Lysine Production Research Articles

Grain Lysine enrichment and improved stress tolerance in rice through protein engineering.

Amino acids are a major source of nourishment for people living in regions where rice is a staple food. However, rice grain is deficient in essential amino acids, such as lysine. The activity of dihydrodipicolinate synthase (DHDPS) enzyme is crucial for lysine production in higher plants, but it is highly regulated through a feedback inhibition by its end product lysine, leading to its limited activity in the grain and resulting in low lysine accumulation. We identified lysine binding sites in the DHDPS enzyme and introduced key mutations to make it lysine feedback insensitive. Using in vivo analysis and functional complementation assays, we confirmed that protein engineering of the DHDPS renders it insensitive to lysine. Expression of mutated DHDPS resulted in 29 % higher lysine and 15 % higher protein accumulation in rice grains than the wild type. Importantly, the lysine content in transgenic grains was maintained in cooked rice. Further, the transgenic plants exhibited enhanced stress tolerance along with better antioxidant levels, improved photosynthesis, and higher grain yield compared to wild type plants. We have shown for the first time in rice that protein engineering of DHDPS can lead to accumulation of lysine in grains and impart abiotic stress tolerance. This approach could improve health in regions with nutrient deficiencies and environmental stressors that challenge food production and human health.

Engineering quorum sensing-based genetic circuits enhances growth and productivity robustness of industrial E. coli at low pH

BackgroundMicrobial organisms hold significant potential for converting renewable substrates into valuable chemicals. Low pH fermentation in industrial settings offers key advantages, including reduced neutralizer usage and decreased wastewater generation, particularly in the production of amino acids and organic acids. Engineering acid-tolerant strains represents a viable strategy to enhance productivity in acidic environments. Synthetic biology provides dynamic regulatory tools, such as gene circuits, facilitating precise expression of acid resistance (AR) modules in a just-in-time and just-enough manner.ResultsIn this study, we aimed to enhance the robustness and productivity of Escherichia coli, a workhorse for amino acid and organic acid production, in industrial fermentation under mild acidic conditions. We employed an Esa-type quorum sensing circuit to dynamically regulate the expression of an AR module (DsrA-Hfq) in a just-in-time and just-enough manner. Through careful engineering of the critical promoter PesaS and stepwise evaluation, we developed an optimal Esa-PBD(L) circuit that conferred upon an industrial E. coli strain SCEcL3 comparable lysine productivity and enhanced yield at pH 5.5 compared to the parent strain at pH 6.8.ConclusionsThis study exemplifies the practical application of gene circuits in industrial environments, which present challenges far beyond those of well-controlled laboratory conditions.

Influence of the Chemical Properties of Cereal Grains on the Structure and Metabolism of the Bacteriome of Rhyzopertha dominica (F.) and Its Development: A Cause-Effect Analysis.

Rhyzopertha dominica causes significant economic losses in stored cereals. Insects' digestive tract microbiome is crucial for their development, metabolism, resistance, and digestion. This work aimed to test whether the different chemical properties of different wheat and barley grain cultivars cause disturbances in insect foraging and rearrangements of the structure of the R. dominica microbiome. The results indicated that grain cultivars significantly influence the microbiome, metabolism, and insect foraging. Most observed traits and microbiome structures were not correlated at the species level, as confirmed by ANOSIM (p = 0.441). However, the PLS-PM analysis revealed significant patterns within barley cultivars. The study found associations between C18:2 fatty acids, entomopathogenic bacteria, an impaired nitrogen cycle, lysine production of bacterial origin, and insect feeding. The antioxidant effects also showed trends towards impacting the microbiome and insect development. The findings suggest that manipulating grain chemical properties (increasing C18:2 and antioxidant levels) can influence the R. dominica microbiome, disrupting their foraging behaviours and adaptation to storage environments. This research supports the potential for breeding resistant cereals, offering an effective pest control strategy and reducing pesticide use in food production.

A comparative guide to expression systems for phage lysin production.

Phage lysins, bacteriophage-encoded enzymes tasked with degrading their host's cell wall, are increasingly investigated and engineered as novel antibacterials across diverse applications. Their rapid action, tuneable specificity, and low likelihood of resistance development make them particularly interesting. Despite numerous application-focused lysin studies, the art of their recombinant production remains relatively undiscussed. Here, we provide an overview of the available expression systems for phage lysin production and discuss key considerations guiding the choice of a suitable recombinant host. We systematically surveyed recent literature to evaluate the hosts used in the lysin field and cover various recombinant systems, including the well-known bacterial host Escherichia coli or yeast Saccharomyces cerevisiae, as well as plant, mammalian, and cell-free systems. Careful analysis of the limited studies expressing lysins in various hosts suggests a host-dependent effect on activity. Nonetheless, the multitude of available expression systems should be further leveraged to accommodate the growing interest in phage lysins and their expanding range of applications.

Efficient synthesis of benzyl-protected cyclic lysine on Pt/m-Al2O3 catalysts and its application in functional nylon-6 copolymers production

Functional nylon-6 polymers, particularly those with special properties such as antibacterial, flame retardant, and specialized thermal properties, have a wide range of applications. However, the efficient synthesis of these polymer monomers has long remained a challenge. Benzyl-protected cyclic lysine (BCL) is a potentially functional monomer, and this article proposes an efficient method for the synthesis of benzyl-protected cyclic lysine on a supported platinum catalyst. Compared to existing techniques, this approach offers higher synthesis efficiency and sustainability. Pt/m-Al2O3 (microspherical Al2O3) catalysts were prepared using a vacuum impregnation method and showed good activity and selectivity in the production of benzyl-protected cyclic lysine. The conversion rate of α-amino-ε-caprolactam (α-ACL) was 100%, and the selectivity of BCL was 96.0%. The Pt/m-Al2O3 catalyst maintained good stability over multiple reaction cycles. The reaction mechanism and thermodynamics of functionalized α-ACL were also investigated. The copolymer synthesized with the prepared BCL showed similar structure and thermal stability to pure nylon-6, indicating possible unique functional properties. This study provides a route for the synthesis of cyclic lysine protected with various groups and its potential applications in the field of polymer chemistry.

A Self-Assembled MOF-Escherichia Coli Hybrid System for Light-Driven Fuels and Valuable Chemicals Synthesis.

The development of semi-artificial photosynthetic systems, which integrate metal-organic frameworks (MOFs) with industrial microbial cell factories for light-driven synthesis of fuels and valuable chemicals, represents a highly promising avenue for both research advancements and practical applications. In this study, an MOF (PCN-222) utilizing racemic-(4-carboxyphenyl) porphyrin and zirconium chloride (ZrCl4) as primary constituents is synthesized. Employing a self-assembly process, a hybrid system is constructed, integrating engineered Escherichia coli (E. coli) to investigate light-driven hydrogen and lysine production. These results demonstrate that the light-irradiated biohybrid system efficiently produce H2 with a quantum efficiency of 0.75% under full spectrum illumination, the elevated intracellular reducing power NADPH is also observed. By optimizing the conditions, the biohybrid system achieves a maximum lysine production of 18.25mg L-1, surpassing that of pure bacteria by 332%. Further investigations into interfacial electron transfer mechanisms reveals that PCN-222 efficiently captures light and facilitates the transfer of photo-generated electrons into E. coli cells. It is proposed that the interfacial energy transfer process is mediated by riboflavin, with facilitation by secreted small organic acids acting as hole scavengers for PCN-222. This study establishes a crucial foundation for future research into the light-driven biomanufacturing using E. coli-based hybrid systems.

Обеспечение продовольственной безопасности на основе развития импортозамещающих производств лизина и других аминокислот методом ферментации

the article presents the results of a study of the processes of ensuring food security based on the development of import-substituting production of lysine and other amino acids using the fermentation method. The dynamics of lysine in the Russian Federation, Belarus, China, and the countries of the European Union were analyzed. Opportunities and promising directions for increasing the production of domestic feed additives used in animal husbandry have been identified.

Metabolic Engineering of Corynebacterium glutamicum for Highly Efficient Production of Ectoine.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

Engineering a Lactobacillus Lysine Riboswitch to Dynamically Control Metabolic Pathways for Lysine Production in Corynebacterium glutamicum.

Knock-out of genes of metabolic pathways is conventionally used in the metabolic engineering of microorganisms, but it is not applicable for genes of essential pathways. In order to avoid undesirable effects caused by gene deletion, it is attractive to develop riboswitches to dynamically control the metabolic pathways of microbial cell factories. In this regard, the aim of this study is to utilize the lysine riboswitch to control gene expressions of the biosynthetic pathways and by-pathways and thus improve lysine production in Corynebacterium glutamicum. To achieve this, a natural lysine riboswitch from Lactobacillus plantarum (LPRS) was first detected and then fused with RFP to test its functionality. After that, engineered lysine-activated (Lys-A) and lysine-repressed (Lys-R) riboswitches were successfully screened by dual genetic selection. Furthermore, the optimized A263 and R152 were applied to control the expression of aspartate kinase III and homoserine dehydrogenase in the lysine-producing strain C. glutamicum QW45, respectively. In contrast with QW45, the growth of the resulting A263-lysC mutant QW48 was similar to that of QW45; however, the growth of the resulting R357-hom mutant QW54 was slightly inhibited, indicating an inhibition of threonine biosynthesis caused by the riboswitch upon binding of intracellular lysine. Importantly, the lysine production of QW48 and QW54 was, respectively, 35% and 43% higher than that of the parent strain QW45, implying more metabolic flux directed into the lysine synthesis pathway. Finally, the engineered A263 and R357 were simultaneously applied to the same mutant QW55, which greatly improved lysine production. Thus, the approach demonstrated in this work could be principally used as a powerful tool to dynamically control any other undesired metabolic pathways.

The Genome of Arsenophonus sp. and Its Potential Contribution in the Corn Planthopper, Peregrinus maidis.

The co-evolution between symbionts and their insect hosts has led to intricate functional interdependencies. Advances in DNA-sequencing technologies have not only reduced the cost of sequencing but, with the advent of highly accurate long-read methods, have also enabled facile genome assembly even using mixed genomic input, thereby allowing us to more easily assess the contribution of symbionts to their insect hosts. In this study, genomic data recently generated from Peregrinus maidis was used to assemble the genome of a bacterial symbiont, Pm Arsenophonus sp. This ~4.9-Mb assembly is one of the largest Arsenophonus genomes reported to date. The Benchmarking Universal Single-Copy Orthologs (BUSCO) result indicates that this Pm Arsenophonus assembly has a high degree of completeness, with 96% of the single-copy Enterobacterales orthologs found. The identity of the Pm Arsenophonus sp. was further confirmed by phylogenetic analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicates a major contribution by Pm Arsenophonus sp. to the biosynthesis of B vitamins and essential amino acids in P. maidis, where threonine and lysine production is carried out solely by Pm Arsenophonus sp. This study not only provides deeper insights into the evolutionary relationships between symbionts and their insect hosts, but also adds to our understanding of insect biology, potentially guiding the development of novel pest control methods.

A novel biochemistry approach combined with MALDI-TOF MS to discriminate Escherichia coli and Shigella species

Escherichia coli and Shigella spp. are closely related, making it crucial to accurately identify them for disease control and prevention. In this study, we utilized MALDI-TOF MS to identify characteristic peaks of decarboxylation products of lysine and ornithine to distinguish between E. coli and Shigella spp. Our findings indicate that the peak at m/z 103.12 ± 0.1 of the product cadaverine from lysine decarboxylase is unique to E. coli, while all Shigella species lack the m/z 103.12 ± 0.1 peak. However, S. sonnei and S. boydii serotype C13 exhibit a specific peak at m/z 89.10 ± 0.1, which is the product of putrescine from ornithine decarboxylase. We were able to correctly identify 97.06% (132 of 136) of E. coli and Shigella isolates and 100% (8 of 8) of S. sonnei isolates using this biochemical-based MALDI-TOF MS detection system. This technology is advantageous for its high-throughput, high quality, and ease of operation, and is of significant value for the diagnosis of E. coli and Shigella-related diseases.

Production of L-lysine under submerged fermentation by <i>Corynebacterium glutamicum</i> using different agricultural plants leaves

Introduction: This study investigated the production of L-lysine using Corynebacterium glutamicum and various leaf extracts (cassava, palm tree, maize, cowpea, cocoyam, and plantain). The study also explored the activities of amylases and proteases, as well as the levels of total soluble proteins, reducing sugars, glucose, flavonoid and phenolic contents, and pH changes.
 Materials and Methods: Different treatments (extract, boiled extract, extract + C. glutamicum, boiled extract + C. glutamicum) were examined for their effects on L-lysine concentration. Additionally, the activities of amylases and proteases, as well as levels of total soluble proteins, reducing sugars, glucose, flavonoid and phenolic contents, and pH changes, were analyzed.
 Results: Maize leaf extract + C. glutamicum exhibited the highest L-lysine concentration (1.771a±0.1 mg/g), while boiled cassava leaf extract showed the lowest concentration (0.023b±0.1 mg/g). Palm tree leaf extract had significantly higher reducing sugar levels compared to other extracts. Boiled plantain leaf extract fermented by C. glutamicum had the highest total soluble protein level (9.5±0.2 mg/g), while cassava leaf extract had the lowest (2.1±1.2 mg/g).
 Conclusion: Submerged fermentation of leaf extracts using C. glutamicum can be utilized for L-lysine production. The study highlights the influence of different leaf extracts and treatments on L-lysine production, as well as on amylase and protease activities, total soluble protein levels, reducing sugars, glucose, flavonoid and phenolic contents, and pH values. These findings provide valuable insights into the potential application of this approach for lysine production.

Synthesis and characterization of the N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) alternate substrate analog N,N-dimethyl-l,l-SDAP

Growing antibiotic resistance by pathogenic bacteria has led to a global crisis. The bacterial enzyme N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) provides a very attractive target for the discovery of a new class of antibiotics, as it resides exclusively in many pathogenic bacterial strains and is a key enzyme in the lysine biosynthetic pathway. This pathway is responsible for the production of lysine as well as meso-diaminopimelate (m-DAP), both of which are required for peptidoglycan cell-wall synthesis, and lysine for peptide synthesis. The enzyme DapE catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP), and due to its absence in humans, inhibition of DapE avoids mechanism-based side effects. We have executed the asymmetric synthesis of N,N-dimethyl-SDAP, an l,l-SDAP substrate analog and an analog of the synthetic substrate of our previously described DapE assay. Previous modeling studies advocated that N,N-dimethyl-SDAP might function as an inhibitor, however the compound behaves as a substrate, and we have demonstrated the use of N,N-dimethyl-SDAP as the substrate in a modified ninhydrin-based DapE assay. Thermal shift experiments of DapE in the presence of N,N-dimethyl-SDAP are consistent with a melt temperature (Tm) shifted by succinate, the product of enzymatic hydrolysis.

Multi-objective optimization of fed-batch bioreactor for lysine production

ABSTRACT Lysine production via the fermentation process is one of the most economical production routes that involves simultaneous conflicting objectives. Two multi-objective differential evolution algorithms MODE-III and MODE-III-IMS are used to obtain the optimal control parameters of the lysine bioreactor. Two conflicting objectives, namely yield and productivity, are studied. Singular and constant feeding policies using both algorithms are studied, and respective Pareto fronts are reported. Tournament selection and penalty constraint handling methods are used, and their performances are compared for both algorithms. The higher bound of feeding rate (2.0 L/s) is found to be the best feeding rate compared to other feeding rates. The MODE-III-IMS algorithm converged to the Pareto front faster than the MODE-III algorithm. The COPRAS method is used to carry out the Pareto ranking, and the best optimal solution is reported. The decision tree method is used to predict and report the best optimal solution with acceptable accuracy.

Enhanced L-lysine Production by UV-irradiated and S-2-aminoethyl-L-cysteine Resistant Mutants Derived from Bacillus species Using Agricultural Products as Carbon and Nitrogen Sources

L-Lysine is essential for human and animal nutrition and may be added to food and feed materials to improve the protein quality. It is utilized in human medicine, in cosmetics and in the pharmaceutical industry, particularly in the formulation of diets with balanced amino acid concentration and in amino acid infusions. Enhanced L-lysine production by UV irradiated and S-2-aminoethyl-L-cysteine resistant (AECR) mutants derived from Bacillus species, using agricultural products as carbon and nitrogen sources was studied. The L-lysine producing bacteria had already been isolated from Nigerian soil. They were purified and identified as B. subtilis PR13, B. subtilis PR9 and B. pumilus SS16, using cultural and biochemical characteristics. UV irradiated mutants were obtained by exposing the parent strains which include B. subtilis PR13, B. subtilis PR9 and B. pumilus SS16, to UV light while, AECR mutants were derived by exposing the parent strains to various concentrations of AEC. The mutants were screened for L-lysine production by cultivating in fermentation media. The results obtained showed that, UV mutants PRMU-8 and PRMU-14 derived from Bacillus subtilis PR13 accumulated higher lysine yield of 2.47 and 2.55 mg/ml compared to the wild type. Also, UV mutant MUPR-4 derived from Bacillus subtilis PR9 recorded a higher lysine yield of 1.77 mg/ml while, enhanced lysine yields of 1.96 and 2.34 mg/ml were observed in UV mutants SSMU-4 and SSMU-9 respectively. AECR mutant PRT-2 derived from Bacillus subtilis PR13, accumulated higher lysine yield of 2.35 mg/ml compared to the wild type. Enhanced lysine production of 1.47 and 1.32 mg/ml was observed in mutants ART-5 and ART-8 derived from B. subtilis PR9 while, mutant SRT-4 derived from B. pumilus SS16 recorded enhanced lysine accumulation of 2.18 mg/ml. The results obtained in the study showed that L-lysine production by some UV irradiated and AECR mutants was enhanced.

The Influence of Vitamins, Antibiotics and Growth Stimulators on L-lysine Production by Bacillus subtilis Using Agricultural Products as Carbon and Nitrogen Sources

L-Lysine is one of the 9 amino acids which are essential for human and animal nutrition. It is a basic building block of all proteins and cannot be synthesized biologically in the body. The influence of vitamins, antibiotics and growth stimulators on L-lysine production by Bacillus subtilis using agricultural products as carbon and nitrogen sources was examined. The L-lysine producing bacteria had already been isolated from Nigerian soil. They were purified and Identified as B. subtilis PR13 and B. subtilis PR9, using cultural and biochemical characteristics. The of influence of vitamins, antibiotics and growth stimulators on L-lysine production by Bacillus subtilis PR13 and 9 was evaluated in 100 ml flasks containing 20 ml fermentation media (FM1 and FM2). The results obtained showed that, an enhanced lysine yield of 3.41 and 1.57 mg/ml by Bacillus subtilis PR13 and Bacillus subtilis PR9, was observed in the presence of 1 µg/ml of biotin and 10 µg/ml respectively. The supplementation of 0.04 units/ml of penicillin, enhanced optimum L-lysine yield of 2.38 mg/ml for B. subtilis PR 13 and 1.64 mg/ml for B. subtilis PR9 respectively. The addition of 0.1% w/v peptone and yeast extract, enhanced optimum L-lysine yield of 2.66 mg/ml for B. subtilis PR 13 and 1.72 mg/ml for B. subtilis PR9 respectively. There was a positive correlation between peptone and L-lysine production by B. subtilis PR13 (r= 0.85) and yeast extract and L- lysine production by B. subtilis PR9 (r= 0.54). The results obtained in the study showed that the supplementation of the fermentation media with some vitamins, antibiotics and growth stimulators enhanced the L-lysine yield of B. subtilis PR13 and B. subtilis PR9.

Purification, crystallization, and X‐ray diffraction analysis of succinyl‐diaminopimelate desuccinylase from Wolbachia endosymbiont of Brugia malayi

AbstractLymphatic filariasis (LF) is a debilitating mosquito‐borne parasitic disease caused by Brugia malayi in humans in the lymphatic system. Although limited drugs are available to treat this disease, these drugs are only effective against the larvae of Brugia malayi. As resistance has been reported to the available drugs, a new antifilarial drug is needed with better prognostic features, a short period of time of use, and efficiency at preventing adult worm survival. In this study, we have targeted succinyl‐diaminopimelate desuccinylase (DapE) from the Wolbachia endosymbiont of Brugia malayi (WBm), which is involved in the production of lysine and meso‐diaminopimelic acid, an essential component for the formation of bacterial cell walls, and plays a vital role in pathogen survival. Deletion of DapE gene is lethal to WBm since the organism has no alternative pathway for lysine biosynthesis. Here, the expression, purification, and crystallization of DapE are reported. The crystals of DapE, with an estimated solvent content of 50.76%, diffract synchrotron X‐rays to a resolution of 2.3 Å and belong to the space group C2221, with unit‐cell parameters of a = 51.78, b = 78.83, and c = 216.20 Å. The X‐ray absorption spectroscopy further confirms that zinc atoms are incorporated in DapE.

Sustainable production of dimethyl-protected cyclic lysine over Pd/m-Al2O3-Si catalysts and its application in synthesis of antibacterial nylon‑6 copolymers

Antibacterial monomers are prerequisites for synthesizing antibacterial polymers, especially during the current COVID-19 pandemic. Dimethyl-protected cyclic lysine (DMCL) is a promising functional monomer for nylon-6 based self-cleaning antibacterial polymers. However, the production of DMCL still faces formidable challenges, such as harsh reaction conditions and low catalyst activities. In this study, we developed a Pd/m-Al2O3-Si catalyst, which exhibited high efficiency in converting α-amino-ε-caprolactam (α-ACL) to DMCL, affording a yield of as high as 97.1% at 100 °C and 1 MPa H2. The lack of Brönsted acid sites on the catalyst surface facilitated the formation of DMCL and suppressed undesirable hydrolysis or cracking by-products from the lactam-based reactant. The recycled experiments showed that Pd/m-Al2O3-Si performed excellent stability and physical strength with essentially no damage to its microspheres after the reaction. The nylon‑6 copolymers produced from the as-synthesized DMCL exhibited similar structure and thermal stability with pure nylon-6, showing great potential in synthesizing the self-cleaning antibacterial polymers. This work provides a sustainable and efficient method for producing DMCL and other lysine-based antibacterial monomers, showing a great prospect for the utilization of bio-based chemicals in synthesizing functional polymers.

Ionic Cooperativity between Lysine and Potassium in the Lysine Riboswitch: Single-Molecule Kinetic and Thermodynamic Studies.

Functionality in many biological systems, including proteins and nucleic acid structures, including protein and nucleic acid riboswitch structures, can depend on cooperative kinetic behavior between multiple small molecule ligands. In this work, single-molecule FRET data on the Bacillus subtilis lysine riboswitch reveals that affinity for the cognate lysine ligand increases significantly with K+, providing evidence for synergism between lysine/K+ binding to the aptamer and successful folding of the riboswitch. To describe/interpret this more complex kinetic scenario, we explore the conventional 4-state ("square") model for aptamer binding as a function of K+. Extension into this additional dimension generates a novel "cube" model for riboswitch folding dynamics with respect to lysine/K+ binding, revealing that riboswitch folding (kfold) and unfolding (kunfold) rate constants increase and decrease dramatically with K+, respectively. Furthermore, temperature-dependent single-molecule kinetic studies indicate that the presence of K+ entropically enhances the transition state barrier to folding but partially compensates for this by increasing the overall exothermicity for lysine binding. We rationalize this behavior as evidence that K+ facilitates hydrogen bonding between the negatively charged carboxyl group of lysine and the RNA, increasing structural rigidity and lowering entropy in the binding pocket. Finally, we explore the effects of cation size with Na+ and Cs+ studies to demonstrate that K+ is optimally suited for bridging interactions between lysine and the riboswitch aptamer domain. Regulation of lysine production and transport, dictated by the riboswitch's ability to recognize and bind lysine, is therefore intimately tied to the presence of K+ in the binding pocket and is strongly modulated by local cation conditions. The results suggest an increase in lysine riboswitch functionality by sensitivity to additional species in the cellular riboswitch environment.

Metabolic heterogeneity and cross-feeding within isogenic yeast populations captured by DILAC

Genetically identical cells are known to differ in many physiological parameters such as growth rate and drug tolerance. Metabolic specialization is believed to be a cause of such phenotypic heterogeneity, but detection of metabolically divergent subpopulations remains technically challenging. We developed a proteomics-based technology, termed differential isotope labelling by amino acids (DILAC), that can detect producer and consumer subpopulations of a particular amino acid within an isogenic cell population by monitoring peptides with multiple occurrences of the amino acid. We reveal that young, morphologically undifferentiated yeast colonies contain subpopulations of lysine producers and consumers that emerge due to nutrient gradients. Deconvoluting their proteomes using DILAC, we find evidence for in situ cross-feeding where rapidly growing cells ferment and provide the more slowly growing, respiring cells with ethanol. Finally, by combining DILAC with fluorescence-activated cell sorting, we show that the metabolic subpopulations diverge phenotypically, as exemplified by a different tolerance to the antifungal drug amphotericin B. Overall, DILAC captures previously unnoticed metabolic heterogeneity and provides experimental evidence for the role of metabolic specialization and cross-feeding interactions as a source of phenotypic heterogeneity in isogenic cell populations.