Graphical abstract
Keywords: Bacillus, Nitrogen metabolism, Regulation, Synthetic biology
Highlights
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Recent understanding of Bacillus nitrogen metabolism was systematically summarized.
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The potential as well as practical applications of nitrogen metabolism regulation in synthetic biology were discussed.
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The future research directions of nitrogen metabolism and synthetic biology based on nitrogen metabolism were prospected.
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More attention needs to be paid to the role of nitrogen metabolism in improving cell factory performance.
Abstract
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.
Introduction
When faced with an extreme environment, bacteria are typically unable to adapt to their environment by adjusting their metabolism. Therefore, it makes bacteria sacrifice some of their metabolic potential in order to maximize their probability of survival [1]. However, a milder external environment gives bacteria a greater regulatory permission threshold to help it achieve a relatively high-quality metabolic state. As a highly refined factory that independently reflects the external nutritional status and carries out corresponding signal transfer for fine internal regulation, systematic regulatory network and logic cannot be ignored and should be strictly followed.
As an essential nutrient, nitrogen participates in the biomass synthesis of proteins, nucleotides, and secondary metabolites for microorganism growth and metabolism [2], as well as the biosynthesis of high value-added products like antibiotics [3], [4], and enzymes [5],.etc. Bacillus is generally regarded as safe, and can use cheap nutrition sources as substrates for fermentation to reduce costs [6]. And as an important part of gram-positive bacteria heterotrophic bacterium, Bacillus has many important applications in fermentation biological manufacturing [7], [8], environmental protection including biological nitrogen removal from waste water[9], it is also used as a component of biological fertilizer to inhibit NH3 volatilization to reduce nitrogen loss in agroecosystems [10], [11]. These applications are primarily based on a relatively complete and mature internal metabolic regulation system, especially for nitrogen metabolism regulation. However, as there are currently few in-depth overviews of the regulation of nitrogen metabolism in Bacillus [12], deeper understanding of this internal regulation mechanism may be necessary to make application of the process more scientific and efficient, as well as explore other suitable fields where it may be applied.
This review summarizes the important metabolic enzymes, genes, and effectors involved in regulation of nitrogen metabolism in Bacillus. Overall, it shows that bacteria respond to disturbances in internal nitrogen homeostasis. In addition, the development of nitrogen metabolism-based metabolic engineering elements and their application to synthetic biology are innovatively summarized, and potential future research is also outlined.
Current understanding of nitrogen metabolism in Bacillus
Nitrogen metabolism tends to be a complex biological transformation process of assimilating external inorganic nitrogen like ammonium, nitrite, nitrate, or urea into organic nitrogen and keeping its concentration relatively stable for direct use, and this process involving complex and fine regulation is gradually being revealed clearly. Its investigation seems to be more inclined to model microorganism photosynthetic cyanobacteria, because it has the typical characteristic of photosynthetic microorganisms, namely, nitrogen fixation. However, little attention has been paid to heterotrophic organisms. Historically, the primary role of these organisms has been the decomposition and mineralization of dissolved and particulate organic nitrogen [13].
In fact, as a non-photosynthetic heterotrophic microorganism, it cannot simply copy the regulation mode of nitrogen sources utilization in photosynthetic autotrophic cyanobacteria. Instead, Bacillus has its own unique logic of responding to the external environment and guiding internal metabolic regulation. A better understanding of this logic can highlight possible applications, which can help maximize its potential and translate theory into actual productivity.
Ammonium: The first step in the utilization of inorganic nitrogen
Ammonium is generally the preferred nitrogen source, although Bacillus can usually obtain a variety of nitrogen sources. Almost all organic nitrogen compounds can be derived from ammonium [14], and most nitrogen sources need to be converted to ammonium before being used in biosynthesis pathways [15]. Ammonium can exist in two forms depending on different external conditions, namely free ammonia (NH3) and ammonium (NH4+). When the concentration of extracellular ammonium ion is high, or at alkaline pH a large fraction of ammonium exists as gaseous ammonia (NH3), which can enter the cells via diffusion. While at low concentration of ammonium or acidic pH, ammonium (NH4+) begins to dominate, its transport is conducted by AmtB (originally called NrgA) due to poor membrane permeability [16], [17].
Bacillus can also utilize nitrite, nitrate, or urea as nitrogen sources, and incorporate these inorganic nitrogen into biomolecules. As mentioned above, they need to be reduced to ammonium first [18]. The transporter exercises the function of transmembrane transport, allowing nitrite and nitrate entry into the cell for assimilation, subsequently, biotransformation to ammonium is accomplished by nitrite reductase [19]. As for urea, it can be directly hydrolyzed into ammonium by urease [20]. Which utilization mode the cell will choose mainly depends on which above-mentioned external nitrogen source is provided. This is the result of re-planning of the metabolic network and pathways, which involves signal transmission, transcription factors, threshold response, gene expression, and many other common factors.
GS-GOGAT cycle: Bioconversion of inorganic nitrogen and distribution
After the ammonium is transported into the cell or converted from these inorganic nitrogen, the transformation of inorganic nitrogen ammonium to organic nitrogen glutamate and glutamine needs to be completed subsequently.
Generally, glutamine and glutamate provide about 20 % and 80 % of nitrogen respectively for the synthesis of biomass and other nitrogen compounds [21], the required bioconversion and this correct distribution ratio are completed by the GS-GOGAT cycle (Fig. 1).
Fig. 1.
GS-GOGAT cycle for ammonium assimilation. The assimilation of ammonium is realized by the formation of glutamine by glutamine synthetase using ammonium and glutamate as substrates. NADPH dependent glutamate synthase catalyze the formation of glutamate from 2-oxoglutarate and glutamine. Catabolic glutamate dehydrogenase catalyze the degradation of glutamate. Enzyme annotation: glutamate dehydrogenase (GudB, RocG), glutamine synthetase (GS), glutamate synthetase (GOGAT).
For the synthesis of glutamine, it is catalyzed by glutamine synthetase (GS, encoded by glnA) using ammonium and intracellular glutamate as substrates. It is the only way to assimilate inorganic nitrogen ammonium into biomass in B. subtilis [16], and is virtually the only enzyme that can perform this function [22], its complete loss causes glutamine auxotrophy.
In general, there are two biosynthetic pathways of glutamate in bacteria. It can be biosynthesized by glutamate dehydrogenase (GDH) or glutamate synthetase (GOGAT, encoded by gltAB). Additionally, GDH can catalyze the catabolism of glutamate utilization or the anabolism of glutamate biosynthesis, such reversible reactions. There are two genes encoding catabolic glutamate dehydrogenase, the rocG gene has been proven to be the major contributor, and the other gene is gudB, which encodes its mutationally-activated form, normally an intrinsically inactive protein [23]. One pathway of synthesized glutamate is from 2-oxoglutarate and ammonium through the catalytic reduction reaction by NADPH dependent anabolic GDH. This pathway only exists when concentration of ammonium is high (>1 mM), due to the low affinity of the enzyme for ammonium [24]. Since there is no anabolic glutamate dehydrogenase in B. subtilis, de novo synthesis of glutamate can only be catalyzed by GOGAT [25]. Two molecules of glutamate will be synthesized by GOGAT by transferring the amide group from glutamine to 2-oxoglutarate. This synthetic strategy seems to be uneconomical, since the assimilation of ammonium consumes ATP, but it has the advantage that it is possible to synthesize glutamate with a very low ammonium concentration.
Glutamate, 2-oxoglutarate, and glutamine can be converted into each other and maintain a relative balance of concentration through the GS-GOGAT cycle by the combined reactions catalyzed by GS, GOGAT, and GDH. This cycle at the crossroads of carbon and nitrogen metabolism, and thus can be considered as an indicator that can determine the strength of two metabolic fluxes. In a given nutritional environment, bacteria should balance and coordinate metabolic branches to achieve high growth rates [26]. As an important catalytic enzyme in the cycle, GS is also active in nitrogen metabolism and exercises important trigger functions [27].
Main regulators and enzyme involved in nitrogen assimilation
Bacterial cells need to maintain intracellular nitrogen stability, not just in a specific branch, or a certain catalytic enzyme, but through global regulation. Recent studies have established that some regulators and enzyme respond to the specific signaling metabolites and coordinate flow through key metabolic intersections (Table 1).
Table 1.
Main contributors of nitrogen metabolism in Bacillus.
Transcription factors or enzyme | Metabolites sensed | Mediator | Function |
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TnrA | Glutamine | Glutamine synthetase–glutamine complex interacts with TnrA | Activate many nitrogen-metabolism genes |
GltC | 2-oxoglutarate; Glutamate |
Glutamate dehydrogenase | Activate gltAB operon |
Glutamine synthetase | Glutamine; NH4+ |
None | Trigger enzyme |
GlnK | ATP and 2-oxoglutarate | None | Sensor of energy state and control NH4+ absorption |
GlnR | Glutamine | Glutamine synthetase | Inactivate glnRA, ureABC and tnrA |
CodY | GTP and BCAAs | None | Activate many carbon‑and nitrogen‑metabolizing genes |
BCAAs: branched‑chain amino acids.
GltC, a mediator to modulate GS-GOGAT cycle
GltC, a DNA-binding transcription factor belonging to the LysR family, usually functions as a transcription activator of gltAB operon. The activator function can be stimulated by 2-oxoglutarate, and indirectly inhibited by excess intracellular glutamate. This indirect inhibition is actually the result of directly weakening GltC binding ability by glutamate dehydrogenase, which catalyzes the decomposition of relatively high glutamate pool to achieve its steady state concentration [28], [29]. In addition, TnrA can also indirectly participate in the regulation of gltAB operon by inhibiting the expression of GltC.
TnrA, usually as a global activator of nitrogen assimilation
TnrA is a global DNA-binding nitrogen regulator belonging to the MerR family [30], [31], a master regulator of nitrogen assimilation primarily serving as an activator, also sometimes as a repressor (for example, inhibiting glnRA operon and gltC) [32]. It has a highly conservative amino acid sequence in Bacillus, and plays its regulatory role by binding to the nucleic acid motif in the active dimer form (Fig. 2A and 2B). Its N-terminal is a DNA-binding domain, and the palindromic consensus sequence recognized is TGTNAN7TNACA [32], [33], which can also be described as TGTNANAWWWTMTNACA. The recognized binding box ususlly located immediately upstream of the −35 region of target promoters [34]. The C-terminal has the ability to combine the PII signal transduction proteins (GlnK) or feedback-inhibited glutamine synthetase (FBI-GS) to exercise specific biological functions and protect from protease degradation.
Fig. 2.
The structure of dimer TnrA and DNA complex and its regulated target genes. (A) Surface model of TnrA-DNA complex. (B) The TnrA-DNA complex. TnrA consists of two structural regions, N-terminal region for DNA-binding, and C-terminal region for GlnK or GS binding. (C) Target genes regulated by TnrA. TnrA activates the transcription of tnrA, ureABC operon, nasBC, and nasDEF operon, and inhibits the transcription of gltC and glnRA operon. The solid lines with hollow arrowheads indicate the expression products of the genes; the solid lines with solid arrowheads and blunted lines indicate positive and negative regulations by transcription factors, respectively. Enzyme annotation: nitrate reductase (NR), nitrite reductase (NiR), glutamate dehydrogenase (GudB, RocG).
There is only one nitrite reductase (NiR) in Bacillus subtilis, which is encoded by the nas operon [19]. TnrA could enhance the assimilation of nitrite and nitrate by activating transcription through binding to the motif in the nasBC and nasDEF promoter [18]. For urease, which can hydrolyze urea to ammonium and carbon dioxide. TnrA can promote the formation of urease by activating transcription of its structural genes ureABC, it makes B. subtilis show great potential for urea decomposition [35]. The common characteristics of these genes are required for the utilization of alternative nitrogen sources (Fig. 2C) [36]. In conclusion, TnrA can enhance the bioconversion of nitrite, nitrate and urea to ammonium, therefore, it can be considered as an activator of nitrogen assimilation under nitrogen limited conditions.
Glutamine synthetase, nitrogen monitor and signal transmitter
Glutamine synthetase (GS) in Bacillus is a large protein containing 12 identical subunits, TnrA can induce pore opening and make dodecamer into tetradecamer [37] (Fig. 3A). Tetradecamer can combine with 14 TnrA molecules through protein–protein interaction to form a tight complex [38], this state inhibits the dimerization of TnrA to greatly reduce the regulatory activity of TnrA (Fig. 3B). The active site of GS is located between the subunit interfaces [39], its enzyme activity can be directly inhibited by high concentrations of glutamine products [40]. The basis for glutamine feedback inhibition is an intimate hydrogen bond network could be formed between the residues Arg62, Glu304, and its products glutamine [41], this special conformation prevents the release of glutamine.
Fig. 3.
Structures of GS, GlnK, and CodY. (A) Surface model of tetradecamer GS; (B) Molecular mechanism for FBI-GS inhibits DNA binding of TnrA. When both subunits of the dimer TnrA bind to the appropriate binding pockets of FBI-GS, the TnrA dimer would be disrupted. (C) Surface model of GlnK–TnrA complex. (D) Cartoon model of the GlnK-ATP complex. (E) Surface model of CodY. (F) Cartoon model of CodY.
GS can be proposed to be a monitor for the nitrogen status [42]. To a certain extent, low or high intracellular glutamine pools is perceived by GS as a limited or excess nitrogen conditions, respectively [43]. It shows two states according to the different nitrogen content, namely the ATP-bound state for catalyzing the formation of glutamine when nitrogen limited, and the binding state (FBI-GS state) for binding with the nitrogen metabolism regulators (like TnrA) for transduction of nitrogen signal when nitrogen excess [44]. Competition between ATP and glutamine for GS is likely to adjust the equilibrium between the two states.
GlnR, a repressor in nitrogen assimilation
Another DNA-binding regulatory protein that belongs to the MerR family is GlnR, its coding gene glnR and the adjacent glnA together form the glnRA operon. GlnR has typical feature of MerR family amino acid sequence in the N-terminal similar to TnrA, but the difference in C-terminal leads to the self-inhibiting of its monomer. This inhibition can be alleviated by C-terminal deletions [45], or combination with FBI-GS which can relieve auto inhibition shift the equilibrium to the DNA-binding active dimer form [37] (Fig. 4A). In most cases, GlnR and TnrA share the same binding site [36], but their regulatory activities respond differently to changes in nitrogen levels [46]. GlnR generally represses gene expression [47], it has three target genes, namely tnrA, ureABC operon, and its own glnRA operon in Bacillus subtilis [31], [48], [49] (Fig. 4B). Based on the above information, it can be concluded that GlnR is a repressor of nitrogen assimilation during nitrogen excess.
Fig. 4.
The schematic model for GlnR. (A) Conversion from self-inhibited state to activated state for DNA-binding by FBI-GS or C-terminal deletions. (B) Target genes regulated by GlnR. GlnR inhibits the transcription of tnrA, glnRA operon, and ureABC operon.
GlnK, a link between nitrogen regulation and energy
GlnK (originally called NrgB) is a signal transduction protein belonging to PII family, which can respond to cellular signals to maintain the cellular homeostasis (Fig. 3C) [16]. Its coding gene glnK invariably acts in conjunction with structural genes amtB to form the nrgAB operon. The native GlnK protein has a trimeric structure that can readily bind ligand ATP or weakly bind 2-oxoglutarate in the absence of divalent cations (Fig. 3D) [17].
In addition, GlnK appears to be able to bind membrane ammonium transporter AmtB to block the transport of ammonium or regulator TnrA responding to nitrogen limitation [17], [37]. Some studies have shown that when in complex with ATP, the GlnK protein loses the ability to bind TnrA [50]. The overlap of binding region seems to explain why the GlnK–ATP complex cannot bind TnrA [37]. The competitive binding of TnrA and ATP with GlnK acts as a sensor and integrator [51], to a certain extent, a correlation between nitrogen regulation and cellular energetic seems to be established.
CodY, a top regulatory of programmed regulation
CodY is a highly conserved protein with a global regulatory effect (Fig. 3 E and 3F) [52]. It controls the expression of more than a hundred genes both in carbon and nitrogen metabolism, which is usually inhibited during rapid growth and induced when cells undergo nutritional deprivation [53] (e.g., sporulation genes). Most genes regulated by CodY have identifiable nucleotide binding motifs (AATTTTCWGAAAATT) that directly bind to CodY in their promoter or coding sequence[54], which guarantees the exercise of its regulatory functions. Two classes of effector molecules can activate CodY as a DNA binding protein in vitro. One is the branched-chain amino acids (BCAAs), which include leucine, isoleucine, and valine (particularly isoleucine and valine), and the other is GTP[55]. The synthesis pathway of BCAAs is repressed by both CodY and the nitrogen regulator TnrA [54]. CodY may have many different forms to regulate the expression of target gene. For example, caused by the changes in intracellular effector concentration pools [56], [57].
BCAAs are the most abundant amino acids in proteins, and maintaining the stability of intracellular pools is essential for efficient protein synthesis. Since CodY can sense intracellular metabolites (GTP, BCAAs), it provides the top layer of general nutritional regulation that can determine the expression rate of central metabolic genes [22], and BCAAs also seem to be widely used by bacteria to monitor their physiological and biochemical status.
One of the main functions of CodY seems to establish a hierarchical utilization of various nitrogen-containing compounds under conditions of nutrient excess, like the carbon catabolite repression (CCR) in the utilization of carbon sources, allowing early consumption of such compounds to have an active effect, and repress the utilization pathways that are less preferred [12], [58]. Thus, CodY is partly responsible for implementing several strategies to adapt to changing nutritional conditions by perceiving the nutritional and energy state of cells [59], [60]. This result is also consistent with the theory that under natural conditions, the external nutritional conditions faced by bacteria are variable, and after a long period of evolution, bacteria have come to have a perfect system of self-regulation to face changes in external nutritional conditions [22]. So far, a good diagram describing nitrogen metabolism of Bacillus can be displayed (Fig. 5).
Fig. 5.
An excellent overview of Bacillus nitrogen metabolism and some applications based on it. Lines with hollow arrowheads indicate biochemical conversion and transmembrane transport of related compounds; Lines with solid arrowheads indicate positive regulation by transcription factors; Blunted lines indicate negative regulation by transcription factors. Dotted Lines with arrowheads indicate CodY is positively regulated by branched-chain amino acids (BCAAs). CATP: the concentration of ATP, high concentration of ATP releases GlnK from the complex formed with AmtB.
Steady state regulation of intracellular nitrogen
Changes in endogenous metabolism may occur on different time scales from changes in the microenvironment [61]. When bacterial cells are in an environment with a rich external nitrogen source, the concentrations of intracellular glutamine are thought to become high, making the trigger enzyme GS feedback-inhibited form FBI-GS, which can both combine the C-terminal of TnrA and GlnR. The FBI-GS-TnrA complex makes TnrA deactivate, owing to losses in its ability to bind to DNA. Meanwhile, the combination of FBI-GS and GlnR folding its C-terminal tail into a helix and preventing auto inhibition effect of GlnR, it gives GlnR ability to form GlnR-DNA complex structure through transient protein–protein interaction. Such effects on two transcription factors can coordinate to reduce nitrogen assimilation. This reduction is manifestation of the economic characteristics of the bacteria cell, because there is no need for it to consume resources and energy for assimilation in an intracellular nutrient-rich state.
As the consumption of nitrogen gradually assimilates into the synthesis of biomass, the concentration of cell will increase. FBI-GS will be difficult to form because the concentration of the glutamine pool will decrease significantly. As nitrogen becomes limited, TnrA will be released from GS, and the genes that enhance nitrogen assimilation will be efficiently expressed due to the restoration of their regulatory activity as an activator. Meanwhile, since GlnR is not supported by FBI-GS, it restores the self-inhibited state that has no regulatory activity to suppress genes related to nitrogen assimilation. Their unique regulation, coordination and competition characteristics play a decisive role in maintaining the steady state of intracellular nitrogen (Fig. 6). To a certain extent, it also confirmed that effective use of the nutrients available in the environment is important, and this is achieved by tightly controlling the major metabolic intersections [22].
Fig. 6.
The mechanism of intracellular nitrogen homeostasis. Transcription factors interact with FBI-GS to inhibit the transcription of nitrogen assimilation genes in nitrogen excess (Left); The transcription factors in the original state activates the transcription of nitrogen assimilation genes in nitrogen limited (Right).
Present and potential application for synthetic biology
Based on the aforementioned in-depth understanding of nitrogen metabolism, it is actually a complex, delicate and rigorous dynamic endogenous regulation process played by promoters, transcription factors, key genes, signal transduction proteins and energy, etc. There may be some areas in which it can play a role.
Synthetic biology is defined as a gene expression system that is as independent of host metabolism as possible, but does engage in minimal interference with host metabolism [62], [63]. Strategies usually focus on targeted modification of rate-limiting steps, such as promoters enhancement [64], ribosome binding sites (RBS) modification [65], pathway diversion [66] to improve the productivity of microbial cell factories [67], [68]. To a certain extent, nitrogen metabolism satisfies the rigid demand of the integration of metabolic components and genetic circuit design to improve production efficiency in synthetic biology, the possibility for its application in the field of synthetic biology has attracted more attention and some practical applications have been carried out. It seems to be an effective supplementary regulation strategy at the nitrogen source, which is different from carbon source level.
Engineering of nitrogen transcription factors
The transcription factor bind to specific DNA sequence (binding site) in target promoter area and exercise function of activating or inhibiting transcription [69], [70]. Generally, transcription factors are important participants in the metabolic network and can dynamically determine the direction of metabolic flux to a certain extent. There is reason to believe that the loss of related transcription factors tends to produce pleiotropic phenotypes rather than a desirable strategy [71], but even so, the application of transcription factor-deficient mutants in synthetic biology has proven to be an exciting achievement. For example, after knockout the gene encoding the nitrogen transcription regulator GlnR, the glnR-deletion host cell shows greater adaptability to low nitrogen, which can enhance the biotransformation of phytosterol [72]. In contrast, the strategy of artificially fine-changing the expression level of transcription factors seems to be more adopted, such as improvement of biosynthesis of riboflavin [73] and bacitracin [74] (Table 2). In order to maximize the yield of the interesting bioproducts, numerous studies have shown that how to artificially change the expression level of regulatory proteins based on their own enhancing or inhibiting effects in the pathway which they are located. In brief, re-direction of metabolic flux to more efficiently synthesize the target product can be achieved with the help of intracellular plasmids with regulatory functions, this means that strengthening the pathways flux of interest; simultaneously, weakening the bypass pathways flux which generate by-products (Fig. 7A).
Table 2.
Application of nitrogen metabolism to improve biosynthesis.
Strategies involved | Target bioproducts | Result | Improvement | Reference |
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Knockout glnR | Androstenedione | Conversion ratio of 75.7 ± 3.5 % to 86.3 ± 3.7 % via phytosterols | 14.00 % | [72] |
Deletion of rocG, gudB, and overexpression of gdh | N-acetylglucosamine | 14.0 to 22.2 g/L | 58.57 % | [93] |
Knock down tnrA and glnR | Riboflavin | 1.67 to 2.28 g/L | 36.73 % | [73] |
Overexpressed amtB, glnA, gdH | N-acetylglucosamine | 16.0 to 27.3 g/L | 70.6 % | [106] |
Overexpression of codY, tnrA, and glnR | Bacitracin | 890.05 to 987.62 U/mL | 10.96 % | [74] |
Overexpression of glnA | l-arginine | 29.8 to 32.8 ± 0.6 g/L | 9.70 % | [94] |
Fig. 7.
Applications of nitrogen transcription factors in synthetic biology. (A) re-direction of metabolic flux by controlling the expression of genes encoding transcription factors. Transcriptional repressor (red color) and activator (blue color) can bind to their respective binding sites (binding site/R and binding site/A) to inhibit and activate the target promoter, respectively. The position of the binding sites in the synthetic pathway of the target product needs to be considered. For example, when binding site/A and binding site/R are located in the promoter of the gene on the interested pathway and bypass pathway, respectively, should overexpress both TFr and TFa in plasmid. If the binding sites are in the opposite position, down-regulate both TFr and TFa in plasmid is necessary. (B) Development of nitrogen-responsive promoters. (C) Application of nitrogen biosensor in genetic circuit. When nitrogen replete, the biosensor that senses the external nitrogen content turns off the key gene for product synthesis to allow cell growth, when the cell accumulates to a certain amount, nitrogen beginning at limited stage, biosensor turns on the key gene for products biosynthesis, and desired bioproducts can be accumulated. (D) Development of artificial transcription factors. Building a random mutational library, then using high-throughput screening method to obtain competitive strains and finally its application. RNAP: RNA polymerase; RBS: ribosome binding site; FACS: fluorescence-activated cell sorting; TFr: gene encoding transcriptional repressor; TFa: gene encoding transcriptional activator. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
However, it should be noted that natural transcription factors are usually unable to meet the requirements of ideal production capacity and efficient transformation tools, due to the limited regulation threshold and response substrate spectrum [75]. Artificial modification can be performed to mitigate such natural deficiency. The mechanism of action seems to stipulate the direction of modification, that is, the combined DNA motif or the transcription factor protein itself. Improvement can be made by artificial mutation in the recognized nucleic acid binding region [76], [77], [78]. For the modification of transcription factors, site-directed mutagenesis strategy is usually adopted to meet the specific needs in application [79], and the understanding of the mechanism can be built at the molecular-level [80], [81], [82].
In addition, building a random mutation library to develop artificial transcription factors is also an effective method. After that, high-potential transcription factors can be screened by using high-throughput screening method like fluorescence-activated cell sorting (FACS). Finally, they can be designed as key elements in genetic circuits and used in synthetic biology [83], [84] (Fig. 7D). Together, these examples demonstrate that the effectiveness and feasibility of transcription factors engineering strategies in improving the productivity of strains.
Wild-type and artificial nitrogen-dependent promoters
With the in-depth study of metabolic regulation, some nitrogen-dependent promoters with special functions have gradually been discovered and efficiently applied. For example, the ammonia-inducible promoters can realize multiple effects that mediate enzyme expression and also adjust the pH value by adding ammonium hydroxide in industrial fermentation [85]. Additionally, some promoters that can re-direction the carbon flow in response to nitrogen deficiencies in the environment have also been unearthed [86], [87]. They can be further engineered into biosensors as regulatory switches that regulate gene expression of key pathways to achieve dynamic regulation in synthetic biology. For example, nitrogen-limitation biosensor which is induced by nitrogen starvation could be developed to improve two-stage production and increases itaconic acid yields [88] (Fig. 7C).
However, there are also some promoters that are not actually used in production, but still remain at the research on mechanism, for example, the promoter of glutaminase which can be induced by glutamine [89], promoter of nitrate reductase [90] or the RNA polymerase group 2 sigma factor SigE [86] can be induced by nitrate, etc (Fig. 7B). For more diverse needs, differential RNA-seq method seems to be an effective means to satisfy researchers in mining more nitrogen-dependent promoters in the genome in response to nitrogen availability [91], [92]. Theoretically, some of them can be used scientifically and efficiently as biological elements after certain structural modification, for example, adjusting response thresholds, expanding or reducing the effectors spectrum. It is gratifying that solid theoretical research is also a process of laying the foundation for their full application.
Strengthening of nitrogen metabolic pathway
An adequate carbon source is usually given to ensure sufficient supply of the carbon skeleton of the product in synthetic biology. However, it is important to note that most biosynthetic pathway of value-added compounds highly depend on global metabolism, strengthening the intensity of nitrogen metabolism to coordinate between them may be another effective way.
Since bacteria have strict regulation to ensure internal nitrogen homeostasis, the homeostasis of a nitrogen donor in an intracellular environment also provides a guarantee for efficient biosynthesis of value-added nitrogen-containing compounds. Even so, the intensity of endogenous or additional nitrogen supply is usually strengthened to further improve the production efficiency of microbial cell factories during synthesis process, for example, the supply of intracellular glutamine is increased by knocking out or overexpressing related genes to increase the titer of N-acetylglucosamine [93] or arginine [94]. In addition to metabolic engineering strategies that supplement endogenous nitrogen metabolism, external supplementation of nitrogen sources to adjust nitrogen content is also effective. Methods include the addition of a low-cost nitrogen source urea instead of expensive aspartate to the medium that enhances the supply of nitrogen to de novo synthesis of high-value ectoine [95], or using urea as a nitrogen source, which has been shown to stimulate itaconate production in some cases [96].
The reason why strengthening nitrogen metabolic pathway is effective, just like bucket effect, when the same water flow is provided (substrate supply), its water storage capacity (the yield of bioproduct) is determined by the relatively shortest board (the weakest metabolism). Great achievement will be made in improving the weak links (nitrogen metabolism) (Fig. 8 A).
Fig. 8.
Visualization models of industrial strains (A) Strengthening and coordination of metabolic modules. Strengthening of nitrogen metabolic pathway to improve the yield of bioproduct and coordinating global metabolic network to further improve. (B) Microbial cell factory model. The product requires a long biosynthetic pathway starting from glucose as the sole substrate, the substrates that directly access multiple pathways can shorten the biosynthetic pathway, and biosynthesis becomes more efficient.
Coordination of global metabolic network
Glucose is usually the preferred carbon source. However, some works have proven that it supports lower growth rates than secondary sugars by using the amino acid like arginine, proline, or glutamate as sole nitrogen source [97]. Even so, bioproduct synthesis is usually implemented in the laboratory and industry with a single organic carbon source as input because of simplicity [98], [99]. Because this ‘bug’ phenomenon can be almost completely avoided, that is, exposure to glucose usually occurs when the nitrogen source is sufficient, in other words, a wider mixture of amino acids that substrates provide are usually encountered in the actual industrial fermentation process. In addition, the use of specific amino acids can increase the productivity of the strain. And with the rapid development of metabolic engineering, mixed carbon sources containing various sugars are often provided for microbes in order to get a high yield of interested products [100]. This strategy has been widely adopted, because the substrates that directly access multiple pathways can meet the timely and economical requirements of biosynthesis, means that, different types of carbon skeletons can be flexibly added to the synthetic pathway in cellular systems. By contrast, when a single sugar is used as a substrate, microbial cell factories are forced to choose a longer synthetic pathway that requires more resources and energy to be allocated, thus, the reduction in product volume naturally becomes reasonable (Fig. 8 B). It shows the fact that improvement can be achieved by coordinating the carbon metabolism module.
Substrates can be transformed into secondary metabolites, energy, electron carriers, and building blocks of macromolecules through metabolic pathways [101]. The essence of this conversion is a product of global metabolism, which is a complex, non-linear, multi-scale and autocatalytic biochemical process. Current metabolic engineering strategies usually funnel metabolic fluxes by “increasing revenue and reducing expenditure”. It is a fact that the use of local optimization and supervision to improve production performance can easily lead to metabolic imbalance [102]. Research methods of microbial metabolism modularization has become a trend to solve the problem, and progress is expected. While, individual metabolic reactions cannot exist independently of the host global metabolism to which it is attached. In this case, coordinating the global metabolism becomes particularly important [103]. It is like nailing the belts tightly to the barrel, which makes board (metabolism module) fit more closely (harmonious), and thus water storage capacity (the yield of bioproduct) can be further improved (Fig. 8 A).
Conclusions and future perspectives
Glutamine metabolism plays a prominent role in the utilization of nitrogen sources by Bacillus, and the maintenance of a relatively suitable and stable intracellular glutamine pool is critical for bacterial cells. During this process, glutamine synthetase detects the pool in real time and transmits dynamic nitrogen signals to transcription factors. Upon receipt of the signals, the transcription factors achieve a structural modification-mediated switch between active and inactive bioregulation, and ultimately adapt the expression levels of their targeted nitrogen assimilation-related pathway genes to the current nitrogen levels. Such a complex and large, yet finely- and efficiently-regulated mechanism ensures a rapid return to homeostatic levels through the synergistic cooperation of various aspects when cells experience an unsuitable nitrogen state. The specific regulatory mechanisms involved can be adapted to synthetic biology through engineering transcription factors or promoters or strengthening and coordinating metabolic modules so that their contributions can be fully realized. It can be broadly described as a new drive at the nitrogen level and is receiving attention and showing unprecedented potential.
A relatively clear theoretical network of nitrogen metabolism has been established through access to relevant information. However, some of the specific local details used to build this network are interesting and deserve to be investigated in depth, such as the fact that when the formation and dissociation of the complexes were monitored in real time, it was found that FBI-GS-TnrA can bind to the nrgAB promoter even in the presence of excess glutamine [44] and non-feedback inhibited GS can also bind weakly to TnrA [50]. These phenomena cannot yet be explained by established theories. Thus, some studies need to be conducted to elucidate these findings. Additionally, transcription factor binding sites are located in other structural genes or promoters on the genome in addition to the target genes mentioned about. Therefore, there are other targets genes for regulation, so a wider range and deeper supervision may exist and requires further study.
Only a few applied research studies have considered the effect of nitrogen metabolism on product enhancement. More extensive development and application of these nitrogen metabolism-related transcription factors or promoters should be implemented to enrich the dynamic regulation tools of metabolic engineering. For relevant components with important applications, efforts should be made to uncover the features that make them exhibit excellent qualities in order to maximize their application value. One example is biosensors, which respond to nitrogen in a complex nitrogen environment. The specific effector of these biosensors is still unknown, so confirmatory experiments should be designed and carried out to investigate the specific effectors. More importantly, once a more in-depth understanding of the relevant mechanisms and a foundation for the application of biosynthesis is obtained, researchers can turn their focus to enriching the types of target products produced by microorganisms and solving the challenge of output differences caused by standardized genetic elements in chassis cells of different genetic backgrounds. In this way, a wide range of microbial host resources could be used more efficiently.
For Nitrogen Catabolite Repression (NCR), good nitrogen sources are easier to use. This phenomenon has been most extensively researched in Saccharomyces cerevisiae as a pioneering organism for nutrition sensing and signaling mechanisms [104]. In order to overcome the inhibitory effect of catabolites, industrial fermentation processes are usually carried out in a fed-batch mode restricted by substrates [105]. In current industrial biotechnology (CIB), sufficient or even excessive nitrogen sources are generally provided to ensure efficient output of nitrogen-containing target products. However, whether NCR also exists in Bacillus still remains to be seen, which makes it challenging to balance productivity and resource conservation. The next generation of industrial biotechnology (NGIB) will need to ensure high output while maintaining economical consumption of its substrate, thereby achieving cost-effective manufacturing. Therefore, further research is necessary to increase its competitiveness.
Compliance with Ethics Requirements
This article does not contain any studies with human or animal subjects.
CRediT authorship contribution statement
Hehe He: Investigation, Visualization, Writing – original draft. Youran Li: Conceptualization, Supervision, Writing – review & editing. Liang Zhang: Resources, Funding acquisition. Zhongyang Ding: Investigation, Funding acquisition. Guiyang Shi: Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by National Key Research & Development Program of China (2018YFA0900504, 2020YFA0907700, and 2018YFA0900300), the National Natural Foundation of China (31401674), the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-22), and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
Biographies
Hehe He obtained his M.S. degree in 2019. And now is studying for his Ph.D. in fermentation engineering in the School of Biotechnology, Jiangnan University. His Ph.D. topic is the global study and regulation of nitrogen metabolism in Bacillus. He is working to gain a deep understanding of the complicated regulatory mechanism of nitrogen metabolism and to develop efficient synthetic biological elements on this basis. His advisors, Youran Li and Guiyang Shi, encouraged him to write this review article to present the current understanding and and future research directions.
Youran Li obtained his Ph.D. degree in fermentation engineering at Jiangnan University in 2014. After that, he became Assistant Professor and Associate Professor in the School of Biotechnology, Jiangnan University. His current research focuses mainly on applied research and technology development in the field of industrial microorganisms, especially development of Bacillus chassis cells and refined metabolic engineering. He began to work on this topic in 2014 and aims to improve the efficiency of biotransformation of cell factories through regulating the nitrogen metabolism pathways in traditional industrial fermentation.
Liang Zhang obtained his Ph.D. degree in fermentation engineering at Jiangnan University in 2005. After that, he became Assistant Professor, Associate Professor, and Full Professor in the School of Biotechnology, Jiangnan University. He served as a cadre of the Ministry of Education, China in 2015, and he is the Director of Principal Office of Jiangnan University from 2019. His current main research focus is on biomanufacturing and environmental bioremediation. He began to work on this topic in 2011 and wish to produce high value-added products and repair environmental pollution through biotechnology.
Zhongyang Ding obtained his Ph.D. degree at Nanjing Agricultural University in 2007 and became Associate Professor in 2008. He was a visiting scholar at The Hong Kong Polytechnic University from 2013 to 2014. Since then, he became Full Professor in the School of Biotechnology, Jiangnan University and he is the Dean of the School of Biotechnology, Jiangnan University from 2018. His current research focuses mainly on development and application of fungal active natural products and enzyme resources. He began to work on this topic in 2008 and aims to realize the broadening and efficient application of biomass resources.
Guiyang Shi obtained his Ph.D. degree in fermentation engineering from Jiangnan University (formerly Wuxi University of Light Industry) in 1995. Since then, he became Assistant Professor, Associate Professor, and Full Professor in the School of Biotechnology, Jiangnan University. He is Academic leader of Science and Technology Innovation Team in Jiangsu Provence, China. His research interests include (i) fermentation and controlling metabolic processes, (ii) separation and purification technology of fermentation products and (iii) wine industry technology. Since 2015, he has focused on studying the regulation mechanisms of the nitrogen metabolism during the traditional fermentation process and the development of synthetic biological elements, such as biosensor.
Footnotes
Peer review under responsibility of Cairo University.
Contributor Information
Youran Li, Email: liyouran@jiangnan.edu.cn.
Guiyang Shi, Email: gyshi@jiangnan.edu.cn.
References
- 1.Chatterji D., Kumar Ojha A. Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol. 2001;4(2):160–165. doi: 10.1016/s1369-5274(00)00182-x. [DOI] [PubMed] [Google Scholar]
- 2.Gobert A., Tourdot-Maréchal R., Sparrow C., Morge C., Alexandre H. Influence of nitrogen status in wine alcoholic fermentation. Food Microbiol. 2019;83:71–85. doi: 10.1016/j.fm.2019.04.008. [DOI] [PubMed] [Google Scholar]
- 3.Li J., Pan Y., Liu G. Disruption of the nitrogen regulatory gene AcareA in Acremonium chrysogenum leads to reduction of cephalosporin production and repression of nitrogen metabolism. Fungal Genet Biol. 2013;61:69–79. doi: 10.1016/j.fgb.2013.10.006. [DOI] [PubMed] [Google Scholar]
- 4.Niehaus E.-M., Kleigrewe K., Wiemann P., Studt L., Sieber C.K., Connolly L., et al. Genetic Manipulation of the Fusarium fujikuroi Fusarin Gene Cluster Yields Insight into the Complex Regulation and Fusarin Biosynthetic Pathway. Chem Biol. 2013;20(8):1055–1066. doi: 10.1016/j.chembiol.2013.07.004. [DOI] [PubMed] [Google Scholar]
- 5.Yasumura A., Abe S., Tanaka T. Involvement of nitrogen regulation in Bacillus subtilis degU expression. J Bacteriol. 2008;190(15):5162–5171. doi: 10.1128/JB.00368-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Gu Y., Xu X., Wu Y., Niu T., Liu Y., Li J., et al. Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications. Metab Eng. 2018;50:109–121. doi: 10.1016/j.ymben.2018.05.006. [DOI] [PubMed] [Google Scholar]
- 7.Xiao F., Li Y., Zhang Y., Wang H., Zhang L., Ding Z., et al. Construction of a novel sugar alcohol-inducible expression system in Bacillus licheniformis. Appl Microbiol Biot. 2020;104(12):5409–5425. doi: 10.1007/s00253-020-10618-8. [DOI] [PubMed] [Google Scholar]
- 8.Mattanovich D., Ivan Nikel P., Zhang Q., Wu Y., Gong M., Zhang H., et al. Production of proteins and commodity chemicals using engineered Bacillus subtilis platform strain. Essays Biochem. 2021;65(2):173–185. doi: 10.1042/EBC20210011. [DOI] [PubMed] [Google Scholar]
- 9.Yang Q., Yang T., Shi Y.i., Xin Y.u., Zhang L., Gu Z., et al. The nitrogen removal characterization of a cold-adapted bacterium: Bacillus simplex H-b. Bioresource Technol. 2021;323:124554. doi: 10.1016/j.biortech.2020.124554. [DOI] [PubMed] [Google Scholar]
- 10.Sun B.o., Bai Z., Bao L., Xue L., Zhang S., Wei Y., et al. Bacillus subtilis biofertilizer mitigating agricultural ammonia emission and shifting soil nitrogen cycling microbiomes. Environ Int. 2020;144:105989. doi: 10.1016/j.envint.2020.105989. [DOI] [PubMed] [Google Scholar]
- 11.Sun B.o., Gu L., Bao L., Zhang S., Wei Y., Bai Z., et al. Application of biofertilizer containing Bacillus subtilis reduced the nitrogen loss in agricultural soil. Soil Biol Biochem. 2020;148:107911. [Google Scholar]
- 12.Fisher S.H. Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference! Mol Microbiol. 1999;32(2):223–232. doi: 10.1046/j.1365-2958.1999.01333.x. [DOI] [PubMed] [Google Scholar]
- 13.Luque-Almagro V.M., Gates A.J., Moreno-Vivian C., Ferguson S.J., Richardson D.J., Roldan M.D. Bacterial nitrate assimilation: gene distribution and regulation. Biochem Soc Trans. 2011;39(6):1838–1843. doi: 10.1042/BST20110688. [DOI] [PubMed] [Google Scholar]
- 14.Suzuki A., Knaff D.B. Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism. Photosynth Res. 2005;83(2):191–217. doi: 10.1007/s11120-004-3478-0. [DOI] [PubMed] [Google Scholar]
- 15.Merrick NWM. Regulation and function of ammonium carriers in bacteria, fungi, and plants. Molecular Mechanisms Controlling Transmembrane Transport 2004:25.
- 16.Detsch C., Stulke J. Ammonium utilization in Bacillus subtilis: transport and regulatory functions of NrgA and NrgB. Microbiology. 2003;149(11):3289–3297. doi: 10.1099/mic.0.26512-0x. [DOI] [PubMed] [Google Scholar]
- 17.Heinrich A., Woyda K., Brauburger K., Meiss G., Detsch C., Stülke J., et al. Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis. J Biol Chem. 2006;281(46):34909–34917. doi: 10.1074/jbc.M607582200. [DOI] [PubMed] [Google Scholar]
- 18.Nakano M.M., Hoffmann T., Zhu Y.i., Jahn D. Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE. J Bacteriol. 1998;180(20):5344–5350. doi: 10.1128/jb.180.20.5344-5350.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Ogawa K., Akagawa E., Yamane K., Sun Z.W., LaCelle M., Zuber P., et al. The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis. J Bacteriol. 1995;177(5):1409–1413. doi: 10.1128/jb.177.5.1409-1413.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Farrugia M.A., Macomber L., Hausinger R.P. Biosynthesis of the Urease Metallocenter. J Biol Chem. 2013;288(19):13178–13185. doi: 10.1074/jbc.R112.446526. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Oh Y.K., Palsson B.O., Park S.M., Schilling C.H., Mahadevan R. Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data. J Biol Chem. 2007;282(39):28791–28799. doi: 10.1074/jbc.M703759200. [DOI] [PubMed] [Google Scholar]
- 22.Sonenshein A.L. Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol. 2007;5(12):917–927. doi: 10.1038/nrmicro1772. [DOI] [PubMed] [Google Scholar]
- 23.Belitsky B.R., Sonenshein A.L. Role and regulation of Bacillus subtilis glutamate dehydrogenase genes. J Bacteriol. 1998;180(23):6298–6305. doi: 10.1128/jb.180.23.6298-6305.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Reitzer L. Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol. 2003;57(1):155–176. doi: 10.1146/annurev.micro.57.030502.090820. [DOI] [PubMed] [Google Scholar]
- 25.Belitsky BR. Biosynthesis of Amino Acids of the Glutamate and Aspartate Families, Alanine, and Polyamines. Bacillus subtilis and Its Closest Relatives: From Genes to Cells. America: Am Soc Microbiol (2001), 203–231.
- 26.Commichau F.M., Karl F., Stülke J. Regulatory links between carbon and nitrogen metabolism. Curr Opin Microbiol. 2006;9(2):167–172. doi: 10.1016/j.mib.2006.01.001. [DOI] [PubMed] [Google Scholar]
- 27.Gunka K., Commichau F.M. Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation. Mol Microbiol. 2012;85(2):213–224. doi: 10.1111/j.1365-2958.2012.08105.x. [DOI] [PubMed] [Google Scholar]
- 28.Stannek L., Thiele M.J., Ischebeck T., Gunka K., Hammer E., Völker U., et al. Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis. Environ Microbiol. 2015;17(9):3379–3390. doi: 10.1111/1462-2920.12813. [DOI] [PubMed] [Google Scholar]
- 29.Commichau F.M., Herzberg C., Tripal P., Valerius O., Stülke J. A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol. 2007;65(3):642–654. doi: 10.1111/j.1365-2958.2007.05816.x. [DOI] [PubMed] [Google Scholar]
- 30.Schreier H.J., Brown S.W., Hirschi K.D., Nomellini J.F., Sonenshein A.L. Regulation of Bacillus subtilis glutamine synthetase gene expression by the product of the glnR gene. J Mol Biol. 1989;210(1):51–63. doi: 10.1016/0022-2836(89)90290-8. [DOI] [PubMed] [Google Scholar]
- 31.Wray L.V., Ferson A.E., Rohrer K., Fisher S.H. a transcription factor required for global nitrogen regulation in Bacillus subtilis. Proc Natl Acad Sci USA. 1996;93(17):8841–8845. doi: 10.1073/pnas.93.17.8841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Yoshida K., Yamaguchi H., Kinehara M., Ohki Y.H., Nakaura Y., Fujita Y. Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box. Mol Microbiol. 2003;49(1):157–165. doi: 10.1046/j.1365-2958.2003.03567.x. [DOI] [PubMed] [Google Scholar]
- 33.Mirouze N., Bidnenko E., Noirot P., Auger S. Genome-wide mapping of TnrA-binding sites provides new insights into the TnrA regulon in Bacillus subtilis. Microbiologyopen. 2015;4(3):423–435. doi: 10.1002/mbo3.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Wray L.V., Zalieckas J.M., Fisher S.H. Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor. J Mol Biol. 2000;300(1):29–40. doi: 10.1006/jmbi.2000.3846. [DOI] [PubMed] [Google Scholar]
- 35.Liu Q., Chen Y., Yuan M., Du G., Chen J., Kang Z., et al. A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine. Appl Environ Microbiol. 2017;83(17) doi: 10.1128/AEM.01258-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Zalieckas J.M., Wray L.V., Fisher S.H. Cross-Regulation of the Bacillus subtilis glnRA and tnrA Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA. J Bacteriol. 2006;188(7):2578–2585. doi: 10.1128/JB.188.7.2578-2585.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Schumacher M.A., Chinnam N.B., Cuthbert B., Tonthat N.K., Whitfill T. Structures of regulatory machinery reveal novel molecular mechanisms controlling B. subtilis nitrogen homeostasis. Genes Dev. 2015;29(4):451–464. doi: 10.1101/gad.254714.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Wray L.V., Zalieckas J.M., Fisher S.H. Bacillus subtilis glutamine synthetase controls gene expression through a protein-protein interaction with transcription factor TnrA. Cell. 2001;107(4):427–435. doi: 10.1016/s0092-8674(01)00572-4. [DOI] [PubMed] [Google Scholar]
- 39.van Rooyen J., Abratt V., Belrhali H., Sewell T. Crystal structure of Type III glutamine synthetase: surprising reversal of the inter-ring interface. Structure. 2011;19(4):471–483. doi: 10.1016/j.str.2011.02.001. [DOI] [PubMed] [Google Scholar]
- 40.Deuel T.F., Prusiner S. Regulation of glutamine synthetase from Bacillus subtilis by divalent cations, feedback inhibitors, and L-glutamine. J Biol Chem. 1974;249(1):257–264. doi: 10.1007/BF01520862. [DOI] [PubMed] [Google Scholar]
- 41.Murray D.S., Chinnam N., Tonthat N.K., Whitfill T., Wray L.V., Fisher S.H., et al. Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism. J Biol Chem. 2013;288(50):35801–35811. doi: 10.1074/jbc.M113.519496. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Schreier H.J., Fisher S.H., Sonenshein A.L. Regulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product. Proc Natl Acad Sci U S A. 1985;82(10):3375–3379. doi: 10.1073/pnas.82.10.3375. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Chubukov V., Gerosa L., Kochanowski K., Sauer U. Coordination of microbial metabolism. Nat Rev Microbiol. 2014;12(5):327–340. doi: 10.1038/nrmicro3238. [DOI] [PubMed] [Google Scholar]
- 44.Hauf K., Kayumov A., Gloge F., Forchhammer K. The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis. J Biol Chem. 2016;291(7):3483–3495. doi: 10.1074/jbc.M115.680991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Wray L.V., Jr., Fisher S.H. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase. Mol Microbiol. 2008;68(2):277–285. doi: 10.1111/j.1365-2958.2008.06162.x. [DOI] [PubMed] [Google Scholar]
- 46.Randazzo P., Aucouturier A., Delumeau O., Auger S. Revisiting the in vivo GlnR-binding sites at the genome scale in Bacillus subtilis. BMC Res Notes. 2017;10(1) doi: 10.1186/s13104-017-2703-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Fisher S.H., Wray L.V. Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes. Proc Natl Acad Sci U S A. 2008;105(3):1014–1019. doi: 10.1073/pnas.0709949105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Fisher, Susan H, Jr. W, Lewis V. Mutations in the Bacillus subtilis glnRA Operon That Cause Nitrogen Source-Dependent Defects in Regulation of TnrA Activity. J Bacteriol. 2002, 184(16):4636–4639. http://doi.org/10.1128/JB.184.16.4636-4639.2002. [DOI] [PMC free article] [PubMed]
- 49.Wray, Lewis, V., Ferson, Amy, E. Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors. J Bacteriol 1997;179(17):5494–5501. [DOI] [PMC free article] [PubMed]
- 50.Kayumov A., Heinrich A., Fedorova K., Ilinskaya O., Forchhammer K. Interaction of the general transcription factor TnrA with the PII-like protein GlnK and glutamine synthetase in Bacillus subtilis. FEBS J. 2011;278(10):1779–1789. doi: 10.1111/j.1742-4658.2011.08102.x. [DOI] [PubMed] [Google Scholar]
- 51.Arcondéguy T., Jack R., Merrick M. P-II signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol Mol Biol R. 2001;65(1):80–105. doi: 10.1128/MMBR.65.1.80-105.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Slack F.J., Serror P., Joyce E., Sonenshein A.L. A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol. 1995;15(4):689–702. doi: 10.1111/j.1365-2958.1995.tb02378.x. [DOI] [PubMed] [Google Scholar]
- 53.Ratnayake-Lecamwasam M., Serror P., Wong K.-W., Sonenshein A.L. Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev. 2001;15(9):1093–1103. doi: 10.1101/gad.874201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Belitsky B.R., Sonenshein A.L. Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution. Proc Natl Acad Sci USA. 2013;110(17):7026–7031. doi: 10.1073/pnas.1300428110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Shivers R.P., Sonenshein A.L. Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids. Mol Microbiol. 2004;53(2):599–611. doi: 10.1111/j.1365-2958.2004.04135.x. [DOI] [PubMed] [Google Scholar]
- 56.Belitsky B.R. Indirect repression by Bacillus subtilis CodY via displacement of the activator of the proline utilization operon. J Mol Biol. 2011;413(2):321–336. doi: 10.1016/j.jmb.2011.08.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Belitsky B.R., Sonenshein A.L. Genetic and biochemical analysis of CodY-binding sites in Bacillus subtilis. J Bacteriol. 2008;190(4):1224–1236. doi: 10.1128/JB.01780-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Molle V., Nakaura Y., Shivers R.P., Yamaguchi H., Losick R., Fujita Y., et al. Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis. J Bacteriol. 2003;185(6):1911–1922. doi: 10.1128/JB.185.6.1911-1922.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Brinsmade S.R., Alexander E.L., Livny J., Stettner A.I., Segrè D., Rhee K.Y., et al. Hierarchical expression of genes controlled by the Bacillus subtilis global regulatory protein CodY. Proc Natl Acad Sci U S A. 2014;111(22):8227–8232. doi: 10.1073/pnas.1321308111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60.Barbieri G., Voigt B., Albrecht D., Hecker M., Albertini A.M., Sonenshein A.L., et al. CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr. J Bacteriol. 2015;197(8):1423–1432. doi: 10.1128/JB.02588-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61.Liu D, Bentley GJ, Chu K, Zhang F. Design of Dynamic Pathways. 2016:165–200.http://doi.org/10.1016/b978-0-444-63475-7.00007-8.
- 62.Mutalik V.K., Guimaraes J.C., Cambray G., Lam C., Christoffersen M.J., Mai Q.-A., et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013;10(4):354–360. doi: 10.1038/nmeth.2404. [DOI] [PubMed] [Google Scholar]
- 63.Singh V. Recent advancements in synthetic biology: current status and challenges. Gene. 2014;535(1):1–11. doi: 10.1016/j.gene.2013.11.025. [DOI] [PubMed] [Google Scholar]
- 64.Gu Y., Lv X., Liu Y., Li J., Du G., Chen J., et al. Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis. Metab Eng. 2019;51:59–69. doi: 10.1016/j.ymben.2018.10.002. [DOI] [PubMed] [Google Scholar]
- 65.Ding N., Yuan Z., Zhang X., Chen J., Zhou S., Deng Y. Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor. Nucleic Acids Res. 2020;48(18):10602–10613. doi: 10.1093/nar/gkaa786. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Shen B., Zhou P., Jiao X., Yao Z., Ye L., Yu H. Fermentative production of Vitamin E tocotrienols in Saccharomyces cerevisiae under cold-shock-triggered temperature control. Nat Commun. 2020;11(1) doi: 10.1038/s41467-020-18958-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.Cui S., Lv X., Wu Y., Li J., Du G., Ledesma-Amaro R., et al. Engineering a Bifunctional Phr60-Rap60-Spo0A Quorum-Sensing Molecular Switch for Dynamic Fine-Tuning of Menaquinone-7 Synthesis in Bacillus subtilis. ACS Synth Biol. 2019;8(8):1826–1837. doi: 10.1021/acssynbio.9b00140. [DOI] [PubMed] [Google Scholar]
- 68.Lo T.-M., Chng S.H., Teo W.S., Cho H.-S., Chang M.W. A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production. Cell Syst. 2016;3(2):133–143. doi: 10.1016/j.cels.2016.07.012. [DOI] [PubMed] [Google Scholar]
- 69.Aravind L., Anantharaman V., Balaji S., Babu M., Iyer L. The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev. 2005;29(2):231–262. doi: 10.1016/j.femsre.2004.12.008. [DOI] [PubMed] [Google Scholar]
- 70.Schreiter E.R., Drennan C.L. Ribbon-helix-helix transcription factors: variations on a theme. Nat Rev Microbiol. 2007;5(9):710–720. doi: 10.1038/nrmicro1717. [DOI] [PubMed] [Google Scholar]
- 71.Ding N., Zhou S., Deng Y.u. Transcription-Factor-based Biosensor Engineering for Applications in Synthetic Biology. Acs. Synth Biol. 2021;10(5):911–922. doi: 10.1021/acssynbio.0c00252. [DOI] [PubMed] [Google Scholar]
- 72.Zhang Y., Zhou X., Wang X., Wang L.u., Xia M., Luo J., et al. Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum. Microb Cell Fact. 2020;19(1) doi: 10.1186/s12934-020-1285-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.You J., Yang C., Pan X., Hu M., Du Y., Osire T., et al. Metabolic engineering of Bacillus subtilis for enhancing riboflavin production by alleviating dissolved oxygen limitation. Bioresource Technol. 2021;333:125228. doi: 10.1016/j.biortech.2021.125228. [DOI] [PubMed] [Google Scholar]
- 74.Cai D., Zhu J., Zhu S., Lu Y.u., Zhang B., Lu K., et al. Metabolic Engineering of Main Transcription Factors in Carbon, Nitrogen, and Phosphorus Metabolisms for Enhanced Production of Bacitracin in Bacillus licheniformis. ACS Synth Biol. 2019;8(4):866–875. doi: 10.1021/acssynbio.9b00005. [DOI] [PubMed] [Google Scholar]
- 75.Koch M., Pandi A., Borkowski O., Batista A.C., Faulon J.-L. Custom-made transcriptional biosensors for metabolic engineering. Curr Opin. Biotech. 2019;59:78–84. doi: 10.1016/j.copbio.2019.02.016. [DOI] [PubMed] [Google Scholar]
- 76.Li Y., Jin K.e., Zhang L., Ding Z., Gu Z., Shi G. Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon. J Agr Food Chem. 2018;66(36):9456–9464. doi: 10.1021/acs.jafc.8b02857. [DOI] [PubMed] [Google Scholar]
- 77.Reuß D.R., Rath H., Thürmer A., Benda M., Daniel R., Völker U., et al. Changes of DNA topology affect the global transcription landscape and allow rapid growth of a Bacillus subtilis mutant lacking carbon catabolite repression. Metab Eng. 2018;45:171–179. doi: 10.1016/j.ymben.2017.12.004. [DOI] [PubMed] [Google Scholar]
- 78.Xu X., Li X., Liu Y., Zhu Y., Li J., Du G., et al. Pyruvate-responsive genetic circuits for dynamic control of central metabolism. Nat Chem Biol. 2020;16(11):1261–1268. doi: 10.1038/s41589-020-0637-3. [DOI] [PubMed] [Google Scholar]
- 79.Zhang Y., Li Y., Xiao F., Wang H., Zhang L., Ding Z., et al. CcpA mutants influence selective carbon source utilization by changing interactions with target genes in Bacillus licheniformis. Syst Microbiol Biomanuf. 2022;2(1):193–207. [Google Scholar]
- 80.Rondon R.E., Wilson C.J. Engineering a New Class of Anti-LacI Transcription Factors with Alternate DNA Recognition. ACS Synth Biol. 2019;8(2):307–317. doi: 10.1021/acssynbio.8b00324. [DOI] [PubMed] [Google Scholar]
- 81.Wu Y., Yang Y., Ren C., Yang C., Yang S., Gu Y., et al. Molecular modulation of pleiotropic regulator CcpA for glucose and xylose coutilization by solvent-producing Clostridium acetobutylicum. Metab Eng. 2015;28:169–179. doi: 10.1016/j.ymben.2015.01.006. [DOI] [PubMed] [Google Scholar]
- 82.Weme R.D.O., Seidel G., Kuipers O.P. Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis. Front Microbiol. 2015;6.1051. doi: 10.3389/fmicb.2015.01051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 83.Cao H., Villatoro-Hernandez J., Weme R.D.O., Frenzel E., Kuipers O.P. Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis. Metab Eng. 2018;49:143–152. doi: 10.1016/j.ymben.2018.08.001. [DOI] [PubMed] [Google Scholar]
- 84.Cao H., Kuipers O.P. Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis. Microb Cell Fact. 2018;17(1) doi: 10.1186/s12934-018-0985-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 85.Shen P., Niu D., Permaul K., Tian K., Singh S., Wang Z. Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis. J Ind Microbiol Biotechnol. 2021;48(5-6) doi: 10.1093/jimb/kuab037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 86.Immethun C.M., DeLorenzo D.M., Focht C.M., Gupta D., Johnson C.B., Moon T.S. Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803. Biotechnol Bioeng. 2017;114(7):1561–1569. doi: 10.1002/bit.26275. [DOI] [PubMed] [Google Scholar]
- 87.Muro-Pastor M.I., Cutillas-Farray Á., Pérez-Rodríguez L., Pérez-Saavedra J., Vega-de Armas A., Paredes A., et al. CfrA, a Novel Carbon Flow Regulator, Adapts Carbon Metabolism to Nitrogen Deficiency in Cyanobacteria. Plant Physiol. 2020;184(4):1792–1810. doi: 10.1104/pp.20.00802. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 88.Elmore J.R., Dexter G.N., Salvachúa D., Martinez-Baird J., Hatmaker E.A., Huenemann J.D., et al. Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion. Nat Commun. 2021;12(1) doi: 10.1038/s41467-021-22556-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 89.Satomura T., Shimura D., Asai K., Sadaie Y., Hirooka K., Fujita Y. Enhancement of glutamine utilization in Bacillus subtilis through the GlnK-GlnL two-component regulatory system. J Bacteriol. 2005;187(14):4813–4821. doi: 10.1128/JB.187.14.4813-4821.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 90.Till P., Toepel J., Bühler B., Mach R.L., Mach-Aigner A.R. Regulatory systems for gene expression control in cyanobacteria. Appl Microbiol Biot. 2020;104(5):1977–1991. doi: 10.1007/s00253-019-10344-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 91.Jäger D., Sharma C.M., Thomsen J., Ehlers C., Vogel J., Schmitz R.A. Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability. Proc Natl Acad Sci USA. 2009;106(51):21878–21882. doi: 10.1073/pnas.0909051106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 92.Ye B.C., Zhang Y., Yu H., Yu W.B., Liu B.H., Yin B.C., et al. Time-resolved transcriptome analysis of Bacillus subtilis responding to valine, glutamate, and glutamine. PLoS ONE. 2009;4(9) doi: 10.1371/journal.pone.0007073. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 93.Niu T., Lv X., Liu Z., Li J., Du G., Liu L. Synergetic engineering of central carbon and nitrogen metabolism for the production of N-acetylglucosamine in Bacillus subtilis. Biotechnol Appl Biochem. 2020;67(1):123–132. doi: 10.1002/bab.1845. [DOI] [PubMed] [Google Scholar]
- 94.Wang Q., Jiang A.n., Tang J., Gao H., Zhang X., Yang T., et al. Enhanced production of L-arginine by improving carbamoyl phosphate supply in metabolically engineered Corynebacterium crenatum. Appl Microbiol Biotechnol. 2021;105(8):3265–3276. doi: 10.1007/s00253-021-11242-w. [DOI] [PubMed] [Google Scholar]
- 95.Ma H., Zhao Y., Huang W., Zhang L., Wu F., Ye J., et al. Rational flux-tuning of Halomonas bluephagenesis for co-production of bioplastic PHB and ectoine. Nat Commun. 2020;11(1) doi: 10.1038/s41467-020-17223-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 96.Otten A., Brocker M., Bott M. Metabolic engineering of Corynebacterium glutamicum for the production of itaconate. Metab Eng. 2015;30:156–165. doi: 10.1016/j.ymben.2015.06.003. [DOI] [PubMed] [Google Scholar]
- 97.Bren A., Park J.O., Towbin B.D., Dekel E., Rabinowitz J.D., Alon U. Glucose becomes one of the worst carbon sources for E.coli on poor nitrogen sources due to suboptimal levels of cAMP. Sci Rep. 2016;6(1) doi: 10.1038/srep24834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 98.Atsumi S., Hanai T., Liao J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature. 2008;451(7174):86–89. doi: 10.1038/nature06450. [DOI] [PubMed] [Google Scholar]
- 99.Ledesma-Amaro R., Nicaud J.-M. Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica. Trends Biotechnol. 2016;34(10):798–809. doi: 10.1016/j.tibtech.2016.04.010. [DOI] [PubMed] [Google Scholar]
- 100.Liu N., Santala S., Stephanopoulos G. Mixed carbon substrates: a necessary nuisance or a missed opportunity? Curr Opin Biotechnol. 2020;62:15–21. doi: 10.1016/j.copbio.2019.07.003. [DOI] [PubMed] [Google Scholar]
- 101.de Jong H., Casagranda S., Giordano N., Cinquemani E., Ropers D., Geiselmann J., et al. Mathematical modelling of microbes: metabolism, gene expression and growth. J R Soc Interface. 2017;14(136):20170502. doi: 10.1098/rsif.2017.0502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 102.Blazeck J., Hill A., Liu L., Knight R., Miller J., Pan A., et al. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat Commun. 2014;5(1) doi: 10.1038/ncomms4131. [DOI] [PubMed] [Google Scholar]
- 103.Kochanowski K, Okano H, Patsalo V, Williamson J, Sauer U, Hwa T. Global coordination of metabolic pathways in Escherichia coli by active and passive regulation. Mol Syst Biol 2021;17(4).http://doi.org/ARTN e1006410.15252/msb.202010064. [DOI] [PMC free article] [PubMed]
- 104.Conrad M., Schothorst J., Kankipati H.N., Van Zeebroeck G., Rubio-Texeira M., Thevelein J.M. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev. 2014;38(2):254–299. doi: 10.1111/1574-6976.12065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 105.Habicher T., John A., Scholl N., Daub A., Klein T., Philip P., et al. Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane-based fed-batch shake flasks. Biotechnol Bioeng. 2019;116(6):1326–1340. doi: 10.1002/bit.26948. [DOI] [PubMed] [Google Scholar]
- 106.Deng C., Lv X., Li J., Zhang H., Liu Y., Du G., et al. Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors. Metab Eng. 2021;67:330–346. doi: 10.1016/j.ymben.2021.07.012. [DOI] [PubMed] [Google Scholar]