Exploration of the Native Sucrose Operon Enables the Development of an Inducible T7 Expression System in Paenibacillus polymyxa.

The use of Paenibacillus polymyxa as an industrial producer is limited by the lack of suitable synthetic biology tools. In this study, we identified a native sucrose operon in P. polymyxa. Its structural and functional relationship analysis revealed the presence of multiple regulatory elements, including four ScrR-binding sites and a catabolite-responsive element (CRE). In P. polymyxa, we established a cascade T7 expression system involving an integrated T7 RNA polymerase (T7P) regulated by the sucrose operon and a T7 promoter. It enables controllable gene expression by sucrose and regulatory elements, and a 5-fold increase in expression efficiency compared with the original sucrose operon was achieved. Further deletion of SacB in P. polymyxa resulted in a 38.95% increase in the level of thermophilic lipase (TrLip) production using the cascade T7 induction system. The results highlight the effectiveness of sucrose regulation as a novel synthetic biology tool, which facilitates exploring gene circuits and enables their dynamic regulation.

Metabarcoding Analysis of Microorganisms Inside Household Washing Machines in Shanghai, China.

Washing machines are one of the tools that bring great convenience to people's daily lives. However, washing machines that have been used for a long time often develop issues such as odor and mold, which can pose health hazards to consumers. There exists a conspicuous gap in our understanding of the microorganisms that inhabit the inner workings of washing machines. In this study, samples were collected from 22 washing machines in Shanghai, China, including both water eluted from different parts of washing machines and biofilms. Quantitative qualitative analysis was performed using fluorescence PCR quantification, and microbial communities were characterized by high-throughput sequencing (HTS). This showed that the microbial communities in all samples were predominantly composed of bacteria. HTS results showed that in the eluted water samples, the bacteria mainly included Pseudomonas, Enhydrobacter, Brevibacterium, and Acinetobacter. Conversely, in the biofilm samples, Enhydrobacter and Brevibacterium were the predominant bacterial microorganisms. Correlation analysis results revealed that microbial colonies in washing machines were significantly correlated with years of use and the type of detergent used to clean the washing machine. As numerous pathogenic microorganisms can be observed in the results, effective preventive measures and future research are essential to mitigate these health problems and ensure the continued safe use of these household appliances.

Emulsion for stabilizing ß-carotene and curcumin prepared directly using a continuous phase of polysaccharide-rich Schizophyllum commune fermentation broth.

In this study, we examined the effect of Schizophyllum commune fermentation broth (SCFB) rich in polysaccharides (SCFP) on the stability and bioaccessibility of ß-carotene and curcumin. An SCFB-stabilized oil-in-water (o/w) emulsion (SCFBe) was prepared using SCFB as the continuous phase, and then evaluated for storage stability using an SCFP-based emulsion (SCFPe) as the control. The findings revealed that SCFBe is more stable at 60 °C than SCFPe, and stratification or droplet size varied at differing pH levels (3-9) and concentrations of Na+ (0.1-0.5 M) and Ca2+ (0.01-0.05 M). Since the absolute value of the zeta potential of SCFBe is much lower at 60 °C than that at 4 °C and 25 °C, a higher temperature (60 °C) may enhance the reactivity of polysaccharides and proteins in SCFB to improve the stability of SCFBe. Both the protective impact of SCFB on functional food molecules and their capacity to block lipid oxidation increased as polysaccharide content improved. The bioaccessibility of ß-carotene after in vitro simulated gastrointestinal digestion is 11.18 %-12.28 %, whereas that of curcumin is 31.64 %-33.00 %. By fermenting edible and medicinal fungi in liquid, we created a unique and environmentally friendly approach for getting food-grade emulsifiers without extraction.

Lignin-degrading enzyme production was enhanced by the novel transcription factor Ptf6 in synergistic microbial co-culture.

Synergistic microbial co-culture has been an efficient and energy-saving strategy to produce lignin-degrading enzymes (LDEs), including laccase, manganese peroxidase, and versatile peroxidase. However, the regulatory mechanism of microbial co-culture is still unclear. Herein, the extracellular LDE activities of four white-rot fungi were significantly increased by 88-544% over monoculture levels when co-cultured with Rhodotorula mucilaginosa. Ptf6 was demonstrated from the 9 million Y1H clone library to be a shared GATA transcription factor in the four fungi, and could directly bind to the laccase gene promoter. Ptf6 exists in two alternatively spliced isoforms under monoculture, namely Ptf6-α (1078 amino acids) containing Cys2/Cys2-type zinc finger and Ptf6-ß (963 amino acids) lacking the complete domain. Ptf6 responded to co-culture by up-regulation of both its own transcripts and the proportion of Ptf6-α. Ptf6-α positively activated the production of most LDE isoenzymes and bound to four GATA motifs on the LDEs' promoter with different affinities. Moreover, Ptf6-regulation mechanism can be applicable to a variety of microbial co-culture systems. This study lays a theoretical foundation for further improving LDEs production and providing an efficient way to enhance the effects of biological and enzymatic pretreatment for lignocellulosic biomass conversion.

Identification of a Novel Lactose-Specific PTS Operon in Bacillus licheniformis and Development of Derivative Artificial Operon Modules.

Bacillus licheniformis plays a crucial role as a microbial host in the food industry and shows promising potential as a probiotic for human intestinal regulation. It exhibits a remarkable ability to utilize lactose as its sole carbon source. Despite its significance, the lactose-related metabolic pathway in this strain remains unclear. In this study, we identified a novel lactose-specific operon (lacDCAB) in B. licheniformis, consisting of the lacD gene that encodes a unique 6-phospho-ß-galactosidase belonging to the GH4 family, and the lacCAB genes encoding a lactose-specific PTS1 system. Notably, we constructed and assessed an array library of transport and catabolic modules specifically for lactose utilization. Among these modules, PDS-lacD-P2-pts1 demonstrated the highest specific lactose consumption rate of 0.64 g/(L·h·OD), which was 8 times higher than that of the control strain. Furthermore, we developed a dual carbon source transport model based on the PDS-lacD-P2-pts1 assembly module, which highlighted efficient coutilization of glucose/sucrose, lactose/sucrose, lactose/galactose, and lactose/2,3-butanediol. This study provides insight into the lactose-specific metabolic pathway of B. licheniformis and presents a promising strategy for enhancing lactose utilization efficiency and mixed carbon source coutilization.

Design of a sorbitol-activated nitrogen metabolism-dependent regulatory system for redirection of carbon metabolism flow in Bacillus licheniformis.

Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.

Mechanisms and biotechnological applications of transcription factors.

Transcription factors play an indispensable role in maintaining cellular viability and finely regulating complex internal metabolic networks. These crucial bioactive functions rely on their ability to respond to effectors and concurrently interact with binding sites. Recent advancements have brought innovative insights into the understanding of transcription factors. In this review, we comprehensively summarize the mechanisms by which transcription factors carry out their functions, along with calculation and experimental-based methods employed in their identification. Additionally, we highlight recent achievements in the application of transcription factors in various biotechnological fields, including cell engineering, human health, and biomanufacturing. Finally, the current limitations of research and provide prospects for future investigations are discussed. This review will provide enlightening theoretical guidance for transcription factors engineering.

A New Mechanism of Carbon Metabolism and Acetic Acid Balance Regulated by CcpA.

Catabolite control protein A (CcpA) is a critical regulator in Gram-positive bacteria that orchestrates carbon metabolism by coordinating the utilization of different carbon sources. Although it has been widely proved that CcpA helps prioritize the utilization of glucose over other carbon sources, this global regulator's precise mechanism of action remains unclear. In this study, a mutant Bacillus licheniformis deleted for CcpA was constructed. Cell growth, carbon utilization, metabolites and the transcription of key enzymes of the mutant strain were compared with that of the wild-type one. It was found that CcpA is involved in the regulation of glucose concentration metabolism in Bacillus. At the same time, CcpA regulates glucose metabolism by inhibiting acetic acid synthesis and pentose phosphate pathway key gene zwF. The conversion rate of acetic acid is increased by about 3.5 times after ccpA is deleted. The present study provides a new mechanism of carbon metabolism and acetic acid balance regulated by CcpA. On the one hand, this work deepens the understanding of the regulatory function of CcpA and provides a new view on the regulation of glucose metabolism. On the other hand, it is helpful to the transformation of B. licheniformis chassis microorganisms.

Highly Efficient Production of UDP-Glucose from Sucrose via the Semirational Engineering of Sucrose Synthase and a Cascade Route Design.

Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.

Construction of a bacteriophage-derived recombinase system in Bacillus licheniformis for gene deletion.

Bacillus licheniformis and its related strains have found extensive applications in diverse industries, agriculture, and medicine. However, the current breeding methods for this strain primarily rely on natural screening and traditional mutagenesis. The limited availability of efficient genetic engineering tools, particularly recombination techniques, has hindered further advancements in its applications. In this study, we conducted a comprehensive investigation to identify and characterize a recombinase, RecT, derived from a Bacillus phage. Remarkably, the recombinase exhibited a 105-fold enhancement in the recombination efficiency of the strain. To facilitate genome editing, we developed a system based on the conditional expression of RecT using a rhamnose-inducible promoter (Prha). The efficacy of this system was evaluated by deleting the amyL gene, which encodes an α-amylase. Our findings revealed that the induction time and concentration of rhamnose, along with the generation time of the strain, significantly influenced the editing efficiency. Optimal conditions for genome editing were determined as follows: the wild-type strain was initially transformed with the genome editing plasmid, followed by cultivation and induction with 1.5% rhamnose for 8 h. Subsequently, the strain was further cultured for an additional 24 h, equivalent to approximately three generations. Consequently, the recombination efficiency reached an impressive 16.67%. This study represents a significant advancement in enhancing the recombination efficiency of B. licheniformis through the utilization of a RecT-based recombination system. Moreover, it provides a highly effective genome editing tool for genetic engineering applications in this strain.

Preparation of water-insoluble lignin nanoparticles by deep eutectic solvent and its application as a versatile and biocompatible support for the immobilization of α-amylase.

As one of the most abundant biopolymers, lignin is a widely available resource. However, its potential largely remains untapped, with most of it ending up as waste from industries like paper production, pulp processing, and bio-refining. The research undertaken in this study focused on the extraction of lignin from agroforestry waste using a deep eutectic solvent (DES) as a carrier for α-amylase immobilization, resulting in high stability and reusability. Several techniques, including Nuclear Magnetic Resonance (NMR), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray Spectroscopy (EDS), and the Brunauer-Emmett-Teller (BET) method were employed to examine the structure and morphology of both the extracted lignin and the immobilized enzyme. The temperature used to recover lignin by DES would affect immobilization efficiency and enzyme loading by influencing its specific surface area, pore size, and volume distribution. Investigations using Nuclear Overhauser Effect Spectroscopy (NOESY) uncovered that the hydroxyl groups in G, H, and S units and the ß-O-4 structure of lignin primarily serve as binding sites for enzyme molecules. Immobilized α-amylase demonstrated a higher pH and thermal stability level, with an optimal pH of 7.0 and temperature of 100 °C, compared to the free enzyme, which exhibited optimal activity at a pH of 6.5 and temperature of 90 °C. Importantly, immobilized α-amylase retained >80 % of its initial activity even after 28 days at room temperature, and it maintained 70 % of its activity after being reused 12 times. These findings strongly suggest that lignin derived from agroforestry residues holds promising potential as a future versatile immobilization material, a prospect integral to society's sustainable development.

N-terminal lid swapping contributes to the substrate specificity and activity of thermophilic lipase TrLipE.

TrLipE is a thermophilic lipase that has potential commercial applications because of its catalytic ability under extreme conditions. Consistent with most lipases, the lid of TrLipE is located over the catalytic pocket, controls the substrate channel to the active center, and regulates the substrate specificity, activity, and stability of the enzyme through conformational changes. TrLipE from Thermomicrobium roseum has potential industrial applications, which is hindered by its weak enzymatic activity. Here, 18 chimeras (TrL1-TrL18) were reconstructed by N-terminal lid swapping between TrLipE and structurally similar enzymes. The results showed that the chimeras had a similar pH range and optimum pH as wild TrLipE but a narrower temperature range of 40-80°C, and TrL17 and the other chimeras showed lower optimum temperatures of 70°C and 60°C, respectively. In addition, the half-lives of the chimeras were lower than those of TrLipE under optimum temperature conditions. Molecular dynamics simulations indicated that chimeras had high RMSD, RMSF, and B-factor values. When p-nitrophenol esters with different chains were used as substrates, compared with TrLipE, most of the chimeras had a low Km and high kcat value. The chimeras TrL2, TrL3, TrL17, and TrL18 could specifically catalyze the substrate 4-nitrophenyl benzoate, with TrL17 showing the highest kcat/Km value of 363.88 ± 15.83 L⋅min-1⋅mmol-1. Mutants were then designed by investigating the binding free energies of TrL17 and 4-nitrophenyl benzoate. The results indicated that single, double, and triple substitution variants (M89W and I206N; E33W/I206M and M89W/I206M; and M89W/I206M/L21I and M89W/I206N/L21I, respectively) presented approximately 2- to 3-fold faster catalysis of 4-nitrophenyl benzoate than the wild TrL17. Our observations will facilitate the development of the properties and industrial applications of TrLipE.

Engineering Saccharomyces cerevisiae YPH499 for Overproduction of Geranylgeraniol.

Optimization of supply and conversion efficiency of geranylgeranyl diphosphate (GGPP) is important for enhancing geranylgeraniol (GGOH) production in Saccharomyces cerevisiae. In this study, first, a strain producing 26.92 ± 1.59 mg/g of dry cell weight squalene was constructed with overexpression of all genes of the mevalonate (MVA) pathway, and an engineered strain producing 597.12 mg/L GGOH at the shake flask level was obtained. Second, through additional expression of PaGGPPs-ERG20 and PaGGPPs-DPP1, and downregulating expression of ERG9, the GGOH titer was increased to 1221.96 mg/L. Then, a NADH HMG-CoA reductase from Silicibacter pomeroyi (SpHMGR) was introduced to alleviate the high dependence of the strain upon NADPH, and the GGOH production was further increased to 1271.14 mg/L. Finally, the GGOH titer reached 6.33 g/L after optimizing the fed-batch fermentation method in a 5 L bioreactor, with a 24.9% improvement from the previous report. This study might accelerate the process of developing S. cerevisiae cell factories for diterpenoid and tetraterpenoid production.

Synergism of Photo-Induced Electron Transfer and Aggregation-Induced Quenching Mechanisms for Highly Sensitive Detection of Silver Ion and Captopril.

Carbon-based nanoprobes, with excellent physicochemical performance and biocompatibility, are a kind of ideal nanomaterial for biosensing. Herein, we designed and prepared novel oxygen-doped nitrogen-enrichment carbon nanoribbons (ONCNs) with an excellent optical performance and uniform morphology, which could be used as a dual-mode fluorescence probe for the detection of Ag+ ion and captopril (Ctl) based on the synergism of photo-induced electron transfer and aggregation-induced quenching mechanisms. By recording the changes in fluorescent intensities of ONCNs, the Ag+ ion and Ctl concentrations can be easily tested in real samples. The results displayed that two good linear relationships existed between the change in fluorescent intensity of ONCNs and the concentrations of Ag+ ion and Ctl in the ranges of 3 µM to 30 µM and 1 µM to 30 µM, with the detection limit of 0.78 µM and 74 nM, respectively. The proposed sensing platform has also been successfully applied for the Ctl analysis in commercial tablet samples based on its high selectivity, proving its value in practical applications.

Preparation and characterization of a laccase-like enzyme from Thermomicrobium roseum.

In this study, a laccase-like gene from Thermomicrobium roseum DSM 5159 (TrLac-like) (NCBI: WP_012642205.1) was recombinantly expressed in Bacillus subtilis WB600. The optimum temperature and pH for TrLac-like were 50 °C and 6.0, respectively. TrLac-like showed high tolerance to mixed systems of water and organic solvents, indicating its potential for large-scale application in various industries. It showed 36.81 % similarity with YlmD from Geobacillus stearothermophilus (PDB:6T1B) in sequence alignment; therefore, 6T1B was employed as the template for homology modeling. To improve catalytic efficiency, amino acid substitutions within 5 Å of the inosine ligand were simulated to reduce the binding energy and promote substrate affinity. Single and double substitutions (44 and 18, respectively) were prepared, and the catalytic efficiency of the mutant A248D was increased to approximately 110-fold that of the wild type, while the thermal stability was maintained. Bioinformatics analysis revealed that the significant improvement in catalytic efficiency could be attributed to the formation of new hydrogen bonds between the enzyme and substrate. With a further decrease in the binding energy, the catalytic efficiency of the multiple mutant H129N/A248D was approximately 14-fold higher than that of the wild type but lower than that of the single mutant A248D. This is possibly because kcat also decreased with the decrease of Km; consequently, the substrate could not be released in time owing to the enzyme with the combination mutation not being able to release the substrate at a high rate.

Biotechnological and food synthetic biology potential of platform strain: Bacillus licheniformis.

Bacillus licheniformis is one of the most characteristic Gram-positive bacteria. Its unique genetic background and safety characteristics make it have important biologic applications in the food industry, including, the biosynthesis of high value-added bioproducts, probiotic functions, biological treatment of wastes derived from food production, etc. In this review, these recent advances are summarized and presented systematically for the first time. In addition, we highlight synthetic biology strategies as a potential driver of developing this strain for wider and more efficient application in the food industry. Finally, we present the current challenges faced and provide our unique perspective on relevant future research directions. In summary, this review will provide an illuminating and comprehensive perspective that will allow an in-depth understanding of B. licheniformis and promote its more effective development in the food industry.

Engineering the Thermostability of Sucrose Synthase by Reshaping the Subunit Interaction Contributes to Efficient UDP-Glucose Production.

The restricted availability of UDP-glucose, an essential precursor that targets oligo/polysaccharide and glycoside synthesis, makes its practical application difficult. Sucrose synthase (Susy), which catalyzes one-step UDP-glucose synthesis, is a promising candidate. However, due to poor thermostability of Susy, mesophilic conditions are required for synthesis, which slow down the process, limit productivity, and prevent scaled and efficient UDP-glucose preparation. Here, we obtained an engineered thermostable Susy (mutant M4) from Nitrosospira multiformis through automated prediction and greedy accumulation of beneficial mutations. The mutant improved the T1/2 value at 55 °C by 27-fold, resulting in UDP-glucose synthesis at 37 g/L/h of space-time yield that met industrial biotransformation standards. Furthermore, global interaction between mutant M4 subunits was reconstructed by newly formed interfaces according to molecular dynamics simulations, with residue Trp162 playing an important role in strengthening the interface interaction. This work enabled effective, time-saving UDP-glucose production and paved the way for rational thermostability engineering of oligomeric enzymes.

Insight into the Cold Adaptation Mechanism of an Aerobic Denitrifying Bacterium: Bacillus simplex H-b.

Psychrophilic bacteria with aerobic denitrification ability have promising potential for application in nitrogen-contaminated wastewater treatment, especially under cold conditions. A better understanding of the cold adaptation mechanism during aerobic denitrification would be beneficial for the practical application of this type of functional bacterium. In this study, Bacillus simplex H-b with good denitrification performance at 5°C was used to investigate the corresponding cold tolerance mechanism. Transcriptomics and nitrogen removal characterization experiments were conducted at different temperatures (5°C, 20°C, and 30°C). At low temperatures, more nitrogen was utilized for assimilation, accompanied by the accumulation of ATP and extracellular polymeric substances (EPS), rather than transforming inorganic nitrogen in the dissimilation pathway. In addition, the proportion of unsaturated fatty acids was higher in strains cultured at low temperatures. At the molecular level, the adjustment of membrane transport, synthesis of cofactors and vitamins, and transcriptional regulators might contribute to the survival of the strain under cold conditions. Moreover, nucleotide precursor synthesis, translation, and oxidative and temperature stress response mechanisms also enhanced the resistance of strain H-b to low temperatures. The results suggest that combining multiple regulatory mechanisms and synergistic adaptation to cold stress enabled the growth and relatively high nitrogen removal rate (27.22%) of strain H-b at 5°C. By clarifying the mechanism of regulation and cold resistance of strain H-b, a theoretical foundation for enhancing the application potential of this functional bacterium for nitrogen-contaminated wastewater treatment was provided. IMPORTANCE The newly isolated aerobic denitrifying bacterium Bacillus simplex H-b removed various forms of inorganic nitrogen (nitrate, nitrite, and ammonium) from wastewater, even when the temperature was as low as 5°C. Although this environmentally functional bacterium has been suggested as a promising candidate for nitrogen-contaminated water treatment at low temperatures, understanding its cold adaptation mechanism during aerobic denitrification is limited. In this study, the cold tolerance mechanism of this strain was comprehensively explained. Furthermore, a theoretical basis for the practical application of this type of functional bacterium for nitrogen removal in cold regions is provided. The study expands our understanding of the survival strategy of psychrophilic bacteria and hence supports their further utilization in wastewater treatment applications.

Pivotal biological processes and proteins for selenite reduction and methylation in Ganoderma lucidum.

Microbial transformations, especially the reduction and methylation of Se oxyanion, have gained significance in recent years as effective detoxification methods. Ganoderma lucidum is a typical Se enrichment resource that can reduce selenite to elemental Se and volatile Se metabolites under high selenite conditions. However, the detailed biological processes and reduction mechanisms are unclear. In this study, G. lucidum reduced selenite to elemental Se and further aggregated it into Se nanoparticles with a diameter of < 200 nm, simultaneously accompanied by the production of pungent, odorous, and volatile methyl-selenium metabolites. Tandem mass tag-based quantitative proteomic analysis revealed thioredoxin 1, thioredoxin reductase (NADPH), glutathione reductase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and cystathionine gamma-lyase as proteins involved in selenite reduction and methylation. Furthermore, the high expression of proteins associated with cell structures that prompted cell lysis may have facilitated Se release. The upregulation of proteins involved in the defense reactions was also detected, reflecting their roles in the self-defense mechanism. This study provides novel insights into the vital role of G. lucidum in mediating Se transformation in the biogeochemical Se cycle and contributes to the application of fungi in Se bioremediation.

Understanding and application of Bacillus nitrogen regulation: A synthetic biology perspective.

BACKGROUND: Nitrogen sources play an essential role in maintaining the physiological and biochemical activity of bacteria. Nitrogen metabolism, which is the core of microorganism metabolism, makes bacteria able to autonomously respond to different external nitrogen environments by exercising complex internal regulatory networks to help them stay in an ideal state. Although various studies have been put forth to better understand this regulation in Bacillus, and many valuable viewpoints have been obtained, these views need to be presented systematically and their possible applications need to be specified. AIM OF REVIEW: The intention is to provide a deep and comprehensive understanding of nitrogen metabolism in Bacillus, an important industrial microorganism, and thereby apply this regulatory logic to synthetic biology to improve biosynthesis competitiveness. In addition, the potential researches in the future are also discussed. KEY SCIENTIFIC CONCEPT OF REVIEW: Understanding the meticulous regulation process of nitrogen metabolism in Bacillus not only could facilitate research on metabolic engineering but also could provide constructive insights and inspiration for studies of other microorganisms.