Deciphering styrene oxide tolerance mechanisms in Gluconobacter oxydans mutant strain.

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L-1 styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses. Physiological changes are one of the main factors responsible for the high OS tolerance in mutant strains. Moreover, the P-type ATPase GOX_RS04415 and the LysR family transcriptional regulator GOX_RS04700 were also verified as critical genes for styrene oxide tolerance. The tolerance mechanisms of OSs can be used in biocatalytic chassis cell factories to synthesize compounds and degrade environmental pollutants. This study provides new insights into the mechanisms underlying the toxicological response to OS stress and offers potential targets for enhancing the solvent tolerance of G. oxydans.

Design-build-test of recombinant Bacillus subtilis chassis cell by lifespan engineering for robust bioprocesses.

Microbial cell factories utilize renewable raw materials for industrial chemical production, providing a promising path for sustainable development. Bacillus subtilis is widely used in industry for its food safety properties, but challenges remain in the limitations of microbial fermentation. This study proposes a novel strategy based on lifespan engineering to design robust B. subtilis chassis cells to supplement traditional metabolic modification strategies that can alleviate cell autolysis, tolerate toxic substrates, and get a higher mass transfer efficiency. The modified chassis cells could produce high levels of l-glutaminase, and tolerate hydroquinone to produce α-arbutin efficiently. In a 5 L bioreactor, the l-glutaminase enzyme activity of the final strain CRE15TG was increased to 2817.4 ± 21.7 U mL-1, about 1.98-fold compared with that of the wild type. The α-arbutin yield of strain CRE15A was increased to 134.7 g L-1, about 1.34-fold compared with that of the WT. To our knowledge, both of the products in this study performed the highest yields reported so far. The chassis modification strategy described in this study can Improve the utilization efficiency of chassis cells, mitigate the possible adverse effects caused by excessive metabolic modification of engineered strains, and provide a new idea for the future design of microbial cell factories.

N-terminal truncation (N-) and directional proton transfer in an old yellow enzyme enables tunable efficient producing (R)- or (S)-citronellal.

(R)-Citronellal is a valuable molecule as the precursor for the industrial synthesis of (-)-menthol, one of the worldwide best-selling compounds in the flavors and fragrances field. However, its biocatalytic production, even from the optically pure substrate (E)-citral, is inherently limited by the activity of Old Yellow Enzyme (OYE). Herein, we rationally designed a different approach to increase the activity of OYE in biocatalytic production. The activity of OYE from Corynebacterium glutamicum (CgOYE) is increased, as well as superior thermal stability and pH tolerance via truncating the different lengths of regions at N-terminal of CgOYE. Next, we converted the truncation mutant N31-CgOYE, a protein involved in proton transfer for the asymmetric hydrogenation of CC bonds, into highly (R)- and (S)-stereoselective enzymes using only three mutations. The mixture of racemic (E/Z)-citral is reduced into the (R)-citronellal with ee and conversion up to 99 % by the mutant of CgOYE, overcoming the problem of the reduction for the mixtures of (E/Z)-citral in biocatalytic reaction. The present work provides a general and effective strategy for improving the activity of OYE, in which the partially conserved histidine residues provide "tunable gating" for the enantioselectivity for both the (R)- and (S)-isomerases.

Microbial production of branched chain amino acids: Advances and perspectives.

Branched-chain amino acids (BCAAs) such as L-valine, L-leucine, and L-isoleucine are widely used in food and feed. To comply with sustainable development goals, commercial production of BCAAs has been completely replaced with microbial fermentation. However, the efficient production of BCAAs by microorganisms remains a serious challenge due to their staggered metabolic networks and cell growth. To overcome these difficulties, systemic metabolic engineering has emerged as an effective and feasible strategy for the biosynthesis of BCAA. This review firstly summarizes the research advances in the microbial synthesis of BCAAs and representative engineering strategies. Second, systematic methods, such as high-throughput screening, adaptive laboratory evolution, and omics analysis, can be used to analyses the synthesis of BCAAs at the whole-cell level and further improve the titer of target chemicals. Finally, new tools and engineering strategies that may increase the production output and development direction of the microbial production of BCAAs are discussed.

Exploring the mechanism of Si-Ni-San against depression by UPLC-Q-TOF-MS/MS integrated with network pharmacology: experimental research.

Background: Depression is becoming an urgent mental health problem. Si-Ni-San has been widely used to treat depression, yet its underlying pharmacological mechanism is poorly understood. Thus, we aim to explore the antidepressant mechanism of Si-Ni-San by chemical analysis and in-silico methods. Methods: Compounds in Si-Ni-San were determined by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Then, bioactive compounds were obtained from Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform and SwissADME, and the potential targets of which were acquired from SwissTargetPrediction. Depression-related targets were collected from GeneCards. The intersection between compound-related targets and depression-related targets were screened out, and the overlapped targets were further performed protein-protein interaction, biological functional and pathway enrichment analysis. Finally, networks of Si-Ni-San against depression were constructed and visualized by Cytoscape. Results: One hundred nineteen compounds in Si-Ni-San were determined, of which 24 bioactive compounds were obtained. Then, 137 overlapped targets of Si-Ni-San against depression were collected. AKT1, PIK3R1, PIK3CA, mTOR, MAPK1 and MAPK8 were the key targets. Furthermore, PI3K-Akt signalling pathway, serotonergic synapse, MAPK signalling pathway and neurotrophin signalling pathway were involved in the antidepressant mechanism of Si-Ni-San. It showed that components like sinensetin, hesperetin, liquiritigenin, naringenin, quercetin, albiflorin and paeoniflorin were the mainly key active compounds for the antidepressant effect of Si-Ni-San. Conclusions: This study demonstrated the key components, key targets and potential pharmacological mechanisms of Si-Ni-San against depression. These results indicate that Si-Ni-San is a promising therapeutic approach for treatment of depression, and may provide evidence for the research and development of drugs for treating depression.

Pre-optimization and one-step preparation of cascade enzymes system with broad substrates by model guidance: Application of chiral L-norvaline and L-phenylglycine biosynthesis.

Cascade biocatalyst systems with catalytic promiscuity can be used for synthesis of a class of chiral chemicals but the optimization of these systems by model guidance is poorly explored. In this study, a cascade system with broad substrate spectrum was characterized and simulated by kinetic model with substrates of DL-Norvaline (DL-Nor) and DL-Phenylglycine (DL-Phg) as examples. To evaluate the optimal cascade system, maximum accumulation of intermediate products and conversion rate in the process were investigated by simultaneous solution of the rate equations for varying enzyme quantities. According to the simulation results, the cascade system was optimized by regulating the expression of D-amino acid oxidase and formate dehydrogenase and was prepared by one-step. The conversion efficiency of DL-Nor and DL-Phg have been significantly improved compared with that of before optimization. Moreover, the total of L-Nor and L-Phg were reached 498.2 mM and 79.5 mM through a gradient fed-batch conversion strategy, respectively.

Combining protein and metabolic engineering strategies for high-level production of L-theanine in Corynebacterium glutamicum.

L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering. Firstly, a γ-glutamylmethylamide synthetase from Paracoccus aminovorans (PaGMAS) was isolated and engineered by computer-aided design, the resulting mutant E179K/N105R improved L-theanine yield by 36.61 %. Subsequently, to increase carbon flux towards L-theanine production, the gene ggt which degrades L-theanine, the gene alaT which participated in L-alanine synthesis, and the gene NCgl1221 which encodes glutamate-exporting protein were deleted. Finally, ppk gene was overexpressed to enhance intracellular ATP production. The reprogramed strain produced 44.12 g/L L-theanine with a yield of 57.11 % and productivity of 1.16 g/L/h, which is the highest L-theanine titer reported by Corynebacterium glutamicum. This study provides an efficient and economical biosynthetic pathway for the industrial production of L-theanine.

High-efficient production of L-homoserine in Escherichia coli through engineering synthetic pathway combined with regulating cell division.

L-Homoserine is an important amino acid as a precursor in synthesizing many valuable products. However, the low productivity caused by slow L-homoserine production during active cell growth in fermentation hinders its potential applications. In this study, strategies of engineering the synthetic pathway combined with regulating cell division were employed in an L-homoserine-producing Escherichia coli strain for efficiently biomanufacturing L-homoserine. First, the flux-control genes in the L-homoserine degradation pathway were omitted to redistribute carbon flux. To drive more carbon flux into L-homoserine production, the phosphoenolpyruvate-pyruvate-oxaloacetate loop was redrawn. Subsequently, the cell division was engineered by using the self-regulated promoters to coordinate cell growth and L-homoserine production. The ultimate strain HOM23 produced 101.31 g/L L-homoserine with a productivity of 1.91 g/L/h, which presented the highest L-homoserine titer and productivity to date from plasmid-free strains. The strategies used in this study could be applied to constructing cell factories for producing other L-aspartate derivatives.

Association detection between multiple traits and rare variants based on family data via a nonparametric method.

Background: The rapid development of next-generation sequencing technologies allow people to analyze human complex diseases at the molecular level. It has been shown that rare variants play important roles for human diseases besides common variants. Thus, effective statistical methods need to be proposed to test for the associations between traits (e.g., diseases) and rare variants. Currently, more and more rare genetic variants are being detected throughout the human genome, which demonstrates the possibility to study rare variants. Yet complex diseases are usually measured as a variety of forms, such as binary, ordinal, quantitative, or some mixture of them. Therefore, the genetic mapping problem can be attributable to the association detection between multiple traits and multiple loci, with sufficiently considering the correlated structure among multiple traits. Methods: In this article, we construct a new non-parametric statistic by the generalized Kendall's τ theory based on family data. The new test statistic has an asymptotic distribution, it can be used to study the associations between multiple traits and rare variants, which broadens the way to identify genetic factors of human complex diseases. Results: We apply our method (called Nonp-FAM) to analyze simulated data and GAW17 data, and conduct comprehensive comparison with some existing methods. Experimental results show that the proposed family-based method is powerful and robust for testing associations between multiple traits and rare variants, even if the data has some population stratification effect.

Construction of a plasmid-free L-leucine overproducing Escherichia coli strain through reprogramming of the metabolic flux.

BACKGROUND: L-Leucine is a high-value amino acid with promising applications in the medicine and feed industries. However, the complex metabolic network and intracellular redox imbalance in fermentative microbes limit their efficient biosynthesis of L-leucine. RESULTS: In this study, we applied rational metabolic engineering and a dynamic regulation strategy to construct a plasmid-free, non-auxotrophic Escherichia coli strain that overproduces L-leucine. First, the L-leucine biosynthesis pathway was strengthened through multi-step rational metabolic engineering. Then, a cooperative cofactor utilization strategy was designed to ensure redox balance for L-leucine production. Finally, to further improve the L-leucine yield, a toggle switch for dynamically controlling sucAB expression was applied to accurately regulate the tricarboxylic acid cycle and the carbon flux toward L-leucine biosynthesis. Strain LEU27 produced up to 55 g/L of L-leucine, with a yield of 0.23 g/g glucose. CONCLUSIONS: The combination of strategies can be applied to the development of microbial platforms that produce L-leucine and its derivatives.

Microbial production of L-methionine and its precursors using systems metabolic engineering.

L-methionine is an essential amino acid with versatile applications in food, feed, cosmetics and pharmaceuticals. At present, the production of L-methionine mainly relies on chemical synthesis, which conflicts with the concern over serious environmental problems and sustainable development goals. In recent years, microbial production of natural products has been amply rewarded with the emergence and rapid development of system metabolic engineering. However, efficient L-methionine production by microbial fermentation remains a great challenge due to its complicated biosynthetic pathway and strict regulatory mechanism. Additionally, the engineered production of L-methionine precursors, L-homoserine, O-succinyl-L-homoserine (OSH) and O-acetyl-L-homoserine (OAH), has also received widespread attention because they can be catalyzed to L-methionine via a high-efficiently enzymatic reaction in vitro, which is also a promising alternative to chemical route. This review provides a comprehensive overview on the recent advances in the microbial production of L-methionine and its precursors, highlighting the challenges and potential solutions for developing L-methionine microbial cell factories from the perspective of systems metabolic engineering, aiming to offer guidance for future engineering.

The flavohaemoprotein hmp maintains redox homeostasis in response to reactive oxygen and nitrogen species in Corynebacterium glutamicum.

BACKGROUND: During the production of L-arginine through high dissolved oxygen and nitrogen supply fermentation, the industrial workhorse Corynebacterium glutamicum is exposed to oxidative stress. This generates reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are harmful to the bacteria. To address the issue and to maintain redox homeostasis during fermentation, the flavohaemoprotein (Hmp) was employed. RESULTS: The results showed that the overexpression of Hmp led to a decrease in ROS and RNS content by 9.4% and 22.7%, respectively, and improved the survivability of strains. When the strains were treated with H2O2 and NaNO2, the RT-qPCR analysis indicated an up-regulation of ammonium absorption and transporter genes amtB and glnD. Conversely, the deletion of hmp gives rise to the up-regulation of eight oxidative stress-related genes. These findings suggested that hmp is associated with oxidative stress and intracellular nitrogen metabolism genes. Finally, we released the inhibitory effect of ArnR on hmp. The Cc-ΔarnR-hmp strain produced 48.4 g/L L-arginine during batch-feeding fermentation, 34.3% higher than the original strain. CONCLUSIONS: This report revealed the influence of dissolved oxygen and nitrogen concentration on reactive species of Corynebacterium glutamicum and the role of the Hmp in coping with oxidative stress. The Hmp first demonstrates related to redox homeostasis and nitrite metabolism, providing a feasible strategy for improving the robustness of strains.

A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis.

ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular-intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.

[Rational metabolic engineering of Corynebacterium glutamicum for efficient synthesis of L-glutamate].

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.

[Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis].

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

[Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum].

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 µmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Multidimensional engineering of Escherichia coli for efficient synthesis of L-tryptophan.

Development of microbial cell factory for L-tryptophan (L-trp) production has received widespread attention but still requires extensive efforts due to weak metabolic flux distribution and low yield. Here, the riboswitch-based high-throughput screening (HTS) platform was established to construct a powerful L-trp-producing chassis cell. To facilitate L-trp biosynthesis, gene expression was regulated by promoter and N-terminal coding sequences (NCS) engineering. Modules of degradation, transport and by-product synthesis related to L-trp production were also fine-tuned. Next, a novel transcription factor YihL was excavated to negatively regulate L-trp biosynthesis. Self-regulated promoter-mediated dynamic regulation of branch pathways was performed and cofactor supply was improved for further L-trp biosynthesis. Finally, without extra addition, the yield of strain Trp30 reached 42.5 g/L and 0.178 g/g glucose after 48 h of cultivation in 5-L bioreactor. Overall, strategies described here worked up a promising method combining HTS and multidimensional regulation for developing cell factories for products in interest.

Efficient 2-O-α-D-glucopyranosyl-sn-glycerol production by single whole-cell biotransformation through combined engineering and expression regulation with novel sucrose phosphorylase from Leuconostoc mesenteroides ATCC 8293.

2-O-α-D-glucopyranosyl-sn-glycerol (2-αGG) is a high value product with wide applications. Here, an efficient, safe and sustainable bioprocesses for 2-αGG production was designed. A novel sucrose phosphorylase (SPase) was firstly identified from Leuconostoc mesenteroides ATCC 8293. Subsequently, SPase mutations were processed with computer-aided engineering, of which the activity of SPaseK138C was 160% higher than that of the wild-type. Structural analysis revealed that K138C was a key functional residue moderating substrate binding pocket and thus influences catalytic activity. Furthermore, Corynebacterium glutamicum was employed to construct microbial cell factories along with ribosome binding site (RBS) fine-tuning and a two-stage substrate feeding control strategy. The maximum production of 2-αGG by these combined strategies reached 351.8 g·L-1 with 98% conversion rate from 1.4 M sucrose and 3.5 M glycerol in a 5-L bioreactor. This was one of the best performance reported in single-cell biosynthesis of 2-αGG, which paved effective ways for industrial-scale preparation of 2-αGG.

Transcriptomics reveals the effect of ammonia nitrogen concentration on Pseudomonas stutzeri F2 assimilation and the analysis of amtB function.

The biological treatment of wastewater with high concentrations of ammonia nitrogen has become a hot research issue, but there are limited reports on the mechanism of ammonia nitrogen utilization by microorganisms. In this paper, a transcriptomic approach was used to investigate the differences in gene expression at 500.0 mg/L (Amo 500) and 100.0 mg/L (Amo 100) ammonium concentrations to reveal the mechanism of ammonia nitrogen removal from water by Pseudomonas stutzeri F2. The transcriptome data showed 1015 (459 up-regulated and 556 down-regulated) differentially expressed genes with functional gene annotation related to nitrogen source metabolism, glycolysis, tricarboxylic acid cycle, extracellular polysaccharide synthesis, energy conversion and transmembrane transport, revealing the metabolic process of ammonium nitrogen conversion to biological nitrogen in P. stutzeri F2 through assimilation. To verify the effect of ammonium transporter protein (AmtB) of cell membrane on assimilation, a P. stutzeri F2-ΔamtB mutant strain was obtained by constructing a knockout plasmid (pK18mobsacB-ΔamtB), and it was found that the growth characteristics and ammonium removal rate of the mutant strain were significantly reduced at high ammonium concentration. The carbon source components and dissolved oxygen conditions were optimized after analyzing the transcriptome data, and the ammonium removal rate was increased from 41.23% to 94.92% with 500.0 mg/L ammonium concentration. The study of P. stutzeri F2 transcript level reveals the mechanism of ammonia nitrogen influence on microbial assimilation process and improvement strategy, which provides a new strategy for the treatment of ammonia nitrogen wastewater.