This review therefore focuses on the roles of organic N metabolites derived from microbes, as well as on the roles of pyridoxal-5′-phosphate -dependent enzymes, which constitute the primary family of enzymes responsible for N metabolism. Additionally, this review highlights potential applications of N metabolites and related gene products developed using synthetic biology to engineer new PGPRs that could improve the sustainability of agriculture. As building blocks of proteins and precursors for diverse metabolites, amino acids are essential N metabolites in all living organisms. Aside from 22 amino acids used to build proteins, a wide variety of non-proteinogenic amino acids are essential in cellular metabolism and physiological maintenance. For example, β-alanine and 5-aminolevulinic acid are building blocks of Coenzyme A and chlorophyll, respectively, and 1-aminocyclopropane- 1-carboxylic acid  is a precursor of ethylene, a phytohormone associated with plant senescence and stress response . Also, L-tryptophan and its analogs are precursors of endogenous auxins , indole-3- butyric acid , and 4-chloroindole-3-acetic acid. These auxins are essential for plant growth and development. Ornithine, citrulline, and γ-aminobutyric acid are precursors for various metabolic networks and are converted to bioactive-specialized metabolites. Canavanine and m-tyrosine are important for alleviating cellular oxidative stress, and betaines, derived from glycine or β-alanine, confer salt tolerance in plants. Also, nicotianamine is a precursor of phytosiderophores, mugineic acid , and its derivatives,hydroponic nft gully for iron chelation. Recently, N-hydroxypipecolic acid was identified as a critical inducer of the systemic acquired resistance immune response in plants.

Proteinogenic L-amino acids can be isomerized to D-amino acids important for biofilm formation. For example, Dalanine and D-glutamate are key molecules in peptidoglycan layers in Gram-negative bacteria.These metabolites may function in communication between biome members and may help microbial producers survive in competitive resource-scarce environments. Decarboxylation of amino acids or amination of carbonyl compounds generates bio-genic amines, which are important in cell signaling. For example, melatonin acts as a hormone in plants and fungi. Although their biological functions are poorly understood, polyamines have been identified as phytohormones coordinating cellular activities and mediating plant–pathogen interaction in plants. For example, researchers found that cadaverine , a polyamine, inhibited root growth by inducing biotin limitation. Microbes and plants often produce complex organic N compounds exhibiting diverse physiological activities. Notably, using alkaloids for their antimicrobial and antiviral effects has been proposed as a sustainable approach to protecting plant health. Some organic N compounds can confer substantial biological activity, even in small quantities. For example, most vitamin-B derivatives serve as organic cofactors , and their deficiency induces metabolic disorders. In addition, a pyrroloquinoline quinone , a cofactor synthesized by some PGPRs, affects PGP traits by modulating redox state as well as through its role in synthesis of sugar acids useful for solubilization of inorganic phosphorus. Moreover, N-containing pigments are essential for photosynthesis, allowing plants to absorb energy from light. In addition, some plants have unique specialized metabolites bearing N and sulfur. Glucosinolates , which are derived from various amino acids, are essential to plant immune systems and can function as natural pesticides. In the next section, we review the biological pathways and enzymes involved in synthesizing important organic N metabolites.

Although modern agriculture relies heavily on chemical fertilizers as an N source, plants can also source bio-available N through ecological relationships with microbes that can directly fix atmospheric nitrogen. N catabolism in microbes proceeds in four phases: fixation, assimilation, distribution, and bio-transformation. Plants can take up N metabolites yielded at any phase, and may proceed with the subsequent phases, utilizing the N metabolites for their development and growth. N-fixation converts N2 into ammonia , which is accessible to most living organisms. Major N-fixation enzymes include dinitrogenase and its reductase, which exploit molybdenum–iron, vanadium–iron, or iron–iron cofactors. The fixed NH4 + molecules enter into the N-assimilation phase for synthesis of organic N compounds, but can also undergo nitrification, anammox, and/or denitrification in the microbial nitrogen cycle, and be converted into diverse inorganic forms. In the N-assimilation phase, NH4 + is incorporated into organic N compounds such as glutamine and glutamate, catalyzed by NADH-dependent glutamate dehydrogenase and ATP-dependent glutamine synthetase. In the case of nitrate, metal-dependent nitrate and nitrite reductases are additionally employed to prepare NH4 +. In the distribution and biotransformation phases, in which assimilated N is converted into primary and specialized metabolites, glutamate synthase and PLPdependent aminotransferases distribute N throughout the metabolic networks. PLP can form a Schiff-base linkage with the amino group. This unique property makes PLP a versatile cofactor for enzymes associated with N metabolism. In fact, PLP-dependent enzymes distribute across five of the seven EC classes: EC1 , EC2 , EC3 , EC4, and EC5 .

Additionally, 1.6% of all reported enzymes in UniProtKB are annotated as PLP-dependent. Most enzymes involved in ATs reactions employ PLP as a cofactor, and 73% of all enzymes predicted to catalyze transaminase reactions have PLP-binding motifs. These observations strongly suggest the core functions of PLP-dependent enzymes in N metabolic networks. N metabolism after the assimilation phase is poorly understood because of the networks’ complexity. In particular, assigning each reaction is difficult because ATs have broader substrate specificity than do most other enzymes, and because function overlaps significantly among different ATs. These metabolic redundancies may be important to create robust N networks for production of essential metabolites whose demands could proportionally shift, depending on plants’ growth and their developmental phases, as well as on their physiological responses to environmental changes. Besides increasing understanding of N-network regulation, designing microbial systems that can help plants shift their optimal N metabolic networks could promote plant growth. Molecules that confer PGP traits may be classified as phytohormones , nutrients , or antimicrobials . Native symbiotic communities produce these molecules, but in addition, recent studies have shown promising results from micro-biome engineering in modulating bio-active N metabolites in order to promote plant growth. In the following section, we discuss potential applications of PLP-dependent enzymes for improving agricultural sustainability via micro-biome engineering. Phytohormones are among the well-known metabolites involved in plant growth and development. They influence plant physiology in many ways. To date, several microbes engineered to produce phytohormones have shown promising ability to promote plant growth. For example, Zúñiga et al. engineered Cupriavidus pinatubonensis to produce the auxin indole acetic acid  under the control of a quorum-sensing signal, enhancing the root growth of Arabidopsis thaliana. In addition to IAA , plants synthesize endogenous auxins and 4-chloroindole-3-acetic acid, which may also be useful if provided by microbes. Recently, the β-subunit of tryptophan synthase , which is involved in biosynthesis of tryptophan via a β-replacement reaction between Lserine and indole,aeroponic tower garden system has emerged as useful for synthesis of aromatic amino acids via artificial evolution. For example, Rix et al. constructed various TrpB variants for tryptophan derivatives using an in vivo high-throughput evolution technique with orthogonal DNA polymerase. Broad substrate specificities of TrpB variants potentially enable production of diverse unnatural auxins. Ethylene, a multifunctional phytohormone involved in plant growth and senescence, can be modulated by ACC deaminase via ACC degradation. This reaction can be used to influence growth and other processes in plants. In one experiment, chickpeas were inoculated with microbes that heterologously expressed ACC deaminase , resulting in growth promotion of chickpea. More recently, endophytic microbes were engineered to display ACC deaminase, which conferred saline and wilt resistances to rice and banana plants, respectively, and also promoted their growth. Chen et al. elucidated the function of L-cysteine desulfhydrase in abscisic acid signaling, which is critical in plant response to drought stress. Hydrogen sulfide generated by L-cysteine desulfhydrase regulates plant signaling pathways through post-translational modification. Ser/Cys synthase , which catalyzes reversible β-replacement between L-serine and L-cysteine, may be an alternative for synthetic control of cellular H2S.

As H2S can lead to both positive and negative effects, depending on its cellular concentration, its precise control based on serine/cysteine metabolism may be a promising strategy for sustainable agriculture. Biogenic amines serve as hormones in plants. Bacteria can be engineered to produce amino acid decarboxylase to produce these amines. For example, putrescine and cadaverine are generated by a single decarboxylation step from ornithine and lysine, respectively. Melatonin is synthesized from 5- hydroxy-L-tryptophan by AADC coupled with serotonin-N-acetyltransferase and acetyl-serotonin O-methyltransferase. Bacterial AADCs with broad substrate specificities have been used to produce diverse monoamines that are precursors of bio-control and bio-stimulation agents such as alkaloids and catecholamines. Aromatic aldehyde synthases , specific clades of AADCs especially lacking a catalytic Tyr residue, can catalyze oxidative deamination of aromatic amino acids to yield VOCs such as phenylacetaldehyde, a nematocidal agent and mediator of plant–insect interaction. These various functions reveal the broad potential of AADCs to provide microbes with bio-stimulation and bio-control activities. In the next section, we review potential uses of PLP dependent enzymes for bio-control and bio-fertilization. Microbes engineered to produce siderophores may help source essential recalcitrant metal ions from fields for use by plants. For example, rhizoferrin is a siderophore produced in fungi and bacteria. Li et al. revealed the biosynthetic route for rhizoferrin from ornithine and citrate by a nonribosomal peptide synthetase-independent siderophore synthetase and PLP-dependent N-citrylornithine decarboxylase. Plants in the Poaceae family were previously engineered to express PLP-dependent nicotianamine ATs to make phytosiderophores, mugineic acid , and its derivatives, resulting in significantly improved ability to acquire metal ions. Pathways for rhizoferrin , phytosiderophores , and other siderophores could also be introduced into microbes such as PGPRs to increase the availability of metal ions. Moreover, combining siderophore-mediated and PQQ-mediated routes in PGPRs could be an attractive approach to enhancing plant mineral uptake. Microbes engineered to produce antibiotics have also proved their ability to protect plants from biotic stresses. Recently, Schaffer et al. and Scott et al. found that L-threonine transaldolase catalyzed formation of β-hydroxy-α-amino acids and was essential for biosynthesis of -2-amino-3-hydroxy-4-butanoate in the plant-associated bacterium Pseudomonas fluorescens. This metabolite has bio-control properties and is also a precursor for the antibacterial agent obafluorin. Xu et al. engineered Pseudomonas sp. to produce LTTA that could catalyze formation of -β-hydroxy-α-amino acids with the phenyl group at the β-position substituted with phenyl-group derivatives. These unnatural specialized metabolites may be used as precursors for diverse antibacterial agents. However, the low stereoselectivity of the engineered LTTA at the β-position limits the utility of this bio-conversion owing to the unknown effect of side products. Nevertheless, this work represents progress toward engineering microbes as novel bio-control agents. Dai et al. discovered a novel fungal BGC for synthesis of antibacterial alkaloids. In this BGC, CuaB exploits PLP as a cofactor to catalyze formation of CeC and CeO bonds via Claisen condensation and oxidation, respectively. This study also determined that ATs CtaB participated in the biosynthesis of an antibiotic peptide, closthioamide , by amination of the aldehyde group. These BGCs may be introduced into symbiotic microbes as bio-control agents. As discussed, PLP-dependent enzymes participate in production of a wide variety of metabolites. However, an excessive supply of specific metabolites may burden plant growth. Therefore, engineering of microbes to produce the metabolites discussed in this section must be carefully balanced. The following section discusses potential synthetic platforms for precisely modulating primary and bioactive-specialized metabolites that could contribute to developing more-sustainable agriculture. Synthetic biology has been used to directly engineer key members of microbiomes that provide plants with consistent benefits. Because symbiosis-based plant breeding and use of molecular biology techniques to engineer microbiomes have been reviewed extensively elsewhere, this section focuses on potential microbial hosts and regulatory circuits for practical applications of PLP-dependent enzymes and related BGCs for sustainable agriculture. As key members of plant-associated microbiomes, methanotrophs have recently gained attention as synthetic platform hosts. Because they natively possess the ability to fix both N2 and CO2, they are attractive targets for adding further PGP traits and establishing as synthetic PGP microbes. Liu et al. recently engineered methanotrophs that could biodegrade herbicide contaminants, and Pham et al. engineered them to produce IAA ; inoculation of wheat with these engineered methanotrophs accelerated plant development. While practical applications need to be developed, use of methane to control cell proliferation in methanotrophs may be a viable strategy for addressing bio-safety concerns. Wang et al. recently reported an O2-insensitive bacterial methane synthesis in which a PLP-dependent AT converted methylamine into methane in the presence of α-ketoglutarate.