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CN-122012655-A - Biosynthesis technology and equipment of adenosine triphosphate

CN122012655ACN 122012655 ACN122012655 ACN 122012655ACN-122012655-A

Abstract

The invention relates to the technical field of biosynthesis, in particular to a biosynthesis process of adenosine triphosphate and equipment thereof. The method comprises the steps of optimizing an adenosine triphosphate synthase gene sequence, constructing an expression vector, introducing the expression vector into a yeast host cell, subpackaging an engineering yeast colony into an orifice plate for parallel culture, detecting acid production and substrate conversion rate data of each micropore, screening elite strains, fermenting the elite strains, dynamically feeding materials through an on-line monitoring and feedback control system, accurately modifying the yeast strains through codon optimization and gene overexpression driven by a strong promoter, further adopting high-throughput screening to match with microminiaturization parallel fermentation and real-time optical detection to quickly identify the elite strains, introducing an automatic control feedback feeding technology in the fermentation process, stabilizing a culture environment in an optimal physiological state through real-time monitoring and dynamic regulation, fully playing production potential of the engineering bacteria, and realizing improvement of adenosine triphosphate yield, substrate conversion rate and product purity.

Inventors

  • XU QINGQING
  • ZHANG JIE
  • GAO GUOQING
  • SUN LUN

Assignees

  • 如东众意化工有限公司

Dates

Publication Date
20260512
Application Date
20260226

Claims (10)

  1. 1. A process for the biosynthesis of adenosine triphosphate, comprising the steps of: s1, strain transformation, namely optimizing an adenosine triphosphate synthase gene sequence, constructing an expression vector, introducing the expression vector into a yeast host cell, and screening to obtain an engineering yeast strain for efficiently synthesizing adenosine triphosphate; s2, high-throughput screening, namely subpackaging the engineering yeast flora into an orifice plate for parallel culture, and screening elite strains by detecting the acid yield and substrate conversion rate of each micropore; S3, fermenting the elite strain, and dynamically feeding materials through an on-line monitoring and feedback control system to maintain an optimal fermentation state; S4, coarse purification, namely collecting thalli after fermentation, crushing, separating and filtering to obtain adenosine triphosphate coarse extract; S5, purifying and concentrating, namely performing chromatographic purification, concentration and drying on the crude extract to obtain a high-purity adenosine triphosphate product.
  2. 2. The process for biosynthesis of adenosine triphosphate according to claim 1, wherein in S1, the expression vector contains a potent promoter and a selection marker.
  3. 3. The process for synthesizing adenosine triphosphate according to claim 2, wherein the potent promoter is one of GAL1 promoter, TEF1 promoter and PGK1 promoter.
  4. 4. The process for biosynthesis of adenosine triphosphate according to claim 1, wherein in S2, the parallel cultivation comprises adding a detection reagent to the microwells of the multiwell plate by an automated system and scanning the data with a high throughput detection platform.
  5. 5. The process for the biosynthesis of adenosine triphosphate according to claim 1, wherein in S3, the parameters monitored on line include pH, dissolved oxygen and substrate concentration; the fed-batch material comprises one or more of acid-base neutralizer, concentrated nutrient substrate or oxygen.
  6. 6. The process for the biosynthesis of adenosine triphosphate according to claim 5, wherein the concentrated nutrient substrate is used for controlling the carbon-nitrogen ratio in fermentation broth.
  7. 7. A pore plate device for the biosynthesis process of adenosine triphosphate according to any one of claims 1 to 6, wherein in S2, the pore plate comprises a plate body (100) and a cover body (200), the cover body (200) comprises a main cover plate (201) and a nested cover (202), the main cover plate (201) and the nested cover (202) are fixedly connected through a nested structure, and a breathable film (203) is clamped between the main cover plate (201) and the nested cover (202).
  8. 8. The apparatus of claim 7, wherein a through hole (204) penetrating the main cover plate (201) is formed in one side of the main cover plate (201), the through hole (204) is matched with an auxiliary hole (205) formed in one side of the nested cover (202), and the through hole (204) and the auxiliary hole (205) together form a ventilation channel.
  9. 9. The well plate apparatus according to claim 7, wherein the plate body (100) is a 96-well plate or 384-well plate.
  10. 10. The apparatus according to claim 7, wherein the gas permeable membrane (203) is one of a polycarbonate membrane and a polyester membrane.

Description

Biosynthesis technology and equipment of adenosine triphosphate Technical Field The invention relates to the technical field of biosynthesis, in particular to a biosynthesis process of adenosine triphosphate and equipment thereof. Background Adenosine triphosphate (adenosine triphosphate, ATP) is a nucleotide molecule formed by connecting adenine, ribose and three phosphate groups, and is called adenosine triphosphate, which belongs to coenzyme substances in the field of biological organic chemistry. The molecule contains two high-energy phosphate bonds, can release energy (7.5-8.6 kcal/gram molecule) during hydrolysis, and is a main direct energy supply substance for organism metabolism, and is called energy communication of cells. The synthesis of the adenosine triphosphate in the prior art generally comprises a chemical synthesis method, an enzyme catalysis method and a biological fermentation method, wherein the specific flow of the biological fermentation method is that high-yield strains are obtained through screening, large-scale batch or fed-batch fermentation culture is carried out in an optimized culture medium, bacterial cells are promoted to grow and accumulate the adenosine triphosphate through controlling environmental parameters such as temperature, pH, dissolved oxygen and the like, bacterial cells are collected after fermentation is finished, and then the adenosine triphosphate is separated and purified from cell extract through a series of downstream processing technologies such as mechanical crushing, centrifugation, membrane filtration, chromatographic separation and the like, and finally the product is obtained through concentration and drying. However, in the existing biological fermentation method, the existing strains are directly screened, strain transformation is not performed, so that the expression efficiency of heterologous adenosine triphosphate synthase genes in yeast hosts is low, the yield of target enzymes is insufficient, the subsequent screening process relies on manual shake flask fermentation with low flux and off-line detection, most high-performance strains are omitted due to the low screening efficiency, the full play of the production potential of engineering bacteria is restricted, and the adenosine triphosphate yield and the substrate conversion rate are far lower than the laboratory level. Disclosure of Invention The invention provides a biosynthesis process of adenosine triphosphate and equipment thereof, which accurately reforms yeast strains through codon optimization and gene over-expression driven by a strong promoter, solves the core problem of insufficient yield of target enzyme caused by low expression efficiency of heterologous genes, further adopts high-throughput screening to replace traditional low-efficiency manual screening, and rapidly identifies elite strains with high acid production and high substrate conversion rate through parallel fermentation and real-time optical detection, effectively avoids omission of excellent genotypes, thereby solving the problems proposed in the background art, namely: The prior art restricts the production potential of engineering bacteria due to the use of strains which are not modified, a low-flux screening method and extensive fermentation control, and leads to the problems of low adenosine triphosphate yield and conversion rate. In order to achieve the above object, the biosynthesis process of adenosine triphosphate and the apparatus thereof comprise the steps of: s1, strain transformation, namely optimizing an adenosine triphosphate synthase gene sequence, constructing an expression vector, introducing the expression vector into a yeast host cell, and screening to obtain an engineering yeast strain for efficiently synthesizing adenosine triphosphate; s2, high-throughput screening, namely subpackaging the engineering yeast flora into an orifice plate for parallel culture, and screening elite strains by detecting the acid yield and substrate conversion rate of each micropore; S3, fermenting the elite strain, and dynamically feeding materials through an on-line monitoring and feedback control system to maintain an optimal fermentation state; S4, coarse purification, namely collecting thalli after fermentation, crushing, separating and filtering to obtain adenosine triphosphate coarse extract; S5, purifying and concentrating, namely performing chromatographic purification, concentration and drying on the crude extract to obtain a high-purity adenosine triphosphate product. In the above-described embodiments, the description will be given of the step S1, in which the gene sequence is rewritten according to the codon usage preference of the yeast cell to ensure efficient translation in the yeast. Subsequently, the optimized gene fragment is precisely inserted into a carefully designed yeast expression vector through a molecular cloning technology to construct a recombinant plasmid, wherein the yeast expression vector