A White-Box Machine Learning Approach for Revealing Antibiotic Mechanisms of Action.

Current machine learning techniques enable robust association of biological signals with measured phenotypes, but these approaches are incapable of identifying causal relationships. Here, we develop an integrated "white-box" biochemical screening, network modeling, and machine learning approach for revealing causal mechanisms and apply this approach to understanding antibiotic efficacy. We counter-screen diverse metabolites against bactericidal antibiotics in Escherichia coli and simulate their corresponding metabolic states using a genome-scale metabolic network model. Regression of the measured screening data on model simulations reveals that purine biosynthesis participates in antibiotic lethality, which we validate experimentally. We show that antibiotic-induced adenine limitation increases ATP demand, which elevates central carbon metabolism activity and oxygen consumption, enhancing the killing effects of antibiotics. This work demonstrates how prospective network modeling can couple with machine learning to identify complex causal mechanisms underlying drug efficacy.

Structural basis for Tpt1-catalyzed 2'-PO4 transfer from RNA and NADP(H) to NAD.

Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2'-phosphate generated by tRNA ligase. Tpt1 also removes the 2'-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1″-PO4, ADP-ribose-2″-PO4, and 2'-OH RNA, and as substrate complexes with 2',5'-ADP and NAD+, that illuminate 2'-PO4 junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2'-PO4-containing metabolites NADP+ and NADPH as substrates for 2'-PO4 transfer to NAD+. A role in 2'-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2'-phosphorylation.

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid.

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

Regulatory Network of the Late-Recruited Primary Decarboxylase C4NADP-ME in Sugarcane.

In agronomically important C4 grasses, efficient CO2 delivery to Rubisco is facilitated by NADP-malic enzyme (C4NADP-ME), which decarboxylates malate in bundle sheath cells. However, understanding the molecular regulation of the C4NADP-ME gene in sugarcane (Saccharum spp.) is hindered by its complex genetic background. Enzymatic activity assays demonstrated that decarboxylation in sugarcane Saccharum spontaneum predominantly relies on the NADP-ME pathway, similar to sorghum (Sorghum bicolor) and maize (Zea mays). Comparative genomics analysis revealed the recruitment of eight core C4 shuttle genes, including C4NADP-ME (SsC4NADP-ME2), in the C4 pathway of sugarcane. Contrasting to sorghum and maize, the expression of SsC4NADP-ME2 in sugarcane is regulated by different transcription factors (TFs). We propose a gene regulatory network for SsC4NADP-ME2, involving candidate TFs identified through gene co-expression analysis and yeast one-hybrid experiment. Among these, ABA INSENSITIVE5 (ABI5) was validated as the predominant regulator of SsC4NADP-ME2 expression, binding to a G-box within its promoter region. Interestingly, the core element ACGT within the regulatory G-box was conserved in sugarcane, sorghum, maize, and rice (Oryza sativa), suggesting an ancient regulatory code utilized in C4 photosynthesis. This study offers insights into SsC4NADP-ME2 regulation, crucial for optimizing sugarcane as a bioenergy crop.

cKMT1 is a New Lysine Methyltransferase That Methylates the Ferredoxin-NADP(+) Oxidoreductase and Regulates Energy Transfer in Cyanobacteria.

Lysine methylation is a conserved and dynamic regulatory posttranslational modification performed by lysine methyltransferases (KMTs). KMTs catalyze the transfer of mono-, di-, or tri-methyl groups to substrate proteins and play a critical regulatory role in all domains of life. To date, only one KMT has been identified in cyanobacteria. Here, we tested all of the predicted KMTs in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and we biochemically characterized sll1526 that we termed cKMT1 (cyanobacterial lysine methyltransferase 1) and determined that it can catalyze lysine methylation both in vivo and in vitro. Loss of cKMT1 alters photosynthetic electron transfer in Synechocystis. We analyzed cKMT1-regulated methylation sites in Synechocystis using a timsTOF Pro instrument. We identified 305 class I lysine methylation sites within 232 proteins, and of these, 80 methylation sites in 58 proteins were hypomethylated in ΔcKMT1 cells. We further demonstrated that cKMT1 could methylate ferredoxin-NADP(+) oxidoreductase (FNR) and its potential sites of action on FNR were identified. Amino acid residues H118 and Y219 were identified as key residues in the putative active site of cKMT1 as indicated by structure simulation, site-directed mutagenesis, and KMT activity measurement. Using mutations that mimic the unmethylated forms of FNR, we demonstrated that the inability to methylate K139 residues results in a decrease in the redox activity of FNR and affects energy transfer in Synechocystis. Together, our study identified a new KMT in Synechocystis and elucidated a methylation-mediated molecular mechanism catalyzed by cKMT1 for the regulation of energy transfer in cyanobacteria.

Sufficient potassium improves inorganic phosphate-limited photosynthesis in Brassica napus by enhancing metabolic phosphorus fractions and Rubisco activity.

Crop photosynthesis (A) and productivity are often limited by a combination of nutrient stresses, such that changes in the availability of one nutrient may affect the availability of another nutrient, in turn influencing A. In this study, we examined the synergistic effects of phosphorus (P) and potassium (K) on leaf A in a nutrient amendment experiment, in which P and K were added individually or in combination to Brassica napus grown under P and K co-limitation. The data revealed that the addition of P gradually removed the dominant limiting factor (i.e. the limited availability of P) and improved leaf A. Strikingly, the addition of K synergistically improved the overall uptake of P, mainly by boosting plant growth, and compensated for the physiological demand for P by prioritizing investment in metabolic pools of P (P-containing metabolites and inorganic phosphate, Pi). The enlarged pool of metabolically active P was partially associated with the upregulation of Pi regeneration through release from triose phosphates rather than replacement of P-containing lipids. This process mitigated P restrictions on A by maintaining the ATP/NADPH and NADPH/NADP+ ratios and increasing the content and activity of Rubisco. Our findings demonstrate that sufficient K increased Pi-limited A by enhancing metabolic P fractions and Rubisco activity. Thus, ionic synergism may be exploited to mitigate nutrient-limiting factors to improve crop productivity.

A High-Throughput Visual Screen for the Directed Evolution of C ß -stereoselectivity of L-threonine Aldolase.

L-Threonine aldolase (L-TA) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible condensation of glycine and aldehydes to form ß-hydroxy-α-amino acids. The combination of directed evolution and efficient high-throughput screening methods is an effective strategy for enhancing the enzyme's catalytic performance. However, few feasible high-throughput methods exist for engineering the Cß-stereoselectivity of L-TAs. Here, we present a novel method of screening for variants with improved Cß-stereoselectivity; this method couples an L-threo-phenylserine dehydrogenase, which catalyzes the specific oxidation of L-threo-4-methylsulfonylphenylserine (L-threo-MTPS), with the concurrent synthesis of NADPH, which is easily detectable via 340-nm UV absorption. This enables the visual detection of L-threo-MTPS produced by L-TA through the measurement of generated NADPH. Using this method, we discover an L-TA variant with significantly higher diastereoselectivity, increasing from 0.98% de (for the wild-type) to 71.9% de.

Exploring fermentative metabolic response to varying exogenous supplies of redox cofactor precursors in selected wine yeast species.

The use of non-Saccharomyces yeasts in winemaking is gaining traction due to their specific phenotypes of technological interest, including their unique profile of central carbon metabolites and volatile compounds. However, the lack of knowledge about their physiology hinders their industrial exploitation. The intracellular redox status, involving NAD/NADH and NADP/NADPH cofactors, is a key driver of yeast activity during fermentation, notably directing the formation of metabolites that contribute to the wine bouquet. The biosynthesis of these cofactors can be modulated by the availability of their precursors, nicotinic acid and tryptophan, and their ratio by that of thiamine. In this study, a multifactorial experiment was designed to assess the effects of these three nutrients and their interactions on the metabolic response of various wine yeast species. The data indicated that limiting concentrations of nicotinic acid led to a species-dependent decrease in intracellular NAD(H) concentrations, resulting in variations of fermentation performance and production of metabolic sinks. Thiamine limitation did not directly affect redox cofactor concentrations or balance, but influenced redox management and subsequently the production of metabolites. Overall, this study identified nicotinic acid and thiamine as key factors to consider for species-specific modulation of the metabolic footprint of wine yeasts.

Nicotinic acid availability impacts redox cofactor metabolism in Saccharomyces cerevisiae during alcoholic fermentation.

Anaerobic alcoholic fermentation, particularly in high-sugar environments, presents metabolic challenges for yeasts. Crabtree-positive yeasts, including Saccharomyces cerevisiae, prefer fermentation even in the presence of oxygen. These yeasts rely on internal NAD+ recycling and extracellular assimilation of its precursor, nicotinic acid (vitamin B3), rather than de novo NAD+ production. Surprisingly, nicotinic acid assimilation is poorly characterized, even in S. cerevisiae. This study elucidated the timing of nicotinic acid uptake during grape juice-like fermentation and its impact on NAD(H) levels, the NAD+/NADH ratio, and metabolites produced. Complete uptake of extracellular nicotinic acid occurred premid-exponential phase, thereafter small amounts of vitamin B3 were exported back into the medium. Suboptimal levels of nicotinic acid were correlated with slower fermentation and reduced biomass, disrupting redox balance and impeding NAD+ regeneration, thereby affecting metabolite production. Metabolic outcomes varied with nicotinic acid concentrations, linking NAD+ availability to fermentation efficiency. A model was proposed encompassing rapid nicotinic acid uptake, accumulation during cell proliferation, and recycling with limited vitamin B3 export. This research enhances the understanding of nicotinic acid uptake dynamics during grape juice-like fermentation. These insights contribute to advancing yeast metabolism research and have profound implications for the enhancement of biotechnological practices and the wine-making industry.

Damage-Associated Molecular Patterns and Systemic Signaling.

Cellular damage inflicted by wounding, pathogen infection, and herbivory releases a variety of host-derived metabolites, degraded structural components, and peptides into the extracellular space that act as alarm signals when perceived by adjacent cells. These so-called damage-associated molecular patterns (DAMPs) function through plasma membrane localized pattern recognition receptors to regulate wound and immune responses. In plants, DAMPs act as elicitors themselves, often inducing immune outputs such as calcium influx, reactive oxygen species generation, defense gene expression, and phytohormone signaling. Consequently, DAMP perception results in a priming effect that enhances resistance against subsequent pathogen infections. Alongside their established function in local tissues, recent evidence supports a critical role of DAMP signaling in generation and/or amplification of mobile signals that induce systemic immune priming. Here, we summarize the identity, signaling, and synergy of proposed and established plant DAMPs, with a focus on those with published roles in systemic signaling.

Redox regulation of NADP-malate dehydrogenase is vital for land plants under fluctuating light environment.

Many enzymes involved in photosynthesis possess highly conserved cysteine residues that serve as redox switches in chloroplasts. These redox switches function to activate or deactivate enzymes during light-dark transitions and have the function of fine-tuning their activities according to the intensity of light. Accordingly, many studies on chloroplast redox regulation have been conducted under the hypothesis that "fine regulation of the activities of these enzymes is crucial for efficient photosynthesis." However, the impact of the regulatory system on plant metabolism is still unclear. To test this hypothesis, we here studied the impact of the ablation of a redox switch in chloroplast NADP-malate dehydrogenase (MDH). By genome editing, we generated a mutant plant whose MDH lacks one of its redox switches and is active even in dark conditions. Although NADPH consumption by MDH in the dark is expected to be harmful to plant growth, the mutant line did not show any phenotypic differences under standard long-day conditions. In contrast, the mutant line showed severe growth retardation under short-day or fluctuating light conditions. These results indicate that thiol-switch redox regulation of MDH activity is crucial for maintaining NADPH homeostasis in chloroplasts under these conditions.

Long-range structural defects by pathogenic mutations in most severe glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common blood disorder, presenting multiple symptoms, including hemolytic anemia. It affects 400 million people worldwide, with more than 160 single mutations reported in G6PD. The most severe mutations (about 70) are classified as class I, leading to more than 90% loss of activity of the wild-type G6PD. The crystal structure of G6PD reveals these mutations are located away from the active site, concentrating around the noncatalytic NADP+-binding site and the dimer interface. However, the molecular mechanisms of class I mutant dysfunction have remained elusive, hindering the development of efficient therapies. To resolve this, we performed integral structural characterization of five G6PD mutants, including four class I mutants, associated with the noncatalytic NADP+ and dimerization, using crystallography, small-angle X-ray scattering (SAXS), cryogenic electron microscopy (cryo-EM), and biophysical analyses. Comparisons with the structure and properties of the wild-type enzyme, together with molecular dynamics simulations, bring forward a universal mechanism for this severe G6PD deficiency due to the class I mutations. We highlight the role of the noncatalytic NADP+-binding site that is crucial for stabilization and ordering two ß-strands in the dimer interface, which together communicate these distant structural aberrations to the active site through a network of additional interactions. This understanding elucidates potential paths for drug development targeting G6PD deficiency.

Redox Properties of Bacillus subtilis Ferredoxin:NADP+ Oxidoreductase: Potentiometric Characteristics and Reactions with Pro-Oxidant Xenobiotics.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be -0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.

Strategies to Maintain Redox Homeostasis in Yeast Cells with Impaired Fermentation-Dependent NADPH Generation.

Redox homeostasis is the balance between oxidation and reduction reactions. Its maintenance depends on glutathione, including its reduced and oxidized form, GSH/GSSG, which is the main intracellular redox buffer, but also on the nicotinamide adenine dinucleotide phosphate, including its reduced and oxidized form, NADPH/NADP+. Under conditions that enable yeast cells to undergo fermentative metabolism, the main source of NADPH is the pentose phosphate pathway. The lack of enzymes responsible for the production of NADPH has a significant impact on yeast cells. However, cells may compensate in different ways for impairments in NADPH synthesis, and the choice of compensation strategy has several consequences for cell functioning. The present study of this issue was based on isogenic mutants: Δzwf1, Δgnd1, Δald6, and the wild strain, as well as a comprehensive panel of molecular analyses such as the level of gene expression, protein content, and enzyme activity. The obtained results indicate that yeast cells compensate for the lack of enzymes responsible for the production of cytosolic NADPH by changing the content of selected proteins and/or their enzymatic activity. In turn, the cellular strategy used to compensate for them may affect cellular efficiency, and thus, the ability to grow or sensitivity to environmental acidification.

Dynamic and Static Regulation of Nicotinamide Adenine Dinucleotide Phosphate: Strategies, Challenges, and Future Directions in Metabolic Engineering.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial cofactor in metabolic networks. The efficient regeneration of NADPH is one of the limiting factors for productivity in biotransformation processes. To date, many metabolic engineering tools and static regulation strategies have been developed to regulate NADPH regeneration. However, traditional static regulation methods often lead to the NADPH/NADP+ imbalance, causing disruptions in cell growth and production. These methods also fail to provide real-time monitoring of intracellular NADP(H) or NADPH/NADP+ levels. In recent years, various biosensors have been developed for the detection, monitoring, and dynamic regulate of the intracellular NADP(H) levels or the NADPH/NADP+ balance. These NADPH-related biosensors are mainly used in the cofactor engineering of bacteria, yeast, and mammalian cells. This review analyzes and summarizes the NADPH metabolic regulation strategies from both static and dynamic perspectives, highlighting current challenges and potential solutions, and discusses future directions for the advanced regulation of the NADPH/NADP+ balance.

Carbon dioxide sensitization delays the postharvest ripening and fatty acids composition of Capsicum fruit by regulating ethylene biosynthesis, malic acid and reactive oxygen species metabolism.

Present study would be significant in the sustenance of quality characters for postharvest storage of Capsicum fruit with CO2-sensitization in biocompatible manner. The present experiment describes effects of CO2 sensitization on delaying postharvest ripening through physiological attributes in Capsicum fruit. The experiment was conducted with acidified bicarbonate-derived CO2 exposure for 2 h on Capsicum fruit, kept under white light at 25 °C through 7 days postharvest storage. Initially, fruits responded well to CO2 as recorded sustenance of greenness and integrity of fruit coat resolved through scanning electron micrograph. Loss of water and accumulation of total soluble solids were marginally increased on CO2-sensitized fruit as compared to non-sensitized (control) fruit. The ethylene metabolism biosynthetic genes like CaACC synthase, CaACC oxidase were downregulated on CO2-sensitization. Accompanying ethylene metabolism cellular respiration was downregulated on CO2 induction as compared to control through 7 days of storage. Fruit coat photosynthesis decarboxylating reaction by NADP malic enzyme was upregulated to maintain the reduced carbon accumulation as recorded on 7 days of storage under the same condition. CO2-sensitization effectively reduced the lipid peroxides as oxidative stress products on ripening throughout the storage. Anti-oxidation reaction essentially downregulates the ROS-induced damages of biomolecules that otherwise are highly required for food preservation during postharvest storage. Thus, the major finding is that CO2-sensitization maintains a higher ratio of unsaturated to saturated fatty acids in fruit coat during storage. Tissue-specific downregulation of ROS also maintained the nuclear stability under CO2 exposure. These findings provide basic as well as applied insights for sustaining Capsicum fruit quality with CO2 exposure under postharvest storage. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01471-4.

A natural fusion of flavodiiron, rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis.

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.

Reduction of bundle sheath size boosts cyclic electron flow in C4 Setaria viridis acclimated to low light.

When C4 leaves are exposed to low light, the CO2 concentration in the bundle sheath (BS) cells decreases, causing an increase in photorespiration relative to assimilation, and a consequent reduction in biochemical efficiency. These effects can be mitigated by complex acclimation syndromes, which are of primary importance for crop productivity but are not well studied. We unveil an acclimation strategy involving the coordination of electron transport processes. First, we characterize the anatomy, gas exchange and electron transport of C4 Setaria viridis grown under low light. Through a purposely developed biochemical model, we resolve the photon fluxes and reaction rates to explain how the concerted acclimation strategies sustain photosynthetic efficiency. Our results show that a smaller BS in low-light-grown plants limited leakiness (the ratio of CO2 leak rate out of the BS over the rate of supply via C4 acid decarboxylation) but sacrificed light harvesting and ATP production. To counter ATP shortage and maintain high assimilation rates, plants facilitated light penetration through the mesophyll and upregulated cyclic electron flow in the BS. This shade tolerance mechanism, based on the optimization of light reactions, is possibly more efficient than the known mechanisms involving the rearrangement of carbon metabolism, and could potentially lead to innovative strategies for crop improvement.

A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Systems metabolic engineering upgrades Corynebacterium glutamicum for selective high-level production of the chiral drug precursor and cell-protective extremolyte L-pipecolic acid.

The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol-1. Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol-1, representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L-1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid.