Method for regulating content ratio of isoleucine to valine in fermentation process and application of method
By regulating the redox potential and aeration rate of the fermentation broth, the problem of valine co-accumulation during isoleucine fermentation was solved, thereby increasing the yield and purity of isoleucine and reducing valine impurities, making it suitable for large-scale production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI HUAHENG BIOTECH CO LTD
- Filing Date
- 2024-11-06
- Publication Date
- 2026-05-08
AI Technical Summary
The co-accumulation of valine and isoleucine during isoleucine fermentation makes it difficult to separate valine impurities from the fermentation broth, affecting the yield and purity of isoleucine.
By adjusting the redox potential of the fermentation broth to -200 to -175 mV, combined with adjusting the aeration rate and stirring speed, the accumulation rate of valine can be controlled, thereby increasing the ratio of isoleucine to valine content.
It achieves high yield and high purity production of isoleucine, reduces valine impurity content, improves fermentation conversion rate, and is suitable for large-scale production.
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Abstract
Description
Technical Field
[0001] This application relates to the field of fermentation, and specifically, it provides a method for regulating the ratio of isoleucine to valine content during fermentation and its application. Background Technology
[0002] L-Isoleucine is one of the eight essential amino acids for the human body. It plays a vital role in promoting protein synthesis and muscle growth, lowering blood ammonia levels, providing energy, maintaining blood circulation, and supporting immune homeostasis. Adults need to supplement with at least 20 mg / kg (body weight) of L-isoleucine daily. Therefore, the demand for L-isoleucine is very high in the amino acid sports energy drinks and fortified wheat flour food industries. Furthermore, L-isoleucine significantly promotes the growth and development of most animals and has auxiliary effects on physiological functions such as milk secretion and the nervous system. However, because animals cannot synthesize it themselves, the demand for L-isoleucine in the animal feed industry is also very high.
[0003] L-Isoleucine is one of the three major branched-chain amino acids (BCAAs), and BCAAs play a crucial role in the growth and development of infants, adolescents, and adults. Furthermore, BCAAs have a good auxiliary effect in the treatment and recovery of diseases such as liver and kidney problems. Therefore, L-isoleucine, often combined with L-valine and L-leucine, is widely used in clinical adjuvant therapy as a BCAA injection or oral nutritional solution, and pharmaceutical-grade L-isoleucine is a high-value-added amino acid.
[0004] There are three main methods for producing L-isoleucine: extraction, chemical synthesis, and fermentation. Extraction and chemical synthesis are difficult to scale up industrially due to limited raw material sources, high production costs, and environmental pollution. Fermentation, however, utilizes the metabolic activity of microorganisms to biosynthesize and accumulate L-isoleucine. It offers advantages such as low raw material costs, mild reaction conditions, and ease of large-scale production, making it the most common method for L-isoleucine production. Fermentation includes two main types: precursor fermentation and direct fermentation. Precursor fermentation uses glucose as the carbon source and adds specific precursors such as α-aminobutyric acid (GABA), α-hydroxybutyric acid (hydroxybutyric acid), α-ketobutyric acid (KBA), and threonine to avoid feedback regulation in the amino acid biosynthesis pathway. These precursors are then effectively converted into L-isoleucine by microorganisms. Direct fermentation leverages the ability of microorganisms to synthesize their own required amino acids. By mutagenesis of specific microorganisms and the selection of auxotrophic and amino acid structural analog resistant mutants, feedback inhibition and repression in metabolic regulation are eliminated, thereby achieving the goal of accumulating L-isoleucine. Currently, most L-isoleucine-producing bacteria are derived from glutamate-producing bacteria (such as *Brugia flavescens*, *Corynebacterium glutamicum*, and *Brugia lactose-fermenting*). However, with the continuous maturation of fermentation technology, the development of genetic engineering technology, and the increase in industrial microbial biochemical information, more superior L-isoleucine-producing bacteria of various genera have been developed. Several companies are currently producing L-isoleucine through fermentation, but problems such as low acid production levels in strains and poor extraction technology of fermentation products still exist. Therefore, improving the fermentation and extraction process of L-isoleucine is of great significance for increasing the domestic production of L-isoleucine and enhancing its market competitiveness.
[0005] Analysis of the metabolic pathways of isoleucine and valine revealed that the de novo synthetic pathways of both amino acids begin with a diversion from pyruvate, and both isoleucine and valine are synthesized simultaneously through three isoenzymes. This results in isoleucine fermentation broth often containing a significant amount of difficult-to-separate valine impurities. Therefore, a fermentation process control strategy is urgently needed to address the co-accumulation of valine and isoleucine during isoleucine fermentation. Summary of the Invention
[0006] This application provides a method for regulating the ratio of isoleucine to valine during fermentation, which includes:
[0007] When the valine content in the fermentation broth is 3.00~5.00 g / L, the redox potential of the fermentation broth is adjusted to -200~-175 mV.
[0008] In one or more embodiments of this application, the redox potential of the fermentation broth is adjusted to -196 to -175 mV.
[0009] In one or more embodiments of this application, the redox potential of the fermentation broth is adjusted by at least one of the following methods: adjusting the aeration rate and adjusting the stirring speed.
[0010] In one or more embodiments of this application, the ratio of isoleucine to valine in the fermentation broth after fermentation is ≥8.00.
[0011] In one or more embodiments of this application, the strains used for fermentation are Escherichia coli, Brevibacterium flavum, Corynebacterium glutamicum, or Brevibacterium lactofermentum.
[0012] In one or more embodiments of this application, the strain used for fermentation is Escherichia coli.
[0013] In one or more embodiments of this application, the method is used to:
[0014] a) Fermentation production of isoleucine;
[0015] b) Increase isoleucine yield and / or isoleucine purity; and / or
[0016] c) Improve the raw material conversion rate in isoleucine production.
[0017] In one or more embodiments of this application, the raw material conversion rate is the conversion rate of glucose to isoleucine and / or the conversion rate of threonine to isoleucine.
[0018] In one or more embodiments of this application, the isoleucine is L-isoleucine or D-isoleucine. Attached Figure Description
[0019] Figure 1 The graphs showing the growth trends of valine and L-isoleucine in Examples 1 and 2 are displayed; where 1 represents Example 1 and 2 represents Example 2. Preservation Instructions
[0020] Cell name: Escherichia coli IVL016
[0021] Preservation Institution: China Center for Type Culture Collection
[0022] Abbreviation for depository institution: CCTCC
[0023] Address: Wuhan University, Wuhan, China (No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province)
[0024] Deposit date: October 16, 2023
[0025] Collection Center Registration Number: CCTCC NO: M 20231916 Detailed Implementation
[0026] The redox potential of a fermentation medium is a comprehensive parameter reflecting various environmental conditions, depending on the medium components, pH, osmotic pressure, temperature, and dissolved oxygen concentration. By adjusting the redox potential of the fermentation medium, it is possible to regulate the metabolic flux distribution, electron flow, biosynthesis of various enzymes / proteins, and intracellular NADH levels in industrial microorganisms.
[0027] In the process of fermenting isoleucine (e.g., L-isoleucine) using threonine and glucose as a combined carbon source, in order to solve the problem of valine growing simultaneously as a byproduct during the fermentation of isoleucine (e.g., L-isoleucine), improve the fermentation conversion rate of isoleucine (e.g., L-isoleucine), and reduce the burden of separating valine from isoleucine during downstream extraction, the inventors of this application have significantly controlled the accumulation rate of valine by controlling the redox potential of the fermentation process in stages, thereby increasing the content ratio of isoleucine (e.g., L-isoleucine) to valine, thus enabling stable and efficient production of high-purity isoleucine (e.g., L-isoleucine).
[0028] This application provides a method for controlling the ratio of isoleucine to valine during fermentation, and the application of this method in any of the following: a) fermentation production of isoleucine; b) increasing isoleucine yield and / or isoleucine purity; and c) increasing the feed conversion rate in isoleucine production.
[0029] Unless otherwise specified, this application will be implemented using conventional microbiological, biochemical, and analytical chemistry techniques in the art.
[0030] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.
[0031] Unless otherwise specified, all laboratory reagents are commercially available products. definition
[0032] As used in this article, the term "oxidation-reduction potential (ORP)" reflects the macroscopic redox properties exhibited by all substances in an aqueous solution. A higher ORP indicates stronger oxidizing power; a lower ORP indicates stronger reducing power. A positive potential indicates that the solution exhibits a certain degree of oxidizing power, while a negative potential indicates that the solution exhibits a certain degree of reducing power. During fermentation, ORP serves as a highly sensitive parameter for real-time monitoring of the fermentation process. ORP reflects the relative strength of oxidizing or reducing power in the fermentation broth and is closely related to the growth status of microorganisms. By monitoring ORP, the fermentation process can be better controlled, ensuring that microorganisms grow and metabolize under optimal conditions.
[0033] As used herein, the term "aeration rate" refers to the volume of air passing through a unit volume of culture medium per minute during fermentation. Aeration rate is usually expressed as the aeration ratio, which is the ratio of the aeration rate per minute to the actual volume of culture medium in the tank.
[0034] This application provides a method for regulating the ratio of isoleucine to valine during fermentation, which includes:
[0035] When the valine content in the fermentation broth is 3.00~5.00 g / L, the redox potential of the fermentation broth is adjusted to -200~-175 mV.
[0036] In one or more embodiments of this application, the redox potential in the fermentation broth is increased from -320 to -280 mV (e.g., -308 mV) to -200 to -175 mV.
[0037] In one or more embodiments of this application, the redox potential in the fermentation broth is increased from -320 to -280 mV (e.g., -308 mV) to -196 to -175 mV.
[0038] In one or more embodiments of this application, the valine content in the fermentation broth can be 3.00~5.00 g / L, for example 3.00, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.30, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36, 3.37, 3.38, 3.39, 3.4 0, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.50, 3.51, 3.52, 3.53, 3.54, 3.55, 3.56, 3.57, 3.58, 3.59, 3.60, 3.61, 3.62, 3.63, 3.64, 3.6 5, 3.66, 3.67, 3.68, 3.69, 3.70, 3.71, 3.72, 3.73, 3.74, 3.75, 3.76, 3.77, 3.78, 3.79, 3.80, 3.81, 3.82, 3.83, 3.84, 3.85, 3.86, 3.87, 3.88, 3.89, 3.9 0, 3.91, 3.92, 3.93, 3.94, 3.95, 3.96, 3.97, 3.98, 3.99, 4.00, 4.01, 4.02, 4.03, 4.04, 4.05, 4.06, 4.07, 4.08, 4.09, 4.10, 4.11, 4.12, 4.13, 4.14, 4. 15, 4.16, 4.17, 4.18, 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4. 40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 4.50, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, 4.60, 4.61, 4.62, 4.63, 4.64, 4. 65, 4.66, 4.67, 4.68, 4.69, 4.70, 4.71, 4.72, 4.73, 4.74, 4.75, 4.76, 4.77, 4.78, 4.79, 4.80, 4.81, 4.82, 4.83, 4.84, 4.85, 4.86, 4.87, 4.88, 4.89, 4.90, 4.91, 4.92, 4.93, 4.94, 4.95, 4.96, 4.97, 4.98, 4.99, 5.00 g / L, or any value or range between any two of the above.
[0039] In one or more embodiments of this application, the valine content in the fermentation broth may be 3.00, 4.07, or 5.00 g / L.
[0040] In one or more embodiments of this application, the redox potential of the fermentation broth can be adjusted to -200 to -175 mV, for example -200, -199, -198, -197, -196, -195, -194, -193, -192, -191, -190, -189, -188, -187, -186, -185, -184, -183, -182, -181, -180, -179, -178, -177, -176, -175 mV, or any value or range between any two of the above values.
[0041] In one or more embodiments of this application, the redox potential of the fermentation broth can be adjusted to -196 to -175 mV, for example -196, -195, -194, -193, -192, -191, -190, -189, -188, -187, -186, -185, -184, -183, -182, -181, -180, -179, -178, -177, -176, -175 mV, or any value or range between any two of the above values.
[0042] In one or more specific embodiments of this application, the redox potential of the fermentation broth can be adjusted from -320 to -280 mV (e.g., -308 mV) to -196 mV.
[0043] In one or more specific embodiments of this application, the redox potential of the fermentation broth can be adjusted from -320 to -280 mV (e.g., -308 mV) to -180 mV.
[0044] In one or more specific embodiments of this application, the redox potential of the fermentation broth can be adjusted from -320 to -280 mV (e.g., -308 mV) to -175 mV.
[0045] In one or more embodiments of this application, the redox potential of the fermentation broth is adjusted by at least one of the following methods: adjusting the aeration rate and adjusting the stirring speed.
[0046] In one or more embodiments of this application, the redox potential of the fermentation broth is adjusted by regulating the aeration rate. In one or more embodiments of this application, the oxygen partial pressure of the culture medium is increased by increasing the aeration rate, thereby increasing the redox potential.
[0047] In one or more embodiments of this application, by adjusting the redox potential, the ratio of isoleucine to valine in the fermentation broth after fermentation / when the fermentation is finished / discharged is ≥8.00, for example, it can be 8.00, 8.01, 8.02, 8.03, 8.04, 8.05, 8.06, 8.07, 8.08, 8.09, 8.10, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 8.20, 8.21, 8.22, 8.23, 8.24, 8.25, 8.26, 8.27, 8.28, 8.29, 8.30, 8.31, 8.32, 8.33, 8.34, 8.35, 8.36, 8. 37, 8.38, 8.39, 8.40, 8.41, 8.42, 8.43, 8.44, 8.45, 8.46, 8.47, 8.48, 8.49, 8.50, 8.51, 8.52, 8.53, 8.54, 8.55, 8.56, 8.57, 8.58, 8.59, 8.60, 8.61, 8. 62, 8.63, 8.64, 8.65, 8.66, 8.67, 8.68, 8.69, 8.70, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.80, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8. 87, 8.88, 8.89, 8.90, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.00, 9.01, 9.02, 9.03, 9.04, 9.05, 9.06, 9.07, 9.08, 9.09, 9.10, 9.11, 9. 12, 9.13, 9.14, 9.15, 9.16, 9.17, 9.18, 9.19, 9.20, 9.21, 9.22, 9.23, 9.24, 9.25, 9.26, 9.27, 9.28, 9.29, 9.30, 9.31, 9.32, 9.33, 9.34, 9.35, 9.36, 9. 37, 9.38, 9.39, 9.40, 9.41, 9.42, 9.43, 9.44, 9.45, 9.46, 9.47, 9.48, 9.49, 9.50, 9.51, 9.52, 9.53, 9.54, 9.55, 9.56, 9.57, 9.58, 9.59, 9.60, 9.61, 9. 62, 9.63, 9.64, 9.65, 9.66, 9.67, 9.68, 9.69, 9.70, 9.71, 9.72, 9.73, 9.74, 9.75, 9.76, 9.77, 9.78, 9.79, 9.80, 9.81, 9.82, 9.83, 9.84, 9.85, 9.86, 9.87, 9.88, 9.89, 9.90, 9.91, 9.92, 9.93, 9.94, 9.95, 9.96, 9.97, 9.98, 9.99, 10.00, 10.01, 10.02, 10.03, 10.04, 10.05, 10.06, 10.07, 10.08, 10.09, 10.10, 10.11, 10.12, 10.13, 10.14, 10.15, 10.16, 10.17, 10.18, 10.19, 10.20, 10.2 10.22, 10.23, 10.24, 10.25, 10.26, 10.27, 10.28, 10.29, 10.30, 10.31, 10.32, 10.33, 10.34, 10.35, 10.36, 10.37, 10.38, 10.39, 10.40, 10.41, 10.42, 10.43, 10.44, 10.45, 10.46, 10.47, 10.48, 10.49, 10.50, or any value or range between any two of the above.
[0048] In one or more embodiments of this application, the strains used for fermentation are Escherichia coli, Brevibacterium flavum, Corynebacterium glutamicum, or Brevibacterium lactofermentum.
[0049] In one or more embodiments of this application, the strain used for fermentation is Escherichia coli, such as Escherichia coli with accession number CCTCC NO: M 20231916.
[0050] In one or more embodiments of this application, the method can be used to:
[0051] a) Fermentation production of isoleucine;
[0052] b) Increase isoleucine yield and / or isoleucine purity; and / or
[0053] c) Improve the raw material conversion rate in isoleucine production.
[0054] In one or more embodiments of this application, the method for producing isoleucine by fermentation may include the steps in the method for regulating the ratio of isoleucine to valine content during fermentation.
[0055] In one or more embodiments of this application, the method for increasing isoleucine production may include the steps in the method for regulating the ratio of isoleucine to valine content during fermentation.
[0056] In one or more embodiments of this application, the method for improving the purity of isoleucine may include the steps in the method for regulating the ratio of isoleucine to valine content during fermentation.
[0057] In one or more embodiments of this application, the method for improving the raw material conversion rate in isoleucine production may include the steps in the method for regulating the ratio of isoleucine to valine content during fermentation.
[0058] In one or more embodiments of this application, the fermentation production may include seed culture and fermentation culture.
[0059] In one or more embodiments of this application, the fermentation culture includes the step of regulating the ratio of isoleucine to valine content during fermentation.
[0060] In one or more embodiments of this application, the seed culture may include primary seed culture and secondary seed culture.
[0061] In one or more embodiments of this application, the culture medium used for the primary seed culture is LB liquid medium.
[0062] In one or more embodiments of this application, the culture medium used for the secondary seed culture comprises the following components: glucose 15-25 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 5-12 g / L, ammonium sulfate 3-10 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, pH 7.0-7.5.
[0063] In one or more specific embodiments of this application, the culture medium used for the secondary seed culture comprises the following components: 25 g / L glucose, 1.0 g / L magnesium sulfate, 8 g / L potassium dihydrogen phosphate, 7 g / L ammonium sulfate, 2.5 mg / L ferrous sulfate, 0.6 mg / L manganese sulfate, 0.7 mg / L zinc acetate, 0.03 mg / L copper chloride, and 1 g / L yeast extract, with the pH adjusted to 7.2 using sodium hydroxide.
[0064] In one or more embodiments of this application, the seed culture conditions are as follows: the strain is inoculated into a primary seed culture medium and cultured at 35-37°C until OD (Organic Degree) is reached. 600Inoculate the culture medium at a ratio of 4-6% (v / v) into secondary shake flasks when the OD reaches 2-4, and incubate at 35-37°C until the OD reaches 2-4. 600 Between 6 and 9.
[0065] In one or more specific embodiments of this application, the seed culture conditions are as follows: the strain is inoculated into a primary seed culture medium and cultured at 37°C until OD (Organic Degree) reaches 0.5%. 600 At step 3, inoculate 4% (v / v) into secondary shake flask culture medium and incubate at 37°C until OD reaches 30%. 600 Up to 6.
[0066] In one or more embodiments of this application, the culture medium used for the fermentation culture comprises the following components: corn steep liquor 3-6 g / L, glucose 25-35 g / L, magnesium sulfate 0.2-0.8 g / L, potassium dihydrogen phosphate 0.6-1.5 g / L, ammonium sulfate 1-5 g / L, diammonium hydrogen phosphate 2-5 g / L, ferrous sulfate 2-5 mg / L, threonine 45-50 g / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, with water as the solvent and a pH of 6.9-7.1.
[0067] In one or more specific embodiments of this application, the culture medium used for the fermentation culture comprises the following components: 4 g / L corn steep liquor, 30 g / L glucose, 0.5 g / L magnesium sulfate, 1.2 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 4 g / L diammonium hydrogen phosphate, 3 mg / L ferrous sulfate, 50 g / L threonine, 0.6 mg / L manganese sulfate, 0.8 mg / L zinc acetate, 0.05 mg / L copper chloride, and 2 g / L yeast extract, with water as the solvent and a pH of 6.9.
[0068] In one or more embodiments of this application, the fermentation culture conditions are as follows: the secondary seed culture is inoculated into the fermentation medium at 8-10% (v / v) and cultured.
[0069] In one or more specific embodiments of this application, the fermentation culture conditions are as follows: the secondary seed culture is inoculated into the fermentation medium at 8% (v / v) and cultured.
[0070] In one or more embodiments of this application, during fermentation, sugar is replenished at a rate of 1.2 to 1.6 g / L / h after the initial sugar is depleted.
[0071] In one or more specific embodiments of this application, during fermentation, sugar is replenished at a rate of 1.6 g / L / h after the initial sugar is depleted.
[0072] In one or more embodiments of this application, when isoleucine is produced by direct fermentation, the raw material conversion rate is the sugar-acid conversion rate, i.e., the conversion rate of glucose to isoleucine; when isoleucine is produced by precursor fermentation, the raw material conversion rate is the sugar-acid conversion rate and / or the conversion rate of threonine to isoleucine.
[0073] In one or more embodiments of this application, the isoleucine is L-isoleucine or D-isoleucine.
[0074] In one or more embodiments of this application, the isoleucine is L-isoleucine.
[0075] In one or more embodiments of this application, the isoleucine content in the fermentation product at the end of fermentation is ≥41.00 g / L, for example, it can be 41.00, 41.01, 41.02, 41.03, 41.04, 41.05, 41.06, 41.07, 41.08, 41.09, 41.10, 41.11, 41.12, 41.13, 41.14, 41.15, 41.16, 41.17, 41.18, 41.19, 41.20, 41.21, 41.22, 41.23, 41.24, 41.25, 41.26, 41.27, 41.28, 41.29, 41.30, 41.31, 41.32, 41. 33, 41.34, 41.35, 41.36, 41.37, 41.38, 41.39, 41.40, 41.41, 41.42, 41.43, 41.44, 41.45, 41.46, 41.47, 41.48, 41.49, 41.50, 41.51, 41.52, 41.53, 41 .54, 41.55, 41.56, 41.57, 41.58, 41.59, 41.60, 41.61, 41.62, 41.63, 41.64, 41.65, 41.66, 41.67, 41.68, 41.69, 41.70, 41.71, 41.72, 41.73, 41.74, 4 1.75, 41.76, 41.77, 41.78, 41.79, 41.80, 41.81, 41.82, 41.83, 41.84, 41.85, 41.86, 41.87, 41.88, 41.89, 41.90, 41.91, 41.92, 41.93, 41.94, 41.95 41.96, 41.97, 41.98, 41.99, 42.00, 42.01, 42.02, 42.03, 42.04, 42.05, 42.06, 42.07, 42.08, 42.09, 42.10, 42.11, 42.12, 42.13, 42.14, 42.15, 42.16 42.17, 42.18, 42.19, 42.20, 42.21, 42.22, 42.23, 42.24, 42.25, 42.26, 42.27, 42.28, 42.29, 42.30, 42.31, 42.32, 42.33, 42.34, 42.35, 42.36, 42.3 7, 42.38, 42.39, 42.40, 42.41, 42.42, 42.43, 42.44, 42.45, 42.46, 42.47, 42.48, 42.49, 42.50, 42.51, 42.52, 42.53, 42.54, 42.55, 42.56, 42.57, 42.58, 42.59, 42.60, 42.61, 42.62, 42.63, 42.64, 42.65, 42.66, 42.67, 42.68, 42.69, 42.70, 42.71, 42.72, 42.73, 42.74, 42.75, 42.76, 42.77, 42.78, 42.7 9. 42.80, 42.81, 42.82, 42.83, 42.84, 42.85, 42.86, 42.87, 42.88, 42.89, 42.90, 42.91, 42.92, 42.93, 42.94, 42.95, 42.96, 42.97, 42.98, 42.99, 43.00 g / L, or any value or range between any two of the above.
[0076] In one or more embodiments of this application, the ratio of isoleucine to valine in the fermentation product of isoleucine is ≥8.00, for example, it can be 8.00, 8.01, 8.02, 8.03, 8.04, 8.05, 8.06, 8.07, 8.08, 8.09, 8.10, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 8.20, 8.21, 8.22, 8.23, 8.24, 8.25, 8.26, 8.27, 8.28, 8.29, 8.30, 8.31, 8.32, 8.33, 8.34, 8.35, 8.36, 8.37, 8.38, or 8.3 9, 8.40, 8.41, 8.42, 8.43, 8.44, 8.45, 8.46, 8.47, 8.48, 8.49, 8.50, 8.51, 8.52, 8.53, 8.54, 8.55, 8.56, 8.57, 8.58, 8.59, 8.60, 8.61, 8.62, 8.63, 8.6 4, 8.65, 8.66, 8.67, 8.68, 8.69, 8.70, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.80, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.8 9, 8.90, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.00, 9.01, 9.02, 9.03, 9.04, 9.05, 9.06, 9.07, 9.08, 9.09, 9.10, 9.11, 9.12, 9.13, 9. 14, 9.15, 9.16, 9.17, 9.18, 9.19, 9.20, 9.21, 9.22, 9.23, 9.24, 9.25, 9.26, 9.27, 9.28, 9.29, 9.30, 9.31, 9.32, 9.33, 9.34, 9.35, 9.36, 9.37, 9.38, 9. 39, 9.40, 9.41, 9.42, 9.43, 9.44, 9.45, 9.46, 9.47, 9.48, 9.49, 9.50, 9.51, 9.52, 9.53, 9.54, 9.55, 9.56, 9.57, 9.58, 9.59, 9.60, 9.61, 9.62, 9.63, 9. 64, 9.65, 9.66, 9.67, 9.68, 9.69, 9.70, 9.71, 9.72, 9.73, 9.74, 9.75, 9.76, 9.77, 9.78, 9.79, 9.80, 9.81, 9.82, 9.83, 9.84, 9.85, 9.86, 9.87, 9.88, 9.89, 9.90, 9.91, 9.92, 9.93, 9.94, 9.95, 9.96, 9.97, 9.98, 9.99, 10.00, 10.01, 10.02, 10.03, 10.04, 10.05, 10.06, 10.07, 10.08, 10.09, 10.10, 10.11, 10.12, 10.13, 10.14, 10.15, 10.16, 10.17, 10.18, 10.19, 10.20, 10.21, 10. 22, 10.23, 10.24, 10.25, 10.26, 10.27, 10.28, 10.29, 10.30, 10.31, 10.32, 10.33, 10.34, 10.35, 10.36, 10.37, 10.38, 10.39, 10.40, 10.41, 10.42, 10.43, 10.44, 10.45, 10.46, 10.47, 10.48, 10.49, 10.50, or any value or range between any two of the above.
[0077] In one or more embodiments of this application, if the redox potential of the fermentation broth is not increased, the accumulation rate of valine in the fermentation broth will continue to rise when the residual sugar is below 1 g / L. This application significantly inhibits the accumulation rate of valine while maintaining the accumulation rate of isoleucine by increasing the redox potential of the fermentation broth, thereby improving the sugar conversion rate of isoleucine and the conversion rate of threonine as a precursor amino acid.
[0078] In one or more embodiments of this application, the method for controlling the ratio of isoleucine to valine during fermentation as described in this application achieves at least one of the following benefits:
[0079] It alleviated the feedback inhibition of threonine dehydratase by the accumulation of isoleucine during the fermentation of isoleucine, which uses threonine as a precursor amino acid.
[0080] As isoleucine accumulates, the flow of pyruvate to valine is reduced, thus achieving the goal of controlling valine growth during isoleucine fermentation.
[0081] After about 50 hours of fermentation, the isoleucine yield can reach over 41 g / L.
[0082] The ratio of isoleucine to valine in the fermentation products is ≥8;
[0083] The method is simple and easy to implement, and effectively reduces the impurity content of valine while increasing isoleucine production; and
[0084] The method described is stable and efficient, and is suitable for large-scale production.
[0085] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments. Example
[0086] The fermentation strain used in this application is the isoleucine-producing strain *Escherichia coli* IVL016, deposited at the China Center for Type Culture Collection on October 16, 2023, with accession number CCTCC NO: M 20231916. This strain was obtained by molecular modification (HHVAL-001, ldhA::ilvIH, tdh::leuDH), combined mutagenesis with UV and ARTP, and then screening for isoleucine oxime resistance, from the L-valine strain HHVAL-001 (CCTCC NO: M 2020321).
[0087] The primary seed culture medium was LB liquid medium.
[0088] The secondary shake flask culture medium consists of the following components: glucose 25 g / L, magnesium sulfate 1.0 g / L, potassium dihydrogen phosphate 8 g / L, ammonium sulfate 7 g / L, ferrous sulfate 2.5 mg / L, manganese sulfate 0.6 mg / L, zinc acetate 0.7 mg / L, copper chloride 0.03 mg / L, and yeast extract 1 g / L, with the pH adjusted to 7.2 using sodium hydroxide.
[0089] The fermentation medium consists of the following components: corn steep liquor 4 g / L, glucose 30 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1.2 g / L, ammonium sulfate 4 g / L, diammonium hydrogen phosphate 4 g / L, ferrous sulfate 3 mg / L, threonine 50 g / L, manganese sulfate 0.6 mg / L, zinc acetate 0.8 mg / L, copper chloride 0.05 mg / L, yeast extract 2 g / L, water as solvent, pH 6.9.
[0090] Unless otherwise stated, the detection methods used in this application are as follows:
[0091] OD value: Measured at a wavelength of 600 nm using a UV-5200 (PC) visible spectrophotometer;
[0092] pH value: Monitored online using an InPro3100i / SG / 325 pH sensor;
[0093] Oxidation-reduction potential value: Online monitoring by InPro3100i / SG / 325 ORP sensor (Dibil bioreactor);
[0094] Glucose content: determined using an SBA-40D biosensor analyzer;
[0095] L-Isoleucine was detected by high-performance liquid chromatography (HPLC). The specific steps are as follows:
[0096] The fermentation broth was sampled and placed in a centrifuge tube. It was centrifuged at 5000 rpm for 1 minute. The supernatant was taken and diluted 100 times. The diluted solution was filtered through a 0.22 μm microporous membrane before being injected for testing.
[0097] Chromatographic conditions: Column: Yuexu Ultimate HILIC Amphion II (4.6×150mm, 5μm); Mobile phase: Acetonitrile: 0.05M potassium dihydrogen phosphate = 3:1; Column temperature: 35℃; Flow rate: 1.0mL / min; Injection volume: 10μL; Detector: UV 206nm;
[0098] The preparation method of the mobile phase (acetonitrile / phosphate mixed solution) is as follows: Weigh 0.05 M potassium dihydrogen phosphate and dissolve it in ultrapure water. Adjust the pH to 3.0 with phosphoric acid. After filtration through a 0.22 μm filter membrane, add the corresponding volume of acetonitrile according to the ratio of acetonitrile:0.05 M potassium dihydrogen phosphate = 3:1. Mix well and sonicate for 20 minutes until no bubbles are present.
[0099] Preparation of standard solutions: Prepare 1 g / L L-isoleucine solution, 1 g / L L-valine solution, and 1 g / L L-threonine solution (all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.), dissolve by sonication, filter through a 0.22 μm membrane, and store at -20℃ for later use.
[0100] Threonine conversion rate = Isoleucine yield / Threonine dosage
[0101] Sugar conversion rate = Isoleucine production / Sugar consumption
[0102] Example 1: 5L control vessel - no adjustment of redox potential
[0103] Pick a single colony from the plate and inoculate it into a primary seed culture medium, then incubate at 37°C until OD500. 600 At step 3, inoculate 4% (v / v) into secondary shake flask culture medium for fermentation, and culture at 37°C until OD reaches 300°C. 600 At step 6, inoculate into a 5L fermenter at 8% (v / v). Initial volume is 2L, rotation speed is 200rpm, and airflow is 0.1vvm. During fermentation, pH is maintained at 6.9 using 12.5% ammonia. After initial sugar depletion, sugar is replenished at a rate of 1.6g / L / h. The time points of glucose and threonine depletion during fermentation are monitored, and the redox potential and OD of the fermentation broth are also measured. 600 The levels of residual sugar, L-isoleucine production, valine production, and threonine residue were measured. Fermentation was continued until the threonine residue reached approximately 3 g / L. The fermentation results are shown in Table 1.
[0104] Example 2: 5L experimental vessel - regulation of redox potential
[0105] Pick a single colony from the plate and inoculate it into a primary seed culture medium, then incubate at 37°C until OD500. 600 At step 3, inoculate 4% (v / v) into secondary shake flask culture medium for fermentation, and culture at 37°C until OD reaches 300°C. 600 At step 6, inoculate 8% (v / v) into a 5L fermenter. Initial volume is 2L, rotation speed is 200rpm, and airflow is 0.1vvm. During fermentation, pH is maintained at 6.9 using 12.5% ammonia. After the initial sugar is depleted, sugar is replenished at a rate of 1.6g / L / h. When valine production reaches approximately 4g / L, increase the airflow to raise the redox potential from -308mV to -196mV. Monitor the time points of glucose and threonine depletion during fermentation, and monitor and measure the redox potential and OD of the fermentation broth. 600 The levels of residual sugar, L-isoleucine production, valine production, and threonine residue were measured. Fermentation was continued until the threonine residue reached approximately 3 g / L. The fermentation results are shown in Table 2.
[0106] From the growth curves of L-isoleucine and valine in Examples 1 and 2, it can be seen that when valine accumulates to about 4 g / L, increasing the redox potential to -196 mV can effectively inhibit the growth rate of valine. Figure 1 Its regulatory mechanism may be related to the expression activity of bacterial enzymes and the conformation of enzyme-substrate binding.
[0107] Table 1. Relevant fermentation data from the 5L control tank in Example 1
[0108]
[0109] Table 2. Relevant fermentation data from the 5L experimental tank in Example 2
[0110]
[0111] Example 3
[0112] The difference between this embodiment and Embodiment 2 is that when the valine yield reaches 3.00 g / L during fermentation, the air volume is increased to raise the redox potential from -308 mV to -175 mV. After being removed from the tank, the yield of L-isoleucine in the fermentation broth is 41.20 g / L, the yield of valine is 3.93 g / L, and the ratio of L-isoleucine to valine is 10.48.
[0113] Example 4
[0114] The difference between this embodiment and Embodiment 2 is that when the valine yield reaches 5.00 g / L during fermentation, the air volume is increased to raise the redox potential from -308 mV to -180 mV. After being removed from the tank, the yield of L-isoleucine in the fermentation broth is 42.40 g / L, the yield of valine is 5.27 g / L, and the ratio of L-isoleucine to valine is 8.04.
[0115] It should be noted that the differences between the data in Tables 1 and 2 before adjusting the redox potential are normal batch variations. The presence of L-isoleucine and valine fermentation yields at 0h is because the strain produces small amounts of L-isoleucine and valine during the two-stage seed culture process before fermentation in the tank. Therefore, the content of L-isoleucine and valine is not zero at 0h during tank fermentation monitoring.
[0116] It is understood that although the inventions described in this application are in the specific forms described above, these inventions are not limited to the specific content described in these specific forms. It will be apparent to those skilled in the art that various equivalent changes can be made to the technical features contained in the inventions described herein without departing from the spirit of the inventions described herein, and all such changes should fall within the scope of the inventions.
Claims
1. Methods for regulating the ratio of isoleucine to valine during fermentation, including: When the valine content in the fermentation broth is 3.00~5.00 g / L, the redox potential of the fermentation broth is adjusted to -200~-175 mV; Preferably, the isoleucine is L-isoleucine.
2. The method of claim 1, wherein the redox potential of the fermentation broth is adjusted to -196 to -175 mV.
3. The method of claim 1 or 2, wherein the redox potential of the fermentation broth is adjusted by at least one of the following methods: adjusting the aeration rate and adjusting the stirring speed.
4. The method of claim 3, wherein the redox potential of the fermentation broth is adjusted by adjusting the aeration rate.
5. The method according to any one of claims 1-4, wherein the ratio of isoleucine to valine in the fermentation broth after fermentation is ≥8.
00.
6. The method according to any one of claims 1-5, wherein the strain used for fermentation is Escherichia coli, Brevibacterium flavum, Corynebacterium glutamicum, or Brevibacterium lactofermentum.
7. The method of claim 6, wherein the strain used for fermentation is Escherichia coli.
8. The method of claim 7, wherein the Escherichia coli is Escherichia coli with accession number CCTCC NO: M 20231916.
9. The method according to any one of claims 1-8, wherein it is used for: a) Fermentation production of isoleucine; b) Increase isoleucine yield and / or isoleucine purity; and / or c) Improve the raw material conversion rate in isoleucine production.
10. The method of claim 9, wherein the raw material conversion rate is the conversion rate of glucose to isoleucine and / or the conversion rate of threonine to isoleucine.