Structures of Toxic Advanced Glycation End-Products Derived from Glyceraldehyde, A Sugar Metabolite.

Advanced glycation end-products (AGEs) have recently been implicated in the onset/progression of lifestyle-related diseases (LSRDs); therefore, the suppression of AGE-induced effects may be used in both the prevention and treatment of these diseases. Various AGEs are produced by different biological pathways in the body. Glyceraldehyde (GA) is an intermediate of glucose and fructose metabolism, and GA-derived AGEs (GA-AGEs), cytotoxic compounds that accumulate and induce damage in mammalian cells, contribute to the onset/progression of LSRDs. The following GA-AGE structures have been detected to date: triosidines, GA-derived pyridinium compounds, GA-derived pyrrolopyridinium lysine dimers, methylglyoxal-derived hydroimidazolone 1, and argpyrimidine. GA-AGEs are a key contributor to the formation of toxic AGEs (TAGE) in many cells. The extracellular leakage of TAGE affects the surrounding cells via interactions with the receptor for AGEs. Elevated serum levels of TAGE, which trigger different types of cell damage, may be used as a novel biomarker for the prevention and early diagnosis of LSRDs as well as in evaluations of treatment efficacy. This review provides an overview of the structures of GA-AGEs.

Development of a Highly Selective NAD+ -Dependent Glyceraldehyde Dehydrogenase and its Application in Minimal Cell-Free Enzyme Cascades.

Anthropogenic climate change has been caused by over-exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell-free enzyme cascade generating pyruvate as the central intermediate, the NAD+ -dependent, selective oxidation of D-glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24-fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi-rational engineering improved the substrate selectivity of the enzyme up to 21-fold, thus minimizing side reactions in the one-pot enzyme cascade. The final biotransformation of D-glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications.

A strategic tool to improve the study of molecular determinants of Alzheimer's disease: The role of glyceraldehyde.

Alzheimer's disease (AD) is the most prevalent form of dementia and is characterized by progressive neurodegeneration leading to severe cognitive, memory, and behavioral impairments. The onset of AD involves a complex interplay among various factors, including age, genetics, chronic inflammation, and impaired energy metabolism. Despite significant efforts, there are currently no effective therapies capable of modifying the course of AD, likely owing to an excessive focus on the amyloid hypothesis and a limited consideration of other intracellular pathways. In the present review, we emphasize the emerging concept of AD as a metabolic disease, where alterations in energy metabolism play a critical role in its development and progression. Notably, glucose metabolism impairment is associated with mitochondrial dysfunction, oxidative stress, Ca2+ dyshomeostasis, and protein misfolding, forming interconnected processes that perpetuate a detrimental self-feeding loop sustaining AD progression. Advanced glycation end products (AGEs), neurotoxic compounds that accumulate in AD, are considered an important consequence of glucose metabolism disruption, and glyceraldehyde (GA), a glycolytic intermediate, is a key contributor to AGEs formation in both neurons and astrocytes. Exploring the impact of GA-induced glucose metabolism impairment opens up exciting possibilities for creating an easy-to-handle in vitro model that recapitulates the early stage of the disease. This model holds great potential for advancing the development of novel therapeutics targeting various intracellular pathways implicated in AD pathogenesis. In conclusion, looking beyond the conventional amyloid hypothesis could lead researchers to discover promising targets for intervention, offering the possibility of addressing the existing medical gaps in AD treatment.

An artificial multienzyme cascade for the whole-cell synthesis of rare ketoses from glycerol.

PURPOSE: In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. METHODS: To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. RESULTS: The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2 g l-1 only from the sole carbon source glycerol. CONCLUSION: This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.

Metagenomic insights into the symbiotic relationship in anammox consortia at reduced temperature.

Anammox as a promising biological nitrogen removal technology has attracted much attention. However, cold temperature would limit its wide application and little is known about the microbial interactions between anammox bacteria (AnAOB) and heterotrophic bacteria at cold temperature. Here, we observed reduced temperature (25-15 °C) promoted the secretion of EPS and thus stimulated bigger size of granular sludge in a laboratory-scale anammox reactor. We further combined co-occurrence network analysis and genome-centered metagenomics to explore the potential interactions between AnAOB and heterotrophic bacteria. Network analysis suggested 22 out of 25 positively related species were reported as definite heterotrophic bacteria in subnetwork of AnAOB. Genome-centered metagenomics analysis yielded 23 metagenomic assembly genomes (MAGs), and we found that Acidobacteriota-affiliated bacteria could biosynthesize most polysaccharides (PS) precursors and contain the most glycosyltransferases and transporters to facilitate exopolysaccharides biosynthesis, together with partial PS precursors produced by AnAOB. AMX1 as the only anammox genome could synthesize most amino acids and cross feed with some heterotrophs to affect the extracellular protein function. Additionally, Bacteroidota, Planctomycetota, Chloroflexota, and Proteobacteria could contribute folate and molybdopterin cofactor for AMX1 to benefit their activity and growth. Superphylum Patescibacteria could survive by cross-feeding with AnAOB and heterotrophic organisms about organic compounds (Glyceraldehyde-3P and lactate). These cross-feedings maintained the stability of anammox reactor performance and emphasize the importance of heterotrophs in anammox system at reduced temperature.

The TKFC Ala185Thr variant, reported as 'null' for fructose metabolism, is fully active as triokinase.

TKFC-encoded triokinase catalyses glyceraldehyde phosphorylation in fructose metabolism and favours lipogenesis in mice. In Tkfc knockouts or knockdowns, fructose oxidation predominates over lipogenesis. The highly prevalent human variant Ala185Thr-Triokinase/FMN cyclase (TKFC) has been reported to be 'null' for fructose metabolism, since Ala185-TKFC rescues the mouse TKFC-deficient phenotype, whereas Ala185Thr-TKFC does not. Such report implies that most humans would display a noncanonical fructose metabolism, but it ignores the well-characterized triokinase activity of Ala185Thr-TKFC. Here, earlier evidence is summarized, along with new evidence that both human variants are equally active in yeast. Therefore, future research on triokinase in the context of human fructose metabolism should consider that Ala185Thr-TKFC is not biochemically 'null'.

Reuterin in the healthy gut microbiome suppresses colorectal cancer growth through altering redox balance.

Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricts colon tumor growth, increases tumor reactive oxygen species, and decreases protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.

Probiotic and Functional Properties of Limosilactobacillus reuteri INIA P572.

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.

Substrate usage determines carbon flux via the citrate cycle in Helicobacter pylori.

Helicobacter pylori displays a worldwide infection rate of about 50%. The Gram-negative bacterium is the main reason for gastric cancer and other severe diseases. Despite considerable knowledge about the metabolic inventory of H. pylori, carbon fluxes through the citrate cycle (TCA cycle) remained enigmatic. In this study, different 13 C-labeled substrates were supplied as carbon sources to H. pylori during microaerophilic growth in a complex medium. After growth, 13 C-excess and 13 C-distribution were determined in multiple metabolites using GC-MS analysis. [U-13 C6 ]Glucose was efficiently converted into glyceraldehyde but only less into TCA cycle-related metabolites. In contrast, [U-13 C5 ]glutamate, [U-13 C4 ]succinate, and [U-13 C4 ]aspartate were incorporated at high levels into intermediates of the TCA cycle. The comparative analysis of the 13 C-distributions indicated an adaptive TCA cycle fully operating in the closed oxidative direction with rapid equilibrium fluxes between oxaloacetate-succinate and α-ketoglutarate-citrate. 13 C-Profiles of the four-carbon intermediates in the TCA cycle, especially of malate, together with the observation of an isocitrate lyase activity by in vitro assays, suggested carbon fluxes via a glyoxylate bypass. In conjunction with the lack of enzymes for anaplerotic CO2 fixation, the glyoxylate bypass could be relevant to fill up the TCA cycle with carbon atoms derived from acetyl-CoA.

3-Hydroxypropionic acid contributes to the antibacterial activity of glycerol metabolism by the food microbe Limosilactobacillus reuteri.

Strains of Limosilactobacillus reuteri are used as starter and bioprotective cultures and contribute to the preservation of food through the production of fermentation metabolites lactic and acetic acid, and of the antimicrobial reuterin. Reuterin consists of acrolein and 3-hydroxypropionaldehyde (3-HPA), which can be further metabolized to 1,3-propanediol and 3-hydroxypropionic acid (3-HP). While reuterin has been the focus of many investigations, the contribution of 3-HP to the antimicrobial activity of food related reuterin-producers is unknown. We show that the antibacterial activity of 3-HP was stronger at pH 4.8 compared to pH 5.5 and 6.6. Gram-positive bacteria were in general more resistant against 3-HP and propionic acid than Gram-negative indicator strains including common food pathogens, while spoilage yeast and molds were not inhibited by ≤ 640 mM 3-HP. The presence of acrolein decreased the minimal inhibitory activity of 3-HP against E. coli indicating synergistic antibacterial activity. 3-HP was formed during the growth of the reuterin-producers, and by resting cells of L. reuteri DSM 20016. Taken together, this study shows that food-related reuterin producers strains synthesize a second antibacterial compound, which might be of relevance when strains are added as starter or bioprotective cultures to food products.

RasGRP2 inhibits glyceraldehyde-derived toxic advanced glycation end-products from inducing permeability in vascular endothelial cells.

Advanced glycation end-products (AGEs) are formed by the non-enzymatic reaction of sugars and proteins. Among the AGEs, glyceraldehyde-derived toxic AGEs (TAGE) are associated with various diseases, including diabetic complications such as diabetic retinopathy (DR). The risk of developing DR is strongly associated with poor glycemic control, which causes AGE accumulation and increases AGE-induced vascular permeability. We previously reported that Ras guanyl nucleotide releasing protein 2 (RasGRP2), which activates small G proteins, may play an essential role in the cell response to toxicity when exposed to various factors. However, it is not known whether RasGRP2 prevents the adverse effects of TAGE in vascular endothelial cells. This study observed that TAGE enhanced vascular permeability by disrupting adherens junctions and tight junctions via complex signaling, such as ROS and non-ROS pathways. In particular, RasGRP2 protected adherens junction disruption, thereby suppressing vascular hyper-permeability. These results indicate that RasGRP2 is an essential protective factor of vascular permeability and may help develop novel therapeutic strategies for AGE-induced DR.

Human serine racemase is inhibited by glyceraldehyde 3-phosphate, but not by glyceraldehyde 3-phosphate dehydrogenase.

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex. Rather, hSR is inhibited by the hGAPDH substrate glyceraldehyde 3-phosphate (G3P) in a time- and concentration-dependent fashion, likely through a covalent reaction of the aldehyde functional group. The inhibition was similar for the two G3P enantiomers but it was not observed for structurally similar aldehydes. We ruled out a mechanism of inhibition based on the competition with either pyridoxal phosphate (PLP) - described for other PLP-dependent enzymes when incubated with small aldehydes - or ATP. Nevertheless, the inhibition time course was affected by the presence of hSR allosteric and orthosteric ligands, suggesting a conformation-dependence of the reaction.

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation.

Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.

Characterization of alditol oxidase from Streptomyces coelicolor and its application in the production of rare sugars.

A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor d-glyceraldehyde was directly produced from glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and d-glyceraldehyde was performed by l-rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phosphate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars d-sorbose and d-psicose. To accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldOS.coe) was expressed in E. coli and the purified AldOS.coe was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes including AldOS.coe was constructed. Under the optimized conditions, it produced 7.9 g/L of d-sorbose and d-psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective method for the synthesis of rare sugars.

Growth of Lactobacillus reuteri DSM17938 Under Two Simulated Microgravity Systems: Changes in Reuterin Production, Gastrointestinal Passage Resistance, and Stress Genes Expression Response.

Extreme factors such as space microgravity, radiation, and magnetic field differ from those that occur on Earth. Microgravity may induce and select some microorganisms for physiological, metabolic, and/or genetic variations. This study was conducted to determine the effects of simulated microgravity conditions on the metabolism and gene expression of the probiotic bacterium Lactobacillus reuteri DSM17938. To investigate microbial response to simulated microgravity, two devices-the rotating wall vessel (RWV) and the random positioning machine (RPM)-were used. Microbial growth, reuterin production, and resistance to gastrointestinal passage were assessed, and morphological characteristics were analyzed by scanning electron microscopy. The expression of some selected genes that are responsive to stress conditions and to bile salts stress was evaluated through real-time quantitative polymerase chain reaction assay. Monitoring of bacterial growth, cell size, and shape under simulated microgravity did not reveal differences compared with 1 × g controls. On the contrary, an enhanced production of reuterin and a greater tolerance to the gastrointestinal passage were observed. Moreover, some stress genes were upregulated under RWV conditions, especially after 24 h of treatment, whereas RPM conditions seemed to determine a downregulation over time of the same stress genes. These results show that simulated microgravity could alter some physiological characteristics of L. reuteri DSM17938 with regard to tolerance toward stress conditions encountered on space missions and could be useful to elucidate the adaptation mechanisms of microbes to the space environment.

Non-enzymatic reaction of carnosine and glyceraldehyde-3-phosphate accompanies metabolic changes of the pentose phosphate pathway.

OBJECTIVES: Carnosine (ß-alanyl-l-histidine) is a naturally occurring dipeptide that selectively inhibits cancer cell growth, possibly by influencing glucose metabolism. As its precise mode of action and its primary targets are unknown, we analysed carnosine's effect on metabolites and pathways in glioblastoma cells. MATERIALS AND METHODS: Glioblastoma cells, U87, T98G and LN229, were treated with carnosine, and metabolites were analysed by gas chromatography coupled with mass spectrometry. Furthermore, mitochondrial ATP production was determined by extracellular flux analysis and reaction products of carnosine were investigated using mass spectrometry. RESULTS: Carnosine decreased the intracellular abundance of several metabolites indicating a reduced activity of the pentose phosphate pathway, the malate-aspartate shuttle and the glycerol phosphate shuttle. Mitochondrial respiration was reduced in U87 and T98G but not in LN229 cells, independent of whether glucose or pyruvate was used as substrate. Finally, we demonstrate non-enzymatic reaction of carnosine with dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. However, glycolytic flux from glucose to l-lactate appeared not to be affected by the reaction of carnosine with the metabolites. CONCLUSIONS: Carnosine reacts non-enzymatically with glycolytic intermediates reducing the activity of the pentose phosphate pathway which is required for cell proliferation. Although the activity of the malate-aspartate and the glycerol phosphate shuttle appear to be affected, reduced mitochondrial ATP production under the influence of the dipeptide is cell-specific and appears to be independent of the effect on the shuttles.

Cascade synthesis of rare ketoses by whole cells based on L-rhamnulose-1-phosphate aldolase.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases demonstrate important values in the production of rare ketoses due to their unique stereoselectivities. As a specific example, we developed an efficient Escherichia coli whole-cell biocatalytic cascade system in which rare ketoses were produced from abundant glycerol and catalyzed by four enzymes based on L-rhamnulose-1-phosphate aldolase (RhaD). For the semicontinuous bioconversion in which D-glyceraldehyde was continuously added, once D-glyceraldehyde was consumed, the final yields of D-sorbose and D-psicose were 15.30 g/L and 6.35 g/L, respectively. Moreover, the maximum conversion rate and productivity of D-sorbose and D-psicose were 99% and 1.11 g/L/h at 8 h, respectively. When L-glyceraldehyde was used instead of the D-isomer, the final yield of L-fructose was 16.80 g/L. Furthermore, the maximum conversion rate and productivity of L-fructose were 95% and 1.08 g/L/h at 8 h, respectively. This synthetic platform was also compatible with other various aldehydes, which allowed the production of many other high-value chemicals from glycerol.

The Human Fecal Microbiota Metabolizes Foodborne Heterocyclic Aromatic Amines by Reuterin Conjugation and Further Transformations.

SCOPE: Heterocyclic aromatic amines (HAAs) are process-induced food contaminants with high mutagenic and/or carcinogenic potential. Although the human gut microbiota is known to affect the metabolism of dietary constituents, its impact on HAA metabolism and toxicity has been little studied. Here, the glycerol-dependent metabolism of seven foodborne HAAs (AαC, Trp-P-1, harman, norharman, PhIP, MeIQx, and MeIQ) by the human fecal microbiota is investigated. METHODS AND RESULTS: As analyzed by HPLC-DAD/FLD, the extent of conversion is strongly dependent on glycerol supplementation and HAA structure. AαC (60-100%) and the 2-aminoimidazoazarenes (up to 58%) are especially prone to microbial conversion. Based on high-resolution MS and/or NMR spectroscopy data, 70 fecal metabolites are identified in total, mainly formed by chemical reactions with one or two molecules of microbially derived reuterin. Moreover, it has been demonstrated that the human fecal microbiota can further transform reuterin adducts by reduction and/or hydroxylation reactions. Upon isolation, some reuterin-induced HAA metabolites appear to be partially unstable, complicating structural identification. CONCLUSION: The formation of microbial metabolites needs to be incorporated into risk assessment considerations for HAAs in human health. In this study, several HAA metabolites, mainly reuterin-dependent, are identified in vitro, providing the basis for future human studies investigating microbial HAA metabolism.

Lactobacillus reuteri NAD(P)H oxidase: Properties and coexpression with propanediol-utilization enzymes for enhancing 3-hydroxypropionic acid production from 3-hydroxypropionaldehyde.

Lactobacillus reuteri metabolizes glycerol through propanediol-utilization (Pdu) pathway to 1,3-propanediol (1,3-PD) via 3-hydroxypropionaldehyde (3-HPA) as intermediate. In the resting cells, the oxidized co-factor obtained in the reaction is regenerated by simultaneous oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP) using propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). We have earlier shown that the use of resting cells of recombinant Escherichia coli expressing the oxidative pathway gives the highest theoretical yield of 1 mol 3-HP per mol 3-HPA but is limited by cofactor depletion. In the present study, the gene encoding the enzyme NAD(P)H oxidase (LreuNox) that utilizes molecular oxygen as substrate, was isolated from L. reuteri and heterologously overexpressed in E. coli. LreuNox has a pH optimum of 6 and exhibits Vmax of 101.1 ± 2.2 U/mg with NADH, which is 30% higher than that for NADPH. Co-expression of LreuNox with PduP, PduL and PduW in E. coli enhances the biocatalytic lifetime as well as productivity at least two-fold compared to that achieved without co-factor regeneration.

Influence of environmental and genetic factors on 3-hydoxypropionaldehyde production by Lactobacillus reuteri.

The influence of environmental factors such as glycerol concentration, time of production, presence of Escherichia coli, and two different strains of Lactobacillus reuteri (ATCC 55730 and ATCC 53608) on 3-hydroxypropionaldehyde (3-HPA) production was analyzed. Additionally, the influence of those factors on gene expression in the 3-HPA production pathway was evaluated. The genes studied were GldC, cbiP, and Lreu_1734. The results of this study showed that the principal environmental factors that influence 3-HPA production are glycerol concentration and Lactobacillus reuteri strain. As glycerol concentration increased, 3-HPA content increased. The greatest 3-HPA concentration (56.6 mM ±5.99) was achieved by L. reuteri ATCC 55730. Gene expression was also affected by environmental factors. Factor that showed the greatest influence were also strain and glycerol concentration. The genes cbiP, GldC, and Lreu_1734 had basal gene expression in glycerol absence; however, glycerol regulated its expression. Glycerol induced overexpression of cbiP and GldC genes (Strain ATCC 53608), probably to ensure its efficient utilization. On the contrary, glycerol concentration suppressed Lre_1734 expression in both analyzed strains, as a mechanism for 3-HPA accumulation. Down-regulation was observed in all the genes tested in strain ATCC 55730, probably due to feedback inhibition by 3-HPA.