms, such as Urh in Saccharomyces cerevisiae (YDR 400w, mitterbauer 2002), rih and Rih from Acinetobacter ammoniagenes (Kim 2006), and the like, can be used to enzymatically hydrolyze cytidine in vitro to produce cytosine.

2B intracellular hydrolysis of cytidine to cytosine

The disadvantage of the extracellular hydrolysis of cytidine in the aforementioned 2A is that a large amount of ribose is produced after cytidine hydrolysis, 0.54 kg of ribose is produced per kg of cytidine, and the market for ribose is small. Thus, cytidine is directly hydrolyzed in the cell to form cytosine and ribose, and the latter is recycled to the synthesis of nucleotide, which is key to reducing the production cost of cytosine. In addition, earlier studies showed that genetically engineered strains produced cytidine at a much faster rate than only cytosine under the same fermentation conditions, and the yield (mole/L) was much higher, which may be detrimental to cytosine production by accumulation of ribose or cytosine. It is known that when glucose and ribose are present in wild-type E.coli, the wild-type E.coli does not use ribose until glucose is consumed and then ribose is reused. In the present application, it was found that E.coli also accumulates D-ribose when producing cytosine using glycerol as a carbon source.

There are many precedents and references to engineering E.coli to use glucose and other sugars simultaneously, but no reference has been made to the simultaneous consumption of ribose by genetically engineered species using glucose. The genome of E.coli has a gene encoding riboregulated RbsR. When ribose is the only carbon source in the culture medium, ribose and RbsR are combined, so that four enzymes related to ribose metabolism, such as ribose transporter enzyme, ribose kinase and the like, are started. Since ribose is already intracellular during the production of cytosine, we phosphorylate ribose to 5-phosphoribosyl by (1) expressing ribose kinase, which can directly generate PRPP from 5-phosphoribosyl pyrophosphate kinase (Prs) into the de novo synthesis pathway of nucleotides; (2) Placing the rbsDACBK operon (www.ecocyc.org) in escherichia coli under a promoter which is not regulated by ribose, so that the gene of the operon can be expressed at any time, thereby enabling the genetically engineered strain to utilize ribose; (3) The rbsR gene is knocked out by using a genetic engineering method, so that the transcriptional regulation of rbsDABCK is relieved. The method for knocking out the rbsR gene is proved in the application, so that the genetically engineered strain can effectively produce cytosine and simultaneously does not accumulate ribose in fermentation liquor.

3. Nucleotide 5-nucleoside phosphatase catalysis CMP synthesis cytosine

Various nucleotide 5-phosphatases are produced in E.coli, and although their specific functions in cells are not well understood, they may be applicable to the synthesis of purines and pyrimidines. The most studied nucleotide 5-phosphonucleosidase in E.coli is the enzyme adenosine 5-phosphate (Amp, uniprot ID: P0AE 12), which catalyzes the synthesis of adenine and ribose 5-phosphate from adenosine 5-phosphate (AMP) (Leung 1980). The nucleotide nucleotidase (nucleotidase) (PpnN, uniprot ID: P0ADR 8) in e.coli has been reported to catalyze the production of corresponding purines or pyrimidines (Sevin 2017) of various monophosphate nucleotides (e.g., AMP, TMP) in vitro. Like Amn, proteins homologous to PpnN are not only widely present in various gram-negative bacteria, but also very highly homologous (more than 85%). Unlike AMP nucleotidase (Amn), the current research on the catalytic activity and function of PpnN in cells is completely lacking, and attempts have been made to overexpress PpnN or Amn to generate adenine in E.coli cells, wherein the overexpressed Amn can accumulate adenine in a culture medium, but the overexpressed PpnN has no adenine accumulation, which indicates that the intracellular PpnN cannot take AMP as a substrate or has low activity on AMP. Thus, prior to the present application, there was no evidence that PpnN could be used as a substrate in cells with pyrimidine nucleotides such as CMP, MMP and TMP. Among other microorganisms, similar nucleotide 5-phosphonucleosidases have also been reported, for example, in vitro studies of BlsM (Grochowski 2006) and MilB (Zhao 2014) of Streptomyces remofaciens ZJU5119 origin, which are involved in the synthesis of Blasticidin (Blastidin) in Streptomyces griseus, have been found to be able to produce cytosine using CMP as a substrate. However, E.coli-derived PpnN did not have any homology to the above Streptomyces-derived MilB and BlsM. It is presumed that the nucleotidase having a different origin exists in nature has a function of hydrolyzing nucleotide monophosphates to release corresponding purines or pyrimidines. It is predicted that if these enzymes and their homologous proteins are expressed in E.coli, an enzyme capable of converting intracellular CMP to cytosine can be found.

4. Others

The abbreviations of Chinese and English words in the application are mainly genes, enzymes and metabolites thereof in escherichia coli, which can be found in www.eccocyc.org webpage, and the abbreviations of English words which are not derived from escherichia coli are found in corresponding documents.

Modern biotechnology, especially those related to synthetic biology and DNA synthesis, can easily obtain mutants of the proteins of the present application, which not only circumvent the present application, but also make it possible to obtain enzymes with higher and better enzyme activities. Meanwhile, with the serial measurement of the genome of a natural organism by human, scientific researchers can easily find proteins homologous to the enzymes mentioned in the present application in these gene libraries, and then heterologously express genes encoding homologous proteins, it is well known that proteins with high homology often have uniform biological functions. It should be considered as an infringement of the present application whether from a library of gene sequences or by mutation (mutation obtained by means of gene synthesis or PCR).

The application provides genetically engineered escherichia coli for the first time in the world, which can ferment and produce cytosine in a large amount, and with further development, the yield of cytosine is gradually improved, and the cost for producing cytosine is further reduced. The pathways and methods of cytosine synthesis disclosed in the present application are readily imitated for cytosine production in other species-Bacillus, corynebacterium, salmonella, yeast and the like.

The beneficial effects are that: the application has the following advantages: the application provides three methods for biologically producing cytosine, in particular, a genetically engineered strain is constructed to be capable of fermenting to produce cytosine alone, or to produce a mixture of cytosine and cytidine (wherein cytidine can be produced by nucleoside hydrolases), and the like. The cytosine fermentation production of the application can completely replace the current production method, thereby realizing the environment-friendly and green cytosine production.

Drawings

FIG. 1 schematic diagram of the cytosine metabolic pathway of E.coli, wherein orotate: orotic acid; OMP: 5-monophosphate acid orotidine; MMP: uridine 5-monophosphate; UDP: uridine 5-diphosphate; UTP: uridine 5-triphosphate; CTP: cytidine 5-triphosphate; CMP: cytidine 5-monophosphate; cdR: cytidine; cyt: cytosine; ribose: d-ribose; R5P-5 ribose phosphate; uracil: uracil; codA: cytosine deaminase; prs: 5-phosphoribosyl diphosphate kinase; PRPP: 5-phosphoribosyl-1-pyrophosphate; upp: uracil phosphoribosyl transferase; (1): ribonucleoside hydrolases; (2): nucleoside phosphorylase; (3): nucleotide 5-phosphonucleosidase;

FIG. 2 is a schematic diagram of the formation of cytosine by hydrolysis CMP of a nucleotide 5-phosphate nucleosidase (nucleosidase);

FIG. 3 is a graph of the optimal assay of buffer concentration in example 8;

FIG. 4 is a graph of the condition optimization test of example 8 pH;

FIG. 5 is a graph of the reaction temperature optimization test of example 8.

Detailed Description

The application will now be further illustrated by means of several specific examples, which are given for illustrative purposes only and are not intended to be limiting.

The technical scheme of the application can be applied to escherichia coli, bacillus subtilis, corynebacterium glutamicum, lactobacillus or other microorganisms, and the technical scheme is further described below by taking escherichia coli as an example.

Example 1 method of Gene knockout in E.coli

The application adopts the method of Datsenko to knock out genes in escherichia coli (Datsenko 2000), and corresponding gene knockout primers are shown in Baba 2006.

Example 2 method for verifying recombinant strains by shake flask fermentation

Verifying that a recombinant strain produces a fermentation medium of cytosine in shake flask fermentation, wherein the fermentation medium comprises 100ml of YC solution, 200ml of 5-fold salt solution, 1ml of TM2 solution, 10mg of ferric citrate, 120mg of anhydrous magnesium sulfate, 111mg of calcium chloride and 1ug of thiamine, deionized water is used for volume fixation, and the 5-fold salt solution comprises 30g of disodium hydrogen phosphate, 15g of potassium dihydrogen phosphate, 2.5g of sodium chloride and 5.0g of ammonium chloride in each liter, and ionized water is used for volume fixation; each liter of the TM2 solution contains 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid and 100ml of hydrochloric acid. The YC solution in the fermentation medium is 2g of glycerol, 0.6g of peptone, 0.2g of yeast powder and 100ml of deionized water; the above solution was sterilized at 115℃for 20-30 minutes.

The shaking flask fermentation process is as follows: firstly inoculating recombinant strain into LB culture medium (Green 2012) containing 50mg/L spectinomycin, placing in a shaking table at 34 ℃ and culturing at 250rpm for overnight; transferring 200 μl of overnight seeds to 2ml LB containing antibiotics, and culturing at 250rpm in a shaker at 34 ℃ for 5 hours until OD600 is about 1; then 2ml of the secondary seeds are all transferred into a shake flask filled with 18ml of fermentation medium M11, the shake flask is placed in a shaking table at 34 ℃ for culturing for 4-6 hours at 250rpm, IPTG is added to a final concentration of 0.1mM, the culture is continued for about 20 hours, 1ml of fermentation liquor is taken for centrifugation (7000 rmp,4 minutes), and the supernatant is taken for detection, and the detection method is shown in example 3.

EXAMPLE 3 HPLC determination of cytidine and cytosine in fermentation broths

Sucking 1ml of fermentation broth into a 2ml centrifuge tube, heating at 100deg.C for 5 min, cooling to room temperature, diluting by a certain multiple, centrifuging through 0.22 μm filter membrane, detecting with High Performance Liquid Chromatography (HPLC), and the parameters of HPLC are as follows: the method comprises the steps of adopting Agilent SB C18.6X105 mM 5 μm, wherein a mobile phase is methanol and 10mM PBS (pH 4.0), the methanol proportion of the mobile phase is 0.01-2.80 min and 2%, the methanol proportion of 2.80-3.50 min is increased from 2% to 10%, the methanol proportion of 3.50-3.60 min is decreased from 10% to 2%, the methanol proportion of 3.60-8.5 min is 2%, and the wavelength is 260nm detected by an ultraviolet detector; the flow rate of the initial mobile phase was 1.0mL/min, the loading of the fermentation broth was 5. Mu.L, and the column temperature was 30 ℃.

EXAMPLE 4 construction of recombinant E.coli incapable of degrading and utilizing cytosine

The cytosine in the microorganism is mainly converted to uracil under the catalysis of cytosine deaminase CodA, and then returned to the synthesis of pyrimidine nucleotides. Thus, in order for microorganisms to accumulate cytosine, cytosine is first utilized in cells

The coding gene codA of the cytosine deaminase which is degraded into uracil and participates in the pyrimidine nucleotide synthesis salvage path is blocked or knocked out, so that the cytosine cannot be deaminated and degraded into uracil, and microorganisms can accumulate the cytosine. In addition, in order to accumulate more cytosine, it is necessary to knock out the transporter gene codB that transports cytosine into the cell, so that cytosine is accumulated outside the cell, which contributes to the improvement of yield and the simplification of the later product purification. The codB and codA are located under the same operator and thus knockdown is performed simultaneously.

In order to enable the recombinant microorganism to accumulate cytosine, the codBA gene in the parent strain HF9 of the cytidine strain HF9/pHS01-nudG-PHM8-pyrE-pyrHm (CGMCC No. 13427) was knocked out together, resulting in the strain HF11K lacking the production of cytosine deaminase and cytosine transporter. The specific experimental process is as follows:

when the codAB is knocked out, pKD4 is used as a template, kan-FRT knocked out fragments containing 50bp homology arms are amplified in codBA-F/codBA-R by using the primer pair in the following table (primer table 1), electrophoresis of the amplified fragments is carried out without impurity bands, column recovery purification (a JieRui gum recovery purification kit) is directly carried out, about 200ng of the obtained fragments are electrically transformed, HF9/pKD46 competent cells are added, uniformly mixed and transferred into a 0.1cm electric shock cup, and an electric shock instrument is used for electric transformation. Electric shock conditions: 200 Ω,25 μF, 1.8kV shock voltage, 5ms shock time, 1mL LB was added immediately after shock, incubated at 37℃for 1h at 200rpm, then plated on LB plates containing 50ng/mL kanamycin, and incubated overnight at 37 ℃. The next day, bacterial liquid PCR verification is carried out on the transformant obtained by electric transformation, and the primers are codBA-200F/Kan-R, kan-F/codBA-200R respectively, so that the construction success of HF11K, namely HF9 (delta codBA:: kan-FRT) is confirmed.

It was then verified whether HF11K degraded cytosine. At the time of verification, 2g/L of cytosine was added to an HF11K inoculated shake flask, and after 24 hours of growth at 34 ℃, the remaining amount of cytosine in the supernatant of the culture broth was detected by HPLC, thereby proving that the recombinant strain HF11K obtained by knocking out codAB could not utilize and degrade cytosine.

TABLE 1 CodBA knock-out and validation primers

EXAMPLE 5 construction of recombinant E.coli for expression of nucleoside hydrolases and nucleoside phosphorylases

When BL21 (DE 3) was used as an expression host, it was found that cytidine was hydrolyzed to cytosine by nucleoside hydrolase, and the uracil content in the reaction solution became high, and the analysis was probably due to the fact that cytosine deaminase CodA in wild-type bacteria was not knocked out, thereby degrading cytosine produced by cytidine to uracil. Thus, in the present application, the gene encoding cytosine deaminase (codA) and the gene encoding cytosine transporter (codB) in BL21 (DE 3) are knocked out simultaneously. Since BL21 (DE 3) and W3110 have the same sequence in this segment of the gene cluster, knockdown can be performed using the primers CodBA-F/CodBA-R in example 4 and the like, and the resulting knockdown strain is digested as follows: BL21 (DE 3, ΔcodBA:: kanFRT) was inoculated into a resistant tube and cultured overnight for chemocompetent preparation, transformed into plasmid pCP20, and plated with Amp-resistant plates. The obtained monoclonal after the overnight culture at 30 ℃ is inoculated into an LB test tube containing 20mg/L Cm, after the culture at 30 ℃ for 4 hours, the obtained monoclonal is transferred into an LB non-resistance liquid culture medium test tube for continuous culture for 4 hours, the temperature is raised to 42 ℃ for the culture overnight, then the LB plates are coated, the obtained monoclonal is respectively spotted on ampicillin, chloramphenicol and kanamycin resistant LB plates and non-resistant LB plates, the success of elimination of the resistant fragments is confirmed, and finally bacterial liquid PCR verification is carried out through a primer codBA-200F/codBA-200R to obtain a recombinant strain BL21 (DE 3, DELTAcodBA) which is used for expressing and preparing hosts of nucleoside hydrolase and nucleoside phosphorylase.

Example 6 screening of nucleoside hydrolases and nucleoside phosphorylases in E.coli to produce cytosine

The E.coli W3110 genome is used as a template, the primers corresponding to the primers shown in the following table (table 2) are used for amplifying the rihA, rihB, rihC gene, the ppnP gene and the like, the obtained fragments are respectively connected with an enzyme-digested pET28a vector, a host TG1 is transformed, and the successfully constructed expression plasmids pET28a-rihA, pET28arihB, pET28a-rihC and pET28a-ppnP are obtained through bacterial liquid PCR and enzyme digestion verification. These plasmids were transformed into E.coli BL21 (DE 3. DELTA. CodBA) strain by electrotransformation, and overexpressed, respectively. Wherein, for example, the gene sequence of rilA is shown in SEQ ID No. 23, and the amino acid sequence is shown in SEQ ID No. 24.

The recombinant strain overexpression flow is as follows: the recombinant E.coli was activated in LB medium (Green 2012) containing 50mg/l kanamycin, and the cultured overnight strains were transferred to 0.8% to 50mL of LB medium containing 50mg/l kanamycin, cultured at 37℃and 200rpm, and when grown to an OD of about 0.8, the final concentration was induced by adding IPTG to 0.4mM, and the temperature was lowered to 30℃to culture, and after overnight culture, the bacterial solution was collected by centrifugation, and cells were suspended in 5mL of 50mM PBS buffer pH 7.0. The recombinant bacterial cells of the above-obtained enzymes were disrupted by sonication under the following conditions: opening 2S, closing 4S,8min,30% power, crushing, centrifuging, respectively carrying out protein electrophoresis detection on supernatant and sediment, and directly using supernatant enzyme liquid after centrifuging for reaction of cytidine conversion to generate cytosine.

TABLE 2 construction of primers for nucleoside hydrolase

Example 7 conversion of cytidine to cytosine Using a nucleoside hydrolase enzyme solution

The various nucleoside hydrolase enzyme solutions obtained above react according to the following reaction system and time: the enzyme solution obtained by the above-mentioned disruption treatment was diluted 50 times, 100ul of the enzyme solution was taken and fed into a reaction tube containing 3ml of a substrate, which was cytidine 15g/L in 10mM PBS buffer pH7.0, and the reaction system was subjected to a reaction at 3.1ml at 35℃overnight, and the results were shown in the following Table.

The results show that the RihA-expressing enzyme solution has the best enzyme activity and the most thorough hydrolysis of cytidine, and the following examples are based on RihA-expressing enzyme solution to demonstrate the feasibility of extracellular enzyme reaction to produce cytosine.

TABLE 3 relative enzyme activities of nucleoside hydrolase hydrolysis

Example 8 optimization of nucleoside hydrolase (RihA) reaction conditions

The nucleoside hydrolase is reacted overnight (reaction is carried out for 18 h) and is completely converted into cytosine, and in order to further shorten the reaction time and improve the conversion efficiency, nucleoside hydrolase (RihA) enzyme solution is selected, and the buffer solution concentration, the reaction pH, the reaction temperature and the like are further optimized. The detailed examination is as follows:

1) Buffer concentration optimization:

the conversion of PBS concentration at various time points at 0mM, 2mM, 10mM, 50mM was examined.

TABLE 4 reaction system

The method comprises the following steps: according to the system, adding a substrate A solution, carrying out a bath at 35 ℃ for 3min, adding 100ul of enzyme solution B, starting timing, stirring at 1200rpm, taking 100ul of reaction solution at different time points, adding 900ul of 0.2% formic acid water, and stopping reaction, wherein the HPLC detection result is shown in FIG. 3: as shown in the results of the table in FIG. 3, the buffer PBS concentration is preferably between 2 and 10 mM.

Meanwhile, running water replaces PBS buffer solution to perform experiments, and cytidine is also successfully converted into cytosine.

2) pH condition optimization:

the conversion at various time points under the conditions of pH5.2, pH5.5, pH5.7, pH6.0, pH6.2, pH6.5, pH7.0, pH7.5 and pH8.0 was examined. The reaction system is shown in Table 4 and the method in 1). The reaction results are shown in FIG. 4: as shown in the results of the table in FIG. 4, the reaction pH is preferably 5.7 to 6.5.

3) Optimizing reaction temperature conditions:

under different temperature conditions, the conversion rates at different time points were examined, and the temperatures of 35 ℃,37 ℃, 40 ℃, 43 ℃, 45 ℃, 47 ℃, 50 ℃, 53 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃ were examined. The reaction system is shown in Table 4 and the method in 1). The reaction results are shown in FIG. 5 below: as shown in the results of the table in FIG. 5, the reaction temperature is preferably 60 to 65 ℃.

In summary, preferred reaction conditions for nucleoside hydrolases (RihA) are: the concentration of the buffer solution PBS is 2-10mM, the reaction pH is 6.0, and the reaction temperature is 60-65 ℃.

Example 9 production of cytosine by addition of nucleoside hydrolase (RihA) after the end of cytidine fermentation

The method of example 2 was followed to carry out the fermentation in a tank to produce cytidine, after the fermentation was completed, a certain amount of nucleoside hydrolase (RihA) enzyme solution was directly added to the fermentation tank, the pH in the tank was adjusted to 6.0 with ammonia, the temperature of the fermentation solution was raised to 60℃and after 10 hours of reaction, sampling and detection were carried out. The detection result shows that cytidine in the fermentation broth is completely hydrolyzed into cytosine.

EXAMPLE 10 construction of recombinant plasmid producing cytosine

The above examples of the in vitro reaction with respect to the enzyme solution of nucleoside hydrolase RihA show that the activity of this enzyme is very high, and therefore, it was designed to construct the rihA gene on pHS01-nudG-PHM8-pyrE-pyrHm and then transform the host HF11 for shake flask fermentation to produce cytosine.

Furthermore, the strain HF9/pHS01-nudG-PHM8-pyrE-pyrHm (CGMCC No. 13427) of the previous patent (application No. 201710003833.7) can produce cytidine up to 20g/L or more in an upper tank fermentation, wherein cytidine is mainly obtained by CMP hydrolysis, which indicates that CMP is sufficient in the production of nucleoside products by this strain, so that it is designed to construct nucleotide phosphatase PpnN (the gene sequence of which is shown in SEQ ID No. 21 and the amino acid sequence of which is shown in SEQ ID No. 22) onto pHS01-nudG-PHM8-pyrE-pyrHm, and it is expected that cytidine can be produced by hydrolyzing CMP directly in the fermentation process without producing cytidine.

For example, the W3110 genome is used as a template, the primers ppnN-F1/ppnN-R1 and rihA-F/rihA-R (see Table 5 below) are used to amplify fragments ppnN and rihA, the obtained PCR products are electrophoretically detected to be free of impurity bands, column recovery and purification (a JieRui gel recovery and purification kit) are directly carried out, the obtained purified fragments are respectively subjected to recombinant cloning construction with SalI digestion and recovery pHS01-nudG-PHM8-pyrE-pyrHm carrier fragments (a Gclonart seamless cloning kit for the Shenzhou gene GBclart), the recombinant cloning reaction solution is transferred to ice in a 45-degree water bath pot for 30min, TG1 competent cells are added, 42-degree heat shock is carried out for 2min, a resuscitating culture medium LB is added in ice bath for 2min, after resuscitating culture is carried out for 1h, a secondary day decoloning culture is carried out on the obtained purified fragments, and enzyme digestion verification is carried out on the obtained plasmid extracts, and finally pHS 01-nudG-PHM-nudG-8-pyrH-hM plasmid is obtained.

TABLE 5 rilA and ppnN-1 amplification primers

EXAMPLE 11 construction of Gene recombinant Strain direct fermentation to cytosine

The expression plasmid pHS01-nudG-PHM8-pyrE-pyrHm-rihA was transformed into the host HF11K to give the recombinant strain HF11K/pHS01-nudG-PHM8-pyrE-pyrHm-rihA. The flask fermentation was carried out in accordance with example 2, and sampling detection was carried out after 24 hours of fermentation, whereby 0.37g/L of cytosine and 0.02g/L of cytidine could be detected. Recombinant strain HF11K/pHS01-nudG-PHM 8-pyrE-pyrHm-rila abbreviated as strain HF11K145, which is classified as Escherichia coli (Escherichia coli), which has been deposited at the chinese microorganism center, address, at 05, 08, 2019: beijing, chaoyang area, north Chen Xi Lu No.1, 3, china academy of sciences microbiological institute, post code: 100101 and the preservation number is CGMCC No.17729.

The expression plasmid pHS01-nudG-PHM8-pyrE-pyrHm-ppnN was transformed into the host HF11K to obtain the recombinant strain HF11K/pHS01-nudG-PHM8-pyrE-pyrHm-ppnN. After shaking flask fermentation was performed according to example 2, and after 24 hours of fermentation, sampling was performed, and it was possible to detect 0.08g/L of cytosine and 0.58g/L of cytidine, and analysis was performed because PHM8 on this plasmid was expressed too high, competed for the same substrate as ppnN, and the enzyme activity was high, so that the accumulated cytosine was very small, and thus plasmid pHS01-nudG-pyrE-pyrHm-ppnN was constructed, and then host HF11 was transformed for shaking flask fermentation to produce cytosine.

For example, the W3110 genome is used as a template, a primer pair ppnN-F2/ppnN-R2 (see table 6 below) is used for amplifying a fragment ppnN-2, the obtained PCR product is subjected to electrophoresis detection without a mixed band, column recovery purification (a JieRui gel recovery purification kit) is directly carried out, the obtained purified fragments are respectively subjected to recombinant cloning construction with HindIII digestion and recovery pHS01-nudG-pyrE-pyrHm carrier fragments (a Gclonart seamless cloning kit for the Shenzhou gene), the recombinant cloning reaction solution is transferred to ice in a water bath with the temperature of 45 ℃ for 30min, TG1 competent cells are added, 42-DEG heat shock is carried out for 2min, a recovery culture medium plasmid LB is added in the ice bath for 2min, 100mg/L of spectinomycin resistant LB plates are centrifugally coated after recovery culture for 1h, cloning culture is carried out the next day, enzyme digestion verification is carried out, and plasmid pHS01-nudG-pyrE-pyrHm-ppnN is finally constructed.

The expression plasmid pHS01-nudG-pyrE-pyrHm-ppnN was transformed into the host HF11K to give the recombinant strain HF11K/pHS01-nudG-pyrE-pyrHm-ppnN. Shaking flask fermentation was performed as in example 2, and sampling detection was performed after 24 hours of fermentation, whereby 0.3g/L of cytosine and 0.5g/L of cytidine could be detected. Recombinant strain HF11K/pHS01-nudG-pyrE-pyrHm-ppnN is abbreviated as strain HF11K182, which is classified as Escherichia coli (Escherichia coli) and has been deposited at the chinese microorganism culture collection at month 08 of 2019 at the general microbiological center, address: beijing, chaoyang area, north Chen Xi Lu No.1, 3, china academy of sciences microbiological institute, post code: 100101 and the preservation number is CGMCC No.17730.

TABLE 6 ppnN-2 amplification primers

Example 12 ribose repressor Gene knockout to facilitate D-ribose metabolism

The disadvantage of the extracellular hydrolysis of cytidine in the aforementioned 2A is that a large amount of ribose is produced after cytidine hydrolysis, 0.54 kg of ribose is produced per kg of cytidine, and the market for ribose is small. Thus, cytidine is directly hydrolyzed in the cell to form cytosine and ribose, and the latter is recycled to the synthesis of nucleotide, which is key to reducing the production cost of cytosine. The genome of E.coli has a gene encoding riboregulated RbsR. When ribose is the only carbon source in the culture medium, ribose and RbsR are combined, so that four enzymes related to ribose metabolism, such as ribose transporter enzyme, ribose kinase and the like, are started. Therefore, we considered to knock out rbsR gene by genetic engineering method, thereby relieving transcriptional regulation of rbsDABCK.

For example, by knocking out the gene encoding the riborepressor RbsR on the basis of the HF11 strain to obtain the strain HF12 (HF 11. DELTA. RbsR), and subjecting pHS01-nudG-PHM8-pyrE-pyrHm-rihA to shaking fermentation in HF11 and HF12, respectively, the results of the detection showed that the difference in the cytosine production was not significant, but a certain amount of ribose could be detected in the fermentation broth of the strain derived from HF11, while substantially no D-ribose could be detected in the fermentation broth after rbsR knocking out, as was the case in the 5L fermenter. The rbsR gene knockout strain can effectively ensure that the genetically engineered strain does not accumulate ribose in fermentation broth while producing cytosine, and can recycle ribose after glucose is used when glucose is used as a raw material in fermentation culture, so that the utilization rate of sugar can be improved, the extraction and purification of products are simplified, and the method has great application prospect.

The foregoing is merely illustrative of the embodiments of this application and it will be appreciated by those skilled in the art that variations may be made without departing from the principles of the application and such variations are not to be regarded as a departure from the scope of the application.

Literature

Baba,T.,et al(2006)Construction of Escherichia coli K-12 inframe,single-gene knockout mutants:the Keio collection.Mol.Syst.Bio.2:2006.0008.

Datsenko,K .A,et al(2000)One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci USA 97(12):6640-6645.

Green,M.R.et al(2012)Molecular Cloning:A Laboratory Manual(Fourth Edition)ISBN 978-1-936113-42-2.

Grochowski L.L.(2006)Characterization of BlsM ,a Nucleotide Hydrolase Involved in Cytosine Production for the Biosynthesis of Blasticidin S.ChemBioChem 7:957-964.

Kim,H.,et al(2006)Genes encoding ribonucleoside hydrolase 1 and 2 from Corynebacterium ammoniagenes.Microbiology 152:1169-1172.

Kuznetsova,E.,et al(2015)Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae Biochemical,structural,and evolutionary insights.J boil.Chem.290(30):18678-18698.

Leung,H.B.et al(1980)Adenylate degradation in Escherichia coli.The role of AMP nucleosidase and properties of the purified enzyme.J Bio Chem 255(22):10867-10874.

Petersen ,C.,et al(2001)The RihA,RihB ,and RihC ribobucleoside hydrolase of Escherichia coli.J .Bio.Chem 276(2):884-894.

Mitterbauer,R.,et al(2001)Saccharomyces cerevisiae URH1(Encoding Uridine-Cytidine N-Ribohydrolase):Functional Complementation by a Nucleoside Hydrolase from a Protozoan Parasite and by a Mammalian Uridine Phosphorylase.Appl Environ Microbiol 68(3):1336-1343.

Sevin,D.C,et al 2017 Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli.Nature Methods 14(2)187-194.

Zhao ,G.,et al(2014) Structure of the N-glycosidase MilB in complex with hydroxymethyl CMP reveals its Arg23 specificially recognizes the substrate and control its entry.Nucleic Acids Research42(12):8115-8124.

Sequence listing

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aatgcggcgg attttttggg tttcaaacag caaaaagggg gaatttcgtg gtgtaggctg 60

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atggcgttgg ataaatgcg 19

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gcagttcatt cagggcaccg 20

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gggcacaaca gacaatcggc 20

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ctggtgccgc gcggcagcca taatggcact gccaattctg ttag 44

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cgacggagct cgaattcgga tcttaagcgt aaaatttcag acgatcagcc 50

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ctggtgccgc gcggcagcca taatggaaaa gagaaaaatt attctggatt gtg 53

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cgacggagct cgaattcgga tcttaatggg ttttgatgta gccgcg 46

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ctggtgccgc gcggcagcca taatgcgttt acctatcttc ctcgatactg a 51

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cgacggagct cgaattcgga tcttacgacg ccagagccag c 41

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ctggtgccgc gcggcagcca taatgcttca aagtaatgag tacttttccg 50

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cgacggagct cgaattcgga tcttacagat agcggcacag ataagag 47

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ctttaatcac ggaataagtc gactttagcg ttaagaagga gatatatatg attacacata 60

ttagcccgct tggc 74

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gatgatggtc gagtcgagtc gattacgtgc agatttcgta gcaaggg 47

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<212> DNA

<213> Artificial sequence

<400> 17

ctttaatcac ggaataagtc gactttagcg ttaagaagga gatatatatg gcactgccaa 60

ttctgttag 69

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<211> 50

<212> DNA

<213> Artificial sequence

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gatgatggtc gagtcgagtc gattaagcgt aaaatttcag acgatcagcc 50

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cgtttaactc gagatctaag ctctttagcg ttaagaagga gatatatatg attacac 57

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gtttttgttc gggcccaagc tttacgtgca gatttcgtag caagg 45

<210> 21

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<212> DNA

<213> Artificial sequence

<400> 21

atgattacac atattagccc gcttggctcc atggatatgt tgtcgcagct ggaagtggat 60

atgcttaaac gcaccgccag cagcgacctc tatcaactgt ttcgcaactg ttcacttgcc 120

gtactgaact ccggtagttt gaccgataac agcaaagaat tgctgtctcg ttttgaaaat 180

ttcgatatta acgtcttgcg ccgtgaacgc ggcgtaaagc tggaactgat taatcccccg 240

gaagaggctt ttgtcgatgg gcgaattatt cgcgctttgc aggccaactt gttcgcggtc 300

ctgcgtgaca ttctcttcgt ttacgggcaa atccataaca ccgttcgttt tcccaacctg 360

aatctcgaca actccgtcca catcactaac ctggtctttt ccatcttgcg taacgctcgc 420

gcgctgcatg tgggtgaagc gccaaatatg gtggtctgct ggggcggtca ctcaattaac 480

gaaaacgagt atttgtatgc ccgtcgcgtc ggaaaccagc tgggcctgcg tgagctgaat 540

atctgcaccg gctgtggtcc gggagcgatg gaagcgccga tgaaaggtgc tgcggtcgga 600

cacgcgcagc agcgttacaa agacagtcgt tttattggta tgacagagcc gtcgattatc 660

gccgctgaac cgcctaaccc gctggtcaac gaattgatca tcatgccaga tatcgaaaaa 720

cgtctggaag cgtttgtccg tatcgctcac ggtatcatta tcttccctgg cggtgtgggt 780

acggcagaag agttgctcta tttgctggga attttaatga acccggccaa caaagatcag 840

gttttaccat tgatcctcac cggcccgaaa gagagcgccg actacttccg cgtactggac 900

gagtttgtcg tgcatacgct gggtgaaaac gcgcgccgcc attaccgcat catcattgat 960

gacgccgctg aagtcgctcg tcagatgaaa aaatcgatgc cgctggtgaa agaaaatcgc 1020

cgtgatacag gcgatgccta cagctttaac tggtcaatgc gcattgcgcc agatttgcaa 1080

atgccgtttg agccgtctca cgagaatatg gctaatctga agctttaccc ggatcaacct 1140

gttgaagtgc tggctgccga cctgcgccgt gcgttctccg gtattgtggc gggtaacgta 1200

aaagaagtcg gtattcgcgc cattgaagag tttggtcctt acaaaatcaa cggcgataaa 1260

gagattatgc gtcgtatgga cgacctgcta cagggttttg ttgcccagca tcgtatgaag 1320

ttgccaggct cagcctacat cccttgctac gaaatctgca cgtaa 1365

<210> 22

<211> 454

<212> PRT

<213> Artificial sequence

<400> 22

Met Ile Thr His Ile Ser Pro Leu Gly Ser Met Asp Met Leu Ser Gln

1 5 10 15

Leu Glu Val Asp Met Leu Lys Arg Thr Ala Ser Ser Asp Leu Tyr Gln

20 25 30

Leu Phe Arg Asn Cys Ser Leu Ala Val Leu Asn Ser Gly Ser Leu Thr

35 40 45

Asp Asn Ser Lys Glu Leu Leu Ser Arg Phe Glu Asn Phe Asp Ile Asn

50 55 60

Val Leu Arg Arg Glu Arg Gly Val Lys Leu Glu Leu Ile Asn Pro Pro

65 70 75 80

Glu Glu Ala Phe Val Asp Gly Arg Ile Ile Arg Ala Leu Gln Ala Asn

85 90 95

Leu Phe Ala Val Leu Arg Asp Ile Leu Phe Val Tyr Gly Gln Ile His

100 105 110

Asn Thr Val Arg Phe Pro Asn Leu Asn Leu Asp Asn Ser Val His Ile

115 120 125

Thr Asn Leu Val Phe Ser Ile Leu Arg Asn Ala Arg Ala Leu His Val

130 135 140

Gly Glu Ala Pro Asn Met Val Val Cys Trp Gly Gly His Ser Ile Asn

145 150 155 160

Glu Asn Glu Tyr Leu Tyr Ala Arg Arg Val Gly Asn Gln Leu Gly Leu

165 170 175

Arg Glu Leu Asn Ile Cys Thr Gly Cys Gly Pro Gly Ala Met Glu Ala

180 185 190

Pro Met Lys Gly Ala Ala Val Gly His Ala Gln Gln Arg Tyr Lys Asp

195 200 205

Ser Arg Phe Ile Gly Met Thr Glu Pro Ser Ile Ile Ala Ala Glu Pro

210 215 220

Pro Asn Pro Leu Val Asn Glu Leu Ile Ile Met Pro Asp Ile Glu Lys

225 230 235 240

Arg Leu Glu Ala Phe Val Arg Ile Ala His Gly Ile Ile Ile Phe Pro

245 250 255

Gly Gly Val Gly Thr Ala Glu Glu Leu Leu Tyr Leu Leu Gly Ile Leu

260 265 270

Met Asn Pro Ala Asn Lys Asp Gln Val Leu Pro Leu Ile Leu Thr Gly

275 280 285

Pro Lys Glu Ser Ala Asp Tyr Phe Arg Val Leu Asp Glu Phe Val Val

290 295 300

His Thr Leu Gly Glu Asn Ala Arg Arg His Tyr Arg Ile Ile Ile Asp

305 310 315 320

Asp Ala Ala Glu Val Ala Arg Gln Met Lys Lys Ser Met Pro Leu Val

325 330 335

Lys Glu Asn Arg Arg Asp Thr Gly Asp Ala Tyr Ser Phe Asn Trp Ser

340 345 350

Met Arg Ile Ala Pro Asp Leu Gln Met Pro Phe Glu Pro Ser His Glu

355 360 365

Asn Met Ala Asn Leu Lys Leu Tyr Pro Asp Gln Pro Val Glu Val Leu

370 375 380

Ala Ala Asp Leu Arg Arg Ala Phe Ser Gly Ile Val Ala Gly Asn Val

385 390 395 400

Lys Glu Val Gly Ile Arg Ala Ile Glu Glu Phe Gly Pro Tyr Lys Ile

405 410 415

Asn Gly Asp Lys Glu Ile Met Arg Arg Met Asp Asp Leu Leu Gln Gly

420 425 430

Phe Val Ala Gln His Arg Met Lys Leu Pro Gly Ser Ala Tyr Ile Pro

435 440 445

Cys Tyr Glu Ile Cys Thr

450

<210> 23

<211> 936

<212> DNA

<213> Artificial sequence

<400> 23

atggcactgc caattctgtt agattgcgac ccaggtcatg acgacgctat cgcaatagtt 60

ctcgccctcg cctcaccaga gcttgatgtc aaagcaatta cgtcttccgc cggaaaccag 120

acaccagaaa aaaccttacg caatgttctg cgtatgctga ccttgcttaa tcgcaccgat 180

attccggtag caggcggcgc ggtaaaaccg ttaatgcgtg agttgattat cgcggacaat 240

gtgcacggcg aaagcggtct cgacggcccg gcattaccgg aaccgacatt cgcaccgcaa 300

aactgtacgg cggtagagct gatggcgaaa acgctgcgtg aaagtgcgga acctgtcacc 360

attgtgtcta ccggaccgca aactaacgtt gccttgctgc tcaatagcca tccggaactg 420

catagcaaaa ttgcccgtat cgtgattatg ggtggcgcta tggggctggg taactggacg 480

cctgcggctg aatttaatat ttacgttgac ccggaagcgg cagaaattgt cttccagtca 540

gggatcccgg tggtgatggc cggtctggat gttactcata aagcacaaat ccacgttgaa 600

gacaccgagc gtttccgcgc gattggtaac cctgtttcaa ccattgttgc cgaactgctg 660

gatttcttcc tcgaatatca taaagacgaa aaatggggct ttgtcggcgc accactgcat 720

gacccatgca ccatcgcctg gctgttgaaa ccggagttat ttacctctgt tgagcgctgg 780

gttggcgtgg aaacacaggg gaaatatacc cagggtatga cggttgttga ttattattat 840

ctgacaggca ataaaccgaa tgccaccgta atggtcgatg ttgatcgtca gggctttgtt 900

gatttactgg ctgatcgtct gaaattttac gcttaa 936

<210> 24

<211> 311

<212> PRT

<213> Artificial sequence

<400> 24

Met Ala Leu Pro Ile Leu Leu Asp Cys Asp Pro Gly His Asp Asp Ala

1 5 10 15

Ile Ala Ile Val Leu Ala Leu Ala Ser Pro Glu Leu Asp Val Lys Ala

20 25 30

Ile Thr Ser Ser Ala Gly Asn Gln Thr Pro Glu Lys Thr Leu Arg Asn

35 40 45

Val Leu Arg Met Leu Thr Leu Leu Asn Arg Thr Asp Ile Pro Val Ala

50 55 60

Gly Gly Ala Val Lys Pro Leu Met Arg Glu Leu Ile Ile Ala Asp Asn

65 70 75 80

Val His Gly Glu Ser Gly Leu Asp Gly Pro Ala Leu Pro Glu Pro Thr

85 90 95

Phe Ala Pro Gln Asn Cys Thr Ala Val Glu Leu Met Ala Lys Thr Leu

100 105 110

Arg Glu Ser Ala Glu Pro Val Thr Ile Val Ser Thr Gly Pro Gln Thr

115 120 125

Asn Val Ala Leu Leu Leu Asn Ser His Pro Glu Leu His Ser Lys Ile

130 135 140

Ala Arg Ile Val Ile Met Gly Gly Ala Met Gly Leu Gly Asn Trp Thr

145 150 155 160

Pro Ala Ala Glu Phe Asn Ile Tyr Val Asp Pro Glu Ala Ala Glu Ile

165 170 175

Val Phe Gln Ser Gly Ile Pro Val Val Met Ala Gly Leu Asp Val Thr

180 185 190

His Lys Ala Gln Ile His Val Glu Asp Thr Glu Arg Phe Arg Ala Ile

195 200 205

Gly Asn Pro Val Ser Thr Ile Val Ala Glu Leu Leu Asp Phe Phe Leu

210 215 220

Glu Tyr His Lys Asp Glu Lys Trp Gly Phe Val Gly Ala Pro Leu His

225 230 235 240

Asp Pro Cys Thr Ile Ala Trp Leu Leu Lys Pro Glu Leu Phe Thr Ser

245 250 255

Val Glu Arg Trp Val Gly Val Glu Thr Gln Gly Lys Tyr Thr Gln Gly

260 265 270

Met Thr Val Val Asp Tyr Tyr Tyr Leu Thr Gly Asn Lys Pro Asn Ala

275 280 285

Thr Val Met Val Asp Val Asp Arg Gln Gly Phe Val Asp Leu Leu Ala

290 295 300

Asp Arg Leu Lys Phe Tyr Ala

305 310

Claims (3)

1. A recombinant microorganism producing cytosine, characterized by: the recombinant microorganism takes CGMCC No.13427 as a female parent and has the following characteristics:

1) The codA gene of the encoding cytosine deaminase and the codB gene of the cytosine transporter in the recombinant microorganism are knocked out simultaneously;

2) The recombinant microorganism overexpresses a nucleoside hydrolase RihA gene, and the amino acid sequence encoded by the RihA gene is shown as SEQ ID No. 24.

2. A recombinant microorganism producing cytosine, characterized by: the recombinant microorganism takes CGMCC No.13427 as a female parent and has the following characteristics:

1) The codA gene of the encoding cytosine deaminase and the codB gene of the cytosine transporter in the recombinant microorganism are knocked out simultaneously;

2) The nucleotide phosphatase PpnN gene is overexpressed in the recombinant microorganism, and the coded amino acid sequence of the PpnN gene is shown as SEQ ID No. 22;

3) The cytidine monophosphate nucleotidase PHM8 gene in the recombinant microorganism is knocked out.

3. A method for producing cytosine using a recombinant microorganism, comprising: the method is to use

The recombinant microorganism of any one of claims 1-2 fermented to produce cytosine.