High Coenzyme Q production 10 Establishment and application of Rhodopseudomonas aeruginosa strains

Abstract

This invention relates to a high-yield coenzyme Q method. 10 The establishment and application of *Rhodotorula globulinii* strains. Compared to rational metabolic engineering, the coenzyme Q of this invention... 10 High-yielding Rhodopseudomonas aeruginosa bacteria were used as a template by the global regulatory factor prrA gene. Through directed evolution, different mutant strains were constructed, and combined with high-throughput screening methods, coenzyme Q was achieved. 10 Increased yield. This invention starts from global regulatory factors and leverages complex intracellular regulatory mechanisms to achieve fine-grained global regulation under minute genomic perturbations, providing a new approach for the engineering modification of strains.

Description

Technical Field

[0001] This invention relates to the field of metabolic engineering; specifically, to high-yield coenzyme Q production. 10 The invention relates to the establishment and application of Rhodopseudomonas aeruginosa strains; more specifically, it relates to a method for developing strains that enhance Coenzyme Q based on directed evolution and high-throughput screening using the global regulatory factor prrA. 10 Methods for increasing production. Background Technology

[0002] Coenzyme Q 10 It is a lipid-soluble quinone compound widely distributed on cell membranes, participating in the respiratory chain, electron transport, and energy metabolism, and acting as an activator of cell metabolism. Reduced Coenzyme Q10 10 It can effectively scavenge free radicals, prevent cardiovascular diseases, and assist in the treatment of side effects caused by statins, and is widely used in functional foods, cosmetics, and pharmaceuticals. Therefore, with the increasing research and development of Coenzyme Q10... 10 The increasing demand further underscores the importance of increasing production.

[0003] Rhodopsycetes are coenzyme Q. 10 The main producing bacteria, and also the model strain for studying bacterial photosynthesis; therefore, it possesses multiple metabolic modes within its cells, capable of obtaining substances and energy through aerobic respiration while also maintaining survival under anaerobic conditions. It is this complex metabolic regulation that is crucial for improving coenzyme modification in bacterial strain engineering. 10 Production volume presents numerous challenges.

[0004] Generally, strain engineering strategies can be divided into two categories: one is the "irrational" method of inducing non-specific gene mutations through treatment with ultraviolet light or chemical mutagens; the other is the "rational" method of regulating known biosynthetic pathways using metabolic engineering. Compared to the time-consuming and labor-intensive nature of "irrational" strategies, rational metabolic engineering can effectively improve microbial production performance and is currently a commonly used method in research. Early rational modification of Rhodopseudomonas aeruginosa mainly focused on coenzyme Q. 10 The biosynthetic pathway of coenzyme Q is achieved through strategies such as the addition of precursor substances, overexpression of rate-limiting enzymes in the synthesis pathway, inhibition or knockout of competitive branching pathways, and cofactor supply and energy rebalancing. 10 High yield.

[0005] Unfortunately, the aforementioned modification strategies, as well as some other existing strategies in the field, are not stable and their effects are not significant in some Rhodotorula globosum bacteria (e.g., HY01). Summary of the Invention

[0006] The purpose of this invention is to provide high-yield coenzyme Q. 10 Methods for constructing Rhodotorula globulinii strains and their applications.

[0007] In a first aspect of the invention, there is a method for establishing or screening high-yield coenzyme Q. 10 The method for treating Rhodotorula globulus includes: (1) targeting and modifying the global regulatory factor prrA in Rhodotorula globulus; (2) culturing the Rhodotorula globulus of (1) and isolating bacteria with significantly high chlorophyll content (higher than the control without prrA modification) to obtain high-yield coenzyme Q. 10 Rhodopseudomonas aeruginosa.

[0008] In one or more preferred embodiments, in (1), the modification includes: randomly mutating the global regulatory factor prrA.

[0009] In one or more preferred embodiments, the mutation is introduced into Rhodopseudomonas aeruginosa via site-specific recombination;

[0010] In one or more preferred embodiments, the random mutation of the global regulatory factor prrA is high-throughput, forming a random mutation library;

[0011] In one or more preferred embodiments, the Rhodotorula globulus is Rhodotorula globulus HY01.

[0012] In one or more preferred embodiments, a mutated sequence resulting from a random mutation of the global regulatory factor prrA is introduced into the genome of Rhodotorula globulus to replace the original prrA gene; preferably, the replacement is achieved by introducing an attB site at the original prrA gene position in the Rhodotorula globulus genome; more preferably, the attB site is introduced using a suicide plasmid (such as pK18mobsacB).

[0013] In one or more preferred embodiments, a prrA mutant sequence (library) is inserted into the attB site of the Rhodotorula globulus genome using a plasmid containing the integrase Int and the conjugation transfer site oriT (e.g., using pUC57 as the plasmid backbone); more preferably, the plasmid is subsequently cleaved by the Cre enzyme.

[0014] In one or more preferred embodiments, the construct or plasmid containing the mutant sequence has an attP site, which, after conjugation transfer, works in conjunction with attB to enable the site-specific introduction of the mutant sequence (as a foreign gene).

[0015] In one or more preferred embodiments, in (2), bacteria with significantly high chlorophyll content are isolated by means selected from the group consisting of: (a) observing the color of Rhodococcus pluvialis or its colonies; (b) determining the chlorophyll content of Rhodococcus pluvialis; and (c) determining the OD value of Rhodococcus pluvialis cultures; preferably, the OD value is 700 nm.

[0016] In one or more preferred embodiments, the method further includes setting up a control group, which is a Rhodopseudomonas spp. that has not been modified with the global regulatory factor prrA in step (1).

[0017] In one or more preferred embodiments, in (a), if the colony color of the Rhodopseudomonas cocci modified with the global regulatory factor prrA is green, then it is (or potentially is) a high-production Coenzyme Q. 10 (a) Rhodopseudomonas cocci; preferably, the darker the green color, the higher the yield; more preferably, the green is dark green; (b) if the chlorophyll content of Rhodopseudomonas cocci modified with the global regulator prrA is statistically higher than that of the control, then it is (or potentially is) a high-yielding coenzyme Q producer. 10 (c) Rhodopseudomonas cocci; or, if the OD value of a culture of Rhodopseudomonas cocci modified with the global regulator prrA is statistically higher than that of the control, then it is (or potentially is) a high-producing coenzyme Q. 10 Rhodopseudomonas aeruginosa.

[0018] In one or more preferred embodiments, after obtaining the Rhodotorula globulus (1), (a) is first performed to isolate and obtain Rhodotorula globulus with a green color; then, for the Rhodotorula globulus obtained by screening, (b) and / or (c) are performed.

[0019] In one or more preferred embodiments, the strain is spread on a plate to observe the color of Rhodotorula globosum or its colonies.

[0020] In one or more embodiments, Rhodotorula globulus bacteria obtained by color observation screening are cultured in deep-well plates.

[0021] In another aspect of the invention, an application of any of the methods described above is provided for establishing or screening high-yield coenzyme Q production. 10 Rhodopseudomonas aeruginosa.

[0022] In one or more preferred embodiments, the Rhodotorula globulus is Rhodotorula globulus HY01.

[0023] In another aspect of the present invention, a method for producing coenzyme Q by bio-fermentation is provided. 10 The method comprises: (a) establishing a high-yield coenzyme Q production system using any of the preceding methods. 10 (a) Rhodophyta globosum; (b) Fermenting the Rhodophyta globosum from (a) to produce coenzyme Q. 10 .

[0024] In another aspect of the invention, a method for establishing or screening high-yield coenzyme Q is provided. 10The kit for Rhodotorula globulus includes: a tool for targeting and modifying the global regulatory factor prrA in Rhodotorula globulus, a chlorophyll analysis tool; preferably, it also includes a tool for constructing mutant libraries.

[0025] In one or more preferred embodiments, the tool for targeting and modifying the global regulatory factor prrA in Rhodotorula spp. includes (but is not limited to): materials for randomly mutating the global regulatory factor prrA, and materials for introducing the random mutation into the Rhodotorula spp. genome; more preferably including: (i) materials for introducing the attB site at the original prrA gene position in the Rhodotorula spp. genome, such as the suicide plasmid pK18mobsacB, or (ii) materials for inserting the prrA mutant sequence (library) into the attB site in the Rhodotorula spp. genome, such as the pUC57 plasmid containing the integrase Int and the conjugation transfer site oriT.

[0026] In one or more preferred embodiments, the chlorophyll analysis tool includes (but is not limited to): a culture plate (capable of culturing bacterial strains and observing cell color), a spectrophotometer, and an enzyme-linked immunosorbent assay (ELISA) reader.

[0027] In another aspect of the present invention, a method for preparing high-yield coenzyme Q is provided. 10 A method for producing Rhodopseudomonas cocci, the method comprising: using Rhodopseudomonas cocci as a starting strain, mutating the global regulatory factor prrA in its genome by mutating position 24 to serine (Ser); preferably, the high-yield coenzyme Q... 10 Compared with the original strain, the yield of the Rhodotorula globulus increased by more than 30%.

[0028] In another aspect of the present invention, a high-yield coenzyme Q is provided. 10 The *Rhodopseudomonas cocci* has a serine (Ser) residue at position 24 of the global regulatory factor *prrA* in its genome; preferably, the high-yield coenzyme Q... 10 Compared with the original strain, the yield of the Rhodotorula globulus increased by more than 30%.

[0029] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0030] Figure 1 Knockout of genes involved in the regulation of photosynthetic gene cluster expression affects coenzyme Q. 10 Impact on production.

[0031] Figure 2 A schematic diagram of mutant library vector design and integrase-mediated recombination.

[0032] Figure 3Plasmid design for HY01ΔprrA strain and validation of ΔprrA strain.

[0033] Figure 4 A schematic diagram of the design, construction, and screening process for mutation libraries.

[0034] Figure 5 Chlorophyll content and coenzyme Q 10 Linear correlation between outputs.

[0035] Figure 6 These are the results of fermentation testing using deep-well plates.

[0036] Figure 7 High-throughput screening for prrA mutant strains. Detailed Implementation

[0037] After in-depth research and analysis, the inventors have revealed a new method for enhancing the coenzyme Q of Rhodopseudomonas aeruginosa. 10 The target for coenzyme Q production regulation. Around this novel target, combined with appropriate metabolic engineering strategies and high-throughput screening methods, coenzyme Q was developed through directed evolution of the global regulator prrA combined with high-throughput screening technology. 10 Obtaining high-yield strains.

[0038] the term

[0039] As used in this invention, the term "site-specific recombination" refers to a type of homologous recombination that relies on the pairing of small homologous sequences and requires the participation of integrase (such as phiC31) and specific recombination sites (such as attB / attP) to ultimately integrate the foreign plasmid into the genome.

[0040] As used in this invention, the terms "introduction" or "transformation" refer to the transfer of exogenous polynucleotides into host cells (in this invention, Rhodopseudomonas aeruginosa). Optionally, the exogenous polynucleotides may be integrated into the host genome.

[0041] As used in this invention, "exogenous" or "heterogeneous" genes or proteins refer to genes or proteins that are not naturally contained in the genome of the native organism (Rhodophyta globosum in this invention), and may be, for example, mutant proteins. Typically, "exogenous" genes or proteins are introduced into the organism through genetic engineering recombination technology.

[0042] High-yield strain screening methods

[0043] Regulating the transcriptional levels of key genes in metabolic pathways is a common strategy in metabolic engineering. However, this strategy often fails to significantly increase the yield of target products or has only a minor effect, and may even lead to imbalances in the intracellular carbon / nitrogen metabolic network and cofactor network of engineered strains. Global transcriptional regulation engineering uses transcription factors with specific functions to activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways. Appropriate action of such factors can potentially enhance the synthesis of target metabolites. Global transcriptional regulation engineering modifies transcription factors to reprogram genes and metabolic networks related to metabolic pathways, regulating gene transcription levels across the entire genome. However, given the numerous intracellular metabolic pathways and the extremely complex metabolic networks, identifying global regulatory factors that precisely regulate a series of specific metabolic processes without affecting / threatening the normal functioning of other undesirable pathways remains a bottleneck in this field.

[0044] Through transcriptome data analysis, the inventors discovered that *Rhodophyta globulinii* produces high levels of coenzyme Q. 10 A connection exists between the expression of photosynthetic gene clusters and other factors. Further analysis suggests that components involved in the regulation of photosynthetic gene clusters, including repressor / derepressor proteins PpsR / appA, the two-component regulatory system PrrB / PrrA, and global regulatory proteins FnrL, CrpK, and MppG, may be potentially related. These regulatory elements work together to influence the expression of photosynthetic gene clusters by sensing external oxygen concentration, light intensity, and intracellular redox status. Besides directly participating in the expression of photosynthesis-related operons, the two-component regulatory system PrrB / PrrA also regulates tetrapyrrole compound synthesis, intracellular iron homeostasis, and energy metabolism-related genes. As a global regulator, prrA can function as both an activator and repressor, participating in the expression regulation of hundreds of intracellular genes. The signaling pathways regulating the expression of photosynthetic gene clusters have been studied most extensively. When oxygen concentration is low, the redox signal intensity flowing through cbb3 oxidase weakens. The membrane protein PrrB, acting as a kinase, senses this change in the cbb3 redox signal. Its histidine residues undergo autophosphorylation, transferring the phosphate group to the regulatory protein PrrA. Phosphorylated PrrA binds tightly to DNA, participating in the transcriptional activation of photosynthetic genes. Conversely, under high oxygen partial pressure, the redox signal intensity flowing through cbb3 oxidase is strong. At this time, cbb3 oxidase generates a strong inhibitory signal, which is transmitted to the PrrB / PrrA two-component system, inhibiting photosynthetic gene transcription. However, in Rhodopseudomonas aeruginosa, current methods for improving coenzyme Q through transcription factor engineering are not yet effective. 10 Production studies have not been reported before. This invention, for the first time, through in-depth omics data analysis combined with experimental analysis, identifies PrrA as a participant in coenzyme Q. 10 Regulated transcription factors, combined with transcription factor engineering to promote coenzyme Q 10 Increased production.

[0045] Transcription factor engineering typically utilizes directed evolution to introduce a large number of mutations, applying selection pressure according to specific needs and purposes to screen for proteins with desired characteristics. Considering the cumbersome and complex process of mutant bacterial breeding, there is an urgent need to develop a simple, rapid, and high-throughput screening technology. Previous research by the inventors revealed that industrial Rhodotorula globulus bacteria spontaneously mutate during fermentation to produce a white mutant strain. Spectroscopic scanning showed that this mutant strain does not produce photosynthetic pigments, its photosynthesis-related genes are silenced, and it only produces a small amount of coenzyme Q. 10 Accumulation; while high-yield coenzyme Q 10 Industrial bacteria (such as HY01) exhibit a high-yield green phenotype, a result of activated photosynthetic genes triggering the expression of photosynthetic pigments such as chlorophyll. This suggests a connection between the photosynthetic system and coenzyme Q. 10 There may be some unknown connection in the accumulation; thus, the levels of coenzyme Q in different fermentation samples may be affected. 10 Yield and chlorophyll content were measured, and analysis revealed coenzyme Q. 10 There was a positive correlation with chlorophyll content, with a linear correlation coefficient as high as 0.97. Based on this, this invention attempts to establish a high-throughput detection system for screening engineered strains, in order to accelerate the acquisition of high-yield strains and improve coenzyme Q. 10 This will increase production capacity and reduce production costs for businesses.

[0046] Based on the aforementioned new discoveries of the inventors, this invention discloses for the first time the interaction between prrA and coenzyme Q. 10 Synthesizing related components allows for targeted modification of prrA in Rhodophyta globulins, for example, by constructing a prrA mutant library from which coenzyme Q can be obtained through screening. 10 High-yielding strains.

[0047] There are various ways to target and modify prrA in Rhodotorula glomerulonephrica, as long as it can alter prrA's expression, activity, modification, or interaction with other factors. These alterations would affect prrA's role in interactions with coenzyme Q. 10 The production-related pathways may also change, and coenzyme Q can be screened based on this. 10 The target strain (a type of Rhodopsycete with excellent traits) whose yield can be increased.

[0048] In a preferred embodiment of the present invention, the targeted modification is based on site-specific recombination, involving random mutation of the global regulatory factor prrA. The random mutation is preferably performed in a high-throughput manner to improve screening efficiency. Preferably, this modification is implemented by preparing a random mutation library and introducing the random mutation into the original prrA position in the Rhodopseudomonas spp. genome (replacing the original prrA gene).

[0049] In a preferred embodiment of the present invention, the substitution is achieved by introducing the attB site at the original prrA gene position in the genome of *Rhodotorula spp.*; more preferably, the attB site is introduced using a suicide plasmid (such as pK18mobsacB). Correspondingly, the construct or plasmid containing the mutant sequence (as a foreign gene) has an attP site, which, after conjugation transfer, works in conjunction with attB to achieve site-specific introduction of the mutant sequence.

[0050] As a preferred embodiment of the present invention, a plasmid containing the integrase Int and the conjugation transfer site oriT (such as pUC57 as the plasmid backbone) is used to insert the prrA mutant sequence (librium) into the attB site of the Rhodotorula globosum genome; preferably, the plasmid is subsequently cleaved by Cre enzyme.

[0051] As a specific embodiment of the present invention, in order to achieve the above objectives, the present invention adopts the following technical solution:

[0052] Step 1: Prepare the mutant sequence of the global regulatory factor prrA;

[0053] Step 2: Design and construct a plasmid library containing the global regulatory factor prrA mutant sequence for genome integration, and transform it into E. coli (such as, but not limited to, S17-1);

[0054] Step 3: The mutant library constructed in Step 2 is conjugated and transferred into the recipient bacterium Rhodotorula glutinis for integration, resulting in strains containing different prrA mutation sites;

[0055] Step 4: The monoclonal strains containing different prrA mutations obtained in Step 3 were fermented in deep well plates, and high-yielding strains were screened by measuring the intracellular chlorophyll content.

[0056] In a preferred embodiment, the preparation of the prrA mutant sequence mainly utilizes an error-prone PCR kit, using the genome of Rhodotorula glutinis as a template, and randomly introducing the mutant sequence during the amplification process.

[0057] In a preferred embodiment, the mutant library construction involves cloning the sequence prepared in the first step into a plasmid vector using a recombinase for subsequent conjugation and transfer.

[0058] In a preferred embodiment, conjugation transfer involves introducing the plasmid constructed in the second step into Rhodotorula glomerulosa with the assistance of Escherichia coli, and then using the integrative elements in the plasmid to construct the Rhodotorula glomerulosa genome.

[0059] In a preferred embodiment, deep-well plate fermentation refers to inoculating the conjugated monoclonal cells into a fermentation medium and culturing them in a 48-well deep-well plate at 32°C and 220 rpm for 36 h.

[0060] It should be understood that the fermentation or culture method of the strains with the prrA mutation site of the present invention is not limited to deep-well plate fermentation. However, deep-well plates are preferred when used for high-throughput screening, and the deep-well plates can be 24-well, 48-well, or 96-well deep-well plates, etc., suitable for batch detection by spectrophotometers / ELISA readers. The deep-well plates can have a square bottom or a round bottom, and preferably their bottoms are permeable.

[0061] In a preferred embodiment of the present invention, in order to observe the color of the strain (e.g., to observe whether it is a green strain), the modified strain is plated and observed after a certain period of growth.

[0062] Furthermore, HPLC can be used to test the product yield / potency of high-yielding strains, thereby identifying high-yielding strains. This achieves a complete high-throughput screening platform from strain culture to product detection. However, when using HPLC for potency detection, samples must be tested one by one, which is time-consuming and does not achieve the purpose of high-throughput detection; it is suitable for later screening stages. For example, after plate screening and enzyme-linked immunosorbent assay (ELISA) screening, HPLC can be used for further verification. HPLC is an optional procedure, but in the overall solution of this invention, only plate screening or ELISA screening is required to identify high-yielding Coenzyme Q. 10 Rhodopseudomonas aeruginosa.

[0063] Understandably, once a target strain of interest is observed, one or more secondary screenings can be conducted.

[0064] Optionally, further verification can be performed through genetic stability experiments. Preferably, high-yielding mutant strains selected through screening can be passaged for one or more generations, and each generation can be subjected to shake-flask fermentation. The fermentation broth can be collected, and the product yield can be measured to determine the genetic stability characteristics.

[0065] High Coenzyme Q production 10 Rhodopsyllosis

[0066] This invention also provides a series of high-yield coenzyme Q methods obtained based on the methods of this invention. 10 The method of this invention can efficiently obtain a class of high-yield coenzyme Q bacteria. 10 These are Rhodopseudomonas spp. strains that can form a cell library from which the most suitable strains for experimental and industrial production of Coenzyme Q can be identified. 10 Modified Rhodopseudomonas spp.

[0067] As a preferred embodiment of the present invention, a specific high-yield coenzyme Q is provided. 10 The Rhodopseudomonas cocci has a serine (Ser) residue at position 24 of the global regulatory factor prrA in its genome; the high-yield Coenzyme Q... 10Compared with the original strain, the yield of the Rhodotorula globulus increased by more than 30%.

[0068] The high-yielding strains obtained through the modifications of this invention can be further improved to obtain derivative strains with even higher target product yields or more optimized enzyme systems, or to express other recombinant proteins. The obtained engineered strains can also be used to establish marker-free gene manipulation techniques.

[0069] Furthermore, the strains of the present invention can serve as the basis for further optimization of derived strains or engineered strains of other recombinant proteins. After constructing a label-free screening system, they can be used as objects for molecular manipulation, such as gene knock-in and knock-out, to carry out targeted genetic modification.

[0070] The modified strain of this invention is a live cell. Once the strain is obtained, it can be mass-produced using methods such as inoculation, passage, and regeneration. This is typically achieved by inoculating the strain into solid agar plates or liquid culture media for expansion culture to obtain the live cells of this invention. The obtained live cells can then be further subjected to laboratory domestication, genetic breeding, and molecular genetic manipulation to obtain mutants and transformants. This invention can also be used as a host cell for heterologous expression.

[0071] Methods known to those skilled in the art can be used to further modify / mutate the living cells of the present invention, resulting in changes in the gene coding, enzyme activity characteristics, and morphology of the living cells.

[0072] The culture media and culture methods used to cultivate the strains of the present invention are not limited to those disclosed in the embodiments of the present invention. Other culture media and culture methods conventionally used to cultivate Rhodophyta globulinii can also be used in the present invention.

[0073] As described above, the fermentation system can be scaled up for industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on their general knowledge to facilitate the growth or production of the strain.

[0074] The main beneficial technical effects of this invention include:

[0075] A novel method for enhancing coenzyme Q in Rhodopseudomonas aeruginosa is proposed for the first time. 10 Target for coenzyme Q production regulation. Focusing on this novel target, and employing appropriate metabolic engineering strategies and high-throughput screening methods, through directed evolution of the global regulator prrA combined with high-throughput screening technology, it is possible to develop a method to achieve coenzyme Q production. 10 Obtaining high-yield strains.

[0076] Compared to the rational design of metabolic engineering, transcription factor engineering fully utilizes the characteristics of prokaryotic regulatory networks. Through minute gene perturbations, it achieves precise regulation of intracellular metabolism at the global level, thereby coordinating different physiological metabolic activities within the cell and increasing the yield of target products. It is more advantageous for strains where metabolic modification is relatively difficult.

[0077] Traditional screening processes are somewhat unpredictable, prone to missing samples, resulting in low efficiency and wasted time, effort, and materials. The method described in this invention is precise, efficient, simple, and high-throughput. This invention can increase the number of samples to be screened at low cost, reduce the workload of screening, and improve screening efficiency.

[0078] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions agreed upon by the manufacturer.

[0079] Culture medium formulation

[0080] Seed culture medium:

[0081] 0.8% yeast extract, 0.3% glucose, 0.2% NaCl, 0.13% KH2PO4, 0.0125% MgSO4, 1.5% agarose. In addition, 15 mg / L biotin, 1 mg / L nicotinic acid, and 1 mg / L thiamine need to be added. Sterilize at 121°C for 20 min.

[0082] Fermentation medium:

[0083] 4% glucose, 0.4% corn steep liquor powder, 0.3% monosodium glutamate, 0.3% (NH4)2SO4, 0.28% NaCl, 0.3% KH2PO4, 0.63% MgSO4, 0.2% CaCO3; additionally add 1 mg / L thiamine, 1 mg / L nicotinic acid, and 15 μg / L biotin, sterilize at 121℃ for 20 min.

[0084] TSB medium:

[0085] Tryptone soy beef broth 30g / L. After preparation, sterilize at 115℃ for 20min.

[0086] In the culture medium of this invention, the final concentration of kanamycin is 50 μg / mL, and the final concentration of nalidixic acid is 7.5 μg / mL.

[0087] Example 1: Screening of Global Regulatory Factors

[0088] Coenzyme Q10 Coenzyme Q is a lipid-soluble quinone compound widely distributed on cell membranes. As an important component of the electron transport chain in the respiratory chain, coenzyme Q... 10 It effectively promotes energy supply, acts as an activator of cell metabolism, and plays an important role in areas such as the scavenging of intracellular free radicals and the prevention of cardiovascular diseases. (About Coenzyme Q) 10 The synthetic pathways for it are relatively well understood, mainly including the branched acid pathway, the mevalonic acid pathway, and the quinone ring modification pathway.

[0089] The cladonic acid pathway uses erythrose-4-phosphate and phosphoenolpyruvate as substrates. Through a series of enzymatic catalysis, cladonic acid is first generated, and then coenzyme Q is synthesized under the action of cladonic acid lyase UbiC. 10 The precursor p-hydroxybenzoic acid of the parent nucleus is the catalytic step, which is generally considered the rate-limiting step in the entire pathway. Simultaneously, the branched acid also participates in the synthesis of various aromatic compounds such as aromatic amino acids and folic acid. The mevalonate pathway uses glyceraldehyde triphosphate and pyruvate as precursors to synthesize the precursors dimethylpropenyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). IPP and DMAPP are extended step-by-step under the catalysis of farnesyl pyrophosphate synthase IspA and decanisoprene pyrophosphate synthase (DPS), ultimately forming a decapolyisoprene side chain. Since IPP and DMAPP are substrates for the synthesis of terpenes such as carotenoids and chlorophyll, these terpenes and their derivatives, as well as coenzyme Q, are also involved in the synthesis of these compounds. 10 The synthesis of this precursor leads to competition for the precursor. Finally, p-hydroxybenzoic acid and decapolyisoprene pyrophosphate are condensed by p-hydroxybenzoic acid isopentenyltransferase (UbiA) in the quinone ring modification pathway, followed by a series of hydroxylases and methyltransferases to yield the final product, coenzyme Q. 10 Among them, p-hydroxybenzoic acid isopentenyltransferase UbiA, methyltransferase UbiE, and UbiG are key enzymes in the quinone ring modification pathway. Furthermore, coenzyme Q10 synthesis requires the participation of multiple cofactors such as NAHDH, NADH, ATP, and SAM. Unfortunately, these potential metabolic modification sites are not significantly effective in some Rhodophyta coccidioidae (e.g., HY01).

[0090] Previous research by the inventors revealed that industrial Rhodotorula glutinis (e.g., HY01) spontaneously mutate during fermentation to produce a white mutant strain. Spectroscopic scanning showed that this mutant strain does not produce photosynthetic pigments, its photosynthesis-related genes are silenced, and it produces only a small amount of coenzyme Q. 10 Accumulation; while high-yield coenzyme Q 10 The high-yielding industrial bacterial mutant exhibits a green phenotype, a color phenotype resulting from the activation of photosynthetic genes, triggering the expression of photosynthetic pigments such as chlorophyll. Therefore, the inventors infer that the photosynthetic system is related to coenzyme Q. 10There may be some unknown connection between the accumulation and the process. Rhodopseudomonas colonies are generally red or light red due to the high content of photosynthetic pigments, but some mutant strains with other phenotypes also exist. These photosynthetic pigments gradually accumulate with cell growth, possessing good antioxidant capacity and protecting cells from oxidative damage. It has been reported that restricting oxygen supply during fermentation is beneficial for Rhodopseudomonas to accumulate coenzyme Q. 10 Under hypoxic or anaerobic conditions, *Rhodophyta globosum* expresses photosynthetic genes, producing photosynthetic pigments and promoting the assembly of the photosynthetic system. The inventors believe this indicates that the photosynthetic system is related to coenzyme Q. 10 There is a potentially close connection between accumulation; and the expression of photosynthetic gene clusters is also closely related to the respiratory chain and electron transport chain.

[0091] Therefore, the inventors first investigated regulatory factors involved in photosynthetic gene cluster expression and electron transport chain. Combined with transcriptome data analysis, a series of potential targets were screened, including the prrB / prrA two-component system, ppsR / appA repressor-antiruption elements, global regulatory proteins FnrL, CrpK, and MppG. By knocking out these regulatory components, it was found that prrA and coenzyme Q... 10 Synthesis is most relevant, such as Figure 1 .

[0092] Example 2: Construction of prrA mutant library

[0093] The construction of the global regulatory factor prrA mutant library consists of two parts: random mutation of the global regulatory factor DNA sequence and design and construction of the cloning vector pUC-Int-oriT.

[0094] (1) Random mutations in the DNA sequence of global regulatory factors

[0095] This invention utilizes an error-prone PCR kit (purchased from Takara) to amplify the prrA sequence using genomic DNA of Rhodotorula hy01 as a template and PRRA-ER-F / R primers. Mutant sequences are randomly introduced during the amplification process. The target fragment is 596 bp in size, and the amplified fragment is purified and recovered.

[0096] Table 1. Commonly Misused PCR Primers

[0097]

[0098] (2) Design and construction of cloning vector pUC-Int-oriT

[0099] The high-yield strain constructed in this invention is used for coenzyme Q. 10To avoid antibiotic contamination during production, this invention uses pUC57 as the plasmid backbone, incorporating the integrase Int and the conjugation transfer site oriT. The prrA-mutated library is inserted into the genome of *Rhodotorula glutinis* using the integration system. The plasmid backbone can be subsequently cleaved by the Cre enzyme, providing a basis for later coenzyme Q. 10 This method facilitates production and avoids yield fluctuations caused by unstable copy numbers of free plasmids. A schematic diagram of its construction is shown below. Figure 2 The linear plasmid backbone can be obtained by amplification using primers pUC-Int-oriT-F / R.

[0100] (3) Statistics on mutation sites and base mutations

[0101] The obtained mutant sequences and linear plasmid vectors were ligated at 50°C for 15 min according to the steps provided in the one-step cloning and recombination kit. The ligation products were transformed into E. coli S17-1 and plated on LB plates supplemented with 50 μg / mL kanamycin for screening. A number of single clones were selected for sequencing, and the mutation sites and the number of base mutations were counted.

[0102] All single clones on the plate were scraped into 1 mL of LB medium, and an equal volume of 30% glycerol was added. The medium was then frozen at -80°C. The one-step cloning and recombination kit was purchased from Nearshore Protein Technology Co., Ltd.

[0103] (4) Construction of Rhodopseudomonas HY01-ΔprrA::attB

[0104] The left and right homologous arms of the prrA gene were amplified using genomic DNA from Rhodopseudomonas HY01 as a template. The primers for amplification of the left and right homologous arms of the prrA gene are shown in Table 2. The pK18mobsacB suicide plasmid (purchased from Addgene) was double-digested with EcoRI / HindIII, and the linearized vector fragment was recovered.

[0105] Using the Bacillus knockout plasmid pK18mobsacB as the backbone plasmid, the construction of the recombinant plasmid pK18mobsacB-del prrA was similar to step (3). The constructed knockout plasmid was transformed into Rhodotorula hy01, and the prrA gene was knocked out using the strain's own recombination system. At the same time, the attB site was introduced, with the sequence: gtgccagggcgtgcccttgggctccccgggcgcg. Its construction diagram and identification of the recombinant plasmid are shown below. Figure 3 .

[0106] Table 2

[0107]

[0108] Example 3: Transformation and Cloning Screening of prrA Mutant Library

[0109] In this embodiment, the transformation of the prrA mutant library and clone screening include the following steps:

[0110] (1) Take the mutant library frozen at -80℃ and transfer it to 5 mL of kanamycin-resistant LB medium at a concentration of 5%, and culture until OD. 700 The OD value was between 0.4 and 0.5. Rhodopseudomonas HY01 was cultured on seed plates. Single colonies of Rhodopseudomonas HY01 grown to 6-7 days were picked and inoculated into TSB-free medium. The culture was carried out at 30°C and 200 rpm until the OD value was between 0.8 and 1.0.

[0111] (2) Take 2 mL of the above-mentioned Escherichia coli S17-1 culture medium (donor bacteria) containing the above-mentioned mutant library and 1 mL of Rhodopseudomonas hy01 culture medium (recipient bacteria), centrifuge at 6000 rpm for 3 min, and discard the supernatant; resuspend the bacterial cells in 1 mL of TSB medium, centrifuge at 6000 rpm for 3 min, and discard the supernatant; add another 1 mL of TSB medium to resuspend the bacterial cells; take the resuspended bacterial solution at a donor / recipient bacterial volume ratio of 1:3 and mix well, keeping the total volume at 400 μL, and drop the mixed bacterial solution onto an antibiotic-free seed plate; incubate at 32℃ upside down for 20-24 h to allow conjugation transfer, so that the recombinant plasmid is introduced into the host bacteria, and then the mutant sequence is integrated into the genome through integrase-mediated recombination; after the conjugation transfer is completed, scrape off the bacterial growth and resuspend it in 1 mL of TSB medium; dilute the obtained bacterial solution and spread it on a seed solid medium containing kanamycin and naphthylpyridinium acid antibiotics; incubate the plate at 32℃ for 5-7 days until conjugates grow.

[0112] After conjugates grew, the monoclonal antibodies on the plate exhibited two different phenotypes: white and green monoclonal antibodies. Based on the coenzyme Q analyzed by the inventors... 10 The link between high yield and photosynthetic gene cluster expression was established, and white monoclonal clones were directly excluded, with only green mutants selected for deep-well plate fermentation. A schematic diagram of the analysis and screening process is shown below. Figure 4 .

[0113] Example 4, Coenzyme Q 10 Correlation study between yield and chlorophyll

[0114] Based on analysis, research, and experimental verification, the inventors have determined that the green phenotype mutant strain of Rhodopseudomonas aeruginosa and coenzyme Q... 10 High yield is correlated. To further demonstrate this correlation, the inventors analyzed the levels of coenzyme Q in different fermentation samples. 10 Yield and chlorophyll content were tested.

[0115] The method for chlorophyll determination is as follows:

[0116] Add 300 μL of bacterial culture to a 48-well plate containing 1 mL of water, centrifuge, and discard the supernatant. Add three times the volume of extraction buffer (acetone:methanol = 7:2, v / v), mix well, and centrifuge again. After centrifugation, use a multipipe to transfer 200 μL of the supernatant to a 96-well microplate and measure the absorbance at 770 nm. Calculate the intracellular bacterial chlorophyll concentration using the following formula:

[0117]

[0118] Bacterial chlorophyll has a maximum absorption peak at 770 nm, and its absorbance at 770 nm is linearly correlated with its concentration. According to Beer-Lambert law, OD... 770 = Extinction coefficient * optical path length * chlorophyll concentration; the bacterial chlorophyll extinction coefficient is 76 mM. -1 cm -1 If the optical path length of the cuvette is 1 cm, the molar concentration of bacterial chlorophyll can be calculated based on OD770.

[0119] Analysis revealed that when chlorophyll content is high, coenzyme Q... 10 The output subsequently showed a relatively high level; further fitting of the correlation revealed a linear correlation coefficient as high as 0.97, such as... Figure 5 As shown.

[0120] This result provides a basis for establishing a high-throughput detection method, namely, indirectly characterizing coenzyme Q by batch determination of chlorophyll content in bacterial strains. 10 Yield.

[0121] Example 5: Deep-well plate fermentation and chlorophyll content detection of prrA mutant strain

[0122] Select zygotes from the mutant library of Rhodotorula globosum that have grown for 6-7 days from the seed plate. Quickly shake the pipette tip in the well until the single clone completely detaches from the tip. The single clone does not need to be broken up. Choose the dark green single clone first. Inoculate it into a 48-well deep plate containing 1200 μL of tertiary fermentation broth. Ferment at 32°C and 200 rpm for 48 hours. At this time, the carbon source in the culture medium will be almost completely consumed.

[0123] When dispensing the tertiary fermentation broth, it is recommended to use a syringe to draw up the broth, as it contains insoluble substances such as calcium carbonate that are prone to precipitation, and to shake it occasionally to mix it well. Deep well plates should be handled with care to avoid cross-contamination.

[0124] 1. Fermentation broth OD 700 Measurement

[0125] After fermentation is complete, add 100 μL of bacterial culture to a 48-well plate. To minimize sample error, mix the fermentation broth with a multi-channel pipette before sampling. Immediately after sampling, take 100 μL of the mixed bacterial culture, add 50 μL of 0.5 M hydrochloric acid, and then add water to bring the volume to 3 ml. Take 200 μL and add it to the microplate. Measure the OD value at a wavelength of 700 nm.

[0126] 2. Chlorophyll determination

[0127] The measurement method is the same as in Example 4 above.

[0128] 3. Coenzyme Q 10 Measurement

[0129] For strains with high chlorophyll content, further precise HPLC quantification can be achieved by adding 400 μL of bacterial culture to a 5 mL brown EP tube, along with 10 μL of 6M hydrochloric acid, 0.8 mL of acetone, and 90 μL of hydrogen peroxide. After shaking well, add 2.7 mL of anhydrous ethanol. The sample filtered through a 0.22 μm filter membrane is then ready for HPLC detection.

[0130] (1) Preparation of standard curve

[0131] Weigh 0.004 g of the standard and place it in a 10 mL brown volumetric flask. Add anhydrous ethanol to the mark and sonicate for 10 min to dissolve. The concentration after dissolution is 0.4 g / L. Dilute by half to 0.2, 0.1, 0.05, 0.025, and 0.0125 g / L. (All standards are stored in brown volumetric flasks. They can be stored at room temperature for half a month. Storage at 4°C may cause sample precipitation. The standard must be re-injected before each injection.)

[0132] (2) HPLC quantitative analysis

[0133] Mobile phase: methanol:ethanol = 65:35, wavelength 275 nm, column temperature 35 ℃, injection volume 20 μL, flow rate 1.5 mL / min, column pressure 130 bar, C18 reversed-phase column (YMC, Japan, column number 102HA70078, 4.6 mm × 150 mm).

[0134] 4. Results

[0135] Deep-hole plate fermentation results are as follows Figure 6 As shown, the OD value and chlorophyll content of the mutant strains were measured. For strains with higher chlorophyll / OD ratios, including mutants A3, A8, C8, and G1, coenzyme Q was further tested. 10 Production volume, in order to verify the production volume.

[0136] The results showed that the mutant strains with higher chlorophyll / OD ratios had higher levels of coenzyme Q. 10The yield was significantly higher. Comparing mutant strains A3, A8, C8, and G1, G1 showed the most significant yield increase compared to the original strain. 10 Production increased by more than 30%.

[0137] Analysis of the G1 strain genome showed no changes in other positions, except for a mutation at one site in the global regulatory factor prrA, where the phenylalanine (Phe) at position 24 was mutated to serine (Ser), resulting in a significant increase in its yield.

[0138] Example 6: High-throughput screening of prrA mutant strains

[0139] To verify the screening effect, the applicant conducted more batches of high-throughput testing using a 48-well deep well plate.

[0140] The control group and experimental group are set up as shown in Table 3.

[0141] Table 3

[0142]

[0143] The strain was inoculated into 48-well deep-well plates, and the operation and detection methods were the same as in Example 5. The results are as follows: Figure 7 The dots in the figure represent the yield of coenzyme Q10 in a single well. Statistical results show that the experimental group was able to consistently obtain high-yielding strains, and the number of high-yielding strains obtained was significantly higher than that in the control group (where only a few or almost no high-yielding strains were obtained).

[0144] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for establishing or screening high-yield coenzyme Q. 10 The method for treating Rhodotorula globulus, characterized in that, include: (1) Targeted modification of the global regulatory factor prrA in Rhodotorula spp.; wherein the Rhodotorula spp. is Rhodotorula spp. HY01; wherein the global regulatory factor prrA is a gene amplified from the genome of Rhodotorula spp. HY01 using SEQ ID NO: 1 and SEQ ID NO: 2 as primers; the modification includes high-throughput random mutation of the global regulatory factor prrA to form a random mutation library; (2) Cultivate the Rhodophyta globosum bacteria from (1), isolate bacteria with significantly high chlorophyll content, and obtain high-yield coenzyme Q from them. 10 The method described above involves random mutation of the global regulatory factor prrA in Rhodopsycetes; this process is high-throughput, resulting in a random mutation library. Bacteria with significantly high chlorophyll content are isolated using the following method: Observe the color of the Rhodopsycetes or their colonies; if the colonies of Rhodopsycetes modified with the global regulatory factor prrA are green, then they are high-producing coenzyme Q. 10 The darker the green color of the Rhodotorula globosum bacteria, the higher the yield.

2. The method as described in claim 1, characterized in that, The mutation was introduced into Rhodotorula globulus via site-specific recombination.

3. The method as described in claim 1, characterized in that, The method also includes determining the chlorophyll content of Rhodophyta globulae. If the chlorophyll content of Rhodophyta globulae modified with the global regulatory factor prrA is statistically higher than that of the control, then it is a high-coenzyme Q producer. 10 Rhodopseudomonas aeruginosa.

4. The method as described in claim 1, characterized in that, The method also includes setting up a control group, which is a Rhodopseudomonas spp. that has not been modified with the global regulatory factor prrA in step (1).

5. The application of the method according to any one of claims 1-4, used to establish or screen high-yield coenzyme Q using Rhodophyton floccosum HY01 as the starting strain. 10 Rhodopseudomonas aeruginosa.

6. A bio-fermentation method for producing coenzyme Q 10 The method is characterized by, The method includes: (a) Establishing a high-yield coenzyme Q production method according to any one of claims 1-4 10 Rhodopseudomonas aeruginosa; (b) Fermenting the Rhodotorula gracilis from (a) to produce coenzyme Q. 10 .

7. A method for preparing high-yield coenzyme Q 10 A method for processing Rhodopseudomonas spp., the method comprising: Using Rhodopseudomonas spp. as the starting strain, the global regulatory factor prrA in its genome was mutated, with position 24 being mutated to serine; the Rhodopseudomonas spp. is Rhodopseudomonas spp. HY01; the global regulatory factor prrA is a protein encoded by a gene amplified from the genome of Rhodopseudomonas spp. HY01 using primers SEQ ID NO: 1 and SEQ ID NO:

2.

8. The method as described in claim 7, characterized in that, The high-yield coenzyme Q 10 Compared with the original strain Rhodotorula glutinis, the yield of the new strain increased by more than 30%.

9. A high-yield coenzyme Q method 10 The originating strain of Rhodopseudomonas aeruginosa is Rhodopseudomonas aeruginosa HY01, and the 24th position of the global regulatory factor prrA in its genome is mutated to a serine residue; the global regulatory factor prrA is a protein encoded by a gene amplified from the genome of Rhodopseudomonas aeruginosa HY01 using SEQ ID NO: 1 and SEQ ID NO: 2 as primers.

10. The high-yield coenzyme Q as described in claim 9 10 Rhodopseudomonas spheroidae, characterized by, The high-yield coenzyme Q 10 Compared with the original strain Rhodotorula glutinis, the yield of the new strain increased by more than 30%.

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