Biosynthesis process of adenosine triphosphate and equipment thereof

Abstract

The invention relates to the technical field of biosynthesis, in particular to a biosynthesis process of adenosine triphosphate and equipment thereof. Comprising the following steps: optimizing an adenosine triphosphate synthetase gene sequence, constructing an expression vector, and introducing the expression vector into a yeast host cell; sub-packaging the engineering yeast group in a pore plate for parallel culture, detecting the acid yield and substrate conversion rate data of each micropore, and screening out elite strains; fermenting elite strains, and dynamically feeding materials by an online monitoring and feedback control system; the yeast strain is subjected to precise transformation through codon optimization and gene overexpression driven by a strong promoter; elite strains are quickly identified by adopting high-throughput screening in cooperation with miniaturized parallel fermentation and real-time optical detection; in the fermentation process, a self-control feedback fed-batch technology is introduced, and a culture environment is stabilized in an optimal physiological state through real-time monitoring and dynamic regulation, so that the production potential of engineering bacteria is fully exerted, and the yield of adenosine triphosphate, the substrate conversion rate and the product purity are improved.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and more specifically, to a biosynthesis process and equipment for adenosine triphosphate. Background Technology

[0002] Adenosine triphosphate (ATP) is a nucleotide molecule composed of adenine, ribose, and three phosphate groups. It is a coenzyme in the field of bioorganic chemistry. Its molecule contains two high-energy phosphate bonds, and hydrolysis releases energy (7.5-8.6 kcal / molecular weight). It is the main direct energy source for metabolism in organisms and is known as the energy currency of cells.

[0003] In existing technologies, the synthesis of adenosine triphosphate (ATP) generally includes chemical synthesis, enzymatic catalysis, and bio-fermentation. The specific process of bio-fermentation involves screening high-yield bacterial strains, conducting large-scale batch or fed-batch fermentation in an optimized culture medium, and controlling environmental parameters such as temperature, pH, and dissolved oxygen to promote cell growth and accumulation of ATP. After fermentation, the cells are collected, and ATP is then separated and purified from the cell extract through a series of downstream processing technologies such as mechanical crushing, centrifugation, membrane filtration, and chromatographic separation. Finally, the product is obtained by concentration and drying.

[0004] However, in existing bio-fermentation methods, existing strains are directly screened without any strain modification. This results in low expression efficiency of heterologous adenosine triphosphate synthase genes in yeast hosts, leading to insufficient production of the target enzyme. Furthermore, the subsequent screening process relies on low-throughput manual shake-flask fermentation and offline detection, resulting in low screening efficiency and the omission of the vast majority of high-performance strains. This restricts the full realization of the production potential of engineered bacteria, causing adenosine triphosphate yield and substrate conversion rate to be far lower than laboratory levels. Summary of the Invention

[0005] This invention provides a biosynthesis process and equipment for adenosine triphosphate (ATP). It precisely modifies yeast strains through codon optimization and strong promoter-driven gene overexpression, solving the core problem of insufficient target enzyme yield due to low heterologous gene expression efficiency. Furthermore, it employs high-throughput screening to replace traditional inefficient manual screening, rapidly identifying elite strains with both high acid production and high substrate conversion rates through parallel fermentation and real-time optical detection. This effectively avoids the omission of superior genotypes, thus solving the problems mentioned in the background art. Existing technologies, due to the use of unmodified strains, low-throughput screening methods, and extensive fermentation control, limit the production potential of engineered bacteria, resulting in low yields and conversion rates of adenosine triphosphate (ATP).

[0006] To achieve the above objectives, the biosynthesis process and equipment for this adenosine triphosphate include the following steps: S1. Strain modification: Optimize the gene sequence of adenosine triphosphate synthase, construct an expression vector and introduce it into yeast host cells, and screen to obtain engineered yeast strains that synthesize adenosine triphosphate efficiently. S2. High-throughput screening: Engineered yeast cells are aliquoted into well plates for parallel culture. Elite strains are screened by detecting the acid production and substrate conversion rate of each well. S3. Fermentation culture: Ferment the elite strain and dynamically add materials through an online monitoring and feedback control system to maintain the optimal fermentation state; S4. Crude purification: Collect the bacterial cells after fermentation, and obtain crude adenosine triphosphate extract by crushing, separating and filtering. S5. Purification and Concentration: The crude extract is purified by chromatography, concentrated and dried to obtain a high-purity adenosine triphosphate product.

[0007] In the above technical solution, step S1 will be explained in detail below. First, the gene sequence is rewritten according to the codon usage preferences of yeast cells to ensure that it can be efficiently translated in yeast. Then, the optimized gene fragment is precisely inserted into a carefully designed yeast expression vector using molecular cloning technology to construct a recombinant plasmid. This yeast expression vector not only contains elements that allow it to exist and replicate stably in yeast cells, but more importantly, it carries a potent promoter, which is one of the GAL1 promoter, TEF1 promoter, or PGK1 promoter, to drive gene overexpression, and an antibiotic resistance gene as a marker for subsequent screening.

[0008] The recombinant plasmid was constructed and introduced into competent cells of the target yeast strain via lithium acetate chemical transformation. The successfully transformed yeast strains acquired the resistance gene on the plasmid and were able to grow on selective culture media containing the corresponding antibiotics, thereby allowing positive transformants to be screened out.

[0009] Finally, these positive clones were rigorously identified using molecular biology techniques such as colony PCR and Western blotting, confirming that the adenosine triphosphate synthase gene had been successfully integrated and overexpressed, thus ultimately obtaining an engineered yeast strain that can concentrate metabolic resources on the efficient synthesis of adenosine triphosphate.

[0010] In S2, genetically modified yeast cells are dispensed into well plates in single-cell form for parallel fermentation. These well plates are placed in an automated incubator that can precisely control temperature, humidity, and vibration frequency to simulate optimal fermentation conditions. After cultivation, the integrated system automatically adds specific biochemical detection reagents to each well, such as pH-sensitive dyes for acidic products or enzyme detection systems that can react with acidic products to produce color and fluorescence. This results in the wells containing high-acid-producing strains exhibiting stronger optical signals. Simultaneously, the conversion rate of the strain can be calculated by detecting the residual amount of substrates (such as glucose) consumed by adenosine triphosphate (ATP) or by directly measuring ATP production. Subsequently, a high-throughput fluorescent microplate reader or spectral detection platform rapidly scans the entire microplate, instantly capturing the light signal intensity of each microwell and converting it into digital acid production and conversion rate data. Finally, this data is transmitted to a computer and analyzed and sorted in real time using specialized software, thereby accurately and efficiently identifying elite strains that excel in acid production and substrate conversion efficiency from tens of thousands of candidate strains, thus enabling early strain screening for fermentation culture.

[0011] In S3, during the fermentation culture stage, the elite strains screened in the high-throughput screening step are fermented. Materials are dynamically fed into the system through an online monitoring and feedback control system. Specifically, the working principle of the feedback control system for dynamic material feeding is as follows: Through precisely installed pH electrodes, dissolved oxygen probes, and substrate biosensors, the dynamic changes in the culture environment within the fermenter are continuously monitored, and these real-time physiological parameters are converted into electrical signals and transmitted to the central process control computer. The computer's built-in intelligent algorithm model immediately compares this real-time data with the preset optimal fermentation trajectory. Once any parameter deviates from the optimal range, such as pH below the set value, insufficient substrate concentration, or excessively low dissolved oxygen level, the actuator is immediately triggered. This triggers an automatic and precise metering pump to add acid-base neutralizers, concentrated nutrient substrates, or introduce pure oxygen to the fermenter for regulation, thereby stabilizing the entire fermentation environment at the optimal physiological state for cell growth and product synthesis. This dynamic balance alleviates the environmental stress on the engineered yeast strain, eliminates substrate inhibition or substrate starvation effects, and, by precisely controlling key factors such as the carbon-nitrogen ratio, maximizes the metabolic flow to the target pathway. This ensures that the exogenously introduced adenosine triphosphate synthase gene can be continuously and efficiently overexpressed, ultimately maximizing adenosine triphosphate yield and substrate conversion rate per unit time and unit substrate consumption.

[0012] Furthermore, it should be further explained that in S1, the expression vector contains a potent promoter and a selection marker. The potent promoter integrated into the expression vector is a core regulatory element that drives the high-level transcription and expression of exogenous genes in the yeast host. Its role is to provide a powerful driving force for the transcription of the adenosine triphosphate synthase gene, ensuring that sufficient mRNA is obtained. The presence of selection markers (such as antibiotic resistance genes) on the vector provides a direct genetic basis for subsequent transformant screening, so that only yeast cells that have successfully introduced the recombinant plasmid can survive and grow on selective culture media containing the corresponding antibiotics, thereby efficiently and accurately screening positive clones from a large number of background cells.

[0013] In S3, the parameters monitored online include pH, dissolved oxygen, and substrate concentration; the fed-batch material includes one or more of acid-base neutralizer, concentrated nutrient substrate, and oxygen, wherein the concentrated nutrient substrate is used to control the carbon-nitrogen ratio in the fermentation broth.

[0014] Secondly, this solution also provides a well plate device for high-throughput screening of the S2 step in the biosynthesis process of adenosine triphosphate. Specifically, the well plate includes a plate body and a cover body. The cover body includes a main cover plate and a nested cover. The main cover plate and the nested cover are fixedly connected by a nested structure. A breathable membrane is sandwiched between the main cover plate and the nested cover. The main cover plate has a through hole on one side, which matches the auxiliary hole on one side of the nested cover. The through hole and the auxiliary hole together form a ventilation channel. The plate is a 96-hole plate or a 384-hole plate. The breathable membrane is made of either polycarbonate membrane or polyester membrane.

[0015] In the above-mentioned perforated plate equipment, the connection structure between the auxiliary holes of the nested plate ensures that the high-strength polycarbonate or polyester film can obtain uniform support without deformation when under pressure. The excellent thermoplasticity of the polycarbonate or polyester film allows the film material to undergo local melting at the interface with the main cover plate and the nested plate under ultrasonic or hot pressing, forming a high-strength, seamless seal, which solves the contradiction between zero leakage and high oxygen permeability in the installation of breathable membrane in one go. Furthermore, the energy from ultrasound or hot pressing selectively causes the contact interface between the polycarbonate or polyester film and the main cover plate and nesting plate to partially melt and diffuse with each other. After cooling, an integrated sealing rib is formed, which physically completely replaces traditional adhesives. This ensures that the seal only occurs at the annular interface around the auxiliary hole and never penetrates the microporous structure of the polycarbonate or polyester film itself, thus fundamentally eliminating the risk of clogging that adhesives may cause. At the same time, since the entire system is completely fused from pure polycarbonate or polyester materials, the introduction of external chemical adhesives is avoided, thereby completely eliminating any chemical contaminants that may be generated and ensuring the purity of the culture system.

[0016] Based on the self-melting sealing method, not only does it not compromise the inherent high air permeability and excellent chemical inertness of polycarbonate or polyester membranes, but it also firmly locks and protects the membrane material through a robust fusion sealing ring. This allows the polycarbonate or polyester membrane to independently cope with acidic metabolites and continuous mechanical vibrations during fermentation, thanks to its strong mechanical strength and chemical corrosion resistance. This achieves a high degree of unity between sealing reliability and membrane functional integrity.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: By precisely modifying yeast strains through codon optimization and strong promoter-driven gene overexpression, the core problem of insufficient target enzyme yield caused by low heterologous gene expression efficiency is fundamentally solved. Furthermore, high-throughput screening is used to replace traditional inefficient manual screening. Through miniaturized parallel fermentation and real-time optical detection, elite strains with both high acid production and high substrate conversion rate are quickly identified, effectively avoiding the omission of excellent genotypes.

[0018] By introducing self-controlled feedback feed technology during fermentation, the culture environment is stabilized at the optimal physiological state through real-time monitoring and dynamic regulation. This completely eliminates limiting factors such as substrate inhibition and acidosis, allowing the production potential of engineered bacteria to be fully realized, ultimately achieving a significant improvement in adenosine triphosphate (ATP) yield, substrate conversion rate, and product purity. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the steps in the biosynthesis method of adenosine triphosphate of the present invention; Figure 2 This is a schematic diagram of the overall structure of the orifice plate device used in S2 of the present invention; Figure 3 This is a partial structural diagram of the orifice plate device used in S2 of the present invention; Figure 4 This is a cross-sectional view of the perforated plate device used in S2 of the present invention.

[0020] The meanings of the labels in the diagram are as follows: 100. Plate body; 200. Cover body; 201. Main cover plate; 202. Nested cover; 203. Breathable membrane; 204. Through hole; 205. Auxiliary hole. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Current technologies, due to the use of unmodified strains, low-throughput screening methods, and extensive fermentation control, limit the production potential of engineered bacteria, resulting in low yields and conversion rates of adenosine triphosphate (ATP). This invention provides a ATP biosynthesis process and equipment, such as... Figure 1 As shown, Example 1: Construction of recombinant yeast strain expressing ATP synthase.

[0023] Strain modification: The gene sequence of adenosine triphosphate synthase was optimized, an expression vector was constructed and introduced into yeast host cells, and engineered yeast strains that synthesize adenosine triphosphate efficiently were screened.

[0024] Here, the steps of strain modification will be broken down and explained in detail: S1.1 Gene Optimization and Synthesis: Based on the codon bias of Saccharomyces cerevisiae, the whole-genome optimization of the adenosine triphosphate synthase gene sequence derived from Thermus thermophilus was performed, and the optimized gene fragment was synthesized and named opt-ATPsyn. Specifically, it should be further explained that choosing Thermus thermophilus as the source of the adenosine triphosphate synthase gene and choosing Saccharomyces cerevisiae as the expression host are both conventional techniques in this field, and both strains can be purchased publicly from domestic and international public microbial culture centers or biological reagent companies. The synthesis of the gene fragment in this step can be achieved using mature gene synthesis techniques in this field.

[0025] S1.2, Expression Vector Construction: The opt-ATPsyn gene fragment was cloned into the multiple cloning site of the yeast expression vector pYES2-CT using restriction endonuclease. In this embodiment, BamHI was preferably used as the endonuclease. The expression vector pYES2-CT was purchased from Ingenium Life Sciences, Inc., USA. This vector carries the GAL1 strong promoter and the URA3 selection marker gene. A recombinant plasmid was constructed and named pYES2-opt-ATPsyn. The successfully constructed recombinant plasmid was verified by DNA sequencing. The DNA sequencer used for DNA verification of the recombinant plasmid can be a MiSeq DNA sequencer manufactured by Inmina (China) Scientific Instruments Co., Ltd.

[0026] S1.3 Yeast transformation: The recombinant plasmid pYES2-opt-ATPsyn was transformed into Saccharomyces cerevisiae INVSC1 competent cells using the lithium acetate method.

[0027] S1.4 Positive clone screening: The transformed bacterial culture was spread on a solid selective medium lacking uracil and incubated upside down at 30°C for 2-3 days. Small colonies that grew were screened. The solid selective medium lacking uracil was subsequently referred to as SD-Ura.

[0028] In addition, in this embodiment, after the positive clone screening step, positive clone verification is required, specifically including: 1. Colony PCR Validation: Twenty single colonies were randomly selected and colony PCR amplification was performed using specific primers for the opt-ATPsyn gene. Eighteen colonies were able to amplify the target band of the expected size. In this step, the analysis of colony PCR products was performed using an electrophoresis system of the PowerPac Basic model manufactured by Bio-Rad Laboratories (Shanghai) Co., Ltd.; colony PCR amplification was performed using a PCR amplification instrument of the T100 model manufactured by Bio-Rad Laboratories (Shanghai) Co., Ltd.

[0029] Immunoblot verification: Three PCR-positive clones were selected and induced in a small-scale culture. The GAL1 promoter was induced with galactose, and the bacterial cells were collected. After cell lysis, the cells were detected by SDS-PAGE and immunoblotting. In this step, immunoblotting verification requires a protein electrophoresis and transfer system, specifically a mini vertical electrophoresis tank (Mini-PROTEAN Tetra) and a transfer system (Trans-Blot Turbo). Both devices were manufactured and provided by Bio-Rad Biomedical Products (Shanghai) Co., Ltd. In addition, a Tanon 5200 chemiluminescence imaging system manufactured by Shanghai Tianneng Technology Co., Ltd. was used to detect the immunoblotting results.

[0030] The results showed a distinct specific band at the expected molecular weight, confirming the successful expression of adenosine triphosphate synthase in yeast cells. The clone with the highest expression level was named the engineered strain Y-ATP01, preserved, and used for subsequent experiments.

[0031] Example 2: Screening elite strains using high-throughput plates.

[0032] High-throughput screening: Engineered yeast cultures were aliquoted into well plates and cultured in parallel. Elite strains were screened by detecting the acid production and substrate conversion rate of each well.

[0033] S2.1, Microbial preparation: The bacterial solution before plating after transformation in Example 1 is appropriately diluted to ensure that each well in the plate theoretically receives no more than 1 cell; the well plate used in this step is the well plate equipment provided in this scheme.

[0034] S2.2, Parallel Fermentation: Using the well plate equipment provided by this invention, add 200 μL of SD-Ura induction medium containing 2% galactose to each well; use an automated dispensing system to dispense the diluted bacterial solution into the well plate; place the well plate in a constant temperature shaker at 30°C and 1000 rpm for 48 hours; the automated dispensing system used in this step is an I.DOT automated liquid handling workstation manufactured by Shenzhen Aidi Biotechnology Co., Ltd.; the constant temperature shaker is a THZ-100 manufactured by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0035] S2.3 High-throughput detection: Acid production detection: After cultivation, 20 μL of pH-sensitive dye was added to each well using an automated system. In this embodiment, bromocresol purple was selected as the pH-sensitive dye. The absorbance value, i.e., the OD590 value, was then detected at a wavelength of 590 nm using a high-throughput microplate reader. The OD590 value is negatively correlated with the pH value of the fermentation broth, i.e., the lower the OD590 value, the higher the acid production. When measuring the OD590 value, a multi-functional microplate reader, model Spark, manufactured by Taikang Life Science (China) Co., Ltd., is required. Substrate conversion assay: Simultaneously, 5 μL of fermentation supernatant was taken, and the residual glucose concentration was measured using a glucose assay kit manufactured by Shanghai Beyotime Biotechnology Co., Ltd. Substrate conversion rate (%) = (Initial glucose concentration - Residual glucose concentration) / Initial glucose concentration × 100%.

[0036] S2.4. Import the OD590 values ​​and substrate conversion rate data detected in all microwells into the analysis software. The software sorts the microwells based on two criteria: high acid production (i.e., low OD590 value) and high substrate conversion rate. Finally, the top ten elite strains were selected from approximately 4,000 microwells. Among them, strain B7 performed best, with an OD590 value 35% lower than the average and a substrate conversion rate of 92%. It was named the elite strain Y-ATP01-ELITE.

[0037] Example 3: Fermentation culture of elite strains and production of adenosine triphosphate.

[0038] S3.1 Fermentation Culture: The elite Saccharomyces cerevisiae engineered strain Y-ATP01-ELITE was inoculated into a 5L fermenter containing 3.0L of chemically defined culture medium, which contained 20 g / L glucose, 5.0 g / L ammonium sulfate, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 1.0 mL / L trace element solution, and 0.1 mL / L vitamin solution. Fermentation was carried out at 30±0.2°C and pH 5.50±0.05. The dissolved oxygen concentration was controlled to be no less than 30% saturation by adjusting the stirring speed and aeration rate. When the residual glucose concentration was below 1.0 g / L, an exponential flow of 600 g / L glucose solution was initiated, and the specific growth rate was controlled at 0.15 h. -1 The yield of adenosine triphosphate in the fermentation broth was determined to be 8.5 g / L by timed detection using high performance liquid chromatography.

[0039] In the above steps, it should be further explained that: the fermenter can be a BIOTECH-5BG model manufactured and provided by Shanghai Baoxing Bio-Equipment Engineering Co., Ltd., which has control functions for temperature, pH, dissolved oxygen (DO), stirring speed, aeration rate and automatic feed; in addition, the high performance liquid chromatography system can be a YSI 2900 series model provided by Agilent Technologies (China) Co., Ltd.

[0040] S3.2 Online monitoring and feedback control: Online monitoring: The pH, dissolved oxygen, and glucose concentration of the fermentation broth are monitored in real time using pH electrodes, dissolved oxygen (DO) probes, and glucose biosensors installed in the tank.

[0041] Feedback control: The control targets are set as follows: pH = 6.0 (±0.1), DO>30%, and glucose concentration maintained at 0.5-2.0 g / L to avoid substrate inhibition and starvation.

[0042] Feed material: When the pH is below 5.9, 2 mol of sodium hydroxide solution is automatically added; when the pH is above 6.1, 2 mol of hydrogen chloride solution is added. When DO is below 30%, the stirring speed and aeration rate will be automatically increased. When the glucose concentration is below 0.5 g / L, the metering pump is started to add 600 g / L of concentrated glucose solution (concentrated nutrient substrate). Through this feedback system, the fermentation environment is always controlled in the optimal state. The metering pump used in this step is manufactured by Shanghai Baoxing Bio-equipment Engineering Co., Ltd.

[0043] Example 4: Extraction and purification of adenosine triphosphate.

[0044] S4.1 Crude purification: Take the fermentation broth from Example 3 and centrifuge at 12000×g for 20 minutes at 4°C to collect the cells; resuspend the cells in pre-cooled 20 mM sodium phosphate buffer, and circulate the cells three times under ice-water bath conditions using a high-pressure homogenizer at 1000 bar pressure; centrifuge the lysate at 15000×g for 30 minutes at 4°C to remove cell debris, collect the supernatant and filter it through a 0.45 μm microporous membrane to obtain crude adenosine triphosphate extract.

[0045] S4.2 Purification and Concentration: The crude extract was loaded onto an anion exchange column and eluted with a linear gradient of 0.1-1.0 M sodium chloride. The main peak of adenosine triphosphate (ATP) was collected. The elution process was monitored using an online UV detector at a wavelength of 259 nm, where ATP exhibits its maximum absorption peak. Based on the UV absorption spectrum, the eluent fraction corresponding to the main absorption peak at 259 nm was collected; this is the fraction rich in ATP. The collected solution was concentrated under reduced pressure at 40°C using a rotary evaporator. Finally, the concentrate was freeze-dried to obtain a high-purity ATP product in the form of a white powder.

[0046] S4.3 Purity test: The purity of the adenosine triphosphate product was determined to be 98.5% by high performance liquid chromatography.

[0047] Comparative Example 1: Using traditional, unmodified strains.

[0048] In this comparative example, the same 5L fermenter, the same chemical composition of the culture medium, and the same fermentation process parameters as in Example 3 were used. The only difference was that the fermentation strain was the unmodified original Saccharomyces cerevisiae INVSC1, which was purchased from Ingenie Life Sciences, Inc., USA, instead of the recombinant engineered strain Y-ATP01 obtained in Example 1.

[0049] After a 48-hour fermentation cycle, a sample of the fermentation broth was taken, centrifuged, and the adenosine triphosphate (ATP) content was determined using the same high-performance liquid chromatography (HPLC) method as in Example 3. The results showed that the final ATP yield was only 0.08 g / L, compared to 8.5 g / L in Example 3. This demonstrates that unmodified wild-type yeast does not possess the ability to efficiently synthesize ATP. Therefore, heterologous expression of the opt-ATPsyn gene, as in Example 1, is a necessary prerequisite and core technological foundation for achieving high ATP production.

[0050] Comparative Example 2: Using the traditional shake-flask screening method.

[0051] In this comparative example, the engineered yeast Y-ATP01 transformation culture from the same source as in Example 2 was used, but instead of its high-throughput plate screening method, conventional shake flask screening was performed.

[0052] Specific steps: The bacterial culture was spread onto SD-Ura plates and incubated at 30°C for 48 hours. Then, 200 single colonies were randomly selected and inoculated into 50 mL shake flasks containing induction medium, and incubated at 30°C and 250 rpm for 48 hours. Subsequently, samples were manually taken one by one, and the pH of the fermentation broth was measured using pH test strips. The residual glucose concentration was also determined offline using a glucose assay kit. Based on acid production (low pH) and substrate consumption, the 10 best-performing clones were selected.

[0053] The best strain from these 10 clones was fermented in a 5L fermenter using the exact same process as in Example 3. High-performance liquid chromatography (HPLC) analysis showed that the final yield of adenosine triphosphate (ATP) was 5.1 g / L, and the glucose-to-ATP conversion rate was 0.255 g / g glucose.

[0054] The experimental results above show that the yield of this strain is significantly lower than that of the elite strain Y-ATP01-ELITE obtained by high-throughput screening in Example 2. These results demonstrate that the high-throughput screening method of this invention, with its extremely high throughput, can extract superior elite strains from a larger library, proving the outstanding advantage of high-throughput screening strategies in maximizing the production potential of engineered strains.

[0055] Comparative Example 3: Batch fermentation, without online feedback control.

[0056] In this comparative example, the elite strain Y-ATP01-ELITE obtained from Example 2 was used for conventional batch fermentation in the same 5L fermenter. 40 g / L glucose was added to the culture medium at once. No online monitoring or feed control was performed during fermentation; only an initial addition of 20 g / L calcium carbonate powder was used to attempt to neutralize the acids produced during fermentation. During fermentation, the pH dropped to 4.8 due to acid accumulation; in the later stages of fermentation, due to glucose depletion and acid stress, a large amount of cell autolysis occurred, leading to premature termination of fermentation. High-performance liquid chromatography (HPLC) analysis showed that the final yield of adenosine triphosphate (ATP) was only 3.2 g / L, and the glucose-to-ATP conversion rate was 0.08 g / g glucose.

[0057] The experimental results above show a stark contrast to the intelligent control fermentation results of Example 3. This demonstrates that even with high-performance elite strains, the lack of real-time online monitoring and dynamic feedback control makes it impossible to maintain the optimal fermentation environment, leading to a sharp decline in yield and conversion rate. This fully reflects the decisive role of the fermentation control strategy of this invention.

[0058] Furthermore, this invention also provides a well plate device for high-throughput screening of the S2 step in a biosynthesis process of adenosine triphosphate, such as... Figures 2-4As shown, specifically, the perforated plate includes a plate body 100 and a cover body 200. The cover body 200 includes a main cover plate 201 and a nested cover 202. The main cover plate 201 and the nested cover 202 are fixedly connected by a nested structure. A breathable membrane 203 is sandwiched between the main cover plate 201 and the nested cover 202. A through hole 204 is provided on one side of the main cover plate 201, which fits into an auxiliary hole 205 provided on one side of the nested cover 202. The through hole 204 and the auxiliary hole 205 together form a ventilation channel. The plate body 100 is a 96-well plate or a 384-well plate. The ventilation membrane 203 is made of either polycarbonate membrane or polyester membrane. When using this plate, the selection can be made according to the screening throughput and cost-effectiveness. A 96-well plate can be used in preliminary screening where more refined culture or higher detection sensitivity is required. A 384-well plate is used when large-scale, ultra-high throughput screening is required to maximize the screening capacity.

[0059] Comparative Example 4: Using conventional adhesive sealing plates.

[0060] In this comparative example, all screening steps, conditions, and detection methods were identical to those in Example 2, except that the specially designed well plate described in Example 2 was replaced with a commercially available ordinary 384-well cell culture plate. The 384-well cell culture plate used an adhesive to fix the breathable sealing membrane, and the commercially available well plate was the 384-well cell culture plate with product catalog number 3707 manufactured and supplied by Corning (Shanghai) Management Co., Ltd.

[0061] After 48 hours of shaking culture, approximately 15% of the microwells experienced sealing film detachment or incomplete sealing, leading to culture medium evaporation and cross-contamination between microwells. When detected at 590 nm wavelength, the OD values ​​of these wells showed significant abnormalities, large dispersion, and low signal-to-noise ratio. The optimal strain selected from this data underwent three rounds of repeated validation in a 5L fermenter, with an average adenosine triphosphate (ATP) yield of 7.2 g / L, and yield fluctuations exceeding 10% between different batches. Therefore, compared to the stable high-yielding strain Y-ATP01-ELITE screened using well plates in Example 2, both performance and reliability were significantly reduced. This indicates that traditional adhesive sealing methods are unsuitable for the acidic environment and vigorous shaking conditions in the high-throughput screening of this invention, leading to screening failure. Therefore, the specially designed well plate device employing adhesive-free hot-melt sealing in this invention is crucial for obtaining reliable and efficient screening results and is an integral part of this screening method system.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biosynthetic process for adenosine triphosphate, characterized in that, Includes the following steps: S1. Strain modification: Optimize the gene sequence of adenosine triphosphate synthase, construct an expression vector and introduce it into yeast host cells, and screen to obtain engineered yeast strains that synthesize adenosine triphosphate efficiently. S2. High-throughput screening: Engineered yeast cells are aliquoted into well plates for parallel culture. Elite strains are screened by detecting the acid production and substrate conversion rate of each well. S3. Fermentation culture: Ferment the elite strain and dynamically add materials through an online monitoring and feedback control system to maintain the optimal fermentation state; S4. Crude purification: Collect the bacterial cells after fermentation, and obtain crude adenosine triphosphate extract by crushing, separating and filtering. S5. Purification and Concentration: The crude extract is purified by chromatography, concentrated and dried to obtain a high-purity adenosine triphosphate product.

2. The biosynthesis process of adenosine triphosphate according to claim 1, characterized in that: In S1, the expression vector contains a potent promoter and a selection marker.

3. The biosynthesis process of adenosine triphosphate according to claim 2, characterized in that: The potent promoter is one of the GAL1 promoter, TEF1 promoter, or PGK1 promoter.

4. The biosynthesis process of adenosine triphosphate according to claim 1, characterized in that: In S2, the parallel culture includes adding detection reagents to the microwells of a multi-well plate using an automated system and scanning and acquiring data using a high-throughput detection platform.

5. The biosynthesis process of adenosine triphosphate according to claim 1, characterized in that: In S3, the parameters monitored online include pH, dissolved oxygen, and substrate concentration; The feed material includes one or more of the following: acid-base neutralizer, concentrated nutrient substrate, or oxygen.

6. The biosynthesis process of adenosine triphosphate according to claim 5, characterized in that: The concentrated nutrient substrate is used to control the carbon-nitrogen ratio in the fermentation broth.

7. A well plate apparatus for the biosynthesis process of adenosine triphosphate as described in any one of claims 1 to 6, characterized in that: In S2, the perforated plate includes a plate body (100) and a cover body (200). The cover body (200) includes a main cover plate (201) and a nested cover (202). The main cover plate (201) and the nested cover (202) are fixedly connected by a nested structure. A breathable membrane (203) is sandwiched between the main cover plate (201) and the nested cover (202).

8. The orifice plate device according to claim 7, characterized in that: The main cover plate (201) has a through hole (204) on one side, which fits into an auxiliary hole (205) on one side of the nested cover (202). The through hole (204) and the auxiliary hole (205) together form a ventilation channel.

9. The orifice plate device according to claim 7, characterized in that: The plate (100) is a 96-well plate or a 384-well plate.

10. The orifice plate device according to claim 7, characterized in that: The breathable membrane (203) is made of either polycarbonate or polyester.

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