Genetically engineered bacterium for co-production of L-threonine and L-valine and fermentation method of genetically engineered bacterium

Abstract

The invention provides a genetically engineered bacterium for co-production of L-threonine and L-valine and a fermentation method of the genetically engineered bacterium, and belongs to the technical field of bioengineering and fermentation, the genetically engineered bacterium co-expresses key genes alsS, leuDH, ilvC and ilvD of an L-valine synthesis pathway and efflux related genes ygaZ and ygaH on plasmids, the expression of the L-valine synthesis pathway is regulated by combining with a promoter, and the L-threonine and L-valine co-production is realized. The genetically engineered bacteria for co-production of the L-threonine and the L-valine are obtained; the invention also discloses a fermentation method of the genetically engineered bacterium. According to the technical scheme provided by the invention, efficient co-production of L-threonine and L-valine is realized, the synthesis efficiency of thalli is improved, and the fermentation production cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a genetically engineered bacterium for co-production of L-threonine and L-valine and a fermentation method thereof. BACKGROUND

[0002] L-threonine and L-valine are both essential amino acids for animals, which play an important role in promoting growth and metabolism. L-threonine is a limiting amino acid for pigs, poultry and other animals, second only to lysine and methionine, and must be supplemented through feed. Threonine can promote animal growth, improve muscle deposition and daily weight gain, and improve protein utilization and feed conversion rate. At the same time, threonine is an important component of immunoglobulin and mucin, which can enhance the immunity and disease resistance of animals. In addition, threonine is involved in the synthesis of intestinal mucosal proteins, which can improve intestinal health, protect intestinal barrier function, and reduce diarrhea and other problems. L-valine belongs to branched-chain amino acids (BCAA), which participates in energy metabolism and muscle synthesis together with leucine and isoleucine. In the diet of broilers and laying hens, valine as a limiting amino acid can directly affect weight gain, egg production rate and egg quality. Supplementing valine can improve the feed intake of animals and improve their production performance under high temperature or stress conditions. Therefore, a reasonable proportion of valine, threonine, lysine and other amino acids can optimize the balance of amino acids and improve protein deposition efficiency.

[0003] Currently, the industrial production of L-threonine and L-valine mainly relies on microbial fermentation of Escherichia coli and Corynebacterium glutamicum. Taking E. coli as an example, the biosynthesis of L-threonine starts from the aspartate pathway, which gradually converts aspartate into threonine through key enzymes such as aspartate kinase and cysteine dehydrogenase. The synthesis of L-valine starts from pyruvate, which is catalyzed by acetyl lactate synthase IlvGM to generate acetyl lactate (alpha-acetyl-alpha-hydroxy valeric acid), followed by reduction dehydrogenation catalyzed by acetyl hydroxy acid isomerase IlvC and dihydroxy acid dehydrase IlvD, and transamination catalyzed by branched-chain amino acid transaminase IlvE to generate valine. The expression of endogenous valine synthesis genes such as ilvGM, ilvD and ilvC in E. coli is inhibited by the feedback of end product valine and other similar products branched-chain amino acids, and the substrate specificity of IlvE is poor, which not only catalyzes the synthesis of valine, but also catalyzes the conversion of leucine and isoleucine, resulting in difficulty in significantly improving the yield of valine.

[0004] In order to improve the yield of valine in the prior art, emphasis is mainly placed on enhancing the flux of pyruvate to acetolactate. As disclosed in the patent with the publication number CN118126917A and the title of a genetically engineered strain for enhancing co-production of L-isoleucine and L-valine and application thereof, overexpression of the acetolactate synthase gene alsS from Bacillus subtilis in Escherichia coli by using an inducible promoter can significantly improve the controllable conversion of carbon flow flux from pyruvate to acetolactate, thereby improving the yield of valine. In addition, it is reported that branched-chain amino acid dehydrogenase LeuDH from Bacillus subtilis is NADH-dependent and can catalyze the transamination step in the valine synthesis pathway under limited oxygen conditions, which can replace IlvE to specifically promote the synthesis of valine. Therefore, reasonable combination of the two exogenous genes and the endogenous valine synthesis gene of Escherichia coli can construct an artificial pathway for efficient synthesis of valine. However, the co-production of L-threonine is not involved in the above technical solutions.

[0005] In addition, the fermentation production processes of L-threonine and L-valine are quite different. L-threonine is an aerobic high-density fermentation with high oxygen demand. In the late fermentation stage, due to the high biomass and vigorous aerobic metabolism, the dissolved oxygen is insufficient, which significantly reduces the production rate of L-threonine and the glucose conversion efficiency. L-valine generally adopts a two-stage fermentation, and the growth and production stages of the bacteria are clearly distinguished. The bacteria need aerobic conditions to ensure rapid growth in the early stage, while the synthesis of L-valine mainly occurs in the limited oxygen stage, which also limits the overall yield of L-valine and the glucose conversion efficiency. In addition, when using a chemical inducible promoter to overexpress key genes to improve the yield of valine, it is necessary to add exogenous chemical inducers (such as IPTG, arabinose, etc.) and ensure the effective expression of proteins under aerobic conditions. However, induction of high-density bacteria will sharply increase the metabolic burden and oxygen consumption of the bacteria, leading to rapid reduction of dissolved oxygen level and damage to protein synthesis. The contradictory requirements of protein expression and valine synthesis for dissolved oxygen conditions further limit the improvement of the yield and efficiency of L-valine. SUMMARY

[0006] Therefore, the present application provides a genetically engineered strain for co-producing L-threonine and L-valine and a fermentation method thereof, which realizes efficient co-production of L-threonine and L-valine, improves the synthesis efficiency of the bacteria, and reduces the cost of fermentation production.

[0007] To achieve the above object, the application provides a genetically engineered bacterium for co-production of L-threonine and L-valine, which co-expresses key genes alsS, leuDH, ilvC, ilvD of L-valine synthetic pathway and efflux-related genes ygaZ, ygaH on a plasmid, and regulates expression of the L-valine artificial synthesis pathway by a promoter to obtain the genetically engineered bacterium for co-production of L-threonine and L-valine.

[0008] The technical solution provided by the application constructs different L-valine artificial high-efficiency synthesis pathways, uses the exogenous alsS gene to efficiently guide the flow of pyruvate precursors to L-valine synthesis, further drives product accumulation by combining the exogenous leuDH gene, and realizes efficient production of L-valine under the regulation of different promoters in cooperation with valine synthesis-related genes ilvC, ilvD and efflux-related genes ygaZ, ygaH, and introduces the L-valine artificial high-efficiency synthesis pathway into an L-threonine production bacterium to form a genetically engineered strain for co-production of L-threonine and L-valine, thereby realizing efficient co-production of L-threonine and L-valine.

[0009] Optionally, the promoter is an inducible promoter, and the genetically engineered bacterium is Escherichia coli.

[0010] Optionally, the inducible promoter includes a chemical inducible promoter and a physical inducible promoter.

[0011] Optionally, the chemical inducible promoter is a Ptrc-type promoter or a Ptrc-Plac-type promoter, and the physical inducible promoter is a Pfnr promoter.

[0012] Optionally, when the Ptrc promoter is used for regulation, the order of the key genes and the efflux-related genes is Ptrc-alsS-ilvD-ilvC-leuDH-ygaZH.

[0013] Optionally, when the Ptrc-Plac promoter is used for regulation, the order of the key genes and the efflux-related genes is Ptrc-ilvD-ilvC-ygaZH-Plac-alsS-leuDH.

[0014] Optionally, when the Pfnr promoter is used for regulation, the order of the key genes and the efflux-related genes is Pfnr-ilvD-ilvC-ygaZH-alsS-leuDH.

[0015] To achieve the above objectives, the present invention also provides a fermentation method for a genetically engineered bacterium that co-produces L-threonine and L-valine. When a physically inducible promoter is used, the fermentation method is a two-stage fermentation. The conditions for the first stage of fermentation include a temperature of 35~38℃ and a dissolved oxygen concentration of ≥25%, and the conditions for the second stage of fermentation are a temperature of 37~42℃ and a dissolved oxygen concentration of ≤10%.

[0016] The technical solution of this invention combines the constructed genetically engineered bacteria with a fermentation process to achieve the co-production of L-threonine and L-valine. The first stage provides sufficient dissolved oxygen concentration to ensure rapid growth of the bacteria and efficient synthesis of L-threonine. When the OD tends to stabilize and the L-threonine synthesis rate decreases significantly, the process transitions to the second stage, where the synthesis of L-valine is initiated by limiting the dissolved oxygen concentration.

[0017] Optionally, when the promoter is of the Pfnr type, the expression of all genes in the L-valine artificial synthesis pathway is initiated after fermentation reaches the second stage. The fermentation conditions for the first stage are: pH 6.5~7.5, temperature 36~38℃, rotation speed 200~1000 r / min, and dissolved oxygen concentration ≥25%; the fermentation conditions for the second stage are: temperature 37~40℃, rotation speed 300~400 r / min, and dissolved oxygen concentration ≤10%.

[0018] The technical solution provided by this invention uses the Pfnr dissolved oxygen-inducible promoter, which is in an inhibited state under aerobic conditions and initiates continuous, high-level expression of the gene based on changes in physical and physiological conditions under oxygen-limited conditions, which is highly compatible with the fermentation production process of L-valine. The induction process is synchronized with the metabolic state of the cells, reducing the metabolic burden and avoiding problems such as sudden drop in dissolved oxygen and acetic acid accumulation encountered in chemically inducible systems. It does not require the addition of expensive exogenous chemical inducers (such as IPTG), is highly efficient and energy-saving, low in cost, and the fermentation process is simplified and easy to scale up, making it very suitable for industrial high-density fermentation production.

[0019] Optionally, when using a chemically inducible promoter, the fermentation method is a two-stage fermentation. The conditions for the first stage fermentation include a temperature of 35-38°C and a dissolved oxygen concentration of ≥25%, and the conditions for the second stage fermentation are a temperature of 37-42°C and a dissolved oxygen concentration of ≤10%. Between the first stage fermentation and the second stage fermentation, there is also an induction expression stage, in which a chemical inducer is added, and the induction expression conditions are the same as those for the first stage fermentation. The duration of the induction expression stage is 2-4 hours.

[0020] Optionally, when the promoter is of the Ptrc type, a high-strength Ptrc promoter induced by IPTG (isopropyl-β-D-thiogalactopyranoside) is used to control the expression of all genes in the L-valine artificial synthesis pathway. The fermentation conditions for the first stage are: pH 6.5~7.5, temperature 36~38℃, rotation speed 200~1000 r / min, and dissolved oxygen concentration ≥25%; the fermentation conditions for the second stage are: temperature 37~40℃, rotation speed 300~400 r / min, and dissolved oxygen concentration ≤10%; an induction expression stage is also included between the first stage fermentation and the second stage fermentation, wherein IPTG is added during the induction expression stage, and the induction expression stage lasts for 2~4 hours.

[0021] Optionally, the conditions for the induction expression stage are: pH 6.5-7.5, temperature 36-38℃, rotation speed 200-1000 r / min, dissolved oxygen concentration ≥25%, and the concentration of IPTG in the culture medium after adding IPTG is 0.1-0.5 mM.

[0022] Optionally, when the promoter is of the Ptrc-Plac type, a high-strength Ptrc promoter and a medium-strength Plac promoter induced by IPTG (isopropyl-β-D-thiogalactoside) are used to control the co-expression of different genes in the L-valine artificial synthesis pathway, respectively. The fermentation conditions for the first stage are: pH 6.5-7.5, temperature 36-38℃, rotation speed 200-1000 r / min, and dissolved oxygen concentration ≥25%; the fermentation conditions for the second stage are: temperature 37-40℃, rotation speed 300-400 r / min, and dissolved oxygen concentration ≤10%; an induction expression stage is also included between the first stage fermentation and the second stage fermentation, wherein IPTG is added during the induction expression stage, and the induction expression stage lasts for 2-4 hours.

[0023] Optionally, the conditions for the induction expression stage are: pH 6.5-7.5, temperature 36-38℃, rotation speed 200-1000 r / min, dissolved oxygen concentration ≥25%, and the concentration of IPTG in the culture medium after adding IPTG is 0.1-0.5 mM.

[0024] Optionally, the dissolved oxygen concentration is controlled by the aeration rate. During the first stage of fermentation, the aeration rate is 2-5 L / min, and during the second stage of fermentation, the aeration rate is 1-2 L / min.

[0025] To achieve the above objectives, the present invention provides a fermentation method for producing L-threonine and L-valine by genetically engineered bacteria that co-produce L-threonine and L-valine, resulting in a mixture of L-threonine and L-valine for use in the feed industry.

[0026] The above-described technical solution of the present invention has at least the following beneficial effects: 1. The technical solution of this invention constructs an artificial synthesis pathway for L-valine and combines different promoter regulation strategies to rationally regulate the expression intensity of related genes and control the directional allocation of carbon flux, thus successfully constructing different genetically engineered strains and obtaining different genetically engineered strains that co-produce L-threonine and L-valine.

[0027] 2. The technical solution provided by this invention constructs an efficient synthesis pathway for L-valine and introduces it into L-threonine-producing bacteria, making the fermentation processes of L-valine and L-threonine complementary. Sufficient dissolved oxygen in the early stage of fermentation can effectively ensure the rapid growth of the bacteria and the efficient synthesis of L-threonine. When the dissolved oxygen is insufficient due to excessively high bacterial density, causing a decrease in the synthesis efficiency of L-threonine, the process switches to oxygen-limited conditions to specifically and efficiently synthesize L-valine, thereby achieving the co-production of L-threonine and L-valine, making full use of the synthetic potential of the bacteria, improving glucose conversion rate, and effectively reducing production costs.

[0028] 3. The genetically engineered bacteria and supporting fermentation process constructed by the technical solution of this invention can achieve the co-production of L-threonine and L-valine, which greatly improves the production efficiency of bacteria and the utilization rate of total sugar; the produced L-threonine and L-valine mixture can be directly used in the feed application field, effectively reducing the fermentation production cost and showing good industrial application prospects. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0030] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and experimental procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, metabolic engineering, and fermentation engineering used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0031] "Genetically engineered bacteria" refers to microbial strains with specific target traits obtained through gene knockout, mutation, gene insertion, or plasmid expression.

[0032] A promoter is a DNA sequence that regulates the transcriptional expression of downstream genes.

[0033] In this instruction manual, "organic acids" generally refers to common organic acids such as acetic acid, lactic acid, and succinic acid produced during fermentation.

[0034] Biomass OD 600 "OD" refers to the absorbance of a solution at a wavelength of 600 nm. 600 This is a standard method for tracking the growth of microorganisms in liquid cultures; using a culture medium without added bacteria as a blank, followed by quantitative incubation of the culture medium containing bacteria. OD 600 It can be detected using commercial instruments.

[0035] A plasmid is a DNA construct that contains a DNA sequence that is operatively linked to a suitable control sequence to express a target gene in a suitable host.

[0036] The relevant culture medium involved in this invention is prepared as follows: LB medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0037] Shake flask seed culture medium: peptone 10 g / L, yeast extract 5 g / L, KH2PO4 1 g / L, pH=6.5-7.5.

[0038] Shake-flask fermentation medium: glucose 40 g / L, corn steep liquor 3 g / L, (NH4)2SO4 10 g / L, KH2PO4 2 g / L, MgSO4 1 g / L, choline chloride 0.2 g / L, FeSO4 0.01 g / L, MnSO4 0.01 g / L, vitamin B1 0.005 g / L, vitamin B3 0.005 g / L, vitamin B6 0.005 g / L, biotin 0.002 g / L, pH=6.5-7.5.

[0039] The fermenter culture medium contains carbon source, nitrogen source, inorganic salts, and nutrient growth factors. The preferred composition is: glucose 20-40 g / L, corn steep liquor 10-30 g / L, yeast extract 1-5 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 2-8 g / L, MgSO4 1-4 g / L, FeSO4 0.01-0.5 g / L, MnSO4 0.01-0.5 g / L, choline chloride 0.01-0.5 g / L, vitamin B1 0.001-0.05 g / L, vitamin B3 0.001-0.05 g / L, vitamin B6 0.001-0.05 g / L, biotin 0.001-0.05 g / L, trace metal salt mother liquor 2 mL / L, pH=6.5-7.5.

[0040] The formula for trace metal mother liquor is as follows: zinc sulfate 1 g / L, manganese sulfate 1 g / L, copper sulfate 0.5 g / L, boric acid 0.15 g / L, ammonium molybdate 0.1 g / L, ferrous sulfate 1 g / L, and cobalt chloride 1.5 g / L.

[0041] The shake-flask fermentation in this invention includes the following steps: Inoculate 0.2 mL of bacterial culture (glycerol-freezed bacteria or fresh bacterial suspension) into a 250 mL Erlenmeyer flask containing 50 mL of shake-flask seed culture medium. Incubate at 220 rpm and 37°C for 8-10 hours. Then, transfer the shake-flask seed culture at a ratio of 5-10% (v:v) to a 150 mL Erlenmeyer flask containing 30 mL of shake-flask fermentation medium. Incubate at 220 rpm and 37°C for 12 hours. After inducing with 0.2 mM IPTG for 2 hours, tighten the cap (using Ptrc or Ptrc-Plac promoters) or directly tighten the cap (using Pfnr promoters) to initiate L-valine synthesis. Continue incubation for 24 hours, and measure OD at different time points. 600 Values ​​and the yield of corresponding amino acids.

[0042] The fermentation process in the fermenter of this invention includes the following steps: Inoculate 1 mL of bacterial culture (glycerol-freezed bacteria or fresh bacterial suspension) into a 1 L Erlenmeyer flask containing 200 mL of shake-flask seed culture medium. After incubating at 220 rpm and 37°C for 8-10 hours, transfer the culture at a rate of 1-10% (v:v) to a 5 L fermenter containing 2 L of fermenter culture medium. Maintain the pH of the fermenter at 6.5-7.5 using ammonia, keep the temperature constant at 37°C, maintain an aeration rate of 2-5 L / min, and control the dissolved oxygen at approximately 15-30%. Maintain the glucose concentration in the fermenter at 0.5-2.0 g / L by adding 50-70% glucose solution. When fermentation has progressed for approximately 18-22 hours, add 0.1 mM IPTG for 2-3 hours of induction (chemical induction) and then transfer to anaerobic fermentation, or directly transfer to anaerobic fermentation (physical induction). Anaerobic fermentation conditions: fixed rotation speed of 300-400 rpm, temperature controlled at 37-40°C, and aeration rate adjusted to 1-2 L / min. Odion oxidase (OD) was measured at regular intervals throughout the fermentation process. 600 Values ​​and the yield of corresponding amino acids.

[0043] The determination of amino acid concentration in this invention includes the following steps: Amino acid standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). 1 mL of fermentation broth was centrifuged at 10000 rpm for 5 min to remove bacterial cells. The filtrate was filtered through a 0.22 μm filter membrane. After appropriate dilution, the amino acid concentration in the sample was determined using OPA-derived high-performance liquid chromatography (HPLC) (https: / / www.agilent.com / library). The HPLC system used was a Shimadzu Nexera LC-40, and the column was an Agilent ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5 μm). The detector was a DAD diode array detector with a detection wavelength of 338 nm and a reference wavelength of 390 nm. The mobile phase composition, ratio, flow rate, and column temperature were all set according to the methods described above.

[0044] Example 1 This invention provides a genetically engineered bacterium that co-produces L-threonine and L-valine, comprising the following steps: Amplifying the acetolactate synthase gene alsS and the branched-chain amino acid dehydrogenase gene leuDH from Bacillus subtilis via PCR, and splicing them together with the L-valine synthesis genes ilvD and ilvC and the branched-chain amino acid efflux gene ygaZH from Escherichia coli onto a pZA vector backbone containing a Ptrc promoter to construct the inducible expression plasmid pZA-Ptrc-alsS-ilvD-ilvC-leuDH-ygaZH for the L-valine artificial synthesis pathway. Introducing the above expression plasmid into an L-threonine-producing bacterium yields a genetically engineered bacterium that co-produces L-threonine and L-valine, namely, a Ptrc-type L-valine artificial synthesis pathway genetically engineered bacterium (Ptrc-type engineered bacterium).

[0045] The Ptrc-type engineered bacteria were shake-flask fermented to verify whether L-threonine and L-valine were produced in the product.

[0046] 0.2 mL of Ptrc-type engineered bacterial culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of shake-flask seed culture medium. After incubation at 220 rpm and 37°C for 8-10 hours, the shake-flask seed culture was transferred at a ratio of 5-10% (v:v) to a 150 mL Erlenmeyer flask containing 30 mL of shake-flask fermentation medium. After incubation at 220 rpm and 37°C for 12 hours, IPTG was added to a final concentration of 0.2 mM. After induction for 2 hours, the flask was capped and incubated for another 24 hours. The fermentation results showed that the yield of L-threonine was 3.5 g / L and the yield of L-valine was 1.9 g / L. It can be seen that the Ptrc-type engineered bacteria prepared by constructing an artificial and efficient synthesis pathway for L-valine in this embodiment are genetically engineered bacteria that co-produce L-threonine and L-valine.

[0047] This invention also provides a fermentation method for a genetically engineered bacterium that co-produces L-threonine and L-valine, comprising the following steps: 1 mL of Ptrc-type engineered bacterial culture is inoculated into a 1 L Erlenmeyer flask containing 200 mL of shake-flask seed culture medium. After culturing at 220 rpm and 37°C for 8-10 hours, the culture is transferred at a 5-10% (v:v) inoculation rate to a 5 L fermenter containing 2 L of fermenter culture medium. The pH is maintained at 6.5-7.5 using ammonia, the temperature at 37°C, the aeration rate at 2-5 L / min, and the dissolved oxygen controlled above 25%. The sugar concentration is maintained at approximately 0.5-2.0 g / L by adding 50% glucose solution. When fermentation has progressed for approximately 16-20 hours, IPTG is added to a final concentration of 0.2 mM. After induction for 2 hours, the fermentation is transferred to aerobic fermentation, with the fermentation speed fixed at 300 rpm, the aeration rate adjusted to 1 L / min, and the dissolved oxygen dropping below 10%. Odion precipitate (OD) is measured periodically throughout the fermentation process. 600 The values ​​and corresponding amino acid yields were recorded. Fermentation results are shown in Table 1.

[0048] Table 1. Results of co-production of threonine and valine in a 5-liter fermenter using Ptrc-type engineered bacteria.

[0049] As shown in Table 1, the bacterial cell growth during the L-threonine synthesis stage is very slow, with an OD of 20 hours. 600 The biomass was only 15.8 g / L, with L-threonine production at 22 g / L and L-valine production at 4.6 g / L. Furthermore, due to the continuous decrease in biomass, IPTG induction was not continued during fermentation. The results showed that the artificial L-valine synthesis pathway exhibited significant background leakage expression without IPTG induction. Moreover, due to the high expression intensity of the Ptrc-type promoter, the highly active acetolactate synthase AlsS concentrated pyruvate into the valine synthesis pathway, severely interfering with normal cell growth and limiting L-threonine synthesis. Optimization of carbon flux allocation at pyruvate metabolic nodes is needed to rationally balance cell growth and metabolism. Example 2 This invention provides a genetically engineered bacterium that co-produces L-threonine and L-valine, comprising the following steps: A dual-promoter strategy is used to combine and separately regulate genes in the L-valine artificial synthesis pathway: a high-intensity expression of a Ptrc-type promoter controls the endogenous genes ilvD, ilvC, and ygaZH; a moderate-intensity expression of a Plac-type promoter controls the key exogenous genes alsS and leuDH; and seamless cloning and assembly yields the inducible expression plasmid pZA-Ptrc-ilvD-ilvC-ygaZH-Plac-alsS-leuDH for the L-valine artificial synthesis pathway. The above expression plasmid is then introduced into an L-threonine-producing bacterium to obtain a genetically engineered bacterium that co-produces L-threonine and L-valine, namely, a Ptrc-Plac type L-valine artificial synthesis pathway genetically engineered bacterium (Ptrc-Plac type engineered bacterium).

[0050] Shake-flask fermentation of the Ptrc-Plac engineered bacteria was performed to verify whether L-threonine and L-valine were produced in the products. The verification method and operation were the same as in Example 1, except that the Ptrc engineered bacteria in Example 1 were replaced with the Ptrc-Plac engineered bacteria of this example. The results showed that the yield of L-threonine was 5.5 g / L and the yield of L-valine was 2.9 g / L.

[0051] It can be seen that the Ptrc-Plac type engineered bacteria prepared in this embodiment are genetically engineered bacteria that co-produce L-threonine and L-valine.

[0052] This invention also provides a fermentation method for a genetically engineered bacterium that co-produces L-threonine and L-valine. The specific operation and steps are the same as in Example 1, except that the Ptrc-type engineered bacterium in Example 1 is replaced with the Ptrc-Plac-type engineered bacterium in this example. The fermentation results are shown in Table 2.

[0053] Table 2. Results of co-production of threonine and valine in a 5-liter fermenter using Ptrc-Plac engineered bacteria.

[0054] Table 2 shows that during the L-threonine synthesis phase from 0 to 20 hours, OD 600 The OD reached approximately 48, with L-threonine production at 50.3 g / L and L-valine production at 3.2 g / L; during the 21-36 hour L-valine synthesis phase, OD... 600The L-threonine production was maintained at around 47 g / L, and the L-valine production was 12.6 g / L. The results indicate that although the artificial L-valine synthesis pathway still exhibited basal leakage expression in the early stages without IPTG induction, the dual-promoter strategy effectively regulated the expression intensity of key enzymes AlsS and LeuDH, thereby rationally balancing the carbon flux allocation at the pyruvate metabolism node. This ensured normal cell growth and L-threonine synthesis in the early stages. Later, the addition of an inducer significantly increased L-valine production after entering the L-valine synthesis stage. The Ptrc-Plac engineered bacteria showed a relatively balanced distribution of expression intensity and metabolic flux, balancing the accumulation of both L-threonine and L-valine.

[0055] Example 3 This invention provides a genetically engineered bacterium that co-produces L-threonine and L-valine, comprising the following steps: obtaining the L-valine artificial synthesis pathway ilvD-ilvC-ygaZH-alsS-leuDH through seamless cloning and assembly, and regulating its expression through the dissolved oxygen-responsive promoter Pfnr to construct the inducible expression plasmid pZA-Pfnr-ilvD-ilvC-ygaZH-alsS-leuDH. The above expression plasmid is then introduced into an L-threonine-producing bacterium to obtain a Pfnr-type L-valine artificial synthesis pathway genetically engineered bacterium (Pfnr-type engineered bacterium).

[0056] Shake-flask fermentation of Pfnr-type engineered bacteria was conducted to verify whether L-threonine and L-valine were produced in the product.

[0057] 0.2 mL of Pfnr-type engineered bacterial culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of shake-flask seed culture medium. After culturing at 220 rpm and 37℃ for 8-10 hours, the shake-flask seed culture was transferred at a ratio of 5-10% (v:v) to a 150 mL Erlenmeyer flask with a cap containing 30 mL of shake-flask fermentation medium. After culturing at 220 rpm and 37℃ for 12 hours, the cap was tightened and the culture was continued for 24 hours. The fermentation results showed that the yield of L-threonine was 6.7 g / L and the yield of L-valine was 5.1 g / L.

[0058] It can be seen that the Pfnr-type engineered bacteria prepared in this embodiment are genetically engineered bacteria that co-produce L-threonine and L-valine.

[0059] This invention also provides a fermentation method for a genetically engineered bacterium that co-produces L-threonine and L-valine, comprising the following steps: 1 mL of Pfnr-type engineered bacterial culture is inoculated into a 1 L Erlenmeyer flask containing 200 mL of shake-flask seed culture medium. After culturing at 220 rpm and 37°C for 8-10 hours, the culture is transferred at a 5-10% (v:v) inoculation rate to a 5 L fermenter containing 2 L of fermenter culture medium. The pH is maintained at 6.5-7.5 using ammonia, the temperature at 37°C, the aeration rate at 2-5 L / min, and the dissolved oxygen controlled above 25%. A 50% glucose solution is added to maintain the sugar concentration at approximately 0.5-2.0 g / L. After approximately 16-20 hours of fermentation, the process is switched to anaerobic fermentation, with the fermentation speed fixed at 300 rpm, the aeration rate adjusted to 1 L / min, and the dissolved oxygen dropping below 10%. Odion oxidative stress (OD) is measured periodically throughout the fermentation process. 600 The values ​​and corresponding amino acid yields were recorded. Fermentation results are shown in Table 3.

[0060] Table 3. Results of co-production of threonine and valine in a 5-liter fermenter using Pfnr-type engineered bacteria.

[0061] Table 3 shows that during the L-threonine synthesis phase from 0 to 20 hours, OD 600 The OD reached approximately 50, with L-threonine production at 48.4 g / L and L-valine production at 2.3 g / L; during the 21-36 hour L-valine synthesis phase, OD... 600 The dissolved oxygen level was maintained at around 50 g / L, with L-threonine production at around 45 g / L and L-valine production at 13.1 g / L. These results indicate that the Pfnr-type engineered bacteria primarily synthesize L-threonine under aerobic conditions in the early stages of fermentation, while effectively activating the artificial L-valine synthesis pathway in the middle and later stages by simply reducing dissolved oxygen levels. This demonstrates good growth-metabolic balance and industrial application potential.

[0062] As can be seen from Examples 1 to 3, the genetically engineered bacteria prepared by constructing an artificial L-valine synthesis pathway, as provided by the present invention, can achieve the co-production of L-threonine and L-valine. When the Pfnr-type engineered bacteria prepared by the present invention co-produces L-threonine and L-valine through fermentation, the process is simple, does not require induced expression, has relatively low cost, and exhibits the highest relative yield of L-threonine and L-valine, demonstrating high synthesis efficiency.

[0063] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium that co-produces L-threonine and L-valine, characterized in that, The genetically engineered bacteria co-expressed key genes of the L-valine synthesis pathway, alsS, leuDH, ilvC, and ilvD, as well as efflux-related genes ygaZ and ygaH, on the plasmid. Combined with promoter regulation of the expression of the L-valine artificial synthesis pathway, a genetically engineered bacterium producing both L-threonine and L-valine was obtained.

2. The genetically engineered bacterium that co-produces L-threonine and L-valine as described in claim 1, characterized in that, The promoter is an inducible promoter, and the genetically engineered bacterium is Escherichia coli.

3. The genetically engineered bacterium that co-produces L-threonine and L-valine as described in claim 2, characterized in that, The inducible promoters include chemically inducible promoters and physically inducible promoters.

4. The genetically engineered bacterium that co-produces L-threonine and L-valine as described in claim 3, characterized in that, The chemically induced promoter is a Ptrc-type promoter or a Ptrc-Plac-type promoter; the physically induced promoter is a Pfnr promoter.

5. The genetically engineered bacterium that co-produces L-threonine and L-valine as described in claim 4, characterized in that, During Ptrc promoter regulation, the order of the key genes and efflux-related genes is Ptrc-alsS-ilvD-ilvC-leuDH-ygaZH.

6. The genetically engineered bacterium that co-produces L-threonine and L-valine as described in claim 4, characterized in that, During the regulation by the Ptrc-Plac promoter, the order of the key genes and efflux-related genes is Ptrc-ilvD-ilvC-ygaZH-Plac-alsS-leuDH.

7. The genetically engineered bacterium that co-produces L-threonine and L-valine as described in claim 4, characterized in that, During Pfnr promoter regulation, the order of the key genes and efflux-related genes is Pfnr-ilvD-ilvC-ygaZH-alsS-leuDH.

8. A fermentation method for a genetically engineered bacterium producing L-threonine and L-valine as described in any one of claims 1 to 3, characterized in that, When using a physically induced promoter, the fermentation method is a two-stage fermentation. The conditions for the first stage of fermentation include a temperature of 35~38℃ and a dissolved oxygen concentration of ≥25%, while the conditions for the second stage of fermentation are a temperature of 37~42℃ and a dissolved oxygen concentration of ≤10%.

9. A fermentation method for a genetically engineered bacterium producing L-threonine and L-valine as described in any one of claims 1 to 3, characterized in that, When using a chemically inducible promoter, the fermentation method is a two-stage fermentation. The conditions for the first stage fermentation include a temperature of 35-38°C and a dissolved oxygen concentration of ≥25%, and the conditions for the second stage fermentation are a temperature of 37-42°C and a dissolved oxygen concentration of ≤10%. Between the first stage fermentation and the second stage fermentation, there is also an induction expression stage. In the induction expression stage, a chemical inducer is added, and the induction expression conditions are the same as those for the first stage fermentation. The duration of the induction expression stage is 2-4 hours.

10. The L-threonine and L-valine mixture obtained by the fermentation method of the genetically engineered bacteria that co-produce L-threonine and L-valine according to any one of claims 8 to 9 is applied in the feed industry.

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