Acetyl coenzyme A synthase mutant and application thereof
By mutating Escherichia coli acetyl-CoA synthase and modifying its amino acid sequence to enhance its anti-acetylation ability, the metabolic inhibition problem caused by acetic acid accumulation was solved, and more efficient acetic acid metabolism was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF MICROBIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2024-11-16
- Publication Date
- 2026-05-19
AI Technical Summary
When Escherichia coli uses glucose as a carbon source, it produces acetic acid as a byproduct, which inhibits bacterial growth and metabolism. The activity of acetyl-CoA synthase is affected by acetylation modification, leading to metabolic imbalance.
We designed an acetyl-CoA synthase mutant, Acs_C mut., and modified the acetyltransferase contact interface by making deletion mutations in the amino acid sequence of Acs. This preserved the ability to catalyze acetic acid and improved the resistance to acetylation. The mutant was then inserted into a plasmid and expressed in E. coli.
It improves acetic acid metabolic activity and anti-acetylation ability, reduces acetic acid accumulation, improves acetic acid metabolic conversion efficiency, and avoids the decrease in enzyme activity caused by acetylation.
Smart Images
Figure CN122060692A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial metabolic engineering technology. Background Technology
[0002] Escherichia coli (E. coli) is one of the most widely used chassis cells in biofermentation due to its rapid growth, clear genetic background, and mature genetic manipulation techniques. However, when E. coli uses glucose as a carbon source, it often produces acetic acid as a byproduct. As the concentration of acetic acid increases, both cell growth and metabolism are severely inhibited. Acetic acid accumulation is a metabolic overflow phenomenon caused by the cell's limited respiratory capacity and TCA cycle, which is accompanied by the inhibition of the in vivo activity of acetyl-CoA synthase (Acs). Acs is a key enzyme in the biometabolism of acetic acid; it can synthesize acetyl-CoA (Ac-CoA) using acetic acid and coenzyme A (CoA) as substrates. Therefore, the activity of Acs enzyme is crucial for the biometabolism of acetic acid.
[0003] Besides the influence of the enzyme itself, the post-translational acetylation modification of Acs is also a significant factor contributing to their reduced activity in vivo. Specifically, in *E. coli*, acetyltransferase (Pat) recognizes leucine (L) at position 641 of Acs and uses Ac-CoA as a co-substrate to acetylate lysine (K) at position 609 of Acs, ultimately rendering Acs inactive. In 2005, Starai et al. verified that the mutant Acs_L641P (derived from *S. enterica*), obtained by mutating leucine at position 641 of Acs, exhibited almost complete resistance to acetylation in vitro, suggesting that its metabolism of acetic acid in vivo could be unaffected by acetylation. However, because this mutant is almost entirely unregulated by acetylation, its application in microbial metabolic engineering may pose a risk of disrupting cellular metabolism. Summary of the Invention
[0004] In view of this, the present invention provides an acetyl-CoA synthase mutant, the amino acid sequence of which is shown in SEQ ID No. 1 in the sequence listing.
[0005] Furthermore, the present invention also provides a plasmid obtained by inserting an acetyl-CoA synthase mutant into pET28a.
[0006] Furthermore, the present invention provides an engineered bacterium obtained by transferring a plasmid into Escherichia coli BL21(DE3).
[0007] The acetyl-CoA synthase mutant provided by this invention significantly enhances both acetic acid metabolic activity and its anti-acetylation ability related to activity regulation. This mutant can, on the one hand, improve the efficiency of acetic acid metabolism in organisms, and on the other hand, help reduce the accumulation of the byproduct acetic acid during biological metabolism. Attached Figure Description
[0008] Figure 1 This is a schematic diagram of the activity test principle.
[0009] Figure 2 This is a graph showing the results of the anti-acetylation ability test. Detailed Implementation
[0010] Example
[0011] The purpose of this invention is to provide an acetyl-CoA synthase mutant: Acs_C mut., and the corresponding amino acid sequence (SEQ ID No. 1).
[0012] When acetic acid is used as a substrate, the K mutant M The value is similar to that of the wild type, k cat The value is approximately three times that of the wild type; this mutant retains some acetylation regulatory functions, while its tolerance to acetylation is significantly improved compared to the wild type.
[0013] The mutants disclosed in this invention can be used for metabolic engineering modification, which can reduce the accumulation of the byproduct acetic acid and improve the efficiency of acetic acid metabolism and transformation by the strain.
[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0015] This invention analyzes the DNA sequence corresponding to Acs and then deletes four bases GAGA from positions 1915 to 1918 of Acs, causing a deletion mutation in Acs. This causes a frameshift mutation in the Acs genome, ultimately resulting in a mutation in the Acs amino acid sequence from EKLLEEKQAIAMPS at positions 639 to 652 to SCLKRSRLSRCHRNPQLPDASR at positions 639 to 661. The mutant is named Acs_C mut.
[0016] Acs_C mut. retains the recognition site at leucine (L) at position 641 of acetyltransferase (Pat, Uniprot ID: P76594, derived from E. coli), but due to changes in the amino acid sequence, the interface between Pat and Acs_C mut. has been significantly altered. The resulting mutant Acs_Cmut. retains its affinity for the catalytic substrate acetic acid, improves catalytic efficiency, and, more importantly, exhibits a substantial increase in acetylation tolerance.
[0017] The DNA sequences of Acs and Acs_C mut. were inserted into the pET28a plasmid, respectively, to fuse an N-terminal 6×His tag during protein expression, ultimately forming pET28a-Acs and pET28a-Acs_Cmut. After verification by sequencing, the obtained plasmids were transformed into E. coli competent cells BL21(DE3) to form engineered bacteria BL21(DE3)-pET28a-Acs and BL21(DE3)-pET28a-Acs_C mut. Single colonies of these engineered bacteria were inoculated into LB broth containing the corresponding antibiotics and cultured overnight at 37°C and 220 rpm to form seed culture. The seed culture was prepared at 10%...
[0018] A (V / V) inoculum was inoculated into an appropriate amount of resistant LB broth to form a fermentation medium. Fermentation was carried out at 37°C and 220 rpm until the cells reached the logarithmic growth phase (OD). 600 When the concentration of the bacterial culture medium was 0.6–0.8%, 0.1 mM of isopropyl-β-D-thiogalactoside (IPTG) was added, and the culture was incubated overnight at 16°C and 220 rpm to induce protein expression. The next day, the bacterial cells were collected by centrifugation, and the cells were disrupted by sonication. Protein purification was performed using Ni-NTA agarose resin-mediated affinity chromatography. Low concentration imidazole (20 mM) was used for washing, and high concentration imidazole (250 mM) was used for elution. The eluent was desalted and purified by dextran gel G-25. Part of the purified protein was flash-frozen in liquid nitrogen and stored at -80°C, and another part was mixed with glycerol 1:1 and stored at -20°C.
[0019] The activities of Acs and Acs_C mut. were determined by coupling malate dehydrogenase (Mdh) and citrate synthase (Cs), and the activities were monitored under ultraviolet spectrophotometry (UV). Mdh uses malate as a substrate and NAD+ as a catalyst. + To produce oxaloacetate for coenzyme, NAD... +Reduced to NADH, an increase in absorbance can be detected at 340 nm. This reaction is reversible and quickly reaches equilibrium. Cs can synthesize citrate from oxaloacetic acid and Ac-CoA as substrates. If Acs successfully converts the substrates acetic acid and CoA to Ac-CoA, then the Mdh-mediated oxidation reaction will restart under the drive of Cs, leading to NADH production. Therefore, in this test system, the activities of Acs and Acs_C mut. are indirectly characterized by detecting the rate of NADH production, i.e., the rate of increase in absorbance at 340 nm. The kinetic test system for Acs and Acs_C mut. is: 50 mM Tris-HCl pH 9.0, 10 mM KCl, 5 mM MgCl2, 0.2 mM DTT, 0.2 mM CoA, 1 mM ATP, 0.025–2 mM Acetate, 5 mM L-Malate, and 0.5 mM NAD. + 2.2 U Mdh, 8.8 U Cs, 5 μM Acs or 1.5 μM Acs-C mut., reaction volume 200 μL.
[0020] The antiacetylation ability of Acs and Acs_C mut. needs to be determined by conjugation with Pat. First, the DNA sequence of Pat is inserted into the pTac-His-MBP-PreScission plasmid, so that the N-terminal 10×His and MBP tags are fused sequentially during protein expression, ultimately forming the expression plasmid pTac-His-MBP-PreScission-Pat. The protein expression, purification, and storage methods for Pat are consistent with those for Acs and its mutants. The purified Pat is first mixed with Acs or Acs_C mut. to prepare the acetylation reaction solution. The reaction system consists of 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.2 mM DTT, 0.2 mM Ac-CoA, 5 μM Pat, and 30 μM Acs or Acs_C mut., with a reaction volume of 100 μL. The acetylation reaction solution was incubated at 25°C, and samples were taken at different time points. The enzyme activities of Acs and Acs_C mut. were then detected in an activity assay system. The activity assay system consisted of 50 mM Tris-HCl (pH 9.0), 10 mM KCl, 5 mM MgCl2, 0.2 mM DTT, 0.2 mM CoA, 1 mM ATP, 2 mM Acetate, 5 mM L-Malate, and 0.5 mM NAD. + 2.2U Mdh, 8.8U Cs, 10μL Acs or Acs_C mut. Acetylation reaction solution.
[0021] Based on the molecular mechanism of Acs' post-translational regulation by Pat, this invention designs the C-terminal amino acid sequence of Acs (derived from E. coli), resulting in the mutant Acs_C mut., which, compared to the wild type, exhibits a significantly enhanced tolerance to acetylation while retaining some acetylation regulatory functions. Furthermore, when using acetic acid as a substrate, it demonstrates a substrate affinity (K0.05) similar to the wild type. M ), and its catalytic constant (k) for the substrate. cat The mutant strain exhibits three times the activity of the wild type. The mutant disclosed in this invention can be used for metabolic engineering, reducing the accumulation of the byproduct acetic acid and improving the efficiency of acetic acid metabolism by the strain. Specifically, it offers the following advantages:
[0022] 1. Improve the acetylation resistance of Acs
[0023] Acs is the main enzyme in microbial strains that metabolizes acetic acid. This invention modifies the protein sequence related to the contact interface between Acs and acetyltransferase Pat, obtaining the mutant Acs_Cmut. It retains the affinity and catalytic ability for the substrate acetic acid, while significantly improving its resistance to acetylation.
[0024] This mutation avoids the problem that Acs is severely acetylated and its activity is greatly reduced when E. coli metabolizes glucose as a carbon source, thus reducing the accumulation of acetic acid, a byproduct of microbial metabolism.
[0025] 2. Enhance Acs' acetic acid metabolism activity
[0026] The mutant Acs_C mut. provided by this invention exhibits a substrate affinity (K0.05) similar to the wild type when using acetic acid as a substrate. M ), and its catalytic constant (k) for the substrate. cat It has three times the catalytic activity of the wild type, thus exhibiting higher catalytic performance in catalyzing acetic acid metabolism. This can reduce the accumulation of acetic acid, a byproduct of microbial metabolism, and can also be used to modify biological metabolic processes using acetic acid as a carbon source, thereby improving the biological metabolic capacity of acetic acid.
[0027] Example 1
[0028] Construction of engineered bacteria BL21(DE3)-pET28a-Acs and BL21(DE3)-pET28a-Acs_C mut. for expressing Acs and Acs_C mut. proteins
[0029] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.
[0030] DNA sequences of acetyl-CoA synthase (Acs, Unipro t ID: P27550) and its mutant Acs_C mut. from *Escherichia coli* were inserted into the middle of the NdeⅠ and HindⅢ restriction sites on plasmid pET28a (Zhuangmeng Biotechnology, ZK159), respectively, enabling the fusion of an N-terminal 6×His tag during protein expression, ultimately forming expression plasmids pET28a-Acs and pET28a-Acs_Cmut., with the amino acid sequence of Acs_C mut. being SEQ ID No. 1. The expression plasmids were chemically transformed into *E. coli* BL21(DE3) (Yisheng Biotechnology, 11804ES80), with a recovery time of 60 min. An appropriate amount of the transformation solution was plated onto LB agar plates containing kanamycin sulfate (50 μg / mL) and incubated overnight at 37°C. The following day, single colonies from the agar plates were selected for colony PCR verification (Qingke Biotechnology, TSE005). Verified single colonies were inoculated into 5 mL of LB broth containing kanamycin sulfate resistance and incubated at 37°C and 200 rpm for 16 h. The turbid bacterial culture was then mixed with 80% glycerol (V / V) at a volume ratio of 7:3 and stored at -80°C to obtain engineered bacteria BL21(DE3)-pET28a-Acs and BL21(DE3)-pET28a-Acs_C mut.
[0031] Example 2
[0032] Expression and purification of Acs and Acs_C mut. proteins
[0033] Streaking of preserved bacterial cultures of engineered bacteria BL21(DE3)-pET28a-Acs and BL21(DE3)-pET28a-Acs_Cmut. onto LB agar plates containing kanamycin sulfate resistance was performed and incubated overnight at 37°C. The next day, single colonies were picked and inoculated into 5 mL of LB broth containing kanamycin sulfate resistance, and incubated overnight at 37°C and 200 rpm to form seed culture. The seed culture was then inoculated at a 10% (v / v) inoculation rate into 1 L of LB broth containing kanamycin sulfate resistance to form fermentation medium. Fermentation was carried out at 37°C and 220 rpm until the cells reached the logarithmic growth phase (OD). 600When the bacterial concentration was 0.6–0.8%, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the cells were cultured overnight at 16°C and 220 rpm to induce protein expression. The next day, the bacterial cells were collected by centrifugation (5000 rpm, 4°C, 20 min). The cells were then suspended in 50 mL of cell disruption buffer (50 mM Tris-HCl pH 8.0, 10 mM imidazole, 400 mM NaCl) and stirred with a magnetic stirrer. Cell disruption was then performed using an ultrasonic cell disruptor. The disrupted solution was centrifuged (8000 rpm, 4°C, 20 min) to obtain the supernatant. The supernatant was slowly added to a gravity column containing 2 mL of Ni-NTA agarose resin (Yisheng Biotechnology, 20503ES10). The resin was equilibrated with 10 column volumes of cell disruption buffer before use. After the supernatant was loaded, five column volumes of low-imidazolium buffer (50 mM Tris-HCl pH 8.0, 20 mM imidazolium, 400 mM NaCl) were added to the gravity column for washing. Then, high-imidazolium buffer (50 mM Tris-HCl pH 8.0, 250 mM imidazolium, 400 mM NaCl) was added for protein elution. The eluent was collected sequentially into five 2 mL centrifuge tubes, with 2 mL collected from each tube. The protein amino acid sequence was entered into Expasy (https: / / www.expasy.org / ), and the protein extinction coefficient and molecular weight were calculated. The protein concentration of each eluent was then evaluated using a micro spectrophotometer. The sample with the highest protein concentration was desalted and purified using a dextran gel G-25 (Stenofan, 17085101). The purification method was strictly followed according to the product instructions, including gravity column purification. The desalting buffer was (50mM Tris-HCl pH 8.0, 100mM NaCl). After determining the protein concentration, the desalted purified solution was divided into two portions. One portion was flash-frozen in liquid nitrogen and stored at -80°C, while the other portion was mixed with glycerol at a 1:1 ratio and stored at -20°C.
[0034] Example 3
[0035] Assay of Acs and Acs_C mut. protein acetic acid metabolism activity
[0036] Acs and Acs_C mut. can catalyze the synthesis of acetyl-CoA from acetic acid and coenzyme A (CoA). This catalytic activity was determined by coupling malate dehydrogenase (Mdh, Inokai, B99894) and citrate synthase (Cs, MedChemExpress, HY-P2739), and the activity was monitored under ultraviolet (UV) spectrophotometry. The testing principle is detailed in the appendix. Figure 1 Mdh uses malic acid as a substrate and NAD+ as a catalyst. + To produce oxaloacetate for coenzyme, NAD...+ It was reduced to NADH (ε = 6.2 mM) -1 cm -1 At 340 nm, an increase in absorbance was detected, but the redox reaction quickly reached equilibrium. Cs can synthesize citrate from oxaloacetic acid and Ac-CoA as substrates. If Acs successfully converts the substrates acetic acid and CoA to Ac-CoA, the Mdh-mediated oxidation reaction will restart under the drive of Cs, continuing to promote NADH production. Therefore, in this test system, the activities of Acs and Acs_C mut. are indirectly characterized by monitoring the rate of NADH production, i.e., the rate of increase in absorbance at 340 nm. The kinetic test system for Acs and Acs_C mut. is: 50 mM Tris-HCl pH 9.0, 10 mM KCl, 5 mM MgCl2, 0.2 mM DTT, 0.2 mM CoA, 1 mM ATP, 0.025–2 mM Acetate, 5 mM L-Malate, and 0.5 mM NAD. + The reaction mixture consisted of 2.2 U Mdh, 8.8 U Cs, 5 μM Acs, or 1.5 μM Acs_C mut., with a reaction volume of 200 μL. After testing, the initial reaction rate of the enzyme at different substrate concentrations was calculated using Beer-Lambert's law, i.e., the reaction rate within 1 min after the reaction was initiated. The relevant data were fitted using nonlinear regression with the Michaelis-Menten equation in Origin 9.0. The kinetic parameters of Acs and Acs_C mut. are shown in Table 1. It can be seen that when acetic acid is used as the substrate, the substrate affinity (K0.0) of Acs_C mut. is relatively high. M The catalytic constant (k) for the substrate did not decrease; on the contrary, it increased. cat The enzyme activity of the mutant was more than three times that of the wild type, indicating that the enzyme activity of the designed mutant has increased to a certain extent.
[0037] Table 1. Dynamic Parameters
[0038]
[0039] Example 4
[0040] Determination of the antiacetylation ability of Acs and Acs_C mut. proteins catalyzing acetic acid metabolism.
[0041] The antiacetylation ability of Acs and Acs_C mut. needs to be determined by coupling with an acetyltransferase (Pat, Uniprot ID: P76594, derived from E. coli).
[0042] First, the Pat DNA sequence was seamlessly cloned and inserted into the MCS region of the pTac-His-MBP-PreScission plasmid (Beyotime, D5001), allowing the protein to be expressed by sequentially fusing the N-terminal 10×His and MBP tags, ultimately forming the expression plasmid pTac-His-MBP-PreScission-Pat. The subsequent construction of the engineered bacterium BL21(DE3)-pTac-His-MBP-PreScission-Pat was consistent with Example 1, and the expression and purification of the Pat protein were consistent with Example 2. The purified Pat was first mixed with Acs or Acs_Cmut. to prepare an acetylation reaction solution. The reaction system consisted of 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.2 mM DTT, 0.2 mM Ac-CoA, 5 μM Pat, and 30 μM Acs or Acs_Cmut., with a reaction volume of 100 μL. The reaction solution was incubated statically at 25℃, and samples were taken at 0.5h, 1h, and 2h to detect the enzyme activity of Acs and Acs_C mut. The activity test system was 50mM Tris-HCl pH 9.0, 10mM KCl, 5mM MgCl2, 0.2mM DTT, 0.2mM CoA, 1mM ATP, 2mM Acetate, 5mM L-Malate, and 0.5mM NAD. + 2.2 U Mdh, 8.8 U Cs, and 10 μL Acs or Acs_Cmut. acetylation reaction solution. It is worth noting that since Pat-mediated acetylation has no hysteresis, the acetylation reaction solution at the 0h time point needs to be prepared separately, differing from the above reaction system in that Pat enzyme is not added. The antiacetylation ability test results of Acs and Acs_Cmut. are shown in the table below and attached. Figure 2 :
[0043]
[0044] Note: U is defined as the amount of enzyme required to convert 1 μmol of substrate in 1 minute.
[0045] It can be seen that after 0.5 h of acetylation, the activity of Acs has decreased to 13% of its original value, while Acs_C mut. still retains 79% of its activity. After 2 h of acetylation, Acs_C mut. still retains 16% of its activity, while the activity of Acs is only 8% of its original value. This indicates that the acetylation tolerance of Acs_C mut. is greatly improved compared with that of wild type.
[0046] In summary, Acs_C mut. not only exhibits improved enzyme activity compared to Acs, but also demonstrates significantly enhanced resistance to acetylation. Therefore, Acs_C mut. can be used for metabolic engineering, both to reduce the accumulation of the byproduct acetic acid and to improve the efficiency of acetic acid metabolism by the strain.
Claims
1. An acetyl-CoA synthase mutant, the amino acid sequence of which is shown in SEQ ID No. 1 of the sequence listing.
2. Plasmids, characterized by, The acetyl-CoA synthase mutant of claim 1 was inserted into pET28a to obtain the mutant.
3. Engineered bacteria, characterized by: The plasmid described in claim 2 was obtained by transforming it into Escherichia coli BL21(DE 3).
4. The application of the acetyl-CoA synthase mutant according to claim 1 in improving the biometabolic activity of acetic acid and resisting acetylation to maintain enzyme activity.
5. The use of the plasmid according to claim 2 in enhancing the biometabolic activity of acetic acid and resisting acetylation to maintain enzyme activity.
6. The use of the engineered bacteria according to claim 3 in improving the bio-metabolic activity of acetic acid and resisting acetylation to maintain enzyme activity.