ArcA mutant and application thereof

Abstract

The invention belongs to the technical field of strain genetic engineering. The invention provides an ArcA mutant which is characterized in that glutamic acid at the 94th site of ArcA protein is mutated into a termination codon. The acetic acid generation of the engineering strain in the fermentation reaction is reduced by about 90%, and the multiplication time is shortened by 10%.

Description

Technical Field

[0001] This invention belongs to the field of strain genetic engineering technology. Background Technology

[0002] The TCA cycle (also known as the citric acid cycle or tricarboxylic acid cycle) plays a vital physiological and metabolic role in microorganisms. It is a key link in cellular metabolism, connecting multiple metabolic pathways such as glycolysis, oxidative phosphorylation, and amino acid and lipid synthesis. The TCA cycle is the core pathway for obtaining energy through aerobic respiration. Through the oxidation of acetyl-CoA, NADH and FADH2 are generated, which then enter the electron transport chain, driving oxidative phosphorylation to generate ATP. Intermediates in the TCA cycle (such as citric acid, α-ketoglutarate, and oxaloacetate) are not only used for energy metabolism but also serve as carbon skeletons in the synthesis of amino acids, biomolecules, and lipids. The TCA cycle connects glycolysis with the electron transport chain, helping to regulate the energy needs of strains under different environmental conditions, particularly the transition between aerobic and hypoxic conditions. Due to its importance, the TCA cycle is regulated at multiple levels to ensure efficient metabolism and survival of strains under various environmental conditions.

[0003] ArcA (Aerobic Respiratory Control Regulator A) is a key regulator of aerobic / hypoxic respiration in E. coli. ArcA and its chaperone protein ArcB form a two-component regulatory system that responds to changes in oxygen levels. More broadly, ArcAB has been shown to respond to changes in intracellular redox levels. Under hypoxic or relatively reducing intracellular conditions, ArcB activates ArcA via autophosphorylation. Subsequently, ArcA is phosphorylated and acts as a transcriptional repressor on various genes related to aerobic respiration. ArcA consists of two main functional domains, activated by phosphorylation of its chaperone protein ArcB. The N-terminal receiver domain (RD) is located at the N-terminus of ArcA, and the C-terminal DNA-binding domain (DBD) contains a DNA-binding structural domain. When ArcA is phosphorylated and activated, the C-terminal DNA-binding domain can bind to the promoter region of target genes, regulating the expression of genes related to aerobic respiration. Phosphorylation of ArcA downregulates the transcription of key enzyme genes in the TCA cycle, reducing TCA cycle activity and thus shifting towards fermentation metabolism. Under aerobic or redox equilibrium conditions, ArcA is in a non-phosphorylated state, the expression of TCA cycle-related genes increases, and the TCA cycle is activated to maximize energy production.

[0004] Under certain stress conditions, the overall transcriptional level of the TCA cycle is downregulated due to factors such as carbon starvation and redox imbalance, which cannot provide sufficient energy and precursor supply. Summary of the Invention

[0005] In view of this, the present invention provides an ArcA mutant. Using this mutant, the efficiency of the TCA cycle can be significantly improved under certain stress conditions, such as when formic acid is the only carbon source.

[0006] The ArcA mutant provided by this invention involves mutating the 94th glutamic acid residue of the ArcA protein into a stop codon. The removal of the DNA-binding domain significantly weakens the direct role of ArcA in global regulation, but it can still participate in redox state sensing and signal transduction, and may affect cellular redox homeostasis through protein-protein interactions. These non-DNA-binding functions allow ArcA to continue functioning as a redox sensor to some extent, even though it no longer directly regulates gene expression.

[0007] Furthermore, an engineered strain, namely engineered strain BW25113* containing the ArcA mutant, is also provided.

[0008] This invention also provides a method for constructing the engineered strain, comprising synthesizing an arcA upstream 500bp - mutated arcA(E94*) sequence - arcA downstream 500bp sequence, and using up-arcA(E94*)-down as a template with pTraget-Cm R -N20 was transferred together with strain BW25113-Cm R The strain BW25113-Cm was obtained after -pCas9. R -pCas9 is used to add up-Cm R -down and pTraget-arcA-N20 were transformed into strain BW25113pCas9; strain BW25113pCas9 was obtained by transforming the pCas9 plasmid into strain BW25113; up-Cm R -down is used to connect the upstream and downstream segments of the arcA gene via the resistance gene Cm. R After being connected, it is obtained; the pTraget-arcA-N20 is obtained by designing the N20 of arcA using online gRNA-n20; the pTraget-Cm R -N20 is a Cm design using online gRNA-n20. R The resistance gene was obtained after N20; the ArcA(E94*) sequence was obtained by mutating the 94th glutamic acid in the ArcA protein into a stop codon.

[0009] Furthermore, the application of ArcA mutants in improving the bioconversion efficiency of formic acid is also provided.

[0010] Furthermore, the application of the engineered strain in reducing acetic acid production is also provided. Attached Figure Description

[0011] Figure 1 This is a comparison diagram of the ArcA amino acid sequences before and after the mutation.

[0012] Figure 2 This is a diagram of the predicted protein structure after mutation.

[0013] Figure 3 The ArcA mutant was used to increase the transcriptional levels of major genes in the TCA cycle. Detailed Implementation

[0014] Example

[0015] This invention utilizes the CRISPR / Cas9 system to mutate the base g to t at position 280 of the arcA gene, introducing a stop codon mutation into the genome. The target region of the arcA gene was amplified by PCR, and the introduction of the stop codon mutation was confirmed by Sanger sequencing. RT-qPCR was used to analyze the transcriptional levels of TCA cycle-related genes. Upregulation of transcriptional levels of acs, gltA, icd, acn, aceAB, sdhC, sucC, fum, mdh, aceEF, and lpdA was found. Experimental results demonstrate that the ArcA inactivation mutation activates the TCA cycle. Under the mutant background, the transcriptional level of the formate dehydrogenase gene fdh was simultaneously detected, showing no significant difference, ensuring that the process of formate consumption to generate NADH is normal. Intracellular energy levels were detected using a kit, revealing the NADH / NAD ratio. + The ratio was enhanced, demonstrating the contribution of TCA cycle activation to energy supply. Evaluation of the growth performance of the strains before and after the mutation revealed a decrease of approximately 90% in acetic acid production and a 10% reduction in doubling time.

[0016] Example 1

[0017] Preparation of engineered strain BW25113*

[0018] First, the proteins encoding the metE and metH genes in E. coli BW25113 that degrade methylenetetrahydrofolate were knocked out to construct a methylenetetrahydrofolate accumulating strain BW25113ΔmetEΔmetH. Based on this, serA and gcvP were knocked out to construct a serine-deficient E. coli strain BW25113ΔmetEΔmetHΔserAΔgcvP.

[0019] The ftfl-fchA-mtdA gene cluster derived from Methylobacterium extorquens AM1 was screened, and a serine-deficient strain was introduced into the strain using plasmid pED31-FFM. The strain recovered growth in M9M medium containing glycine, formic acid, and glucose, demonstrating the patency of the formic acid-to-methylenetetrahydrofolate synthesis module. The formic acid-to-methylenetetrahydrofolate synthesis module P1 was obtained.

[0020] On the other hand, based on BW25113ΔmetEΔmetH, glyA was knocked out to construct a glycine growth auxotroph strain, BW25113ΔmetEΔmetHΔglyA. Since the reverse reaction of the E. coli endogenous glycine cleavage system could not support the growth of glycine auxotrophic strains, an overexpression plasmid, pAD-gcvTHP, was constructed. The endogenous promoter of gcvTHP was replaced with LacO1 to avoid endogenous regulation by E. coli, and the RBS sequence was optimized. Plasmids containing different RBS strengths were transformed into strains evaluating glycine synthesis capacity, and the growth capacity of the strains was measured. The optimal glycine synthesis plasmid, pAD31-gcvTHP, was obtained and served as module P2 for CO2 fixation and glycine synthesis using methylenetetrahydrofolate.

[0021] Wild-type Escherichia coli can grow using serine as the sole carbon source and generate pyruvate using the endogenous gly A-encoded serine hydroxymethyltransferase and sdaA and sdaB-encoded serine deaminases. However, the growth is slow. In order to construct a more efficient pyruvate generation module, the overexpression plasmid pA C-sdaA was constructed and transformed into knockout strains for growth testing.

[0022] Overexpression of the sdaA gene accelerated the growth of all strains, with the fastest-growing strain being BW25113ΔsdaB.

[0023] To reduce the burden caused by the presence of plasmids in the strain, the sdaA gene was integrated into the sdaB gene position, thus constructing the serine utilization enhanced strain BW25113sdaB::sdaA.

[0024] Using serine as the sole carbon source, the utilization of this strain showed that its growth was significantly higher than that of the wild-type strain. It was used as a starting strain for subsequent experiments, containing the P3 module for glycine-to-pyruvate synthesis.

[0025] The formate dehydrogenase PseFD H, with the highest NADH regeneration activity, was screened in 20% CO2 air and integrated as a formate energy supply module into the genome of strain BW25113sdaB::sdaA to obtain strain BW25113sdaB::sdaAfadM::PseFdh. Based on this, formate assimilation modules P1 and P2 were introduced to construct a complete formate-trophic strain BW25113sdaB::sdaAfadM::PseFdh pED31-FFM / pAD31-gcvTHP; this strain was named engineered strain BW25113. * .

[0026] Example 2

[0027] CRISPR-Cas9-based ArcA point mutation method

[0028] (1) Transform the pCas9 plasmid into the target strain, pick single clones, culture overnight at 30℃, transfer the inoculum to fresh LB medium at a 1:100 ratio, add 10% 1M L-ara and induce culture for 2 hours until OD. 600 Approximately 0.6, prepared as competent cells for later use.

[0029] (2) Amplify 500bp upstream and downstream fragments of the arcA gene, respectively. Then design appropriate primers to connect the upstream and downstream fragments with the resistance gene Cm. R Connecting them together gives us up-Cm R -down segment.

[0030] (3) Design suitable N20 for arcA using online gRNA-N20 and synthesize primer pTraget-arcA-N20. Amplify with pTraget-arcA-N20 and pTarget-R, transform into DH5α, select single clones for sequencing, and obtain the correct plasmid pTraget-arcA-N20 for later use.

[0031] (4) Up-Cm R -down and pTraget-arcA-N20 were transformed into strain BW25113pCas9, cultured overnight in a 30°C biochemical incubator, and single colonies were picked and plated on Cm. R The single colonies that grow on the plate are sent for sequencing to verify their correctness, and the target strain is obtained.

[0032] (5) The correctly identified single-clone strain was induced to express the plasmid using 0.3 mM IPTG. After culturing for approximately 12 hours, the plasmid was streaked in three zones on a plate. Single clones were selected to verify the loss of pTarget-arcA-N20, and a second colony PCR was performed to verify the result. The single-clone strain BW25113-Cm that lost the plasmid but had a correct colony PCR result was obtained. R -pCas9.

[0033] (7) Same process design Cm R Resistance genes N20 and pTraget-Cm R -N20 plasmid

[0034] (8) After synthesizing the “arcA upstream 500bp - mutated arcA(E94*) sequence - arcA downstream 500bp”, the up-arcA(E94*)-down sequence was used as a template and pTraget-Cm R -N20 was transferred together with strain BW25113-Cm R -pCas9, incubated overnight in a 30℃ biochemical incubator, and then picked and placed in Cm R The flat plate is not long, and there is no cm. R The single clones grown on the plate, whose sequencing verification is correct, are the target strains.

[0035] (9) Induce expression of the correctly identified single-clone strain with 0.3 mM IPTG, and after culturing for approximately 12 hours, streak the culture onto a plate in three zones. Select single clones to verify pTarget-Cm. R The loss of -N20 was investigated, and a second colony PCR was performed to verify the results, yielding single clones that had lost the plasmid but had correct colony PCR results.

[0036] (10) Select single clones of the bacteria that lost N20, incubate them overnight at 42℃ to verify the loss of pCas9, and obtain the antibiotic-free target engineered strain BW25113*ArcA(E94*).

[0037] Example 3

[0038] ArcA mutant comprehensively regulates the transcriptional upregulation of major genes in the TCA cycle.

[0039] A mutation at glutamate position 94 of the ArcA protein to a stop codon completely removes its DNA-binding domain, potentially leading to the loss of transcriptional repression activity. To verify this, we measured the transcription of major genes in the TCA cycle; the data are shown in Table 2.

[0040] RNA extraction: Culture *E. coli* to the logarithmic growth phase and collect 2 OD bacterial sludge. Extract total RNA using an RNA extraction kit (Trizol). Ensure high RNA sample quality by confirming RNA concentration and purity (A260 / A280 ratio between 1.8 and 2.0) using a spectrophotometer or bioanalyzer.

[0041] Reverse transcription: Genomic DNA was removed from the RNA sample by processing with the FastKing cDNA first-strand synthesis kit, and total RNA was transcribed into cDNA.

[0042] Primer design: Specific primers for TCA circulating genes (such as gltA, sdhA, fumA, mdh, etc.) were designed using NCBI-Analyze-Primer-BLAST, and rpoA and 16S rRNA were used as internal reference genes to standardize the expression level of target genes.

[0043] Reaction system and amplification: The reaction system was prepared using the Talent Quantitative Detection Kit (SYBR Green). Amplification was performed using a two-step method (95℃ denaturation, 60℃ annealing and extension) for 40 cycles.

[0044] The experimental results are attached. Figure 3

[0045] Example 4

[0046] The application of ArcA mutants in formic acid biotransformation strains improves formic acid conversion efficiency and reduces acetic acid accumulation.

[0047] The inhibition of the TCA cycle under conditions of carbon starvation when formic acid is used as both a carbon source and energy source, and redox imbalance caused by formic acid dehydrogenase consuming formic acid to generate NADH for energy, was investigated. The results are shown in Table 1.

[0048] Table 1. Comparison of acetic acid concentration during fermentation of strains before and after mutation.

[0049]

[0050] Table 2. Main transcriptional status of TCA cycle-related genes in strains before and after mutation.

[0051]

[0052] The protein of the present invention has:

[0053] 1. Relieve ArcA's inhibition of the TCA cycle

[0054] This invention completely eliminates the transcriptional repression of TCA cycle-related genes by truncating the DNA-binding domain of mutated ArcA, and significantly enhances the activity of the TCA cycle.

[0055] This mutation improves metabolic flux in E. coli under one-carbon metabolic conditions, particularly the expression of key enzymes in the citrate cycle (such as succinate dehydrogenase gltA and malate dehydrogenase), greatly enhancing the stability and efficiency of the TCA cycle in high NADH environments.

[0056] 2. Improve redox balance

[0057] Formate dehydrogenase generates NADH by consuming formate, leading to an intracellular NADH / NAD ratio. + An imbalance in the ratio of NADH to NADH inhibits cellular metabolic activity. This invention restores intracellular redox balance and maintains normal metabolic function by enhancing the TCA cycle and promoting further oxidation of NADH.

[0058] This method helps alleviate redox stress when formic acid is the sole carbon source, ensuring energy metabolism balance and cellular homeostasis.

[0059] 3. Simple technology and easy to operate

[0060] This invention uses CRISPR / Cas9 gene editing technology to achieve the loss of Arc A function through precise point mutations. It is simple to operate and has high feasibility and stability.

[0061] The mutation sites are clearly defined and do not involve complex gene recombination or overexpression operations, which reduces the technical difficulty and cost in experiments and industrialization.

Claims

1. ArcA mutants, characterized by: The 94th glutamic acid in the ArcA protein is mutated to a stop codon.

2. Engineered strains, characterized by: Engineered strain BW25113 containing the ArcA mutant of claim 1 * .

3. The method for constructing engineered strains according to claim 2, characterized in that, Synthesize the 500bp upstream of arcA, the mutated arcA(E94*) sequence, and the 500bp downstream of arcA. Then, use the up-arcA(E94*)-down sequence as a template with pTraget-Cm. R -N20 was transferred together with strain BW25113-Cm R Obtained after using -pCas9; The strain BW25113-Cm R -pCas9 is used to add up-Cm R -down and pTraget-arcA-N20 were transformed into strain BW25113pCas9; strain BW25113pCas9 was obtained by transforming the pCas9 plasmid into strain BW25113; up-Cm R -down is used to connect the upstream and downstream segments of the arcA gene via the resistance gene Cm. R The pTraget-arcA-N20 is obtained by connecting the links; the pTraget-arcA-N20 is obtained by designing the N20 of arcA using online gRNA-n20. pTraget-Cm R -N20 is a Cm design using online gRNA-n20. R Obtained after N20 of the resistance gene; The arcA(E94*) sequence was obtained by mutating the 94th glutamic acid in the ArcA protein to a stop codon.

4. The application of the ArcA mutant according to claim 1 in reducing acetic acid production.

5. The application of the engineered strain according to claim 2 in reducing acetic acid production.

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