A malate:quinone oxidoreductase mutant and its use in the production of l-tryptophan

Abstract

The application relates to a malate:quinone oxidoreductase mutant and application thereof in production of L-tryptophan, and belongs to the technical field of genetic engineering. In order to solve the technical problem that the activity of wild type Mqo is often limited by natural catalytic efficiency, expression level or coupling degree with an electron transfer chain, leading to insufficient electron transfer rate, low quinone reduction efficiency and difficulty in meeting the requirement of ATP for efficient synthesis of L-tryptophan, the application provides an Mqo mutant, which is obtained by mutating alanine at the 176th position into threonine and mutating alanine at the 105th position into valine on the basis of wild type Mqo. It is verified through fermentation that knocking out the wild type Mqo and back-supplementing the Mqo mutant in Escherichia coli can improve the yield of L-tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a malic acid:quinone oxidoreductase mutant and its application in the production of L-tryptophan. Background Technology

[0002] Malate:quinone oxidoreductase (Mqo) is a membrane-bound enzyme widely found in bacteria and other microorganisms. It catalyzes the oxidation of malate to oxaloacetate, simultaneously transferring electrons to the quinone pool (such as ubiquinone or menadione), thus connecting the tricarboxylic acid cycle (TCA cycle) and the electron transport chain (ETC). This process not only drives the continuous operation of the TCA cycle but also directly participates in ATP synthesis by promoting the formation of a proton transmembrane gradient, making it crucial for microorganisms to maintain energy homeostasis and carbon source adaptation in variable environments.

[0003] In microbial fermentation production, the efficient synthesis of L-tryptophan requires substantial amounts of ATP and reducing power. However, the activity of wild-type Mqo is often limited by its natural catalytic efficiency, expression level, or degree of coupling with the electron transport chain, leading to insufficient electron transport rates and low quinone reduction efficiency, which in turn affects ATP production. Existing strategies typically enhance cellular energy charge indirectly through global metabolic engineering or cofactor regulation, but these methods are less targeted, prone to causing metabolic imbalances, and have limited effectiveness in improving ATP supply. Furthermore, while directly enhancing the expression of ETC components may increase ATP production, it is often accompanied by problems such as reactive oxygen species accumulation or impaired cell growth. Therefore, a more targeted and efficient strategy is urgently needed to optimize electron transport flux by directionally modifying key energy metabolism enzymes such as Mqo, thereby effectively supporting the synthesis of high-energy-consuming products such as L-tryptophan. Summary of the Invention

[0004] To address the technical challenge that the activity of wild-type Mqo is often limited by its natural catalytic efficiency, expression level, or degree of coupling with the electron transport chain, leading to insufficient electron transport rate and low quinone reduction efficiency, thus hindering the efficient ATP synthesis of L-tryptophan, this invention provides an Mqo mutant. This mutant is obtained by mutating alanine at position 176 to threonine and alanine at position 105 to valine, based on wild-type Mqo. Fermentation validation of this invention confirms that knocking out wild-type Mqo and integrating the Mqo mutant in *E. coli* can increase L-tryptophan production.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a malate:quinone oxidoreductase mutant that increases L-tryptophan production, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] A second objective of this invention is to provide a gene encoding the above-mentioned malic acid:quinone oxidoreductase mutant.

[0007] In one embodiment of the present invention, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0008] A third objective of this invention is to provide a recombinant vector containing the above-mentioned coding gene.

[0009] A fourth objective of this invention is to provide recombinant bacteria containing the above-described coding gene or the above-described recombinant vector.

[0010] A fifth objective of this invention is to provide the application of the above-mentioned malic acid:quinone oxidoreductase mutant in the production of L-tryptophan.

[0011] A sixth objective of this invention is to provide the application of the above-mentioned encoding gene in the production of L-tryptophan.

[0012] A seventh object of the present invention is to provide the application of the above-described recombinant vector in the production of L-tryptophan.

[0013] The eighth object of the present invention is to provide the application of the above-mentioned recombinant bacteria in the production of L-tryptophan.

[0014] The ninth objective of this invention is to provide a method for increasing L-tryptophan production, wherein the method uses Escherichia coli CGMCC NO.11073 as the starting strain, knocks out wild-type malate:quinone oxidoreductase and replenishes the above-mentioned malate:quinone oxidoreductase mutant.

[0015] The beneficial effects of this invention are: Based on Escherichia coli with accession number CGMCC NO.11073, this invention knocks out wild-type Mqo and reintroduces the Mqo mutant. Fermentation verification confirmed that, compared with Escherichia coli CGMCC NO.11073, the genetically engineered strain with Mqo mutation can increase L-tryptophan production, with an increase in L-tryptophan concentration of 11.94-13.73%. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be noted that the embodiments mentioned below are only for explaining this invention and are not intended to limit the scope of this invention. The embodiments mentioned below are only some embodiments of this invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of this invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, any embodiments obtained by other skilled personnel without creative effort are protected by this invention.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0018] The Escherichia coli used in this embodiment of the invention is Escherichia coli (E. coli) Escherichia coli CGMCCNO.11073, published in Chinese Patent No. CN118389394B, entitled "A Genetically Engineered Bacterium for Increasing L-Tryptophan Production and Its Construction Method and Application".

[0019] The amino acid sequence of the malic acid:quinone oxidoreductase mutant (MqoA176TA105V) provided by the present invention is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene of the mutant is shown in SEQ ID NO.2.

[0020] The CRISPR Cas9 gene editing plasmids used in this invention are p118 and p26D. The nucleotide sequence of plasmid p118 is shown in SEQ ID NO.3. This plasmid contains the Cas9 protein, X-Red recombination key element, and the SacB sucrose reverse screening gene. It is resistant to carbamate and uses p15A as the replicon. The nucleotide sequence of plasmid p26D is shown in SEQ ID NO.4. This plasmid contains gRNA, gRNA scaffold, and the repair template required for editing. It is resistant to ampicillin and uses PSC101 as the replicon.

[0021] Example 1: Construction of genetically engineered bacteria with malic acid:quinone oxidoreductase mutant 1. Constructing a wild-type malic acid:quinone oxidoreductase knockout strain (1) Construction of gene-editing plasmids for knocking out wild-type malic acid:quinone oxidoreductase The genome of Escherichia coli CGMCC NO.11073 was extracted, and PCR amplification of the repair template was performed using two primer pairs: mqo-up-F (SEQ ID NO.5) and mqo-up-R (SEQ ID NO.6), and mqo-down-F (SEQ ID NO.7) and mqo-down-R (SEQ ID NO.8). The amplification products were 585 bp and 517 bp in size, respectively. Using the p26D gene editing vector as a template, the vector fragment was amplified using primers vector-mqo-F (SEQ ID NO.9) and Vetor-mqo-R (SEQ ID NO.10) to obtain vector-mqo, which was 3177 bp in size. PCR amplification was performed using Vazyme 2×Phanta Flash MasterMix under the following conditions: 98℃ for 5 min; 98℃ for 10 s, 56℃ for 25 s, 72℃ for 5 s / kb, for 30 cycles; 72℃ for 5 min; and stored at 4℃.

[0022] After amplification, the fragments were recovered using the OmegaE.ZNA Gel Extraction Kit. During vector fragment amplification, the fragments were digested with Thermo DpnI at 37°C for 15 min before gel extraction. The three fragments obtained above were ligated using the NEBUILDER HiFi DNA assembly master mix according to the manufacturer's instructions. The ligation products were transformed into *E. coli* TOP10 or DH5α competent cells, plated onto LB agar plates containing 50 mg / mL ampicillin resistance, and incubated upside down at 30°C for 12–16 h. Single colonies were then selected. Single colonies were validated using primers p26D-test-F (SEQ ID NO.11) and p26D-test-R (SEQ ID NO.12). The Novizan P131-02 Green Taq Mix kit was used. The PCR reaction program was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 20 s, 57℃ annealing for 20 s, 72℃ extension for 1 min 30 s, for a total of 35 cycles; 72℃ extension for 5 min. Fragments of 1402 bp were selected and sent to Sangon Biotech for sequencing. Transformants with correct sequencing were preserved and plasmids were extracted and named pXY850.

[0023] (2) Construction of wild-type malic acid:quinone oxidoreductase knockout gene-edited strain CGMCC NO.11073 strain was prepared into electrochemically competent cells and transformed into the p118 plasmid. Correct transformants were screened and further prepared into electrochemically competent cells, which were then transformed into the pXY850 plasmid. The cells were cultured upside down at 30°C in LB solid medium containing three antibiotics: tetracycline (50 mg / mL), ampicillin (50 mg / mL), and kanamycin (50 mg / mL). After 12-16 h of culture, a single colony was inoculated into 5 mL of double-antibiotic LB medium, with the addition of IPTG (1 mM) and L-arabinose (20 mM). The culture was induced at 30°C and 220 rpm for 5 h. The bacterial culture was then diluted to 10... -6 200 μL of the culture was evenly spread on double-antibiotic solid LB medium. After culturing for 12-16 h, single colonies were collected for colony PCR verification. Using primers mqo-genome-test-F (SEQ ID NO.13) and mqo-genome-test-R (SEQ ID NO.14), the amplified fragment size was 1102 bp. PCR products with the correct fragment size were selected and sent to Sangon Biotech for sequencing. Transformants with correct sequencing were deposited. Then, the gene-editing plasmid was lost. The correctly sequenced strain was inoculated into 5 mL of tetracycline-resistant LB medium containing 200 μL of 50% sucrose solution. The culture was incubated at 37℃ and 220 rpm for 12-16 h. The bacterial culture was then diluted to 10... -6 Take 200 μL and spread it evenly on tetracycline-resistant solid LB medium. After incubation for 12-16 h, take single colonies for point verification. Streak single colonies on tetracycline-resistant solid LB medium plates, tetracycline-resistant and kanamycin-resistant solid LB medium plates, and tetracycline-resistant and ampicillin-resistant solid LB medium plates. Select colonies that do not grow on tetracycline-resistant and kanamycin-resistant solid LB medium plates, tetracycline-resistant and ampicillin-resistant solid LB medium plates, and grow in tetracycline-resistant medium. Preserve them and name the strain XYWb216.

[0024] 2. Construction of genetically engineered bacteria with malic acid-replenishing quinone oxidoreductase mutants. The MqoA176TA105V mutant was integrated into the yciQ site. (1) Construction of gene-editing plasmids for compensating for malic acid:quinone oxidoreductase mutants The genome of strain XYWb216 was extracted, and PCR amplification of the repair template was performed using two primer pairs: yciQ-up-F (SEQ ID NO.15) and yciQ-up-R (SEQ ID NO.16), and yciQ-down-F (SEQ ID NO.17) and yciQ-down-R (SEQ ID NO.18), with amplification product sizes of 567 bp and 600 bp, respectively. Using the coding gene sequence of the malate:quinone oxidoreductase mutant MqoA176TA105V (SEQ ID NO.2) as a template, the target gene was amplified using primers mqo*-F (SEQ ID NO.19) and mqo*-R (SEQ ID NO.20), with an amplification product size of 1702 bp. Using the p26D gene editing vector as a template, vector-yciQ-F (SEQ ID NO.21) and Vetor-yciQ-R (SEQ ID NO.18) were used for amplification. Primers (NO.22) were used to amplify the vector fragment, and the amplification product was 3185 bp in size. PCR amplification was performed using Vazyme 2×Phanta FlashMaster Mix under the following conditions: 98℃ for 5 min; 98℃ for 10 s, 56℃ for 25 s, 72℃ for 5 s / kb, for 30 cycles; 72℃ for 5 min; and stored at 4℃.

[0025] After amplification, the fragments were recovered using the OmegaE.ZNA Gel Extraction Kit. During vector fragment amplification, the fragments were digested with Thermo DpnI at 37°C for 15 min before gel extraction. The four fragments obtained were ligated using the NEBUILDER HiFi DNA assembly master mix according to the manufacturer's instructions. The ligation products were transformed into *E. coli* TOP10 or DH5α competent cells, plated onto LB agar plates containing 50 mg / mL ampicillin resistance, and incubated upside down at 30°C for 12–16 h. Single colonies were then selected. Single colonies were validated using primers p26D-test-F (SEQ ID NO.11) and p26D-test-R (SEQ ID NO.12). The Novizan P131-02 Green Taq Mix kit was used. The PCR reaction program was: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 20 s, 57℃ annealing for 20 s, 72℃ extension for 1 min 30 s, for a total of 35 cycles; 72℃ extension for 5 min. A 3683 bp fragment was selected and sent to Sangon Biotech for sequencing. Transformants with correct sequencing were preserved, and plasmids were extracted and named pXY801.

[0026] (2) Construction of genetically engineered bacteria with malic acid-replenishing quinone oxidoreductase mutants The strain XYWb216 was prepared into electrochemically competent cells and transformed into the p118 plasmid. Correct transformants were screened and further prepared into electrochemically competent cells, which were then transformed into the pXY801 plasmid. The cells were cultured upside down at 30°C in LB solid medium containing three antibiotics: tetracycline (50 mg / mL), ampicillin (50 mg / mL), and kanamycin (50 mg / mL). After 12-16 h of culture, a single colony was inoculated into 5 mL of double-antibiotic LB medium, and IPTG (1 mM) and L-arabinose (20 mM) were added. The culture was induced at 30°C and 220 rpm for 5 h. The bacterial culture was then diluted to 10... -6 Take 200 μL and spread it evenly on double-antibiotic solid LB medium. After culturing for 12-16 h, take a single colony for colony PCR verification. Using primers yciQ-genome-test-F (SEQ ID NO.23) and yciQ-genome-test-R (SEQ ID NO.24), the amplified fragment size is 3108 bp. Select PCR products with the correct fragment size and send them to Sangon Biotech for sequencing. The correctly sequenced transformants are preserved.

[0027] Then, the gene-editing plasmid was removed, and the correctly sequenced strain was inoculated into 5 mL LB medium containing only tetracycline resistance, supplemented with 200 μL of 50% sucrose solution. The culture was incubated at 37°C and 220 rpm for 12–16 h, and the culture was diluted to 10⁻⁶. -6 Take 200 μL and spread it evenly on tetracycline-resistant solid LB medium. After incubation for 12-16 h, take single colonies for point verification. Streak single colonies on tetracycline-resistant solid LB medium plates, tetracycline-resistant and kanamycin-resistant solid LB medium plates, and tetracycline-resistant and ampicillin-resistant solid LB medium plates. Select colonies that do not grow on tetracycline-resistant and kanamycin-resistant solid LB medium plates, tetracycline-resistant and ampicillin-resistant solid LB medium plates, and grow in tetracycline-resistant medium. Preserve them and name the strain XYWb222.

[0028] Example 2: Application of genetically engineered bacteria with malic acid:quinone oxidoreductase mutant in L-tryptophan production. 1. Shake-flask fermentation experiment The seed culture medium used in this experiment consisted of the following components: 22.5 g / L dipotassium hydrogen phosphate, 9 g / L potassium dihydrogen phosphate, 15 g / L yeast extract, 4 g / L ammonium sulfate, 2 g / L magnesium sulfate, 25 g / L glucose, with the remainder being water, and a pH of 7.15. The fermentation medium used in this experiment consisted of the following components: potassium dihydrogen phosphate 3.5 g / L, dipotassium hydrogen phosphate 3.5 g / L, disodium hydrogen phosphate 6 g / L, ammonium sulfate 4 g / L, ferrous sulfate 0.1 g / L, citric acid monohydrate 2.25 g / L, yeast extract 2 g / L, propanesulfonic acid 50 g / L, 3.2 mL of feedstock, magnesium sulfate 0.4 g / L, and glucose 28 g / L. Formula for ingredient 3: 0.003 g / L manganese sulfate, 0.005 g / L zinc sulfate, 0.0005 g / L copper sulfate pentahydrate, 0.003 g / L cobalt chloride hexahydrate, with the balance being water.

[0029] This experiment involved shake-flask fermentation of Escherichia coli CGMCC NO.11073 and XYWb222, respectively. The specific methods are as follows: 1) Strain activation: After removing the bacterial culture from the -80℃ freezer and allowing it to thaw naturally, use an inoculation loop to apply two loops of the culture to an LB agar plate under sterile conditions. Incubate at 36.5℃ upside down for 16 hours.

[0030] 2) Inoculum: The shaker is pre-sterilized and preheated with UV light. The cultured plates are removed, and under aseptic conditions, a loopful of inoculum is scraped and inoculated into a seed culture flask. After inoculation, the flask opening is completely sealed with gauze and secured with a rubber band. The UV light on the shaker is turned off, and the seed culture flask is placed in the shaker and incubated at 36.5℃ and 170 rpm. OD 660 Transplanting is possible after approximately 8-10 days.

[0031] 3) Fermentation begins: Remove the cultured seed shake flask and, under sterile conditions, transfer 5 mL of bacterial culture into the fermentation shake flask, bringing the final volume to 50 mL. Wrap the fermentation shake flask and place it on a shaker. Incubate at 36.5℃ and 200 rpm for approximately 20-24 hours.

[0032] 4) Judgment of the shake bottle: Shake the flask before the sugar is completely consumed; when shaking the flask, ensure that the pH is within a suitable range and that the residual sugar has not been completely consumed.

[0033] The tryptophan content was determined by HPLC, and the fermentation results are shown in Table 1.

[0034] Table 1 Results of shake-flask fermentation

[0035] As can be seen from Table 1, strain XYWb222 has a higher tryptophan production compared to CGMCC NO.11073.

[0036] 2. Fermentation test in a 10 L fermenter Escherichia coli CGMCC NO.11073 and XYWb222 were fermented in 10 L fermenters respectively. The fermentation medium formula is shown in Table 2. The specific fermentation process is as follows: 1) Seed activation: Inoculate the glycerol bacteria into the seed culture medium (the same as the seed culture medium in shake flask fermentation) at an inoculation amount of 2% and incubate at 200 rpm until the OD value reaches 4-8.

[0037] 2) Fermentation inoculation: Inoculate the seed liquid and base sugar together into a 10 L fermenter with an inoculation amount of 5% and a base sugar of 20 g, and adjust the pH to 6.5-7.2.

[0038] 3) Fermentation process control: The fermentation temperature is 36.5℃. After the base sugar is consumed, the sugar is fed in at a concentration of 50%, and the residual sugar concentration is maintained below 0.05%. The fermentation time is 60 h.

[0039] Table 2. Formulation of fermentation medium

[0040] The composition of the trace element mother liquor is as follows: 38.08 g / L ferrous sulfate heptahydrate, 2.37 g / L manganese sulfate monohydrate, 10 g / L sodium sulfate, 3.2 g / L zinc sulfate, 2 g / L cobalt chloride hexahydrate, 0.3 g / L anhydrous copper sulfate, balance water.

[0041] The composition of the vitamin stock solution is as follows: 0.025 g / L VB12, 0.055 g / L VH, 0.255 g / L VB1, with the balance being water.

[0042] Fermentation results showed that after 60 h of fermentation, the OD of the fermentation broth obtained by inoculating strain XYWb222 was [data missing]. 660nm The value was 78, the L-tryptophan concentration was 49.97 g / L, and the sugar-acid conversion rate was 22.45%. Compared with Escherichia coli CGMCC NO.11073, the L-tryptophan concentration increased by 10.08%.

[0043] It is evident that strain XYWb222 exhibits a significant advantage in acid production capacity compared to the control strain CGMCC NO.11073. Therefore, strain XYWb222 possesses superior potential for industrial fermentation applications.

[0044] The above description is only a preferred embodiment 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 malate:quinone oxidoreductase mutant that increases L-tryptophan production, characterized in that, The amino acid sequence of the malic acid:quinone oxidoreductase mutant is shown in SEQ ID NO.

1.

2. A gene encoding a gene, characterized in that, The encoding gene encodes the malic acid:quinone oxidoreductase mutant of claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector contains the coding gene as described in claim 2 or 3.

5. A recombinant bacterium, characterized in that, The recombinant bacteria contains the coding gene of any one of claims 2 or 3 or the recombinant vector of claim 4.

6. The application of the malic acid:quinone oxidoreductase mutant according to claim 1, characterized in that, The application involves using the malic acid:quinone oxidoreductase mutant to produce L-tryptophan.

7. The application of the coding gene according to any one of claims 2 or 3, characterized in that, The application involves using the encoding gene to produce L-tryptophan.

8. The application of the recombinant vector according to claim 4, characterized in that, The application involves using the recombinant vector to produce L-tryptophan.

9. The application of the recombinant bacteria according to claim 5, characterized in that, The application involves using the recombinant bacteria to produce L-tryptophan.

10. A method for increasing L-tryptophan production, characterized in that, The method uses Escherichia coli CGMCC NO.11073 as the starting strain, knocks out wild-type malic acid:quinone oxidoreductase and replenishes the malic acid:quinone oxidoreductase mutant as described in claim 1.

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